Compositions, methods, systems and / or kits for detecting antimicrobial resistance in bacteria

Abstract

To provide a correct detection method of carbapenemase production.SOLUTION: There is provided a method for determining existence of carbapenemase of zero, one or a plurality of Ambler classes expressed by enteric bacteria, the method comprising: a step for providing a sample including the enteric bacteria; a step for applying the enteric bacteria in a test sample for at least four test compositions for a duration, each of the at least four test compositions includes a growth medium and an antibiotic agent, and at least one of the at least four test compositions further comprises at least one carbapenemase inhibitor; and a step for detecting existence or inhibition of growth of the enteric bacteria in each of the at least four test compositions after the duration, for determining existence of carbapenemase of zero, one or a plurality of Ambler classes expressed by enteric bacteria.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure generally relates to detection tests, including compositions, methods, systems, and / or kits, for the detection of bacteria using enzymes that confer resistance to drugs. Certain embodiments of the disclosure relate to detection tests, including compositions, methods, systems, and / or kits, for the detection and / or identification of carbapenemase-producing Gram-negative bacteria. [Background technology]

[0002] Carbapenemase-producing Gram-negative bacteria pose a major and serious threat to public health worldwide, with few next-generation antibiotic options available for use against these pathogens. Pharmaceutical companies are currently targeting a large number of new antibiotics in the pipeline, but none have coverage across the full range of carbapenemase enzyme types (classes) that these bacteria can acquire.

[0003] Accurate detection of carbapenemase production and differentiation of β-lactamase classes are important for determining antimicrobial therapy, epidemiology, and infection control measures. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 5,922,593 [Patent Document 2] U.S. Patent No. 6,096,272 [Patent Document 3] U.S. Patent No. 6,372,485 [Patent Document 4] U.S. Patent No. 7,115,384 [Patent Document 5] U.S. Patent No. 9,304,141 [Patent Document 6] US Patent Application Publication No. 2009 / 0142796 Summary of the Invention

[0005] One embodiment includes a method for determining the presence of zero, one, or more Ambler-class carbapenemases expressed by Enterobacteriaceae, comprising: providing a sample containing Enterobacteriaceae; applying the Enterobacteriaceae in the test sample to a plurality of at least four test compositions over a sustained period of time, each of the plurality of at least four test compositions comprising a growth medium and an antibiotic, and at least one of the at least four test compositions further comprising at least one carbapenemase inhibitor; and determining the presence of zero, one, or more Ambler-class carbapenemases expressed by the Enterobacteriaceae by detecting the presence or inhibition of growth of the Enterobacteriaceae in each of the plurality of at least four test compositions after the sustained period of time. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprise, consist of, or consist essentially of a first concentration of TEM and an Ambler-class B carbapenemase inhibitor. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler A carbapenemase inhibitor. In any of the embodiments disclosed herein, the antibiotic in at least one test composition comprise, consist of, or consist essentially of a second concentration of DOR.In any of the embodiments disclosed herein, the method may include determining that one or more Ambler class carbapenemases expressed by the Enterobacteriaceae are class D by detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of TEM and an Ambler class B carbapenemase inhibitor. In any of the embodiments disclosed herein, the method may include determining that one or more Ambler class carbapenemases expressed by the Enterobacteriaceae are class A by detecting: inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of TEM and an Ambler class B carbapenemase inhibitor; the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; and inhibition of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor.

[0006] In any of the embodiments disclosed herein, the method may include determining that one or more Ambler class carbapenemases expressed by the Enterobacteriaceae are class B by detecting inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of TEM at a first concentration as the antibiotic and further comprise an Ambler class B carbapenemase inhibitor; inhibition of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of DOR at a first concentration, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; and the presence of growth in a fourth test composition, wherein the antibiotic comprises, consist of, or consist essentially of DOR at a second concentration. In any of the embodiments disclosed herein, the method may include determining that one or more Ambler class carbapenemases expressed by the Enterobacteriaceae are class D by detecting inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of TEM at a first concentration and an Ambler class B carbapenemase inhibitor; in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of DOR at a first concentration, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; and in a fourth test composition, wherein the antibiotic comprises, consist of, or consist essentially of DOR at a second concentration.

[0007] In any of the embodiments disclosed herein, the method may include determining the presence of one or more Ambler class carbapenemases expressed by the Enterobacteriaceae, where the Ambler class is not identified, but includes determining the presence of growth in a first test composition, where the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of TEM and an Ambler class B carbapenemase inhibitor, and the presence of growth in a second test composition, where the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR The presence of growth is determined by detecting the presence of growth in a third test composition of the plurality of at least four test compositions, the antibiotics and inhibitors comprising, consisting of, or consisting essentially of a first concentration of MEM as the antibiotic, and further comprising an Ambler class C carbapenemase inhibitor and an Ambler class A carbapenemase inhibitor.

[0008] In any of the embodiments disclosed herein, the method may comprise determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by the Enterobacteriaceae by detecting inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of TEM, an Ambler class B carbapenemase inhibitor; the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; and the presence of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a third concentration of MEM and an Ambler class C carbapenemase inhibitor.In any of the embodiments disclosed herein, the method includes inhibiting growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of TEM and an Ambler class B carbapenemase inhibitor; the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; and the presence of growth in a third test composition of the plurality of at least four test compositions, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor. The method may further include determining a lack of confidence in identifying one or more Ambler class carbapenemases expressed by the Enterobacteriaceae by detecting the presence of growth in a fifth test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor, and inhibition of growth in a fifth test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of MEM, and an Ambler class C carbapenemase inhibitor.In any of the embodiments disclosed herein, the method includes inhibiting growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of TEM and an Ambler class B carbapenemase inhibitor; the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; and the presence of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor. and the inhibitor comprises, consists of, or consists essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor, and the presence of growth in a fifth test composition, wherein the antibiotic and inhibitor comprise, consists of, or consists essentially of a third concentration of MEM and an Ambler class C carbapenemase inhibitor, thereby determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by the Enterobacteriaceae.

[0009] One embodiment includes a method for determining the presence of zero, one, or more Ambler class carbapenemases expressed by Enterobacteriaceae, the method comprising: providing a sample containing Enterobacteriaceae; applying the Enterobacteriaceae in the test sample to a plurality of at least four test compositions over a sustained period of time, each of the plurality of at least four test compositions comprising a growth medium and an antibiotic, and at least one of the at least four test compositions further comprising at least one carbapenemase inhibitor; and determining the presence of zero, one, or more Ambler class carbapenemases expressed by the Enterobacteriaceae by detecting the presence or inhibition of growth of the Enterobacteriaceae in each of the plurality of at least four test compositions after the sustained period of time. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler A carbapenemase inhibitor. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a second concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler A carbapenemase inhibitor.In any of the embodiments disclosed herein, the method may include determining that one or more Ambler class carbapenemases expressed by the Enterobacteriaceae are class A by detecting inhibition of growth in a first test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor, and an inhibition of growth in a second test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of a second concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor. In any of the embodiments disclosed herein, the method may include determining that one or more Ambler class carbapenemases expressed by the Enterobacteriaceae are class B by detecting inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor, and the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a second concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor.In any of the embodiments disclosed herein, the method may include determining that the one or more Ambler class carbapenemases expressed by the Enterobacteriaceae are class A by detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor, and detecting inhibition of growth in a third test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor. In any of the embodiments disclosed herein, the method may include determining that one or more Ambler class carbapenemases expressed by the Enterobacteriaceae are class D by detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor at a first concentration; the presence of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor at a first concentration; and inhibition of growth in a fourth test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a first concentration.

[0010] In any of the embodiments disclosed herein, the method may comprise determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by the Enterobacteriaceae by detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor at a first concentration; the presence of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor at a first concentration; and the presence of growth in a fourth test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a first concentration. In any of the embodiments disclosed herein, the method may include determining a lack of confidence in identifying one or more Ambler class carbapenemases expressed by the Enterobacteriaceae by detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor at a first concentration; the presence of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor at a first concentration; and the presence of growth in a fourth test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a first concentration.

[0011] In any of the embodiments disclosed herein, the method may include applying the Enterobacteriaceae in the test sample to a plurality of at least five test compositions over a sustained period of time, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a first concentration of MEM and an Ambler class C carbapenemase inhibitor. In any of the embodiments disclosed herein, the method may include determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by the Enterobacteriaceae by detecting the presence of growth in the first test composition, wherein the antibiotic and inhibitor comprises, consists of, or consists essentially of the first concentration of MEM and an Ambler class C carbapenemase inhibitor. In any of the embodiments disclosed herein, the method may include determining the absence of one or more Ambler class A, B, or D carbapenemases expressed by Enterobacteriaceae by detecting inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM and an Ambler class C carbapenemase inhibitor. In any of the embodiments disclosed herein, the method may include a method for identifying zero, one, or more Ambler class carbapenemases expressed by non-fermenting bacteria, the method comprising providing a sample containing non-fermenting bacteria, applying the non-fermenting bacteria in the test sample to a test composition comprising growth medium and an antibiotic and a carbapenemase inhibitor for a sustained period of time, and determining the presence of zero, one, or more Ambler class carbapenemases expressed by the non-fermenting bacteria by detecting the presence or inhibition of growth of the non-fermenting bacteria in the test composition after the sustained period of time.In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a third concentration of DOR and an Ambler class C carbapenemase inhibitor. In any of the embodiments disclosed herein, the method may include determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by the non-fermenting bacteria by detecting the presence of growth in the test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR and an Ambler class C carbapenemase inhibitor. In any of the embodiments disclosed herein, the method may include determining the absence of one or more Ambler class A, B, or D carbapenemases expressed by the non-fermenting bacteria by detecting inhibition of growth in a test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR and an Ambler class C carbapenemase inhibitor.

[0012] In any of the embodiments disclosed herein, the method may further include a method for determining the presence of zero, one or more Ambler-class carbapenemases expressed by non-fermentative bacteria, the method comprising the steps of providing a sample containing non-fermentative bacteria; applying the non-fermentative bacteria in the test sample to a plurality of at least three test compositions over a sustained period of time, wherein each of the plurality of at least three test compositions comprises a growth medium and an antibiotic, and at least one of the at least three test compositions further comprises at least one carbapenemase inhibitor; and determining the presence of zero, one or more Ambler-class carbapenemases expressed by the non-fermentative bacteria by detecting the presence or inhibition of growth of the non-fermentative bacteria in each of the plurality of at least three test compositions after the sustained period of time.

[0013] One embodiment includes a method for determining the presence of zero, one, or more Ambler-class carbapenemases expressed by non-fermentative bacteria, comprising: providing a sample containing non-fermentative bacteria; applying the non-fermentative bacteria in the test sample to a plurality of at least three test compositions over a sustained period of time, each of the plurality of at least three test compositions comprising a growth medium and an antibiotic, and at least one of the at least three test compositions further comprising at least one carbapenemase inhibitor; and determining the presence of zero, one, or additional one or more Ambler-class carbapenemases expressed by the non-fermentative bacteria by detecting the presence or inhibition of growth of the non-fermentative bacteria in each of the plurality of at least three test compositions after the sustained period of time. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler-class C carbapenemase inhibitor, and an Ambler-class B carbapenemase inhibitor. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a fifth concentration of DOR, and an Ambler class C carbapenemase inhibitor and an Ambler class D carbapenemase inhibitor.In any of the embodiments disclosed herein, the method may include determining one or more Ambler class carbapenemases expressed by the non-fermenting bacterium as Class B by detecting inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor. In any of the embodiments disclosed herein, the method may include determining one or more Ambler class carbapenemases expressed by the non-fermenting bacteria as class D by detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and inhibition of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.In any of the embodiments disclosed herein, the method may comprise determining one or more Ambler class carbapenemases expressed by the non-fermenting bacteria as class A by detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor at a third concentration; the presence of growth in a second test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a fourth concentration; and inhibition of growth in a third test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a fifth concentration. In any of the embodiments disclosed herein, the method may comprise determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by the non-fermenting bacterium by detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor at a third concentration; the presence of growth in a second test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a fourth concentration; and the presence of growth in a third test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a fifth concentration.In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor. In any of the embodiments disclosed herein, the method includes detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor. and The method may include determining that the one or more Ambler class carbapenemases expressed by the non-fermenting bacteria are either class A, B, or D by detecting the presence of growth in a second test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

[0014] In any of the embodiments disclosed herein, the method may include determining that the one or more Ambler class carbapenemases expressed by the non-fermenting bacterium are class D by detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and inhibition of growth in a second test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. In any of the embodiments disclosed herein, the method may include determining that the one or more Ambler class carbapenemases expressed by the non-fermenting bacterium are class B by detecting inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor.In any of the embodiments disclosed herein, the method may include determining a lack of confidence in identifying one or more Ambler class carbapenemases expressed by the non-fermenting bacterium by detecting inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor, respectively, and an inhibition of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor. In any of the embodiments disclosed herein, the method may include determining that the one or more Ambler class carbapenemases expressed by the non-fermenting bacterium are either class A, B, or D by detecting inhibition of growth in a first test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and in a third test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor.

[0015] In any of the embodiments disclosed herein, the method may include applying, over a sustained period, to a plurality of at least four test compositions to the non-fermenting bacteria in the test sample, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a fifth concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. In any of the embodiments disclosed herein, the method may include detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. and the presence of growth in a fourth test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of a fifth concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.In any of the embodiments disclosed herein, the method may include determining that the one or more Ambler class carbapenemases expressed by the non-fermenting bacterium are class A by detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor at a third concentration; the presence of growth in a second test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a fourth concentration; and inhibition of growth in a fourth test composition, wherein the antibiotic and inhibitors comprise, consist of, or consist essentially of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a fifth concentration.

[0016] In any of the embodiments disclosed herein, the method may further comprise determining whether the bacteria in the sample are enteric, non-fermentative, or both.

[0017] In any of the embodiments disclosed herein, the first concentration of TEM is about 6 μg / ml to about 128 μg / ml, about 32 μg / ml to about 128 μg / ml, about 32 μg / ml to about 80 μg / ml, or about 64 μg / ml. In any of the embodiments disclosed herein, the first concentration of DOR is about 0.006 μg / ml to about 0.75 μg / ml, about 0.03125 μg / ml to about 0.1 μg / ml, or about 0.0625 μg / ml or about 0.06 μg / ml. In any of the embodiments disclosed herein, the second concentration of DOR is about 0.0125 μg / ml to about 2 μg / ml, 0.0625 μg / ml to about 0.25 μg / ml, or about 0.125 μg / ml. In any of the embodiments disclosed herein, the third concentration of DOR is about 0.1 μg / ml to about 400 μg / ml, about 0.5 μg / ml to about 3 μg / ml, or about 1 μg / ml. In any of the embodiments disclosed herein, the fourth concentration of DOR is about 0.2 μg / ml to about 40 μg / ml, about 0.5 μg / ml to about 4 μg / ml, or about 2 μg / ml. In any of the embodiments disclosed herein, the fifth concentration of DOR is about 0.03125 μg / ml to about 80 μg / ml, about 2 μg / ml to about 24 μg / ml, or about 8 μg / ml. In any of the embodiments disclosed herein, the first concentration of MEM is 0.03125 μg / ml to 1 μg / ml, 0.03125 μg / ml to 0.125 μg / ml, 0.015625 μg / ml to 0.125 μg / ml, about 0.006 μg / ml to about 0.60 μg / ml, about 0.015 μg / ml to about 0.24 μg / ml, about 0.03 μg / ml to about 0.25 μg / ml, about 0.03 μg / ml to about 0.2 μg / ml, about 0.0625 μg / ml, or about 0.060 μg / ml. In any of the embodiments disclosed herein, the second concentration of MEM is about 0.015625 μg / ml to about 0.125 μg / ml, about 0.003 μg / ml to about 0.3 μg / ml, about 0.0075 μg / ml to about 0.12 μg / ml, about 0.01 μg / ml to about 0.12 μg / ml, or about 0.03 μg / ml.In any of the embodiments disclosed herein, the third concentration of MEM is about 0.0125 μg / ml to about 5 μg / ml, about 0.125 μg / ml to about 1 μg / ml, or about 0.5 μg / ml. In any of the embodiments disclosed herein, the fourth concentration of MEM is about 0.4 μg / ml to about 40 μg / ml, about 1 μg / ml to about 16 μg / ml, about 2 μg / ml to about 8 μg / ml, or about 4 μg / ml.

[0018] In any of the embodiments disclosed herein, the Ambler class D carbapenemase inhibitor comprises a compound selected from the group consisting of AVI, clavulanic acid, boronic acid, tazobactam, sulbactam, vaborbactam (RPX-7009), and BLI-489. In any of the embodiments disclosed herein, the Ambler class B carbapenemase inhibitor is a metal chelator. In any of the embodiments disclosed herein, the Ambler class B carbapenemase inhibitor comprises a compound selected from the group consisting of EDTA, DPA, and deferoxamine. In any of the embodiments disclosed herein, the Ambler class C carbapenemase inhibitor comprises a compound selected from the group consisting of CLOX, dicloxacillin, and flucloxacillin. In any of the embodiments disclosed herein, the Ambler class A carbapenemase inhibitor comprises a compound selected from the group consisting of vaborbactam (RPX-7009), AVI, clavulanic acid, boronic acid, tazobactam, sulbactam, and BLI-489. In any of the embodiments disclosed herein, the Ambler class D carbapenemase inhibitor comprises a compound selected from the group consisting of BLI, AVI, clavulanic acid, boronic acid, tazobactam, sulbactam, vaborbactam, and (RPX-7009).

[0019] In any of the embodiments disclosed herein, the Ambler class B carbapenemase inhibitor in combination with a first concentration of TEM and / or a first concentration of DOR comprises, consists of, or consists essentially of EDTA. In any of the embodiments disclosed herein, the Ambler class C carbapenemase inhibitor comprises, consists of, or consists essentially of CLOX. In any of the embodiments disclosed herein, the Ambler class A carbapenemase inhibitor in combination with a first concentration of MEM and / or a second concentration of MEM comprises, consists of, or consists essentially of RPX. In any of the embodiments disclosed herein, the Ambler class B carbapenemase inhibitor in combination with a first concentration of MEM and / or a third concentration of DOR comprises, consists of, or consists essentially of DPA. In any of the embodiments disclosed herein, the Ambler class D carbapenemase inhibitor in combination with the first concentration of DOR and / or the fifth concentration of DOR comprises, consists of, or consists essentially of AVI. In any of the embodiments disclosed herein, the Ambler class D carbapenemase inhibitor in combination with the fourth concentration of MEM comprises, consists of, or consists essentially of the first concentration of BLI. In any of the embodiments disclosed herein, the concentration of EDTA is about 0.025 mg / ml to about 10 mg / ml, about 0.05 mg / ml to about 1.25 mg / ml, or about 0.25 mg / ml. In any of the embodiments disclosed herein, the concentration of CLOX is about 0.0025 mg / ml to about 40 mg / ml, about 0.020 mg / ml to about 0.5 mg / ml, or about 0.1 mg / ml. In any of the embodiments disclosed herein, the concentration of RPX is about 0.2 μg / ml to about 320 μg / ml, about 1.5 μg / ml to about 40 μg / ml, or about 8 μg / ml. In any of the embodiments disclosed herein, the concentration of DPA is about 0.018 mg / ml to about 1.8 mg / ml, about 0.07 mg / ml to about 0.73 mg / ml, or about 0.178 mg / ml.In any of the embodiments disclosed herein, the concentration of AVI is about 0.1 μg / ml to about 40 μg / ml, about 0.5 μg / ml to about 20 μg / ml, or about 4 μg / ml. In any of the embodiments disclosed herein, the concentration of BLI is about 0.1 μg / ml to about 200 μg / ml, about 1 μg / ml to about 25 μg / ml, or about 5 μg / ml.

[0020] In any of the embodiments disclosed herein, the duration for detecting the presence or inhibition of growth is less than about 24 hours, less than about 18 hours, less than about 16 hours, or less than about 14 hours. In any of the embodiments disclosed herein, the duration for detecting the presence or inhibition of growth of Enterobacteriaceae is about 6 to about 8 hours. In any of the embodiments disclosed herein, the duration for detecting the presence or inhibition of growth of Enterobacteriaceae is about 7 hours. In any of the embodiments disclosed herein, the duration for detecting the presence or inhibition of growth of non-fermentative bacteria is about 8 to about 11 hours. In any of the embodiments disclosed herein, the duration for detecting the presence or inhibition of growth of non-fermentative bacteria is about 10 hours. In any of the embodiments disclosed herein, the Enterobacteriaceae comprises bacteria selected from the group consisting of Klebsiella pneumoniae, Escherichia coli, and Enterobacter aerogenes. In any of the embodiments disclosed herein, the non-fermenting bacteria comprises a bacterium selected from the group consisting of Pseudomonas aeruginosa and Acinetobacter baumannii complex.

[0021] In any of the embodiments disclosed herein, detecting the presence or inhibition of proliferation is not performed by imaging changes in cell morphology.

[0022] One embodiment includes a system for carrying out any of the methods of the embodiments disclosed herein, the system including: a plurality of compartments, each of the plurality of compartments comprising a test composition according to any of the preceding claims; a means for providing a sample comprising Enterobacteriaceae, non-fermentative bacteria, or both to the plurality of compartments; an apparatus for obtaining a first signal from the plurality of compartments provided with Enterobacteriaceae, non-fermentative bacteria, or both; an incubator for incubating the plurality of compartments provided with Enterobacteriaceae, non-fermentative bacteria, or both for a sustained period; an apparatus for obtaining a second signal from the plurality of compartments containing Enterobacteriaceae, non-fermentative bacteria, or both; a detector for detecting the presence or inhibition of growth in the plurality of compartments provided with Enterobacteriaceae, non-fermentative bacteria, or both by comparing the first signal and the second signal; a computer for generating a result output from the detector; and an analyzer for interpreting the result output. In any of the embodiments disclosed herein, the plurality of compartments comprises compartments selected from the group consisting of wells, plates, and tubes. In any of the embodiments disclosed herein, the system includes a BD Phoenix panel and / or system.

[0023] One embodiment includes a kit for identifying one or more Ambler-class carbapenemases expressed by Enterobacteriaceae and / or non-fermenting bacteria, the kit comprising a substrate or panel having a plurality of compartments, each of the plurality of compartments comprising a test composition according to any of the embodiments disclosed herein. In any of the embodiments disclosed herein, the substrate comprises at least three, or at least four, different test compositions. In any of the embodiments disclosed herein, the kit comprises a second substrate comprising a plurality of compartments, each of the plurality of compartments comprising a test composition according to any of the methods of claims 1-90, wherein at least one test composition in the plurality of test compositions in the first substrate is different from the plurality of test compositions in the second substrate.

[0024] In any of the embodiments disclosed herein that include a plurality of test compositions, the test compositions comprise, consist of, or consist essentially of a test composition selected from the test compositions disclosed in Boxes 1-14. In any of the embodiments disclosed herein, the test compositions comprise, consist of, or consist essentially of the test compositions disclosed in Boxes 1-5. In any of the embodiments disclosed herein, the test compositions comprise, consist of, or consist essentially of the test compositions disclosed in Boxes 1, 6, 7, 3, 8, and 9. In any of the embodiments disclosed herein, the test compositions comprise, consist of, or consist essentially of the test compositions disclosed in Boxes 10, 11, 12, and 13. In any of the embodiments disclosed herein, the test compositions comprise, consist of, or consist essentially of the test compositions disclosed in Boxes 10, 11, 12, 13, and 14. In any of the embodiments disclosed herein, the test compositions comprise, consist of, or consist essentially of the test compositions disclosed in Boxes 1, 6, 7, 3, and 9. In any of the embodiments disclosed herein, the test composition comprises, consists of, or consists essentially of the test compositions disclosed in Boxes 1, 6, 7, 9, 3, and 10. In any of the embodiments disclosed herein, the test composition comprises, consists of, or consists essentially of the test compositions disclosed in Boxes 1, 6, 7, 3, 9, 10, 11, 12, and 14. In any of the embodiments disclosed herein, the test composition comprises, consists of, or consists essentially of the test compositions disclosed in Boxes 1, 10, 11, 12, and 14. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 shows boxplots of TEM GAM data for Enterobacteriaceae expressing either class A, class B or class D carbapenemases. [Figure 2]FIG. 1 shows box plots of TEM GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B or class D carbapenemases. [Figure 3] FIG. 1 shows boxplots of TEM / CLOX / EDTA GAM data for Enterobacteriaceae expressing either class A, class B, or class D carbapenemases. [Figure 4] FIG. 1 shows box plots of TEM / CLOX / EDTA GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 5] FIG. 1 shows boxplots of TEM / CLOX / EDTA GAM data for Enterobacteriaceae expressing either class A, class B, or class D carbapenemases. [Figure 6] FIG. 1 shows box plots of TEM / CLOX / EDTA GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 7] FIG. 1 shows boxplots of MEM / CLOX GAM data for Enterobacteriaceae expressing either class A, class B, or class D carbapenemases. [Figure 8] FIG. 1 shows box plots of MEM / CLOX GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 9] FIG. 1 shows boxplots of MEM / CLOX / DPA GAM data for Enterobacteriaceae expressing either class A, class B, or class D carbapenemases. [Figure 10] FIG. 1 shows box plots of MEM / CLOX / DPA GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 11]FIG. 1 shows boxplots of MEM / CLOX / RPX GAM data for Enterobacteriaceae expressing either class A, class B, or class D carbapenemases. [Figure 12] FIG. 1 shows box plots of MEM / CLOX / RPX GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 13] FIG. 1 shows boxplots of DOR / CLOX / AVI GAM data for Enterobacteriaceae expressing either class A, class B, or class D carbapenemases. [Figure 14] FIG. 1 shows box plots of DOR / CLOX / AVI GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 15] FIG. 1 shows boxplots of DOR / CLOX / EDTA GAM data for Enterobacteriaceae expressing either class A, class B, or class D carbapenemases. [Figure 16] FIG. 1 shows box plots of DOR / CLOX / EDTA GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 17] FIG. 1 shows boxplots of DOR GAM data for Enterobacteriaceae expressing either class A, class B, or class D carbapenemases. [Figure 18] FIG. 1 shows box plots of DOR GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 19] FIG. 1 shows boxplots of DOR / CLOX GAM data for Enterobacteriaceae expressing either class A, class B, or class D carbapenemases. [Figure 20]FIG. 1 shows box plots of DOR / CLOX GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 21] FIG. 1 shows boxplots of DOR / CLOX / DPA GAM data for Enterobacteriaceae expressing either class A, class B, or class D carbapenemases. [Figure 22] FIG. 1 shows box plots of DOR / CLOX / DPA GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 23] FIG. 1 shows boxplots of MEM / CLOX / BLI GAM data for Enterobacteriaceae expressing either class A, class B, or class D carbapenemases. [Figure 24] FIG. 1 shows box plots of MEM / CLOX / BLI GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 25] FIG. 1 shows a flowchart of an embodiment of an algorithm for Gram-negative enteric bacteria. [Figure 26] FIG. 1 shows a flowchart of an embodiment of an algorithm for Gram-negative enteric bacteria. [Figure 27] FIG. 1 shows a flowchart of an embodiment of an algorithm for Gram-negative enteric bacteria. [Figure 28] FIG. 1 shows a flow chart of an embodiment of an algorithm for non-fermenting Gram-negative bacilli. [Figure 29] FIG. 1 shows a flow chart of an embodiment of an algorithm for non-fermenting Gram-negative bacilli. [Figure 30] FIG. 1 shows a flow chart of an embodiment of an algorithm for non-fermenting Gram-negative bacilli. [Figure 31]FIG. 1 shows box plots of MEM GAM and MEM / CLOX GAM data for Enterobacteriaceae expressing class C carbapenemases. [Figure 32] FIG. 1 shows a flowchart of an embodiment of an algorithm for classification of Enterobacteriaceae into classes A, B, or D. [Figure 33] FIG. 1 shows a flow chart of an embodiment of an algorithm for classification of nonfermenters into class B or D. [Figure 34] FIG. 1 shows a flowchart of an embodiment of an algorithm for classification of Enterobacteriaceae into classes A, B, or D. [Figure 35] FIG. 1 shows a flow chart of an embodiment of an algorithm for classification of non-fermenting strains into classes A, B or D. [Figure 36] FIG. 1 shows a flow chart of an embodiment of the algorithm for Enterobacteriaceae and non-fermenting strains. [Figure 37] FIG. 1 shows a flowchart of an embodiment of an algorithm for classification of Enterobacteriaceae and non-fermenting strains and Enterobacteriaceae. [Figure 38] FIG. 1 shows a flowchart of an embodiment of an algorithm for classification of Enterobacteriaceae and non-fermentative strains and Enterobacteriaceae and non-fermentative strains. [Figure 39] FIG. 1 shows a flow chart of an embodiment of an algorithm for classification of Enterobacteriaceae and non-fermentative and non-fermentative strains. DETAILED DESCRIPTION OF THE INVENTION

[0026] Rising antibiotic resistance and dwindling antibiotic supplies are creating a global public health crisis in which an increasing number of patients are infected with Gram-negative bacteria that are completely or nearly completely antibiotic-resistant. Carbapenemase-producing organisms (CPOs) are becoming a driving force behind the emergence of untreatable pathogens that not only threaten the treatment of bacterial infections, but also the use of antibiotics to protect patients undergoing cancer chemotherapy, transplant surgery, cardiac surgery, joint replacement surgery, and even childbirth.

[0027] Rapid and accurate detection of CPO is a major challenge for clinical laboratories. Unlike most bacterial infections, optimal therapy for CPO infections requires at least two active antibiotics to prevent the emergence and spread of antibiotic-resistant bacteria and patient death. Therefore, physicians may only have one opportunity to select an effective therapy for these infections. Therefore, it is important for laboratories to rapidly and accurately detect CPO to alert physicians to the need for combination therapy.

[0028] Most laboratories currently use inaccurate phenotypic carbapenemase detection tests that require overnight incubation. A minority use accurate but inconvenient phenotypic tests or more expensive PCR-based tests that have several unresolved accuracy issues. Current phenotypic tests are not automated. Therefore, there is a need for rapid diagnostic tests to advance the detection and control of antimicrobial-resistant bacteria.

[0029] The currently commercially available bioMérieux Rapidec® Carba NP test is a manual, stand-alone test that detects but does not classify carbapenemases. Therefore, there is also a therapeutic need to classify carbapenemases into molecular groups.

[0030] Disclosed herein are novel detection tests, including compositions, methods, systems, and / or kits, for detecting CPOs and further identifying and classifying Ambler class carbapenemase enzymes expressed by bacteria. In one embodiment, these novel CPO detection tests are incorporated into a previously developed BD Phoenix Gram-Negative Identification (ID) / Antimicrobial Susceptibility Test (AST) panel for detecting carbapenemase-expressing bacteria. In some embodiments, the detection tests can be applied to all Gram-negative bacteria (e.g., clinical isolates) in a sample to further identify Ambler class carbapenemases in the sample.

[0031] In some embodiments, the detection test incorporates one or more antibiotics and, optionally, one or more inhibitors in a test that allows for more accurate and rapid identification of one or more Ambler-class carbapenemases expressed by bacteria. The one or more antibiotics inhibit the growth of Gram-negative bacteria in a sample. However, if the Gram-negative bacteria express one or more Ambler-class carbapenemases and are therefore resistant to one or more antibiotics, one or more inhibitors may be included to allow for more accurate and rapid identification of the Ambler-class carbapenemases expressed by the bacteria. In some embodiments, the identification test involves exposing the sample to only one antibiotic or antibiotic combination, with or without an inhibitor. In other embodiments, the test may involve exposing portions of the sample to multiple different antibiotics, with or without inhibitors, in multiple wells so that the sample is tested against more than one antibiotic or antibiotic combination, with or without inhibitors, in a single test. These multiple combinations are typically run in parallel, with each combination in a separate well so that a portion of the sample is exposed to all of the combinations at the same time, although it is also possible to run tests by exposing portions of the sample to different combinations in succession. As described herein, multiple wells containing a particular antibiotic with or without an inhibitor can be run for a given sample (e.g., duplicates, triplicates, etc. of a particular combination of antibiotic and inhibitor).

[0032] At least four Ambler classes of β-lactamases are known: classes A, B, C, and D. However, only classes A, B, and D are considered carbapenemases. Infections caused by CPOs producing one class of carbapenemase may be susceptible to antibiotics, whereas infections caused by CPOs producing another class of carbapenemase may be less susceptible to the same antibiotic. For example, Ambler class A carbapenemases are candidates for therapy with the new antibiotic ceftazidime / avibactam, while class B-producing CPOs are inherently resistant to this drug. Therefore, distinguishing between CPOs producing class A and class B carbapenemase is expected to improve patient management. Thus, there is an urgent unmet need for rapid, accurate, and convenient detection and classification of CPOs.

[0033] In some embodiments, the detection test incorporates a combination of one or more antibiotics and one or more inhibitors into the test, allowing for more accurate and rapid identification of Ambler class A carbapenemase.

[0034] In some embodiments, the detection test incorporates a combination of one or more antibiotics and one or more inhibitors into the test, allowing for more accurate and rapid identification of Ambler class B carbapenemase.

[0035] In some embodiments, the detection test may incorporate a combination of one or more antibiotics and one or more inhibitors into the test, allowing for more accurate identification of Ambler Class C β-lactamases.

[0036] In some embodiments, the detection test incorporates a combination of one or more antibiotics and one or more inhibitors into a test that allows for more accurate and rapid identification of Ambler class D carbapenemase. For example, the detection test incorporates temocillin (TEM), a carboxypenicillin antibiotic, and a selective inhibitor into a single test that allows for more accurate and rapid identification of Ambler class D carbapenemase enzymes.

[0037] The various detection tests provided herein can be combined with an automated detection system that utilizes one or more algorithms to automate the phenotypic detection of bacterial carbapenemase expression, and optionally the Ambler classification of bacterial carbapenemase expression.

[0038] (BD Phoenix panels and systems) Microbiological diagnostic testing and systems for microbial identification (ID) and antimicrobial sensitivity determination (AST) are described, for example, in patents (see, e.g., U.S. Patent Nos. 6,111,149; ... and 6,111,149), and published applications (see, e.g., U.S. Patent No. 6,111,149), which are incorporated herein in their entireties. These references disclose a panel and system called the BD Phoenix Gram-negative Identification (ID) / Antimicrobial Sensitivity Testing (AST) Panel and System (BD Phoenix Panel and System) for ID / AST of microorganisms and their sensitivity to one or more antibiotics. The BD Phoenix Panel and System is suitable for various AST determination methods. For example, alamarBlue™, a redox-buffered redox indicator, is added and mixed into the AST inoculum fluid immediately before adding the microbial sample to be tested by the instrument. Visible and UV light sources are used to obtain readings corresponding to red, green, blue, and fluorescent wavelengths of light (see, e.g., U.S. Patent No. 6,111,149), which is incorporated herein in its entirety.

[0039] The BD Phoenix panel and system includes a substrate with multiple test wells adapted to receive bacteria suspended in a liquid medium and specific combinations of reagents (e.g., antibiotics with or without inhibitors). The bacterial response to the specific combinations of reagents in the panel is measured by installing a panel instrumentation system containing multiple light sources (e.g., visible and UV sources) emitting different wavelengths (e.g., red, green, blue, and fluorescent wavelengths). This instrumentation system can receive the panel and perform microbial ID / AST based on colorimetric and / or fluorometric detection (see, e.g., U.S. Patent No. 6,244,629, incorporated herein in its entirety). Based on the results of the BD Phoenix panel and system, the sensitivity of the microorganism (e.g., Gram-negative bacteria) to antibiotics is determined. In addition, the BD Phoenix panel and system can distinguish between Enterobacteriaceae and non-fermenting bacteria.

[0040] It is expected that one of ordinary skill in the art will readily understand the state of the art by reviewing the above-mentioned patents and applications. It is also expected that one of ordinary skill in the art will recognize the improvements over the state of the art, such as existing BD Phoenix panels and systems, that the novel detection tests, including the compositions, methods, systems, and kits disclosed herein, provide.

[0041] [BD Phoenix™ CPO Detect] Provided herein is the novel BD Phoenix™ CPO Detect, also known as the Phoenix™ CPO Detect, which includes a detection test that expands on the BD Phoenix panel and system by including detection of CPO. The CPO Detect offers a rapid, highly sensitive, and specific, algorithm-based, automated detection test for the detection and identification of bacteria that express one or more classes of carbapenemases.

[0042] The BD Phoenix™ CPO Detect detection test expands the BD Phoenix panel and system by combining one or more antibiotics, one or more inhibitors of various classes of carbapenemases, and one or more detection reagents for specifically identifying the class of carbapenemase expressed by bacteria.

[0043] To identify whether one or more class A, B, and D carbapenemases are expressed by bacteria, one or more antibiotics are used to inhibit the growth of Gram-negative bacteria expressing one or more class A, B, and D carbapenemases in a sample. In some embodiments, if the Gram-negative bacteria is resistant to one or more antibiotics because it expresses one or more Ambler class carbapenemases, one or more inhibitors are used to identify the Ambler class carbapenemases. For more accurate and rapid differentiation and identification of Ambler class carbapenemases, one or more antibiotics and one or more inhibitors are typically used in a test that includes samples run in multiple wells containing different combinations of antibiotics with or without inhibitors in different wells.

[0044] In some embodiments, the BD CPO Detect can provide two results: (1) an initial detection-based positive / negative result for carbapenemase detection, and (2) a follow-up classification of the positive isolate from step (1) according to the molecular class of the carbapenemase. In contrast, the bioMerieux Rapidec® Carba NP test provides only an initial detection-based positive / negative result, and this level of analysis is the current standard for commercially available phenotypic tests.

[0045] As used herein, in the context of an initial detection-based positive / negative result for carbapenemase detection, the "sensitivity" of a test or the "sensitivity" of detection is defined as the percent of CPOs detected in the positive / negative phase of the test.

[0046] As used herein, in the context of initial detection-based positive / negative results for carbapenemase detection, the "specificity" of detection or test "specificity" of detection is defined as the percentage of carbapenemase-negative isolates that are correctly identified as such in the positive / negative phase of the test.

[0047] As used herein, in the context of classifying positive isolates according to the molecular class of carbapenemase, a classification result is considered positive if it is either an accurate classification or an untypeable but positive result. Detecting a carbapenemase, even if not classified, is important and highly beneficial for patient management. Accurately classifying a carbapenemase increases the value of the result. If the carbapenemase belongs to class A, ceftazidime / avibactam is a potential candidate for therapy. Detection of class B carbapenemase contraindicates ceftazidime / avibactam therapy because class B CPOs are inherently resistant to this drug. For infections caused by class D carbapenemase-producing strains, there is currently no clear indication for ceftazidime / avibactam therapy. An accurate negative result is also a positive outcome for guiding patient management and infection control.

[0048] Misclassification of carbapenemase as class B or class D is considered unhelpful but relatively harmless. This misclassification does not devalue carbapenemase detection but may delay consideration of ceftazidime / avibactam therapy until sensitivity results are available. A "no answer" result is also unhelpful in that it confers neither benefit nor harm.

[0049] Consequences considered potentially harmful include incorrect classification of a class B carbapenemase as a class A carbapenemase, which could result in a patient receiving ineffective ceftazidime / avibactam therapy. False-negative results are also considered potentially harmful because the consequences of undetected CPO could be the administration of ineffective therapy and / or the failure to implement infection control measures.

[0050] Non-limiting examples of bacteria, antibiotics, inhibitors, and detection reagents are provided herein, as are non-limiting examples of concentration ranges for the antibiotics and inhibitors. However, one skilled in the art will readily recognize that the detection tests can be adapted to run with other bacteria, antibiotics, inhibitors, and detection reagents, and can also run with other concentration ranges for the antibiotics and inhibitors.

[0051] (antibiotics) Non-limiting examples of antibiotics include temocillin (TEM), doripenem (DOR), or meropenem (MEM). TEM (6 beta-(2-carboxy-2-thien-3-ylacetamido)-6 alpha-methoxypenicillanate disodium) is a carboxypenicillin that is stable against hydrolysis by chromosomal and plasmid beta-lactamases, including extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases. TEM is currently used in Belgium and the UK to treat multidrug-resistant Enterobacteriaceae.

[0052] In some embodiments, the concentration range of TEM in the detection tests provided herein is or is approximately 6 μg / ml to 1024 μg / ml. In some embodiments, the concentration range of TEM in the detection tests provided herein is or is approximately 12 μg / ml to 512 μg / ml (FIGS. 1-6). Other concentration ranges are or are approximately 32 μg / ml to 124 μg / ml, and in some embodiments, have a concentration of about 64 μg / ml. In some embodiments, the concentration range of TEM in the detection tests provided herein is or is approximately 32 μg / ml to 100 μg / ml. In some embodiments, the concentration range of TEM in the detection tests provided herein is or is approximately 32 μg / ml to 75 μg / ml. In some embodiments, the concentration range of TEM in the detection tests provided herein is or is approximately 55 μg / ml to 75 μg / ml.

[0053] MEM is an ultra-broad-spectrum injectable antibiotic used to treat a wide variety of infections. It is a beta-lactam, a subgroup of carbapenems. It effectively penetrates many tissues and body fluids, including cerebrospinal fluid, bile, heart valves, lungs, and peritoneal fluid. MEM is bactericidal but bacteriostatic against Listeria monocytogenes. Like other beta-lactam antibiotics, it inhibits bacterial cell wall synthesis.

[0054] In some embodiments, the concentration range of MEM in the detection tests provided herein is or is approximately 0.0039 μg / ml to 128 μg / ml. In some embodiments, the concentration range of MEM in the detection tests provided herein is or is approximately 0.0078 μg / ml to 64 μg / ml (FIGS. 7-12, 23, and 24). In some embodiments, the concentration range of MEM in the detection tests provided herein is or is approximately 0.0156 μg / ml to 64 μg / ml (FIG. 31). Another concentration range is from about 0.016 μg / ml to about 1 μg / ml, and in some embodiments, has a concentration of about 0.0625 μg / ml.

[0055] DOR is an ultra-broad spectrum injectable antibiotic. It is a beta-lactam and belongs to the carbapenem subgroup. DOR can be used for bacterial infections, such as complicated abdominal infections, hospital-acquired pneumonia, and complicated urinary tract infections, including kidney infections associated with sepsis. DOR reduces the process of cell wall growth, ultimately leading to the complete annihilation of infectious bacterial cells.

[0056] In some embodiments, the concentration range of DOR in the detection tests provided herein is or is approximately 0.0078 μg / ml to 128 μg / ml. In some embodiments, the concentration range of DOR in the detection tests provided herein is or is approximately 0.0156 μg / ml to 64 μg / ml (FIGS. 13-22). Other concentration ranges are or are approximately 0.0313 μg / ml to 4 μg / ml, and in some embodiments, have a concentration of about 1 μg / ml.

[0057] Non-limiting examples of other antimicrobial agents include CLOX, EDTA, and RPX7009, avibactam, BLI-489, and DPA.

[0058] In some embodiments, the concentration range of CLOX is at or about 40 μg / ml to 160 μg / ml, and in some embodiments has a concentration of about 100 μg / ml.

[0059] In some embodiments, the concentration range of EDTA is at or about 100 μg / ml to 400 μg / ml, and in some embodiments has a concentration of about 250 μg / ml.

[0060] In some embodiments, the concentration range of RPX7009 is at or about 3 μg / ml to 15 μg / ml, and in some embodiments has a concentration of about 8 μg / ml.

[0061] In some embodiments, the concentration range of avibactam is at or about 1 μg / ml to 10 μg / ml, and in some embodiments has a concentration of about 4 μg / ml.

[0062] In some embodiments, the concentration range of BLI-489 is at or about 1 μg / ml to 10 μg / ml, and in some embodiments has a concentration of about 5 μg / ml.

[0063] In some embodiments, the concentration range of DPA is at or about 50 μg / ml to 400 μg / ml, and in some embodiments has a concentration of about 178 μg / ml.

[0064] (Amble class carbapenemase) Carbapenemases are β-lactamase enzymes (β-lactamases) with diverse hydrolytic activity. They are capable of hydrolyzing penicillins, cephalosporins, monobactams, and carbapenems. The rapid spread of these enzymes in clinically important bacteria, such as Enterobacteriaceae and non-fermenting bacteria, such as Acinetobacter and Pseudomonas, poses a significant threat to public health.

[0065] Carbapenemases belong to two major families, distinguished by the hydrolysis mechanism (either zinc or serine) at their active site. Classification based on amino acid homology (Ambler classification) has resulted in four major classes: Ambler classes A, B, C, and D.

[0066] Ambler class A carbapenemases contain the amino acid serine in their active site. Bacteria expressing Ambler class A carbapenemases are sensitive to mechanism-based inhibitors. Mechanism-based inhibition is an irreversible form of enzyme inhibition that occurs when, during "normal" catalytic reactions, the enzyme binds to a substrate analog and forms an irreversible complex with it via covalent bonds. Non-limiting examples of class A carbapenemases include KPC (e.g., KPC-like, KPC-2, or KPC-3), NMC-A, IMI, and SME enzymes.

[0067] Ambler class B carbapenemases contain a metallic zinc in their active site. Bacteria expressing Ambler class B carbapenemases are sensitive to chelating agents that bind to and remove zinc (a metal ion) from the active site of the class B carbapenemase. Non-limiting examples of class B carbapenemases (metallo-β-lactamases) include NDM (e.g., NDM-like or NDM-1), GIM, SPM (e.g., SPM-like or SPM-1), IMP (e.g., IMP-like or IMP-1), and VIM (e.g., VIM-like or VIM-1) enzymes.

[0068] Like Ambler class A carbapenemases, Ambler class C β-lactamases contain the amino acid serine in their active site. However, they do not hydrolyze carbapenems. Overexpression of Ambler class C β-lactamase in bacteria does not render them insensitive to carbapenems; therefore, it is not a carbapenemase. Nevertheless, carbapenem resistance can occur if other mutations, including loss of porin in the outer membrane or activation of efflux pumps, are present. Overexpression of Ambler class C β-lactamase in bacteria renders them insensitive to broad-spectrum cephalosporins.

[0069] Although bacteria expressing Ambler class C β-lactamases (referred to herein for convenience as class C carbapenemases) may be susceptible to carbapenems, they may nonetheless be nonsusceptible to carbapenems by other mechanisms. To detect class A, B, and D carbapenemases, phenotypic tests must selectively render class C carbapenemase-expressing bacteria susceptible; otherwise, false-positive interpretations may result.

[0070] Like Ambler class A and C, Ambler class D carbapenemases also contain the amino acid serine at their active site. However, at present, class D carbapenemases have no known common specific inhibitors. Therefore, phenotypic testing for the identification of class D carbapenemases is typically performed indirectly by determining that resistance is not due to class A, B, or C, leaving class D as a tentative identification. For example, phenotypic testing for the identification of class D carbapenemases is performed indirectly through the process of elimination of other Ambler class carbapenemases. Non-limiting examples of class D carbapenemases include OXA-23, 40, 48, 58, 72, 181, and 232 enzymes.

[0071] In some cases, more than one class of carbapenemases may be produced by an organism, for example, in some embodiments, two, three, or four classes of carbapenemases are produced by the organism.

[0072] Non-limiting examples of non-carbapenemase resistance mechanisms include ESBLs (e.g., CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-12, CTX-M14, CTX-M-15, CTX-M-15-like, CTX-M-28, SHV ESBLs, SHV-5, SHV-5-like, SHV-12, SHV-12-like, SHV-18, TEM ESBLs, OXA-45), AmpC (including overproducers) (e.g., plasmid-mediated AmpC, e.g., ACT-1, ACT-like, CMY (CMY-like, CMY-2, CMY-2-like), CMY-16, DHA-1, DHA-like, FOX-1, FOX-5, LAT-4, MIR-like, MOX-1, K1), broad-spectrum β-lactamases, and porin mutants.

[0073] (Carbapenemase inhibitors and discriminators) Non-limiting classes of inhibitors of carbapenemases include mechanism-based inhibitors, chelating agents and β-lactam antibiotics.

[0074] Non-limiting examples of mechanism-based inhibitors include β-lactamase inhibitors, including, but not limited to, boronic acid-based inhibitors, vaborbactam (RPX7009), BLI-489, CLOX, clavulanate, tazobactam, or avibactam.

[0075] In addition, bacteria expressing Ambler class A carbapenemases are typically susceptible to temocillin at lower concentrations than most bacteria expressing Ambler class B or class D carbapenemases.

[0076] Bacteria expressing Ambler class A carbapenemases are susceptible to TEM at lower concentrations, e.g., at TEM concentrations ranging from about 6 μg / ml to about 12 μg / ml (Figure 1), whereas class D expressing bacteria typically exhibit elevated MICs to TEM and are therefore susceptible to significantly higher concentrations of TEM, e.g., above about 128 μg / ml (Figure 1).

[0077] Therefore, concentrations of temocillin that inhibit the growth of class A-expressing bacteria are not expected to inhibit the growth of class D-expressing bacteria, and temocillin can be used to distinguish between bacteria that express class A and class D carbapenemases.

[0078] Non-limiting examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and dipicolinic acid (DPA), which bind to and sequester metal ions.

[0079] Bacteria expressing Ambler class B carbapenemase are susceptible to EDTA at 250 μg / ml (Figures 3, 4, 15, and 16), to EDTA at 280 μg / ml (Figures 5 and 6), and to DPA at 180 μg / ml (Figures 9, 10, 21, and 22).

[0080] A non-limiting example of a discriminator for class C β-lactamases is cloxacillin (CLOX), a penicillin derivative useful for treating infections caused by Staphylococci. Bacteria expressing class C β-lactamases are susceptible to CLOX at concentrations of approximately 100 μg / ml, while bacteria expressing classes A, B, or D typically are not, making it possible to distinguish class C from classes A, B, and D (Figure 31).

[0081] (BD Phoenix™ CPO Detect Detection Test) The BD Phoenix™ CPO Detect Detection Test can be used as a qualitative in vitro diagnostic test to phenotypically detect the expression of carbapenemase in bacteria. In addition to providing detection of carbapenemase-expressing bacteria, it further distinguishes the type of carbapenemase enzyme as Ambler Class A, Class B, or Class D. It is anticipated that the BD Phoenix™ CPO Detect Detection Test will enable clinical laboratories to identify bacterial expression of Ambler class carbapenemase in addition to testing all Gram-negative bacteria isolated from patient samples for isolate identity and antibiotic susceptibility.

[0082] In some embodiments, non-limiting examples of samples may include one or more of blood, urine, stool, sputum, saliva, etc. The sample is collected from a human, one or more companion animals, or one or more commercially important animals. In some embodiments, the human, one or more companion animals, or one or more commercially important animals may have a bacterial infection. The bacterial infection may be caused by enterobacteria or non-fermentative bacteria. In some embodiments, the bacteria may be other than enterobacteria or non-fermentative bacteria.

[0083] Non-limiting examples of Enterobacteriaceae include Klebsiella pneumoniae, Escherichia coli, and Enterobacter aerogenes.

[0084] Non-limiting examples of non-fermenting bacteria include Pseudomonas aeruginosa and Acinetobacter baumannii complex strains.

[0085] Those skilled in the art will appreciate that BD Phoenix™ CPO Detect can be adapted to bacteria other than enterobacteria and non-fermenting bacteria.

[0086] This procedure provides a simplified method for accurately identifying carbapenemase production, along with Ambler classification identification necessary for appropriate antibiotic treatment and monitoring, thereby enabling appropriate patient isolation from other non-infected patients. The proposed test can be incorporated with conventional sensitivity tests (ASTs) and therefore does not require additional testing or costs. The test also provides rapid identification of carbapenemase while running ASTs. This is expected to save time and money for hospitals and patients, as a separate test for CPO does not need to be ordered by physicians.

[0087] The detection test comprises multiple wells. In some embodiments, the input in each well is a combination of a sample containing one or more bacteria, one or more antibiotics, optionally one or more inhibitors, and one or more detection reagents. A suitable control for a detection test may comprise multiple wells, where each well contains a sample containing one or more bacteria, one or more antibiotics, one or more inhibitors, and / or one or more detection reagents. In some embodiments, the sample is run twice, three times, or more times, depending on each type of well (e.g., depending on the particular antibiotic / inhibitor combination).

[0088] Figures 1-24 and 31 show non-limiting embodiments of detection tests for Enterobacteriaceae and non-fermenting bacteria using a range of concentrations of one or more antibiotics and one or more inhibitors. These figures illustrate the concentrations of antibiotics tested (in μg / ml) along the x-axis (not all concentrations are tested in any of the figures), and the amount of bacterial growth in the sample along the y-axis. Boxes illustrate the median, interquartile range, minimum and maximum values ​​that are not outliers, and asterisks represent single outliers. Each figure has panels for class A, class B, and class D-producing bacteria, as well as a panel for non-carbapenemase-producing bacteria (NEG). In some embodiments, the detection tests described herein are not performed by imaging changes in cell morphology.

[0089] As an example, Figure 1 illustrates the testing of various Enterobacteriaceae grown in the presence of various concentrations of TEM. As shown in Figure 1, Class A and NEG bacteria are more susceptible to TEM than Class B or D. At a concentration of 24 μg / ml, nearly all Class A Enterobacteriaceae strains tested are inhibited by TEM, while Class D is unaffected until concentrations reach 192 μg / ml. Nearly all NEG Enterobacteriaceae are susceptible to the lowest concentrations of TEM. Tested Class B Enterobacteriaceae begin to show susceptibility at 48 μg / ml, as evidenced by a decrease in the mean growth line, and at 384 μg / ml, growth of most Class B Enterobacteriaceae is inhibited. Figures 1-24 and 31 demonstrate the results of numerous combinations of antibiotics with and without inhibitors that can be used to distinguish between Class A, B, D, and NEG Enterobacteriaceae and non-fermenting bacteria.

[0090] In some embodiments, the detection test may include multiple wells containing Enterobacteriaceae and TEM ranging in concentration from about 12 μg / ml to about 512 μg / ml (FIG. 1).

[0091] In some embodiments, the detection test may include multiple wells containing non-fermenting bacteria and TEM ranging in concentration from about 12 μg / ml to about 512 μg / ml (FIG. 2).

[0092] In some embodiments, the detection test may include multiple wells containing Enterobacteriaceae, 0.1 mg / ml CLOX, 250 μg / ml EDTA, and TEM ranging in concentration from about 12 μg / ml to about 512 μg / ml (FIG. 3).

[0093] In some embodiments, the detection test may include multiple wells containing non-fermenting bacteria, 0.1 mg / ml CLOX, 250 μg / ml EDTA, and TEM ranging in concentration from about 12 μg / ml to about 512 μg / ml (FIG. 4).

[0094] In some embodiments, the detection test may include multiple wells containing Enterobacteriaceae, 0.1 mg / ml CLOX, 280 μg / ml EDTA, and TEM ranging in concentration from about 12 μg / ml to about 512 μg / ml (FIG. 5).

[0095] In some embodiments, the detection test may include multiple wells containing non-fermenting bacteria, 0.1 mg / ml CLOX, 280 μg / ml EDTA, and TEM ranging in concentration from about 12 μg / ml to about 512 μg / ml (FIG. 6).

[0096] In some embodiments, the detection test may include multiple wells containing Enterobacteriaceae, 0.1 mg / ml CLOX, and MEM ranging in concentration from about 0.0078 μg / ml to about 64 μg / ml (FIG. 7).

[0097] In some embodiments, the detection test may include multiple wells containing non-fermenting bacteria, 0.1 mg / ml CLOX, and MEM ranging in concentration from about 0.0078 μg / ml to about 64 μg / ml (FIG. 8).

[0098] In some embodiments, the detection test may include multiple wells containing Enterobacteriaceae, 0.1 mg / ml CLOX, 0.18 mg / ml DPA, and MEM ranging in concentration from about 0.0078 μg / ml to about 64 μg / ml (FIG. 9).

[0099] In some embodiments, the detection test may include multiple wells containing non-fermenting bacteria, 0.1 mg / ml CLOX, 0.18 mg / ml DPA, and MEM ranging in concentration from about 0.0078 μg / ml to about 64 μg / ml (FIG. 10).

[0100] In some embodiments, the detection test may include multiple wells containing Enterobacteriaceae, 0.1 mg / ml CLOX, 8 μg / ml RPX, and MEM ranging in concentration from about 0.0078 μg / ml to about 64 μg / ml (FIG. 11).

[0101] In some embodiments, the detection test may include multiple wells containing non-fermenting bacteria, 0.1 mg / ml CLOX, 8 μg / ml RPX, and MEM ranging in concentration from about 0.0078 μg / ml to about 64 μg / ml (FIG. 12).

[0102] In some embodiments, the detection test may include multiple wells containing Enterobacteriaceae, 0.1 mg / ml CLOX, 4 μg / ml AVI, and DOR ranging in concentration from about 0.0156 μg / ml to about 64 μg / ml (FIG. 13).

[0103] In some embodiments, the detection test may include multiple wells containing non-fermenting bacteria, 0.1 mg / ml CLOX, 4 μg / ml AVI, and DOR ranging in concentration from about 0.0156 μg / ml to about 64 μg / ml (FIG. 14).

[0104] In some embodiments, the detection test may include multiple wells containing Enterobacteriaceae, 0.1 mg / ml CLOX, 0.25 mg / ml EDTA, and DOR ranging in concentration from about 0.0156 μg / ml to about 64 μg / ml (FIG. 15).

[0105] In some embodiments, the detection test may include multiple wells containing non-fermenting bacteria, 0.1 mg / ml CLOX, 0.25 mg / ml EDTA, and DOR ranging in concentration from about 0.0156 μg / ml to about 64 μg / ml (FIG. 16).

[0106] In some embodiments, the detection test may include multiple wells containing Enterobacteriaceae and DOR ranging in concentration from about 0.0156 μg / ml to about 64 μg / ml (FIG. 17).

[0107] In some embodiments, the detection test may include multiple wells containing non-fermenting bacteria and DOR ranging in concentration from about 0.0156 μg / ml to about 64 μg / ml (FIG. 18).

[0108] In some embodiments, the detection test may include multiple wells containing Enterobacteriaceae, 0.1 mg / ml CLOX, and DOR ranging in concentration from about 0.0156 μg / ml to about 64 μg / ml (FIG. 19).

[0109] In some embodiments, the detection test may include multiple wells containing non-fermenting bacteria, 0.1 mg / ml CLOX, and DOR ranging in concentration from about 0.0156 μg / ml to about 64 μg / ml (FIG. 20).

[0110] In some embodiments, the detection test may include multiple wells containing Enterobacteriaceae, 0.1 mg / ml CLOX, 0.18 mg / ml DPA, and DOR ranging in concentration from about 0.0156 μg / ml to about 64 μg / ml (FIG. 21).

[0111] In some embodiments, the detection test may include multiple wells containing non-fermenting bacteria, 0.1 mg / ml CLOX, 0.18 mg / ml DPA, and DOR ranging in concentration from about 0.0156 μg / ml to about 64 μg / ml (FIG. 22).

[0112] In some embodiments, the detection test may include multiple wells containing Enterobacteriaceae, 0.1 mg / ml CLOX, 5 μg / ml BLI, and MEM ranging in concentration from about 0.0078 μg / ml to about 64 μg / ml (FIG. 23).

[0113] In some embodiments, the detection test may include multiple wells containing non-fermenting bacteria, 0.1 mg / ml CLOX, 5 μg / ml BLI, and MEM ranging in concentration from about 0.0078 μg / ml to about 64 μg / ml (FIG. 24).

[0114] In some embodiments, the detection test may include multiple wells containing Enterobacteriaceae, 0.1 mg / ml CLOX, and MEM ranging in concentration from about 0.0156 μg / ml to about 64 μg / ml (FIG. 31).

[0115] In a detection test, a determination of whether a bacterium is susceptible to one or more antibiotics provided herein is achieved based on whether the bacterium grows or does not grow in the presence of a particular concentration of the antibiotic. If the bacterium is non-susceptible to one or more antibiotics, identification of one or more classes of carbapenemase expressed by the bacterium that confer non-susceptibility to the antibiotic is achieved by using one or more carbapenemase inhibitors or discriminators provided herein.

[0116] Non-limiting examples (Examples 1-4) of detection tests for identifying the expression of one or more Ambler class carbapenemases by enteric or non-fermentative bacteria are provided. The determination of whether a sample contains enteric or non-fermentative bacteria can be made by methods known in the art, for example, by biochemical tests including spot oxidase tests, MALDI-TOF, and Phoenix ID systems. The concentrations of antibiotics and inhibitors disclosed in the following examples are exemplary and non-limiting. Other concentrations or ranges of acceptable concentrations are disclosed in the present disclosure, including the drawings.

[0117] In some embodiments, the BD Phoenix™ CPO Detector includes a well (or optionally multiple identical wells) containing an input sample comprising one or more bacteria, one or more detection reagents, and one or more antibiotics, with or without one or more carbapenemase inhibitors. In some embodiments, the well contains one of the combinations disclosed in Table 0.1 below, including one or more antibiotics, with or without one or more carbapenemase inhibitors.

[0118] [Table 1]

[0119] In any of combinations 1-10 provided in Table 0.1, the concentration of CLOX is or is approximately in the range of 2.5 μg / ml to 40,000 μg / ml, or the concentration of CLOX is or is approximately in the range of 20 μg / ml to 500 μg / ml. In some embodiments, the concentration of CLOX is or is approximately in the range of 20 μg / ml to 150 μg / ml, the concentration of CLOX is or is approximately in the range of 150 μg / ml to 250 μg / ml, the concentration of CLOX is or is approximately in the range of 250 μg / ml to 350 μg / ml, or the concentration of CLOX is or is approximately in the range of 350 μg / ml to 500 μg / ml, and in some embodiments has a concentration of or is approximately 100 μg / ml. In some embodiments, the concentration of CLOX is or is approximately in the range of 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 5000, 10,000, or 40,000, or within a range defined by any two of the above values.

[0120] In any of combinations 1-10 provided in Table 0.1, the concentration of AVI is or is approximately in the range of 0.5 μg / ml to 20 μg / ml. In some embodiments, the concentration of AVI is or is approximately in the range of 0.5 μg / ml to 5 μg / ml, the concentration of AVI is or is approximately in the range of 5 μg / ml to 10 μg / ml, the concentration of AVI is or is approximately in the range of 10 μg / ml to 15 μg / ml, or the concentration of AVI is or is approximately in the range of 15 μg / ml to 10 μg / ml, and in some embodiments, has a concentration of 4 μg / ml. In some embodiments, the concentration of AVI is or is approximately in the range of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 7.5, 10, 12.5, 15, 17.5, or 20 μg / ml, or within a range defined by any two of the above values.

[0121] In any of combinations 1-10 provided in Table 0.1, the concentration of BLI-489 is or is approximately in the range of 1 μg / ml to 25 μg / ml. In some embodiments, the concentration of BLI-489 is or is approximately in the range of 1 μg / ml to 5 μg / ml, the concentration of BLI-489 is or is approximately in the range of 5 μg / ml to 10 μg / ml, the concentration of BLI-489 is or is approximately in the range of 10 μg / ml to 17.5 μg / ml, or the concentration of BLI-489 is or is approximately in the range of 17.5 μg / ml to 25 μg / ml, and in some embodiments has a concentration of 5 μg / ml. In some embodiments, the concentration of BLI-489 is at or in the range of about 1, 2.5, 3, 3.5, 4, 4.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, or 25 μg / ml, or within a range defined by any two of the above values.

[0122] In any of combinations 1-10 provided in Table 0.1, the concentration of DPA is or is approximately in the range of 35 μg / ml to 900 μg / ml. In some embodiments, the concentration of DPA is or is approximately in the range of 35 μg / ml to 150 μg / ml, the concentration of DPA is or is approximately in the range of 150 μg / ml to 300 μg / ml, the concentration of DPA is or is approximately in the range of 300 μg / ml to 650 μg / ml, or the concentration of DPA is or is approximately in the range of 650 μg / ml to 900 μg / ml, and in some embodiments has a concentration of 178 μg / ml. In some embodiments, the concentration of DPA is at or in the range of about 35, 70, 140, 178, 200, 280, 350, 450, 560, 640, 730, 820, or 900 μg / ml, or within a range defined by any two of the above values.

[0123] In any of combinations 1-10 provided in Table 0.1, the concentration of EDTA is or is approximately in the range of 50 μg / ml to 1250 μg / ml. In some embodiments, the concentration of EDTA is or is approximately in the range of 50 μg / ml to 250 μg / ml, the concentration of EDTA is or is approximately in the range of 250 μg / ml to 500 μg / ml, the concentration of EDTA is or is approximately in the range of 500 μg / ml to 750 μg / ml, or the concentration of EDTA is or is approximately in the range of 750 μg / ml to 1250 μg / ml, and in some embodiments, has a concentration of 250 μg / ml. In some embodiments, the concentration of EDTA is at or in the range of about 50, 75, 150, 200, 250, 300, 350, 500, 600, 750, 1000, or 1250 μg / ml, or within a range defined by any two of the foregoing values.

[0124] In any of combinations 1-10 provided in Table 0.1, the concentration of RPX7009 is or is approximately in the range of 1.5 μg / ml to 40 μg / ml. In some embodiments, the concentration of RPX7009 is or is approximately in the range of 1.5 μg / ml to 3 μg / ml, the concentration of RPX7009 is or is approximately in the range of 3 μg / ml to 15 μg / ml, the concentration of RPX7009 is or is approximately in the range of 15 μg / ml to 25 μg / ml, or the concentration of RPX7009 is or is approximately in the range of about 25 μg / ml to 40 μg / ml, and in some embodiments has a concentration of 8 μg / ml. In some embodiments, the concentration range of RPX7009 is or is approximately in the range of 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5 or 15 μg / ml, or within a range defined by any two of the above values.

[0125] In Combination 1, the concentration of DOR is at or about the range of 0.0625 μg / ml to 0.25 μg / ml. In some embodiments, the concentration of DOR is at or about the range of 0.0125 μg / ml to 1.25 μg / ml, the concentration of DOR is at or about the range of 0.0625 μg / ml to 0.0825 μg / ml, the concentration of DOR is at or about the range of 0.0825 μg / ml to 0.125 μg / ml, the concentration of DOR is at or about the range of 0.125 μg / ml to 0.175 μg / ml, or the concentration of DOR is at or about the range of 0.175 μg / ml to 0.25 μg / ml. In some embodiments, the concentration of DOR is at or within a range of about 0.0125, 0.0625, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.5, 0.75, 1.0, or 1.25 μg / ml, or within a range defined by any two of the foregoing values.

[0126] In Combination 2, the concentration of DOR is or is approximately in the range of 0.5 μg / ml to 4 μg / ml. In some embodiments, the concentration of DOR is or is approximately in the range of 0.1 μg / ml to 40 μg / ml, the concentration of DOR is or is approximately in the range of 0.1 μg / ml to 10 μg / ml, the concentration of DOR is or is approximately in the range of 0.2 μg / ml to 20 μg / ml, the concentration of DOR is or is approximately in the range of 0.5 μg / ml to 4 μg / ml, the concentration of DOR is or is approximately in the range of 0.5 μg / ml to 1 μg / ml, the concentration of DOR is or is approximately in the range of 1 μg / ml to 2 μg / ml, the concentration of DOR is or is approximately in the range of 2 μg / ml to 3 μg / ml, or the concentration of DOR is or is approximately in the range of 3 μg / ml to 4 μg / ml. In some embodiments, the concentration of DOR is at or within a range of about 0.1, 0.15, 0.2, 0.25, 0.5, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or 40 μg / ml, or within a range defined by any two of the above values.

[0127] In Combination 3, the concentration of DOR is or is approximately in the range of 0.03125 μg / ml to 16 μg / ml, or 0.02 μg / ml to 600 μg / ml. In some embodiments, the concentration of DOR is or is approximately in the range of 0.03126 μg / ml to 1 μg / ml, the concentration of DOR is or is approximately in the range of 1 μg / ml to 4 μg / ml, the concentration of DOR is or is approximately in the range of 4 μg / ml to 8 μg / ml, or the concentration of DOR is or is approximately in the range of 8 μg / ml to 16 μg / ml. In some embodiments, the concentration of DOR is at or within a range of about 0.03125, 0.0625, 0.1, 0.5, 0.75, 1, 2, 4, 5, 6, 8, 10, 12, 14, 16, 24, 32, 40, 48, 56, 60, 80, or 100 μg / ml, or within a range defined by any two of the above values. In some embodiments, the concentration of DOR is at or within a range of about 0.006 μg / ml to 0.6 μg / ml. In some embodiments, the concentration of DOR is or is approximately in the range of 0.03125 μg / ml to 0.0625 μg / ml, the concentration of DOR is or is approximately in the range of 0.015 μg / ml to 0.24 μg / ml, the concentration of DOR is or is approximately in the range of 0.0625 μg / ml to 0.0775 μg / ml, the concentration of DOR is or is approximately in the range of 0.0775 μg / ml to 0.1 μg / ml, or the concentration of DOR is or is approximately in the range of 0.1 μg / ml to 0.125 μg / ml. In some embodiments, the concentration of DOR is at or approximately in the range of 0.03, 0.03125, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.125, 0.15, 0.2, 0.4, 0.5, or 0.6 μg / ml, or within a range defined by any two of the foregoing values.

[0128] In Combination 4, the concentration of DOR is or is approximately in the range of 0.5 μg / ml to 2 μg / ml. In some embodiments, the concentration of DOR is or is approximately in the range of 0.1 μg / ml to 10 μg / ml, the concentration of DOR is or is approximately in the range of 0.25 μg / ml to 4 μg / ml, the concentration of DOR is or is approximately in the range of 0.5 μg / ml to 0.75 μg / ml, the concentration of DOR is or is approximately in the range of 0.75 μg / ml to 1 μg / ml, the concentration of DOR is or is approximately in the range of 1 μg / ml to 1.5 μg / ml, or the concentration of DOR is or is approximately in the range of 1.5 μg / ml to 2 μg / ml. In some embodiments, the concentration of DOR is at or in the range of about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 1, 1.25, 1.5, 1.75, or 2 μg / ml, or within a range defined by any two of the foregoing values.

[0129] In Combination 5, the concentration of DOR is or is approximately in the range of 0.03125 μg / ml to 0.125 μg / ml. In some embodiments, the concentration of DOR is or is approximately in the range of 0.03125 μg / ml to 0.0625 μg / ml, the concentration of DOR is or is approximately in the range of 0.0625 μg / ml to 0.0775 μg / ml, the concentration of DOR is or is approximately in the range of 0.0775 μg / ml to 0.1 μg / ml, or the concentration of DOR is or is approximately in the range of 0.1 μg / ml to 0.125 μg / ml. In some embodiments, the concentration of DOR is or is approximately in the range of 0.006 μg / ml to 0.6 μg / ml. In some embodiments, the concentration of DOR is or is approximately in the range of 0.015 μg / ml to 0.24 μg / ml. In some embodiments, the concentration of DOR is at or within a range of about 0.006, 0.01, 0.015, 0.03, 0.03125, 0.04, 0.05, 0.06, 0.0625, 0.07, 0.08, 0.09, 0.1, 0.115, 0.12, 0.125, 0.15, 0.2, 0.4, 0.5, or 0.6 μg / ml, or within a range defined by any two of the foregoing values.

[0130] In Combination 6, the concentration of MEM is or is approximately in the range of 0.03125 μg / ml to 1 μg / ml. In some embodiments, the concentration of MEM is or is approximately in the range of 0.0125 μg / ml to 5 μg / ml, the concentration of MEM is or is approximately in the range of 0.03125 μg / ml to 0.0625 μg / ml, the concentration of MEM is or is approximately in the range of 0.0625 μg / ml to 0.125 μg / ml, the concentration of MEM is or is approximately in the range of 0.125 μg / ml to 0.5 μg / ml, the concentration of MEM is or is approximately in the range of 0.125 μg / ml to 2 μg / ml, or the concentration of MEM is or is approximately in the range of 0.5 μg / ml to 1 μg / ml. In some embodiments, the concentration of MEM is at or about 0.0125, 0.03, 0.03125, 0.0625, 0.075, 0.1, 0.125, 0.25, 0.5, 0.6, 0.625, 0.7, 0.725, 0.8, 0.875, 0.9, 1, 1.5, 2, 2.5, or 5 μg / ml, or within a range defined by any two of the above values. In some embodiments, the concentration of MEM is at or about 0.006 μg / ml to 0.6 μg / ml. In some embodiments, the concentration of MEM is or is approximately in the range of 0.03125 μg / ml to 0.0625 μg / ml, the concentration of MEM is or is approximately in the range of 0.015 μg / ml to 0.24 μg / ml, the concentration of MEM is or is approximately in the range of 0.0625 μg / ml to 0.0775 μg / ml, the concentration of MEM is or is approximately in the range of 0.0775 μg / ml to 0.1 μg / ml, or the concentration of MEM is or is approximately in the range of 0.1 μg / ml to 0.125 μg / ml.In some embodiments, the concentration of MEM is at or in the range of about 0.03, 0.03125, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.125, 0.15, 0.2, 0.4, 0.5, or 0.6 μg / ml, or within a range defined by any two of the above values.

[0131] In Combination 7, the concentration of MEM is or is approximately in the range of 2 μg / ml to 8 μg / ml. In some embodiments, the concentration of MEM is or is approximately in the range of 0.4 μg / ml to 40 μg / ml, the concentration of MEM is or is approximately in the range of 1 μg / ml to 16 μg / ml, the concentration of MEM is or is approximately in the range of 2 μg / ml to 4 μg / ml, the concentration of MEM is or is approximately in the range of 4 μg / ml to 6 μg / ml, the concentration of MEM is or is approximately in the range of 6 μg / ml to 7.5 μg / ml, or the concentration of MEM is or is approximately in the range of 7.5 μg / ml to 8 μg / ml. In some embodiments, the concentration of MEM is at or in the range of about 0.4, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 12, 16, 20, 25, 30, 35, or 40 μg / ml, or within a range defined by any two of the above values.

[0132] In Combination 8, the concentration of MEM is or is approximately in the range of 0.03125 μg / ml to 0.125 μg / ml. In some embodiments, the concentration of MEM is or is approximately in the range of 0.03125 μg / ml to 0.0625 μg / ml, the concentration of MEM is or is approximately in the range of 0.0625 μg / ml to 0.0775 μg / ml, the concentration of MEM is or is approximately in the range of 0.0775 μg / ml to 0.1 μg / ml, or the concentration of MEM is or is approximately in the range of 0.1 μg / ml to 0.125 μg / ml. In some embodiments, the concentration of MEM is or is approximately in the range of 0.006 μg / ml to 0.6 μg / ml. In some embodiments, the concentration of MEM is or is approximately in the range of 0.015 μg / ml to 0.24 μg / ml. In some embodiments, the concentration of MEM is at or within a range of about 0.006, 0.01, 0.015, 0.03, 0.03125, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.125, 0.15, 0.2, 0.4, 0.5, or 0.6 μg / ml, or within a range defined by any two of the foregoing values.

[0133] In Combination 9, the concentration of MEM is at or about the range of 0.015625 μg / ml to 0.125 μg / ml. In some embodiments, the concentration of MEM is at or about the range of 0.03125 μg / ml to 0.0625 μg / ml, the concentration of MEM is at or about the range of 0.0625 μg / ml to 0.0775 μg / ml, the concentration of MEM is at or about the range of 0.0775 μg / ml to 0.1 μg / ml, or the concentration of MEM is at or about the range of 0.1 μg / ml to 0.125 μg / ml. In some embodiments, the concentration of MEM is or is approximately in the range of 0.006 μg / ml to 0.6 μg / ml, the concentration of MEM is or is approximately in the range of 0.015 μg / ml to 0.24 μg / ml, the concentration of MEM is or is approximately in the range of 0.003 μg / ml to 0.3 μg / ml, or the concentration of MEM is or is approximately in the range of 0.0075 μg / ml to 0.12 μg / ml. In some embodiments, the concentration of MEM is at or within a range of about 0.003, 0.0075, 0.01, 0.015, 0.01, 0.015625, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.125, 0.15, 0.2, 0.3, 0.4, 0.5, or 0.6 μg / ml, or within a range defined by any two of the foregoing values.

[0134] In combination 10, the concentration of TEM is at or about 32 μg / ml to 128 μg / ml. In some embodiments, the concentration of TEM is at or about 24 μg / ml to 128 μg / ml, the concentration of TEM is at or about 32 μg / ml to 75 μg / ml, the concentration of TEM is at or about 32 μg / ml to 50 μg / ml, the concentration of TEM is at or about 50 μg / ml to 75 μg / ml, the concentration of TEM is at or about 75 μg / ml to 100 μg / ml, or the concentration of TEM is at or about 100 μg / ml to 128 μg / ml. In some embodiments, the concentration of TEM is at or in the range of about 32, 40, 50, 60, 64, 70, 75, 80, 90, 100, 110, 120, or 128 μg / ml, or within a range defined by any two of the above values.

[0135] The algorithms provided herein are exemplary and non-limiting, and a person skilled in the art can design an algorithm based on any combination of the boxes provided in the algorithms described herein to obtain the required information regarding CPO detection and / or Ambler classification of carbapenemases.

[0136] For example, in some embodiments, in Examples 10.1 through 10.4, each "box" in the algorithm represents a test location (e.g., a well, or optionally an average of several identical wells) of a detection test provided herein that includes an input sample containing one or more bacteria, one or more detection reagents, and one or more antibiotics, with / without one or more carbapenemase inhibitors, and the CPO detection and / or classification wells of the algorithm for Enterobacteriaceae can be permuted and / or combined with the CPO detection and / or classification wells of the algorithm for non-fermentative bacteria to achieve detection of CPO Enterobacteriaceae, CPO non-fermentative bacteria, or both, and / or Ambler classification of Enterobacteriaceae, non-fermentative bacteria, or both.

[0137] In some embodiments, the wells contain one of the combinations comprising one or more antibiotics with / without one or more carbapenemase inhibitors disclosed in Table 0.1. In some embodiments, the test comprises at least two wells, where one well is for the detection of CPO enterobacteria and one well is for the detection of CPO non-fermenting bacteria (e.g., Figure 36).

[0138] In some embodiments, the test comprises at least six wells, where one well is for detection of CPO Enterobacteriaceae, one well is for detection of CPO non-fermenting bacteria, and four wells are for Ambler classification of carbapenemases produced by Enterobacteriaceae (e.g., Figure 37). Thus, in some embodiments, at least four wells allow for Ambler classification of carbapenemases produced by Enterobacteriaceae (e.g., Figure 37). In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by Enterobacteriaceae is two to five. In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by Enterobacteriaceae is two, three, four, five, or more.

[0139] In some embodiments, the test comprises at least nine wells, where one well is for detection of CPO Enterobacteriaceae, one well is for detection of CPO non-fermentative bacteria, four wells are for Ambler classification of carbapenemases produced by Enterobacteriaceae, and three wells are for Ambler classification of carbapenemases produced by non-fermentative bacteria (e.g., Figure 38). Thus, in some embodiments, at least four wells allow Ambler classification of carbapenemases produced by Enterobacteriaceae, at least three wells allow Ambler classification of carbapenemases produced by non-fermentative bacteria, and at least seven wells allow Ambler classification of carbapenemases produced by Enterobacteriaceae and non-fermentative bacteria (e.g., Figure 38). In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by Enterobacteriaceae is 2 to 5, and the number of wells used for Ambler classification of carbapenemases produced by non-fermentative bacteria is 2 to 4. In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by Enterobacteriaceae is 2, 3, 4, 5, or more, and the number of wells used for Ambler classification of carbapenemases produced by non-fermentative bacteria is 2, 3, 4, 5, or more. In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by Enterobacteriaceae and non-fermentative bacteria is 4 to 9. In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by Enterobacteriaceae and non-fermentative bacteria is 4, 5, 6, 7, 8, 9, or more.

[0140] In some embodiments, the test comprises at least five wells, where one well is for detection of CPO Enterobacteriaceae, one well is for detection of CPO non-fermentative bacteria, and three wells are for Ambler classification of carbapenemases produced by non-fermentative bacteria (e.g., Figure 39). Thus, in some embodiments, at least three wells allow for Ambler classification of carbapenemases produced by non-fermentative bacteria (e.g., Figure 39). In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by non-fermentative bacteria is two to four. In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by non-fermentative bacteria is two, three, four, or more.

[0141] In some embodiments, the number of wells for Ambler classification of carbapenemase is 9, regardless of whether the carbapenemase is produced by enteric or non-fermentative bacteria, where 4 wells are for non-fermentative bacteria and 5 wells are for enteric bacteria (e.g., Figure 38). [Example]

[0142] Example 1 (Identification of class A carbapenemase expression) (intestinal bacteria) If Enterobacteriaceae grows in the presence of 0.06 μg / ml MEM and 0.1 mg / ml CLOX, does not grow in the presence of 0.06 mg / ml MEM, 0.1 mg / ml CLOX, 0.18 mg / ml DPA, and does not grow in the presence of 0.03 mg / ml MEM, 0.1 mg / ml CLOX, 8 μg / ml RPX, the detection test indicates the expression of a class A carbapenemase by the bacterium.

[0143] If Enterobacteriaceae grows in the presence of 0.06 μg / ml MEM and 0.1 mg / ml CLOX, grows in the presence of 0.06 mg / ml MEM, 0.1 mg / ml CLOX, and 0.18 mg / ml DPA, and does not grow in the presence of 0.06 mg / ml MEM, 0.1 mg / ml CLOX, and 8 μg / ml RPX, the detection test indicates the expression of a class A carbapenemase by the bacterium.

[0144] If Enterobacteriaceae grows in the presence of 0.06 μg / ml MEM and 0.1 mg / ml CLOX, does not grow in the presence of 64 μg / ml TEM and 0.25 mg / ml EDTA, grows in the presence of 0.06 μg / ml DOR, 0.1 mg / ml CLOX, and 0.25 mg / ml EDTA, and does not grow in the presence of 0.06 μg / ml MEM, 0.1 mg / ml CLOX, and 8 μg / ml RPX, the detection test indicates the expression of a class A carbapenemase by this bacterium.

[0145] (non-fermenting bacteria) If a non-fermenting bacterium grows in the presence of 1 μg / ml DOR and 0.1 mg / ml CLOX, grows in the presence of 1 μg / ml DOR, 0.1 mg / ml CLOX, and 0.18 mg / ml DPA, grows in the presence of 4 μg / ml MEM, 0.1 mg / ml CLOX, and 5 μg / ml BLI, and does not grow in the presence of 8 μg / ml DOR, 0.1 mg / ml CLOX, and 4 μg / ml AVI, the detection test indicates the expression of a class A carbapenemase by this bacterium.

[0146] Example 2 (Identification of class B carbapenemase expression) (intestinal bacteria) If Enterobacteriaceae grows in the presence of 0.06 μg / ml MEM and 0.1 mg / ml CLOX, does not grow in the presence of 0.06 μg / ml MEM, 0.1 mg / ml CLOX, and 0.18 mg / ml DPA, and grows in the presence of 0.03 μg / ml MEM, 0.1 mg / ml CLOX, and 8 μg / ml RPX, the detection test indicates the expression of a class B carbapenemase by this bacterium.

[0147] If Enterobacteriaceae grows in the presence of 0.06 μg / ml MEM and 0.1 mg / ml CLOX, does not grow in the presence of 64 μg / ml TEM and 0.25 mg / ml EDTA, does not grow in the presence of 0.06 μg / ml DOR, 0.1 mg / ml CLOX, 0.25 mg / ml EDTA, and grows in the presence of 0.125 μg / ml DOR, the detection test indicates the expression of a class B carbapenemase by this bacterium.

[0148] (non-fermenting bacteria) If a non-fermenting bacterium grows in the presence of 1 μg / ml DOR and 0.1 mg / ml CLOX, but does not grow in the presence of 1 μg / ml DOR, 0.1 mg / ml CLOX, and 0.18 mg / ml DPA, the detection test indicates expression of a class B carbapenemase by the bacterium; growth in 2 μg / ml DOR and 0.1 mg / ml CLOX indicates expression of class B.

[0149] If a non-fermenting bacterium grows in the presence of 1 μg / ml DOR and 0.1 mg / ml CLOX, but does not grow in 1 μg / ml DOR, 0.1 mg / ml CLOX, and 0.18 mg / ml DPA, the detection test indicates expression of a class B carbapenemase by the bacterium; growth in 2 μg / ml DOR and 0.1 mg / ml CLOX indicates expression of class B.

[0150] Example 3 (Identification of class D carbapenemase expression) (intestinal bacteria) If Enterobacteriaceae grows in the presence of 0.06 μg / ml MEM, 0.1 mg / ml CLOX, grows in the presence of 0.06 μg / ml MEM, 0.1 mg / ml CLOX, 0.18 mg / ml DPA, grows in the presence of 0.06 μg / ml MEM, 0.1 mg / ml CLOX, 8 μg / ml RPX, and does not grow in the presence of 0.06 μg / ml DOR, 0.1 mg / ml CLOX, 4 μg / ml AVI, the detection test indicates the expression of a class D carbapenemase by this bacterium.

[0151] If Enterobacteriaceae grows in the presence of 0.06 μg / ml MEM, 0.1 mg / ml CLOX, and in the presence of 64 μg / ml TEM, 0.25 mg / ml EDTA, the detection test indicates the expression of class D carbapenemase by this bacterium.

[0152] If Enterobacteriaceae grows in the presence of 0.06 μg / ml MEM and 0.1 mg / ml CLOX, does not grow in the presence of 64 μg / ml TEM and 0.25 mg / ml EDTA, does not grow in the presence of 0.06 μg / ml DOR, 0.1 mg / ml CLOX, 0.25 mg / ml EDTA, and does not grow in the presence of 0.125 μg / ml DOR, the detection test indicates the expression of a class D carbapenemase by this bacterium.

[0153] (non-fermenting bacteria) If a non-fermenting bacterium grows in the presence of 1 μg / ml DOR, 0.1 mg / ml CLOX, grows in the presence of 1 μg / ml DOR, 0.1 mg / ml CLOX, 0.18 mg / ml DPA, and does not grow in the presence of 4 μg / ml MEM, 0.1 mg / ml CLOX, and 5 μg / ml BLI, the detection test indicates the expression of a class D carbapenemase by this bacterium.

[0154] If a non-fermenting bacterium grows in the presence of 1 μg / ml DOR, 0.1 mg / ml CLOX, grows in the presence of 1 μg / ml DOR, 0.1 mg / ml CLOX, 0.18 mg / ml DPA, and does not grow in the presence of 4 μg / ml MEM, 0.1 mg / ml CLOX, and 5 μg / ml BLI, the detection test indicates the expression of a class D carbapenemase by this bacterium.

[0155] Example 4 (Identification of class A, B, or D carbapenemase expression) (intestinal bacteria) If Enterobacteriaceae grows in the presence of 0.06 μg / ml MEM, 0.1 mg / ml CLOX, does not grow in the presence of 64 μg / ml TEM, 0.25 mg / ml EDTA, grows in the presence of 0.06 μg / ml DOR, 0.1 mg / ml CLOX, 0.25 mg / ml EDTA, grows in the presence of 0.06 μg / ml MEM, 0.1 mg / ml CLOX, 8 μg / ml RPX, and grows in the presence of 0.5 μg / ml MEM, 0.1 mg / ml CLOX, the detection test indicates the expression of class A, B, and / or D carbapenemases by this bacterium.

[0156] (non-fermenting bacteria) If a non-fermenting bacterium grows in the presence of 1 μg / ml DOR, 0.1 mg / ml CLOX, grows in the presence of 1 μg / ml DOR, 0.1 mg / ml CLOX, 0.18 mg / ml DPA, and grows in the presence of 4 μg / ml MEM, 0.1 mg / ml CLOX, 5 μg / ml BLI, the detection test indicates the expression of class A, B, and / or D carbapenemases by this bacterium.

[0157] If a non-fermenting bacterium grows in the presence of 1 μg / ml DOR, 0.1 mg / ml CLOX, grows in the presence of 1 μg / ml DOR, 0.1 mg / ml CLOX, 0.18 mg / ml DPA, grows in the presence of 4 μg / ml MEM, 0.1 mg / ml CLOX, 5 μg / ml BLI, and grows in the presence of 8 μg / ml DOR, 0.1 mg / ml CLOX, 4 μg / ml AVI, the detection test indicates the expression of class A, B, and / or D carbapenemases by this bacterium.

[0158] (incubation duration) The detection test is carried out for a predetermined incubation period. In some embodiments, the incubation period is the time it takes for the detection reaction to complete. In some embodiments, the incubation period is predetermined and defined by the user. The incubation time for the detection test may range from about 3 hours to about 16 hours. Thus, the result of the detection test, whether there is growth or not, is obtained within the time frame defined by the incubation time.

[0159] Conventional assays (e.g., plate-based assays) require at least 16 hours to about 24 hours or more for identification of antibiotic-resistant bacteria. In contrast, the present disclosure provides a more rapid detection test. For example, the time frame for the detection test in each well is 15 minutes to 3 hours or approximately in the range. In some embodiments, the duration of the detection test may range from about 5 hours to about 10 hours. In some embodiments, the duration of the detection test for Enterobacteriaceae ranges from about 6 hours to about 8 hours. In some embodiments, the duration of the detection test for Enterobacteriaceae ranges from about 5 hours to about 7 hours. In some embodiments, the duration of the detection test for non-fermentative bacteria ranges from about 8 hours to about 11 hours. In some embodiments, the duration of the detection test for non-fermentative bacteria ranges from about 7 hours to about 14 hours.

[0160] One or more detectors are provided that measure the results of the detection test by determining the presence or absence of bacterial growth in the presence of various combinations of one or more antibiotics and one or more inhibitors. The detectors periodically measure the results of the detection test until a predetermined incubation period has elapsed, followed by detection. The detectors detect growth or absence of growth in a rapid, automated manner. For example, the time frame for the detectors to measure the results of the detection test in multiple wells may be about 5 to about 10 minutes.

[0161] In some embodiments, the detector can analyze the results of the detection tests in multiple wells sequentially. In some embodiments, the detector can analyze the results of the detection tests in multiple wells simultaneously. Analyzing the results of the detection tests in multiple wells simultaneously is more efficient.

[0162] The detection test in each of the plurality of wells is redox-based, and one or more detection reagents in the well allow for redox-based detection of the absence or presence of proliferation. In some embodiments, the detection test in each of the plurality of wells to detect proliferation is based on turbidity in each of the plurality of wells. In some embodiments, the detection test in each of the plurality of wells to detect proliferation is based on a combination of redox and turbidity in each of the plurality of wells.

[0163] Oxidation-reduction reactions are well known in the art and include chemical reactions that change the oxidation state of atoms. Oxidation-reduction reactions involve the transfer of electrons between two or more chemical species. When one or more electrons are transferred from a chemical species, the chemical species is oxidized, and when one or more electrons are transferred to a chemical species, the chemical species is reduced. Turbidity-based growth detection is well known in the art. A non-limiting example of turbidity-based growth detection includes measuring the absorbance of light at a wavelength of 600 nm.

[0164] Non-limiting examples of redox reactions include combination, decomposition, substitution, oxidation, and disproportionation type redox reactions. In some embodiments, redox reactions may be based on changes in pH, color, etc.

[0165] The detector analyzes the results of the detection test in the multiple wells by detecting the outcome of the redox reaction in the wells. The detector analyzes the outcome of the redox reaction in a rapid, automated manner.

[0166] In some embodiments, the outcome of the detection test is growth of one or more bacteria in the plurality of wells. In some embodiments, the outcome of the detection test is no growth of one or more bacteria in the sample in the plurality of wells.

[0167] The assays disclosed so far can only be used for enterobacteria. In contrast, in some embodiments, the detection test can be used for enterobacteria. In some embodiments, the detection test can be used for non-fermentative bacteria. In some embodiments, the detection test can be used for both enterobacteria and non-fermentative bacteria.

[0168] In some embodiments, the detection test can be amenable to automation to allow rapid differentiation and identification of different carbapenemase classes, hi some embodiments, the detection test is combined with an algorithm that automatically performs phenotypic detection of carbapenemase production and Ambler classification of carbapenemases within a few hours.

[0169] One or more algorithms process the data from the one or more detectors to interrogate the results in the wells. The algorithm takes approximately 1 to 10 minutes to process the data. Based on the presence or absence of bacterial growth in the wells, the algorithm provides an output of growth or no growth in the wells. In some embodiments, the time frame for the entire algorithm is about 6 to about 12 hours. In some embodiments, the time frame for the entire algorithm is about 5 to about 7 hours.

[0170] Although this disclosure refers throughout to tests being performed in "wells," one of skill in the art will recognize that numerous test locations are suitable for the tests disclosed herein, and therefore "wells" are non-limiting. For example, microtiter plates, cuvettes, test tubes, or any other suitable structure known in the art can be used.

[0171] Non-limiting examples of algorithms are provided in Examples 6 (Figure 25) through 10 (Figure 30). Each "box" in the algorithm represents an assay well in the detection tests provided herein.

[0172] (BD Phoenix™ CPO Detect Algorithm) In some embodiments, one or more algorithms are provided that allow for rapid, automated identification of carbapenemase-expressing bacteria, along with identification of Ambler class carbapenemases. Incorporation of the algorithms into an automated platform has resulted in high levels of accuracy and improved time to results.

[0173] In some embodiments, a computer or computer system is provided that uses one or more of the algorithms provided herein to analyze and interpret the results of a detection test obtained using CPODetect. For example, the computer queries the detection test results obtained in multiple wells and provides an output based on the results (e.g., growth or no growth) from the queried detection tests as defined in the algorithm. The detection test results provided to the system are either growth positive (G) or no growth (NG) in one or more wells of the detection test within a predetermined time frame. Based on the result (growth or no growth) provided for the queried well, the system proceeds to the next query as defined in the algorithm. The system queries multiple test results until it reaches an output point in the algorithm, at which point the system generates an output result.

[0174] Non-limiting examples of algorithms are provided below (Examples 5-10). Each "box" in the algorithm represents a well (or optionally an average of several identical wells) of a detection test provided herein, including an input sample containing one or more bacteria, one or more detection reagents, and one or more antibiotics, with or without one or more carbapenemase inhibitors. For example, Box 1 in FIG. 25 represents a well of a detection test containing a combination of an input sample containing one or more Enterobacteriaceae, one or more detection reagents, 0.06 μg / ml MEM, and 0.1 mg / ml CLOX. As discussed, a determination of whether a sample contains Enterobacteriaceae or non-fermenting bacteria can be made by methods known in the art, for example, by biochemical tests, including spot oxidase tests, MALDI-TOF, and Phoenix ID systems. The determination can be made before or after the sample is run through the test. In the example algorithms presented herein, the determination of whether a sample contains enteric or non-fermentative bacteria is made either before or after the steps of the algorithm presented. In some embodiments, the algorithms presented herein can be run without first determining whether a sample contains enteric or non-fermentative bacteria. If a test is not suitable to provide a determined CPO detection and / or classification of the type of bacteria (e.g., non-fermentative or enteric) after the test has been run, the test results can simply be ignored.

[0175] Example 5 The algorithm flowchart illustrated in Figure 25 is used to determine whether a sample contains Enterobacteriaceae that produce class A, B, or D carbapenemase. Box 1 represents a well containing an input sample combination including one or more Enterobacteriaceae, 0.06 μg / ml MEM, 0.1 mg / ml CLOX, and one or more detection reagents. As shown in Figure 25 for Enterobacteriaceae, the system can query the result of the detection test in Box 1. If the result of the test in Box 1 is growth (G), the system reports a positive output result indicating the presence of Enterobacteriaceae that produce class A, B, or D carbapenemase in the sample. If the result of the test in Box 1 is no growth (NG), the reported output result is negative, i.e., the sample does not contain Enterobacteriaceae that produce class A, B, or D carbapenemase. As discussed herein, reporting of results depends on determining that the bacteria being tested are enteric, either before or after the test is attempted, to ensure that the appropriate algorithm is used depending on the type of bacteria present.

[0176] Example 6 (Identification of carbapenemase classes in Enterobacteriaceae) 26 illustrates a flowchart of an embodiment of an algorithm for determining whether Enterobacteriaceae in a sample produce carbapenemase, and if so, which class. As shown in FIG. 26 for Enterobacteriaceae, if the system queries the results of the test in Box 1 and growth is reported, it is expected to proceed to query the results of the test in Box 2. Box 2 represents a well containing an input sample combination including one or more Enterobacteriaceae, 0.06 μg / ml MEM, 0.1 mg / ml CLOX, 0.18 mg / ml DPA, and one or more detection reagents. If the system queries the results of the test in Box 2 and no growth is reported, it is expected to proceed to query the results of the test in Box 5. Box 5 represents a well containing an input sample combination including one or more Enterobacteriaceae, 0.03 μg / ml MEM, 0.1 mg / ml CLOX, 8 μg / ml RPX, and one or more detection reagents. If the system interrogates box 5 and reports no growth, it is expected to terminate the interrogation and output the result that the sample contains Enterobacteriaceae that express a class A carbapenemase. If the system interrogates box 2 and reports no growth, it is expected to interrogate the result of the test in box 5. If the system interrogates box 5 and reports growth, it is expected to terminate the interrogation and output the result that the sample contains bacteria that express a class B carbapenemase. If the system interrogates box 2 and reports growth, it is expected to interrogate for the result of the test in box 3. Box 3 represents a well containing an input sample combination that includes one or more Enterobacteriaceae, 0.06 μg / ml MEM, 0.1 mg / ml CLOX, 8 μg / ml RPX, and one or more detection reagents. If the system interrogates box 3 and reports no growth, it is expected to terminate the interrogation and output the result that the sample contains Enterobacteriaceae that express a class A carbapenemase.If the system interrogates box 3 and growth is reported, it is expected to proceed to interrogate the result in box 4. Box 4 represents a well containing an input sample combination including one or more Enterobacteriaceae, 0.06 μg / ml DOR, 0.1 mg / ml CLOX, 4 μg / ml AVI, and one or more detection reagents. If the system interrogates box 4 and no growth is reported, it is expected to terminate the interrogation and output a result that the sample contains Enterobacteriaceae that express a class D carbapenemase. If the system interrogates box 4 and growth is reported, it is expected to terminate the interrogation and output a result that it was not possible to determine which class of carbapenemase the Enterobacteriaceae express.

[0177] As discussed herein, reporting of results depends on determining that the bacteria being tested are enteric, either before or after the test is attempted, to ensure that the appropriate algorithm is used depending on the type of bacteria present.

[0178] Example 7 (Identification of carbapenemase classes in Enterobacteriaceae) 27 illustrates a flowchart of an embodiment of an algorithm for determining whether Enterobacteriaceae in a sample produce carbapenemase, and if so, which class. As shown in FIG. 27 for Enterobacteriaceae, if the system queries the result of the test in Box 1 and growth is reported, it is expected to proceed to query the result of the test in Box 6. Box 6 represents a well containing an input sample combination including one or more Enterobacteriaceae, 64 μg / ml TEM, 0.25 mg / ml EDTA, and one or more detection reagents. If the system queries the result of the test in Box 6 and no growth is reported, it is expected to proceed to query the result of the test in Box 7. Box 7 represents a well containing an input sample combination including one or more Enterobacteriaceae, 0.06 μg / ml DOR, 0.1 mg / ml CLOX, 0.25 mg / ml EDTA, and one or more detection reagents. If the system queries the result of the test in Box 7 and growth is reported, it is expected to proceed to query the result of the test in Box 3. If the algorithm queries the result of the test in Box 3 and no growth is reported, it is expected to terminate the query and output the result that the sample contains Enterobacteriaceae that express a class A carbapenemase. If the system queries the result of the test in Box 6 and no growth is reported, it is expected to proceed to query the result of the test in Box 7. If the algorithm queries the result of the test in Box 7 and no growth is reported, it is expected to proceed to query the result of the test in Box 9. Box 9 represents a well containing an input sample combination that includes one or more Enterobacteriaceae, a DOR of 0.125 μg / ml, and one or more detection reagents. If the system queries the result of the test in Box 9 and growth is reported, it is expected to terminate the query and output the result that the sample contains Enterobacteriaceae that express a class B carbapenemase.If the system queries the result of the test in Box 6 and growth is reported, it is expected to terminate the query and output the result that the sample contains Enterobacteriaceae that express a class D carbapenemase. If the system queries the result of the test in Box 9 and no growth is reported, it is expected to terminate the query and output the result that the sample contains Enterobacteriaceae that express a class D carbapenemase. If the system queries the result of the test in Box 3 and growth is reported, it is expected to proceed to query the result of the test in Box 8. Box 8 represents a well containing an input sample combination including one or more Enterobacteriaceae, 0.5 μg / ml MEM, 0.1 mg / ml CLOX, and one or more detection reagents. If the system queries the result of the test in Box 8 and growth is reported, it is expected to terminate the query and output the result that the sample contains Enterobacteriaceae that express one or more class A, B, or D carbapenemases. If the system queries the results of the test in Box 8 and no growth is reported, it will terminate the query and it is expected to output the result that it was not possible to determine which class of carbapenemase the Enterobacteriaceae expresses.

[0179] As discussed herein, reporting of results depends on determining that the bacteria being tested are enteric, either before or after the test is attempted, to ensure that the appropriate algorithm is used depending on the type of bacteria present.

[0180] Example 8 FIG. 28 illustrates a flowchart of an embodiment of an algorithm for determining whether a sample contains non-fermenting bacteria that produce class A, B, or D carbapenemase. Box 10 represents a well containing an input sample combination including one or more Enterobacteriaceae, 1 μg / ml DOR, 0.1 mg / ml CLOX, and one or more detection reagents. As shown in FIG. 28 for non-fermenting bacteria, the system can query the result of the detection test in box 10. If the result of the test in box 10 is growth (G), the system reports a positive output result indicating the presence of Enterobacteriaceae that produce class A, B, or D carbapenemase in the sample. If the result of the test in box 10 is no growth (NG), the reported output result is negative, i.e., the sample does not contain non-fermenting bacteria that produce class A, B, or D carbapenemase. As discussed herein, reporting of results depends on determining that the bacteria being tested are non-fermenting, either before or after the test is attempted, to ensure that the appropriate algorithm is used depending on the type of bacteria present.

[0181] Example 9 Identification of carbapenemase classes in non-fermenting bacteria 29 illustrates a flowchart of an embodiment of an algorithm for determining whether non-fermentative bacteria in a sample produce carbapenemase, and if so, which class. As shown in FIG. 29 for non-fermentative bacteria, if the system queries the result of the test in box 10 and growth is reported, it is expected to proceed to query the result of the test in box 11. Box 11 represents a well containing an input sample combination including one or more non-fermentative bacteria, 1 μg / ml DOR, 0.1 mg / ml CLOX, 0.18 mg / ml DPA, and one or more detection reagents. If the system queries the result of the test in box 11 and no growth is reported, it is expected to query the result of the test in box 13. Box 13 represents a well containing an input sample combination including one or more non-fermentative bacteria, 2 μg / ml DOR, 0.1 mg / ml CLOX, and one or more detection reagents. If the system queries the result of the test in box 13 and growth is reported, it is expected to terminate the query and output a result that the sample contains non-fermentative bacteria expressing a class B carbapenemase. If the system queries the result of the test in box 11 and growth is reported, it is expected to proceed to query the result of the test in box 12. Box 12 represents a well containing an input sample combination including one or more non-fermentative bacteria, 4 μg / ml MEM, 0.1 mg / ml CLOX, 5 μg / ml BLI, and one or more detection reagents. If the system queries the result of the test in box 12 and no growth is reported, it is expected to terminate the query and output a result that the sample contains non-fermentative bacteria expressing a class D carbapenemase. If the system queries the result of the test in box 12 and growth is reported, it is expected to terminate the query and output a result that the sample contains non-fermentative bacteria expressing one or more class A, B, or D carbapenemases.If the system queries the results of the test in Box 13 and no growth is reported, it is expected to terminate the query and output the result that it was unable to determine which class of carbapenemase the non-fermenting bacteria expresses.

[0182] As discussed herein, reporting of results depends on determining that the bacteria being tested are non-fermenting, either before or after the test is attempted, to ensure that the appropriate algorithm is used depending on the type of bacteria present.

[0183] Example 10 Identification of carbapenemase classes in non-fermenting bacteria FIG. 30 illustrates a flowchart of an embodiment of an algorithm for determining whether non-fermentative bacteria in a sample produce carbapenemase, and if so, which class. As shown in FIG. 30 for non-fermentative bacteria, if the system interrogates box 10 and detects growth, it is expected to interrogate box 11. If the system interrogates box 11 and detects growth, it is expected to interrogate box 12. If the system interrogates the result of the test in box 12 and detects growth, it is expected to interrogate the result of the test in box 14. Box 14 represents a well containing an input sample combination including one or more non-fermentative bacteria, 8 μg / ml DOR, 0.1 mg / ml CLOX, 4 μg / ml AVI, and one or more detection reagents. If the system interrogates the result of the test in box 14 and reports no growth, it is expected to terminate the interrogation and output a result that the sample contains non-fermentative bacteria expressing class A carbapenemase. If the system queries the result of the test in Box 11 and no growth is reported, it is expected to proceed to query the result of the test in Box 13. If the system queries the result of the test in Box 13 and growth is reported, it is expected to terminate the query and output the result that the sample contains a non-fermenting bacterium that expresses a class B carbapenemase. If the system queries the result of the test in Box 11 and growth is reported, it is expected to proceed to query the result of the test in Box 12. If the system queries the result of the test in Box 12 and no growth is reported, it is expected to terminate the query and output the result that the sample contains a non-fermenting bacterium that expresses a class D carbapenemase. If the system queries the result of the test in Box 12 and growth is reported, it is expected to proceed to query the result of the test in Box 14.If the system queries the result of the test in Box 14 and growth is reported, it is expected to terminate the query and output the result that the sample contains non-fermenting bacteria that express one or more of class A, B, or D carbapenemases. If the system queries the result of the test in Box 13 and no growth is reported, it is expected to terminate the query and output the result that it was not possible to determine which class of carbapenemase the non-fermenting bacteria expresses.

[0184] As discussed herein, reporting of results depends on determining that the bacteria being tested are non-fermenting, either before or after the test is attempted, to ensure that the appropriate algorithm is used depending on the type of bacteria present.

[0185] Example 10.1 (Carbapenemase detection in Enterobacteriaceae and non-fermenting strains) FIG. 36 shows a flowchart of an embodiment of an algorithm for CPO detection of Enterobacteriaceae and non-fermenting strains. As shown in FIG. 36 for Enterobacteriaceae, the system can query the result of the detection test in Box 1. If the result of the test in Box 1 is growth, the system reports a positive output result indicating the presence of Enterobacteriaceae that produce class A, B, or D carbapenemase in the sample. If the result of the test in Box 1 is no growth (inhibition), the reported output result is negative, i.e., the sample does not contain Enterobacteriaceae that produce class A, B, or D carbapenemase. As shown in FIG. 36 for non-fermenting bacteria, the system can query the result of the detection test in Box 10. If the result of the test in Box 10 is growth, the system reports a positive output result indicating the presence of non-fermenting bacteria that produce class A, B, or D carbapenemase in the sample. If the result of the test in Box 10 is no growth (inhibition), the reported output result is negative, i.e., the sample does not contain non-fermenting bacteria that produce class A, B, or D carbapenemase.

[0186] As discussed herein, reporting of results depends on determining that the bacteria being tested are enteric and / or non-fermenting, either before or after the test is attempted, to ensure that the appropriate algorithm is used depending on the type of bacteria present.

[0187] Example 10.2 (Carbapenemase detection in Enterobacteriaceae and non-fermenting strains and Ambler classification of Enterobacteriaceae) FIG. 37 shows a flowchart of an embodiment of an algorithm for CPO detection of Enterobacteriaceae and non-fermenting strains and classification of Enterobacteriaceae. The algorithm in FIG. 37 is based on the algorithm in FIG. 36. As shown in FIG. 37 for Enterobacteriaceae, the system can query the result of the detection test in Box 1. If the result of the test in Box 1 is growth, the system reports a positive output result indicating the presence of Enterobacteriaceae that produce class A, B, or D carbapenemase in the sample. If the result of the test in Box 1 is no growth (inhibition), the reported output result is negative, i.e., the sample does not contain Enterobacteriaceae that produce class A, B, or D carbapenemase. As shown in FIG. 37 for non-fermenting bacteria, the system can query the result of the detection test in Box 10. If the result of the test in Box 10 is growth, the system reports a positive output result indicating the presence of non-fermenting bacteria that produce class A, B, or D carbapenemase in the sample. If the result of the test in Box 10 is no growth (inhibition), the reported output result is negative, i.e., the sample does not contain non-fermenting bacteria that produce class A, B, or D carbapenemase.

[0188] The algorithm in Figure 37 further enables determination of the class of carbapenemase produced by Enterobacteriaceae. As shown in Figure 37 for Enterobacteriaceae, if the system queries the result of the test in Box 1 and growth is reported, it is expected to proceed to query the result of the test in Box 6. If the system queries Box 6 and growth is reported, it is expected to terminate the query and output the result that the sample contains Enterobacteriaceae that express a class D carbapenemase. If the system queries Box 6 and no growth (inhibition) is reported, it is expected to proceed to query the result of the test in Box 7. If the system queries Box 7 and no growth (inhibition) is reported, it is expected to proceed to query the result of the test in Box 9. If the system queries Box 9 and no growth (inhibition) is reported, it is expected to terminate the query and output the result that the sample contains Enterobacteriaceae that express a class D carbapenemase. If the system queries Box 7 and growth is reported, it is expected to query the result of the test in Box 3. If the system queries box 3 and reports no growth (inhibition), it is expected to terminate the query and output the result that the sample contains Enterobacteriaceae that express a class A carbapenemase. If the system queries box 9 and reports growth, it is expected to terminate the query and output the result that the sample contains Enterobacteriaceae that express a class B carbapenemase. If the system queries box 3 and reports growth, it is expected to terminate the query and output the result that it was not possible to determine which class of carbapenemase the Enterobacteriaceae express.

[0189] As discussed herein, reporting of results depends on determining whether the bacteria being tested are Enterobacteriaceae and / or non-fermenting bacteria, either before or after the test is attempted, to ensure that the appropriate algorithm is used depending on the type of bacteria present.

[0190] Example 10.3 (Carbapenemase detection and Ambler classification for Enterobacteriaceae and non-fermenting strains) Figure 38 shows a flow chart of an embodiment of an algorithm for CPO detection and classification of Enterobacteriaceae and non-fermentative strains. The algorithm in Figure 38 is based on the algorithm in Figure 37.

[0191] In addition to the procedures and results described with respect to Figure 37, the algorithm in Figure 38 further allows for the determination of the class of carbapenemase produced by non-fermentative bacteria. As shown in Figure 38 for Enterobacteriaceae, if the system queries the result of the test in box 10 and growth is reported, it is expected to proceed to query the result of the test in box 11. If the system queries box 11 and no growth (inhibition) is reported, it is expected to terminate the query and output the result that the sample contains non-fermentative bacteria expressing a class B carbapenemase. If the system queries box 11 and growth is reported, it is expected to proceed to query the result of the test in box 12. If the system queries box 12 and no growth (inhibition) is reported, it is expected to terminate the query and output the result that the sample contains non-fermentative bacteria expressing a class D carbapenemase. If the system queries box 12 and growth is reported, it is expected to proceed to query the result of the test in box 14. If the system interrogates box 14 and reports no growth (inhibition), it is expected to terminate the interrogation and output the result that the sample contains non-fermenting bacteria that express a class A carbapenemase. If the system interrogates box 14 and reports growth, it is expected to terminate the interrogation and output the result that it was unable to determine which class of carbapenemase the non-fermenting bacteria expresses.

[0192] As discussed herein, reporting of results depends on determining that the bacteria being tested are enteric and / or non-fermenting, either before or after the test is attempted, to ensure that the appropriate algorithm is used depending on the type of bacteria present.

[0193] Example 10.4 (Carbapenemase detection for Enterobacteriaceae and non-fermentative strains and Ambler classification for non-fermentative strains) Figure 39 shows a flow chart of an embodiment of an algorithm for CPO detection of Enterobacteriaceae and non-fermenting strains and classification of non-fermenting strains. The algorithm in Figure 39 is the same as Figure 38 except that the part of the algorithm for classification of Enterobacteriaceae is not included.

[0194] Example 11 (BD Phoenix™ CPO Detect Test vs. bioMerieux Rapidec® Carba NP Test) The data in this example relate to a study designed to assess the performance of the BD Phoenix™ CPO Detect to meet current clinical needs. As disclosed herein, the BD Phoenix™ CPO Detect test is designed to be integrated into a sensitivity panel to provide both CPO detection and carbapenemase classification, reducing operator time and accelerating carbapenemase reporting. The comparison test was the bioMerieux Rapidec® Carba NP test, a currently available stand-alone test that detects carbapenemases but does not classify them. Therefore, the BD Phoenix™ CPO Detect IUO panel and the bioMerieux Rapidec® Carba NP test were compared with respect to accuracy and impact on workflow.

[0195] Example 11.1: Procedure The study was performed at the BD Life Sciences, Sparks, MD, laboratory, with BD research staff providing laboratory and computing support. GKID Inc. prepared the inoculum for both studies and interpreted all bioMérieux Rapidec® Carba NP tests. BD staff were not involved in any aspect of the bioMérieux Rapidec® Carba NP tests. Both studies were blinded and performed according to the manufacturer's recommendations. The inoculum was prepared from overnight growth on BD blood agar plates adjacent to imipenem discs used to enhance retention of carbapenemase-encoding plasmids in unstable isolates.

[0196] The bioMérieux Rapidec® Carba NP test was sometimes difficult to interpret. The manufacturer's definition of a positive test was "significant color variation" between test and test control wells. This definition was problematic because it did not provide a clear demarcation between significant and insignificant color variation. For example, no significant color variation was observed using E. cloacae 0164 (IMI class A carbapenemase), which would be expected to result in a positive test; E. coli 0104 (KPC class A carbapenemase), which would be expected to result in a positive test; E. coli 0058 (ESBL), which would be expected to result in a negative test; and Klebsiella pneumoniae G1673 (CMY-2 plasmid-mediated AmpC), which would be expected to result in a negative test. For this reason, borderline results were interpreted as both positive (interpretation 1) and negative (interpretation 2). This provided two sets of bioMérieux Rapidec® Carba NP results.

[0197] Example 11.2 Isolates A total of 294 isolates plus three quality control strains were tested. The test isolates consisted of 236 isolates belonging to Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii that produced a single carbapenemase, seven that produced two carbapenemases, and 51 negative controls. Tables 1.1, 1.2, and 1.3 provide a summary of the isolate types (number of isolates belonging to each species, plus resistance mechanism grouping). Table 1.3 provides mechanism symbols for Tables 1.1–1.3.

[0198] Isolates were obtained from:

[0199] FDA / CDC challenge panel of carbapenemase- and non-carbapenemase-producing Gram-negative bacilli;

[0200] Well-characterized isolates of carbapenemase- and non-carbapenemase-producing Gram-negative bacilli provided by GKID Inc.; and

[0201] ATCC quality control isolates:

[0202] Klebsiella pneumoniae BAA-1705 (positive, KPC) - used for both tests.

[0203] Klebsiella pneumoniae ATCC 700603 (negative) - used for both tests.

[0204] E. coli ATCC BAA2452 (positive, NDM-1) - used with BD Phoenix™ CPO Detect only.

[0205] These were not routine clinical isolates. They were chosen to provide an extreme test of diagnostic capability. The reference standard was prior characterization by molecular, phenotypic, and biochemical testing. There were 110 producers of class A carbapenemases, including KPC, NMC-A, IMI, and SME enzymes; 91 producers of class B carbapenemases (metallo-β-lactamases), including NDM, GIM, SPM, IMP, and VIM enzymes; 35 producers of class D carbapenemases, including OXA-23, 40, 48, 58, 72, 181, and 232; and seven isolates producing two carbapenemases. Fifty-one negative controls (35 AmpC and 16 other non-AmpC) produced ESBLs, AmpC (including overproducers), K1, broad-spectrum β-lactamases, and porin mutants.

[0206] [Table 2]

[0207] [Table 3-1]

[0208] [Table 3-2]

[0209] [Table 4]

[0210] Example 11.3: Sensitivity of detection of all carbapenemases Given the extreme diagnostic difficulties of some of the isolates tested, the results obtained with both the BD Phoenix™ CPODetect and bioMérieux Rapidec® Carba NP were outstanding in terms of overall sensitivity for the detection of all carbapenemases types.

[0211] The BD Phoenix™ CPO Detect achieved a sensitivity of 97.1% (236 of 243 CPOs detected). The sensitivity for the bioMerieux Rapidec® Carba NP test was 98.8% using interpretation 1 and 97.1% using interpretation 2.

[0212] Example 11.4: Sensitivity of Detection of Molecular Classes of Carbapenemases The detection sensitivity of both the BD Phoenix™ CPO Detect and bioMérieux Rapidec® Carba NP was very good for each molecular class of carbapenemase. The BD Phoenix™ CPO Detect discontinued the panel for one isolate, P. aeruginosa G15303. Repeat testing of this isolate is expected to be available in routine clinical laboratories shortly.

[0213] (Sensitivity for detecting class A carbapenemase) The BD Phoenix™ CPO Detect achieved a sensitivity of 97.3% (107 for 110 isolates) detection of class A carbapenemase producers. The bioMerieux Rapidec® Carba NP achieved a sensitivity of 100.0% (110 for 110 isolates) using interpretation 1 and 98.2% (108 for 110 isolates) using interpretation 2. Detection of 97.3% of class A producers in this extremely challenging evaluation is a significant achievement.

[0214] Regarding the sensitivity of BD Phoenix™ CPO Detect for Class A detection, the following three KPC (Class A) producers gave false negative results:

[0215] C. freundii G1706 - This isolate had a relatively low ertapenem MIC of 1 μg / ml (most CPOs were >1 μg / ml). The meropenem MIC (0.25 μg / ml) was unusually low for CPOs. The imipenem MIC was significantly elevated (2 μg / ml), but not within the resistance range. This type of CPO is difficult to detect in tests that cannot detect carbapenem hydrolysis. This would not have raised suspicions if meropenem had been the only carbapenem tested.

[0216] KPC-4-producing K. pneumoniae G1511-KPC-4 is a weakly active enzyme. Because of the significantly elevated MICs (ertapenem > 1; imipenem 4; meropenem 2 μg / ml), the isolate is not expected to be incorrectly reported as susceptible to carbapenems. This makes it an extremely difficult isolate to confirm as a CPO for most phenotypic tests.

[0217] K. oxytoca 0147 - This isolate would not be inappropriately reported as carbapenem-susceptible because it had high, off-scale carbapenem MICs: ertapenem >1; imipenem >8; meropenem >8 μg / ml. It is unclear why its carbapenemase-producing potential was not recognized.

[0218] These three isolates are less common types of CPO, likely due to the limited data currently available to generate a robust algorithm, and two of them are expected to be reported as clearly not susceptible to carbapenems and therefore not candidates for carbapenem therapy.

[0219] (Sensitivity for detecting class B carbapenemase) The BD Phoenix™ CPO Detect achieved a sensitivity of 95.6% (87 of 91 isolates) of detection of class B producers. The bioMerieux Rapidec® Carba NP achieved a sensitivity of 98.9% (90 of 91 isolates) using interpretations 1 and 2.

[0220] Regarding the sensitivity of the BD Phoenix™ CPO Detect for class B detection, four class B (metallo-β-lactamase)-producing strains yielded false-negative results. Three (two P. aeruginosa and one P. mirabilis) had high carbapenem MICs and would not be expected to be reported as carbapenem-susceptible. Their phenotypes resembled those conferred by non-carbapenemase mechanisms, which may have made them difficult to recognize as CPOs. A fourth isolate, E. cloacae G1691, had a low MEM MIC and may not have raised suspicion as a CPO if it had been the only carbapenem tested.

[0221] The four isolates were as follows:

[0222] Despite the elevated MIC of IMP-8-producing E. cloacae G1691:ERT>1;IMP4; and MEM 0.5 μg / ml, the unusual phenotype of this organism, particularly the low but elevated MIC of MEM, may have contributed to the false-negative test.

[0223] VIM-producing P. aeruginosa G15557 and VIM-2-producing P. aeruginosa had the same phenotype: ERT > 1; IMP > 8; MEM 4 μg / ml, a frequently encountered phenotype associated with reduced OprD porin production, which may have contributed to the false-negative results.

[0224] The fourth isolate was an IMP-27-producing Proteus mirabilis. The carbapenem MICs of ERT > 1; IMP8; MEM > 8 μg / ml were distinct from this species' inherently reduced susceptibility to imipenem but not to other carbapenems. The atypical phenotype would be expected to raise suspicion of carbapenemase production, but IMP-27 is a carbapenemase that is extremely difficult to detect by phenotypic testing.

[0225] (Sensitivity for detecting class D carbapenemase) Both BD Phoenix™ CPO Detect and bioMerieux Rapidec® Carba NP were excellent at detecting class D carbapenemase production by Acinetobacter spp. and Enterobacteriaceae.

[0226] BD Phoenix™ CPO Detect achieved a sensitivity of 100% (35 of 35 isolates) detection of class D producers. This is particularly unprecedented given that class D producers present the most difficult diagnostic challenge, as class D carbapenemases have only weak activity and are very difficult or nearly impossible to detect with some current tests.

[0227] bioMerieux Rapidec® Carba NP achieved 94.3% (33 of 35 isolates) using interpretations 1 and 2. bioMerieux Rapidec® Carba NP missed two OXA-48-like producers.

[0228] (Sensitivity of detection of isolates producing two carbapenemases) All seven isolates producing two carbapenemases were reported as carbapenemase positive by both BD Phoenix™ CPO Detect and bioMerieux Rapidec® Carba NP.

[0229] Example 11.5. Specificity of detection of all carbapenemases The extremely difficult nature of the negative control isolates contributed to the lower than usual specificity. The BD Phoenix™ CPO Detect yielded a specificity of 68.6% (35 for 51 isolates). The bioMérieux Rapidec® Carba NP yielded a specificity of 60.8% (31 for 51 isolates) using interpretation 1 and 78.4% (40 for 51 isolates) using interpretation 2.

[0230] Both tests had problems with both AmpC-producing and non-AmpC-producing strains. For AmpC-producing strains, the BD Phoenix™ CPO Detect yielded a specificity of 74.3% (26 of 35 isolates), and the bioMérieux Rapidec® Carba NP yielded a specificity of 57.1% (20 of 35 isolates) using interpretation 1 and 77.1% (27 of 35 isolates) using interpretation 2.

[0231] For other non-AmpC-producing strains, the BD Phoenix™ CPO Detect yielded a specificity of 43.8% (7 for 16 isolates), and the bioMerieux Rapidec® Carba NP yielded a specificity of 62.5% (10 for 16 isolates) using interpretations 1 and 2.

[0232] Table 2 lists the isolates that produced false-positive results, a characterization of their resistance mechanisms, and their carbapenem MICs. False-positive results due to high-level AmpC production are problematic for many carbapenemase detection tests. High-level AmpC production per se does not explain false-positive results when using the BD Phoenix™ CPO Detect test. This test correctly called negative for E. coli G1634 and G1700, which are very high-level AmpC producers. This tends to rule out AmpC production as an explanation for false-positive results in other AmpC-producing strains. Similarly, it is unlikely that the false-positive results for ESBL-producing strains were caused by ESBL production itself. A more likely explanation is that the false-positive results for these isolates were due to porin mutations. A less likely explanation is the production of broad-spectrum AmpC, which hydrolyzes carbapenems. Another possible explanation is chelator inhibition of class B carbapenemase detection tests. This can result in tests of ESBL- or AmpC-producing strains yielding false positives for class B carbapenemase production.

[0233] The identification of the isolates listed in Table 2 adds support for the possibility of porin mutations as an explanation. Fifteen of the 17 isolates were Klebsiella pneumoniae, Escherichia coli, and Enterobacter spp. Among these, Klebsiella pneumoniae is the most common member of the Enterobacteriaceae family in which porin mutations increase the MICs of carbapenems, particularly ertapenem and meropenem. E. coli and Enterobacter spp. are also relatively prone to porin mutations. In Table 2, all isolates had elevated, off-scale MICs of at least one carbapenem, and most had off-scale MICs of all carbapenems. Confirmation of porin mutations is usually not attempted because it is tedious, expensive, and technically challenging. Distinguishing porin mutants from carbapenemase-producing strains is best achieved by testing for the presence or absence of carbapenem hydrolysis.

[0234] False-positive results for P. mirabilis G1745 can sometimes be corrected using software editing. This isolate is representative of a species with imipenem MICs that are characteristically higher than those of ertapenem and meropenem. The elevated imipenem MIC alone for this isolate is unlikely to be caused by a carbapenemase.

[0235] [Table 5]

[0236] Example 11.6: Classification of Carbapenemases The classification of carbapenemases into molecular classes A, B, and D has therapeutic importance. It is also useful for infection control, epidemiology, and surveillance. Four BD Phoenix™ CPO Detect algorithms were analyzed for their ability to classify carbapenemase-producing types.

[0237] The bioMerieux Rapidec® Carba NP test cannot classify carbapenemases. Currently, the ability of the BD Phoenix™ CPO Detect to classify carbapenemases is unparalleled. There are no standards for evaluating this type of test. Indeed, any correct carbapenemase classification may have clinical benefit.

[0238] Algorithm 1 classified Enterobacteriaceae into class A, B, or D (Figure 32). Algorithm 1 classified non-fermentative strains only into class B or D (Figure 33). Algorithm 2 classified both Enterobacteriaceae (Figure 34) and non-fermentative strains (Figure 35) into class A, B, or D. Algorithm 3 is the same as Algorithm 1, except that BD Phoenix™ CPO Detect can produce a "no answer" result (Figure 26 for Enterobacteriaceae and Figure 29 for non-fermentative strains). Algorithm 4 is the same as Algorithm 2, except that BD Phoenix™ CPO Detect can produce a "no answer" result (Figure 27 for Enterobacteriaceae and Figure 30 for non-fermentative strains).

[0239] Algorithms 1-4 (Figures 26, 27, 29, 30, and 32-35) are exemplary and non-limiting. The antibiotic concentrations provided in Algorithms 1-4 (Figures 26, 27, 29, 30, and 32-35) were within the antibiotic concentration ranges disclosed in Table 2.1. In Algorithms 1-4 (Figures 26, 27, 29, 30, and 32-35) and Table 2.1, the concentrations of CLOX were 100 μg / mL, DPA was 178 μg / mL, AVI was 4 μg / mL, BLI was 5 μg / mL, EDTA was 250 μg / mL, and RPX was 8 μg / mL.

[0240] [Table 6]

[0241] Algorithms 1-4 (FIGS. 26, 27, 29, 30, and 32-35) operate as previously described in FIGS. 25-30. For example, each "box" in Algorithms 1-4 (e.g., box 1 in FIG. 32) represents a well (or optionally an average of several identical wells) of a detection test provided herein that contains an input sample containing one or more bacteria, one or more detection reagents, and one or more antibiotics, with or without one or more carbapenemase inhibitors. The detection test result provided to the system is either positive growth (G) or no growth (NG) in one or more wells of the detection test within a predetermined time frame. Based on the result (growth or no growth) provided for the queried well, the system proceeds to the next query as defined in the algorithm. The system queries multiple test results until it reaches an output point in the algorithm, at which point it generates an output result.

[0242] Table 3 summarizes the results of the algorithm for all isolates except for the seven dual carbapenemase-producing strains. The left side of Table 3 lists the successful results, i.e., correct classifications, correct negative results, unclassified carbapenemase (untyped), and the total correct detections regardless of whether they were classified or unclassified (i.e., assigned to columns “A, B, D, or untyped” / untyped).

[0243] [Table 7]

[0244] All algorithms performed well, identifying at least 80% of class A-producing CPOs. Algorithms 2 and 4 correctly classified 91 of 110 class A-producing strains (82.7%), slightly more accurately than algorithms 1 and 3 (81.2%). The incidence of misclassification was very low, and, clinically important, none of the isolates misclassified as class A producers were class B producers. Only one isolate, OXA-40-producing A. baumannii G1734, produced a false-positive class A result using algorithms 2 and 4. Three isolates, CTX-M-9-producing E. coli 0086, CMY-producing P. mirabilis G1745, and AmpC-overproducing M. morganii G1751, produced false-positive class A results using algorithms 1 and 3. The high level of accuracy in identifying class A-producing CPO indicates that the availability of ceftazidime / avibactam therapy fulfills an important, yet currently unmet, clinical need.

[0245] All algorithms correctly classified 63 of 91 (69.2%) Class B-producing strains. This is useful for identifying when ceftazidime / avibactam should not be used. This could ultimately save lives by preventing patients from receiving ineffective ceftazidime / avibactam therapy. Consequences of a false-positive Class B classification include potentially delaying the initiation of effective ceftazidime / avibactam therapy or alternative anti-CPO therapy. Generally, these are expected to be non-life-threatening consequences that may only apply until additional testing (e.g., molecular) is performed. Algorithms 2 and 4 yielded 13 false-positive results, more than twice the number of false-positives in Algorithms 1 and 3. Taken together, the BD Phoenix™ CPO Detect's performance in identifying Class B-producing strains offers a significant potential for clinical benefit and minimal potential for placing patients at serious risk.

[0246] Algorithm 2 correctly classified most class D-producing strains, i.e., 31 of 35 isolates (88.6%). Of all carbapenemases, these are the most difficult to detect, let alone classify. Performance of all algorithms on class D-producing strains was excellent. False-positive class D calls can lead to unnecessary isolation. Algorithms 2 and 4, with four incorrect calls each, outperformed algorithms 1 and 3, which each produced 20 false-positive calls. The "no answer" result for algorithms 3 and 4 is also unhelpful in that it confers neither benefit nor harm.

[0247] All algorithms correctly reported 33 of 51 carbapenemase-negative isolates (64.7%) as negative. In routine clinical practice, where diagnostic difficulty would be significantly lower than in this study, the percentage of correct negative results would be significantly higher.

[0248] In summary, all algorithms correctly classified at least 80% of class A and class D carbapenemases and nearly 70% of class B carbapenemases. This is an important achievement and a major advance in phenotypic testing. Overall, algorithm 2 was slightly better than the other algorithms in correctly classifying carbapenemases, and it also provided the best test.

[0249] Tables 4.1 and 4.2 show the carbapenemase-producing and noncarbapenemase-producing isolates that resulted in inaccurate classifications and their carbapenem MICs, respectively. In the context of the highly difficult-to-detect CPOs in this study, the false-negative rate of 7 out of 244 tested CPOs (2.9%) is not surprising. While no carbapenemase detection test is perfect, it would be desirable to reduce the false-negative rate to 1%. Apart from these 7 false-negative results by each algorithm, the other inaccurate classifications are minimally likely to cause harm.

[0250] [Table 8]

[0251] [Table 9]

[0252] Example 11.7. Algorithm Performance for Non-Fermenting Strains vs. Enterobacteriaceae The algorithms described in Example 11.6 were tested for their ability to classify nonfermentative strains and Enterobacteriaceae that produce a single carbapenemase. Nonfermentative strains, such as Pseudomonas aeruginosa and A. baumannii, may be unexpected reservoirs of class A and class B carbapenemases, and A. baumannii may also harbor endogenous class D carbapenemases and have acquired other transmissible class D carbapenemases. Accurate detection of carbapenemases produced by nonfermentative strains is an important yet technically challenging task, as other mechanisms of carbapenem resistance may result in the same phenotype as carbapenemases.

[0253] As shown in Tables 5.1 and 5.2, classification of carbapenemases in Enterobacteriaceae achieved a higher level of accuracy than for nonfermentative strains. However, the large number of isolates and differences between each group of organisms in the type of β-lactamase production made the comparison less than ideal. This is reflected in the large differences in the numbers obtained for class A producers (105 for Enterobacteriaceae compared to 5 for nonfermentative strains), negative control isolates (51 for Enterobacteriaceae compared to 0 for nonfermentative strains), and total number of CPOs (185 for Enterobacteriaceae compared to 51 for nonfermentative strains).

[0254] [Table 10]

[0255] [Table 11]

[0256] Algorithm 2 appeared to be the best overall algorithm for both groups of organisms. It performed well with class A producers, correctly classifying all five in the non-fermenting group and 81.9% in the Enterobacteriaceae group. It correctly classified Enterobacteriaceae class B producers (73.3% correct) better than non-fermenting strains (61.3%), and was also more accurate in classifying Enterobacteriaceae class D producers (90.9% vs. 78.6%).

[0257] Example 11.7: Algorithm performance for two carbapenemase-producing isolates The seven isolates producing two carbapenemases were: A. baumannii 0063:OXA-23+OXA-40 A. baumannii 0083:OXA-23+NDM Klebsiella pneumoniae 0068:OXA-181+NDM Klebsiella pneumoniae 0153:OXA-232+NDM Klebsiella pneumoniae G15406:OXA-181+NDM E. cloacae G6809: KPC-18+VIM-1 E. cloacae G6810: KPC-18+VIM-1.

[0258] Table 6 shows the distribution of carbapenemase classifications for each algorithm.

[0259] [Table 12]

[0260] Each isolate was correctly reported as carbapenemase-positive during the positive / negative phase of testing. Algorithms 1, 2, and 4 assigned all isolates to either a molecular class or to the carbapenemase-positive untypeable category. Algorithms 2 and 4 classified five of the seven isolates as producers of a specific carbapenemase class, while algorithms 1 and 3 each assigned only one isolate to a specific class. Algorithm 3 assigned five isolates to the "no answer" category.

[0261] In each case in which the algorithm assigned a CPO to a specific class of carbapenemase production, it was either the correct class for one of the two carbapenemases, or, in the case of A. baumannii 0063, which produced two class D carbapenemases, it was correct for both carbapenemases. Although there were not enough isolates to analyze the classification trends for dual carbapenemase producers, it seemed like a viable choice for assigning a carbapenemase to class D.

[0262] Example 11.8: Workflow Comparison The BD Phoenix™ CPO Detect required less manual time than the bioMerieux Rapidec® Carba NP test, and this test did not include wait time because it did not require operator intervention after loading the panel onto the instrument. The manual time per test for the BD Phoenix™ CPO Detect was 1 minute 34 seconds, compared to 2 minutes 3 seconds for a positive (i.e., complete) test after the 30-minute incubation period and 2 minutes 24 seconds for a negative test at 30 minutes, thus requiring additional processing and incubation. Table 7 provides a summary of the workflow analysis.

[0263] [Table 13]

[0264] Example 11.9: Perspective / Summary / Conclusion This example provides the results of a study designed to compare the ability of the automated BD Phoenix CPO Detect test and the bioMerieux Rapidec® Carba NP test to detect and classify carbapenemase-producing organisms (CPOs). The BD Phoenix™ CPO Detect test is an innovative test integrated with a susceptibility panel for detecting and classifying carbapenemases. The bioMerieux Rapidec® Carba NP test is a stand-alone carbapenemase detection test. A selection of 294 research isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii presented significant diagnostic challenges. These isolates have previously been characterized by molecular, phenotypic, and biochemical tests for the type of β-lactamase production. Both tests were blinded and performed according to the manufacturer's recommendations.

[0265] This study presented a challenging evaluation of the ability of the BD Phoenix™ CPO Detect and bioMérieux Rapidec® Carba NP tests to detect carbapenemases. Both tests demonstrated very high sensitivity. The 100% detection of class D-producing CPOs by the BD Phoenix™ CPO Detect was impressive and should be recognized as an impressive achievement, as these are the most difficult of all carbapenemases to detect. The extremely challenging nature of carbapenemase-negative isolates contributed to the lower than usual specificity. In routine clinical use, the isolate types that caused false-positive tests are rarely encountered, and this specificity should be significantly higher.

[0266] The BD CPO Detect can provide two results: a positive / negative result for carbapenemase detection, followed by a classification of positive isolates according to the molecular class of the carbapenemase. In the positive / negative phase of testing, both tests demonstrated high sensitivity for carbapenemase detection (97.1% for the BD Phoenix™ CPO Detect and 97.1% to 98.8% for the bioMérieux Rapidec® Carba NP test). Both tests demonstrated lower specificity than usual in this study due to the extremely challenging nature of carbapenemase-negative isolates.

[0267] The BD Phoenix™ CPO Detect is the first automated test capable of detecting carbapenemases and can be incorporated into routine susceptibility testing. This represents a major technological advancement, as it avoids the need for individual decision-making regarding whether a carbapenemase detection test should be established. The test can also assign carbapenemases to different molecular classes. In the current study, the BD Phoenix™ CPO Detect demonstrated a high ability to detect and differentiate between CPOs producing class A carbapenemase and those producing class B carbapenemase. This diagnostic feature is clinically important for determining the appropriateness of ceftazidime / avibactam as a potential treatment option. Three of the four algorithms investigated correctly classified more than 90% of carbapenemases as either type A, type B, type D, or untyped, with algorithm 2 performing slightly better.

[0268] During the classification phase of the study, the BD Phoenix™ CPO Detect correctly classified more than 90% of carbapenemases as either class A, B, D, or untypeable positive carbapenemases. This demonstrated a high ability to detect and distinguish between CPOs producing class A carbapenemase and those producing class B carbapenemase, a diagnostic feature that is clinically important for determining the appropriateness of ceftazidime / avibactam as a potential treatment option. The bioMérieux Rapidec® Carba NP test did not have the ability to classify carbapenemases. Overall, the BD Phoenix™ CPO Detect is an entirely new type of phenotypic test with a range of capabilities that have no equivalent in currently commercially available tests. This represents a significant advancement in meeting an important clinical need.

[0269] Production of multiple carbapenemases is currently rare, and its detection presents a significant diagnostic and therapeutic challenge. Investigations are currently hampered by the scarcity of available isolates of this type. Until better tests become available, it is important for current studies to provide results that protect patients from inappropriate ceftazidime / avibactam therapy for infections caused by multiple carbapenemase-producing strains. In this study, two E. cloacae isolates producing KPC-18+VIM-1 were classified as producers of class B carbapenemases, thereby correctly indicating a contraindication to therapy with ceftazidime / avibactam. Two A. baumannii isolates that produced both class B and class D carbapenemases were both classified as class D producers. This result also prevented patients from receiving inappropriate ceftazidime / avibactam therapy.

[0270] Example 12: Multicenter evaluation of the BD Phoenix™ CPO Detect test on the BD Phoenix™ automated microbiology system for the detection and typing of carbapenemase-producing organisms in clinical isolates The objective of this study was to evaluate the performance of the BD Phoenix™ CPO Detect test (CPO Detect) (BD Life Sciences, Sparks MD), a growth-based carbapenemase screening assay described in Example 11, to detect and classify carbapenemase production by clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii.

[0271] A total of 1,034 newly frozen isolates, including 722 Enterobacteriaceae and 312 nonfermenting strains (Pseudomonas aeruginosa and Acinetobacter baumannii), were evaluated for carbapenemase production by the BD Phoenix™ CPODetect test at three medical centers. Isolates were evaluated in parallel by a modified carbapenemase inactivation method (mCIM) and meropenem and ertapenem MICs as reference methods. Ambler classification of carbapenemases (class A, B, or D) was determined by multiplex PCR performed by BD. Percent positive and negative agreement (PPA and NPA, respectively) between CPODetect results and reference method results was determined. Discordant results were repeated in duplicate on the BD Phoenix system and the appropriate reference method. The data are presented in Table 8.1 (for Enterobacteriaceae), Table 8.2 (for non-fermentative strains) and Table 8.3 (Enterobacteriaceae and non-fermentative strains combined).

[0272] [Table 14]

[0273] [Table 15]

[0274] [Table 16]

[0275] These results show that for Enterobacteriaceae, the PPA was 99.4% (when the reference system detected a carbapenemase and Phoenix detected one), 96.0% NPA (when the reference system did not detect a carbapenemase and Phoenix did not), and 98.2% classification accuracy (when both Phoenix and the reference system were positive, provided a classification, and Phoenix was correct).For nonfermenting strains, the PPA was 96.0% (when the reference system detected a carbapenemase and Phoenix detected one), 95.0% NPA (when the reference system did not detect a carbapenemase and Phoenix did not), and 96.3% classification accuracy (when both Phoenix and the reference system were positive, provided a classification, and Phoenix was correct). The combined results were a PPA of 98.3% (when the reference system detects a carbapenemase and %Phoenix detects a carbapenemase), an NPA of 95.7% (when the reference system does not detect a carbapenemase and %Phoenix does not detect a carbapenemase), and a classification accuracy of 97.7% (when both Phoenix and the reference system are positive and provide a classification and %Phoenix is ​​correct).

[0276] Results are provided for 1,034 compliant clinical isolates tested and analyzed for carbapenemase detection by CPODetect. After discrepant analysis, PPA and NPA were 99.4% and 96.0%, respectively, for Enterobacteriaceae. Sixteen false positives (2.2%) and two false negatives (0.3%) were observed. For nonfermentative strains, PPA and NPA were 96.0% and 95.0%, respectively, with eight false positives (2.6%) and six false negatives (1.9%). Among the compliant isolates tested, 385 CPODetect results were compared with multiplex PCR for carbapenemase classification. Overall class accuracy was 98.2% (272 / 277) for Enterobacteriaceae and 96.3% (104 / 108) for nonfermentative strains.

[0277] The BD Phoenix™ CPO Detect test is easily integrated into the BD Phoenix automated AST testing system and provides a novel and reliable method for detecting and typing carbapenemases from Enterobacteriaceae, Pseudomonas aeruginosa, and A. baumannii.

[0278] (abbreviation) CLOX cloxacillin

[0279] EDTA Ethylenediaminetetraacetic acid

[0280] DPA dipicolinic acid

[0281] RPX vaborbactam (RPX-7009)

[0282] AVI avibactam

[0283] BLI BLI (BLI-489, beta-lactamase inhibitor)

[0284] DOR Doripenem

[0285] MEM Meropenem

[0286] TEM Temocillin

[0287] GAM Generalized Additive Model

[0288] ERT ertapenem

[0289] IPM imipenem.

[0290] (definition) As used herein, MIC refers to the minimum inhibitory concentration.

[0291] As used herein, GAM refers to generalized additive model, which is the conversion of instrument readings into measurements of growth.

[0292] As used herein, section headings are for organizational purposes only and should not be construed as limiting the subject matter described in any way. All references and similar materials cited in this application, including but not limited to patents, patent applications, papers, books, treatises, and internet web pages, are expressly incorporated by reference in their entirety for all purposes. In the event that the definition of a term in an incorporated reference differs from the definition provided in the present teachings, the definition provided in the present teachings shall prevail. The implicit "about" before temperatures, concentrations, times, etc. discussed in the present teachings is intended to recognize that small, insubstantial deviations fall within the scope of the present teachings herein.

[0293] In this application, the use of the singular includes the plural unless specifically stated otherwise. Similarly, the use of "comprise," "comprises," "including," "contain," "contains," "containing," "include," "includes," and "comprising" is not intended to be limiting.

[0294] As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0295] As used herein, "about" means that a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length varies from a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length by more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%.

[0296] While the present invention has been disclosed in the context of certain embodiments and examples, it is expected that those skilled in the art will appreciate that the present invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the invention, as well as obvious modifications and equivalents thereof. In addition, while numerous variations of the invention have been shown and described in detail, other modifications are within the scope of the invention and are expected to become readily apparent to those skilled in the art based on this disclosure. It is also expected that various combinations or subcombinations of specific features and aspects of the embodiments can be made and still fall within the scope of the invention. It is to be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another to form various modes or embodiments of the disclosed invention. Accordingly, it is intended that the scope of the invention disclosed herein should not be limited by the specific disclosed embodiments described above.

[0297] It should be understood, however, that this detailed description, while indicating embodiments of the present invention, is given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art.

[0298] The terms used in the description set forth herein are not intended to be construed in any limiting or restrictive manner. Rather, the terms are utilized solely in conjunction with detailed descriptions of embodiments of systems, methods, and related components. Furthermore, embodiments may include numerous novel features, none of which may be solely responsible for desired properties or be considered essential to practicing the inventions described herein. The present invention also includes the following inventions. 1. A method for determining the presence of zero, one or more Ambler class carbapenemases expressed by Enterobacteriaceae, comprising: providing a sample containing the gut bacteria; applying the Enterobacteriaceae in a test sample to a plurality of at least four test compositions over a sustained period of time, wherein each of the plurality of at least four test compositions comprises a growth medium and an antibiotic, and at least one of the at least four test compositions further comprises at least one carbapenemase inhibitor; determining the presence of zero, one or more Ambler-class carbapenemases expressed by said Enterobacteriaceae by detecting the presence or inhibition of growth of said Enterobacteriaceae in each of said plurality of at least four test compositions after said sustained period; A method comprising: 2. The method of 1, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a first concentration of TEM and an Ambler class B carbapenemase inhibitor. 3. The method of 1 or 2, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor. 4. The method of any one of 1-3, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor. 5. The method of any one of 1-4, wherein the antibiotic in at least one test composition comprises, consists of, or consists essentially of a second concentration of DOR. 6. The method of any one of 1-5, comprising determining that the one or more Ambler class carbapenemases expressed by the Enterobacteriaceae are class D by detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of TEM and an Ambler class B carbapenemase inhibitor. 7. The method of any one of 1 to 6, comprising determining that the one or more Ambler-class carbapenemases expressed by the Enterobacteriaceae are class A by detecting: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of TEM and an Ambler class B carbapenemase inhibitor; the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; Inhibition of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor. 8. The method of any one of 1 to 7, comprising determining that the one or more Ambler-class carbapenemases expressed by the Enterobacteriaceae are class B by detecting: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprises, consists of, or consists essentially of a first concentration of TEM as the antibiotic, and further comprises an Ambler class B carbapenemase inhibitor; Inhibition of growth in a second test composition, wherein the antibiotic and inhibitor comprises, consists of, or consists essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; The presence of growth in a fourth test composition, wherein said antibiotic comprises, consists of, or consists essentially of a second concentration of DOR. 9. The method of any one of 1 to 8, comprising determining that the one or more Ambler-class carbapenemases expressed by the Enterobacteriaceae are class D by detecting: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of TEM and an Ambler class B carbapenemase inhibitor; Inhibition of growth in a second test composition, wherein the antibiotic and inhibitor comprises, consists of, or consists essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; Inhibition of growth in a fourth test composition, wherein the antibiotic comprises, consists of, or consists essentially of a second concentration of DOR. 10. The method of any one of 1 to 9, comprising determining the presence of one or more Ambler class carbapenemases expressed by the Enterobacteriaceae (wherein the Ambler class is not identified) by detecting: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of TEM and an Ambler class B carbapenemase inhibitor; the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; The presence of growth in a third test composition of the plurality of at least four test compositions, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM as the antibiotic, and further comprise an Ambler class C carbapenemase inhibitor and an Ambler class A carbapenemase inhibitor. 11. The method of any one of 1 to 10, comprising determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by the Enterobacteriaceae by detecting: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprises, consists of, or consists essentially of TEM, an Ambler class B carbapenemase inhibitor at a first concentration; the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; The presence of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor. 12. The method of any one of 1-11, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a third concentration of MEM and an Ambler class C carbapenemase inhibitor. 13. The method of any one of 1 to 12, comprising determining that there is no answer regarding identifying the one or more Ambler class carbapenemases expressed by the Enterobacteriaceae by detecting: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of TEM and an Ambler class B carbapenemase inhibitor; the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; the presence of growth in a third test composition of the plurality of at least four test compositions, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor; Inhibition of growth in a fifth test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of MEM at a third concentration and an Ambler class C carbapenemase inhibitor. 14. The method of any one of 1 to 13, comprising determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by the Enterobacteriaceae by detecting: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of TEM and an Ambler class B carbapenemase inhibitor; the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; the presence of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor; The presence of growth in a fifth test composition, wherein said antibiotic and inhibitor comprises, consists of, or consists essentially of a third concentration of MEM and an Ambler class C carbapenemase inhibitor. 15. The method of 1, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor. 16. The method of any one of 1 and 15, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor. 17. The method of any one of 1 and 15-16, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 18. The method of any one of 1 and 15-17, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a second concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor. 19. The method of any one of 1 and 15-18, comprising determining that the one or more Ambler-class carbapenemases expressed by the Enterobacteriaceae are class A by detecting: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor; Inhibition of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a second concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor. 20. The method of any one of 1 and 15-19, comprising determining that the one or more Ambler-class carbapenemases expressed by the Enterobacteriaceae are class B by detecting: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor; The presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a second concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor. 21. The method of any one of 1 and 15 to 20, comprising determining that the one or more Ambler-class carbapenemases expressed by the Enterobacteriaceae are class A by detecting: detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor; and Inhibition of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor. 22. The method of any one of 1 and 15 to 21, comprising determining that the one or more Ambler-class carbapenemases expressed by the Enterobacteriaceae are class D by detecting: the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor; the presence of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor; Inhibition of growth in a fourth test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 23. The method of any one of 1 and 15 to 22, comprising determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by the Enterobacteriaceae by detecting: the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor; the presence of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor; The presence of growth in a fourth test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 24. The method of any one of 1 and 15 to 22, comprising determining that there is no answer regarding identifying the one or more Ambler class carbapenemases expressed by the Enterobacteriaceae by detecting: the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor; the presence of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor; The presence of growth in a fourth test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 25. The method of any one of 1-24, comprising applying the Enterobacteriaceae in a test sample to a plurality of at least five test compositions over a sustained period of time, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a first concentration of MEM and an Ambler class C carbapenemase inhibitor. 26. The method of any one of 1 and 25, comprising determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by Enterobacteriaceae by detecting the presence of growth in a first test composition, The method, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM and an Ambler class C carbapenemase inhibitor. 27. The method of any one of 1 and 25, comprising determining the absence of one or more Ambler class A, B, or D carbapenemases expressed by the Enterobacteriaceae by detecting inhibition of growth in a first test composition, The method, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a first concentration of MEM and an Ambler class C carbapenemase inhibitor. 28. The method of any one of 1 to 27, further comprising a method for identifying zero, one or more Ambler class carbapenemases expressed by a non-fermenting bacterium, the method comprising: providing a sample containing non-fermenting bacteria; applying the non-fermenting bacteria in a test sample to a growth medium and a test composition comprising an antibiotic and a carbapenemase inhibitor for a sustained period of time; determining the presence of zero, one or more Ambler-class carbapenemases expressed by the non-fermenting bacteria by detecting the presence or inhibition of growth of the non-fermenting bacteria in the test composition after the sustained period; A method comprising: 29. The method of 28, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a third concentration of DOR and an Ambler class C carbapenemase inhibitor. 30. The method of claim 28 or 29, comprising determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by a non-fermenting bacterium by detecting the presence of growth in the test composition, The method, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR and an Ambler class C carbapenemase inhibitor. 31. The method of claim 28 or 29, comprising determining the absence of one or more Ambler class A, B, or D carbapenemases expressed by a non-fermenting bacterium by detecting inhibition of growth in a test composition, The growth inhibition wherein the antibiotic and inhibitor comprises, consists of, or consists essentially of a third concentration of DOR and an Ambler class C carbapenemase inhibitor. 32. The method of any one of 1 to 31, further comprising a method for determining the presence of zero, one or more Ambler class carbapenemases expressed by a non-fermenting bacterium, the method comprising: providing a sample containing non-fermenting bacteria; applying the non-fermenting bacteria in a test sample to a plurality of at least three test compositions over a sustained period of time, each of the plurality of at least three test compositions comprising a growth medium and an antibiotic, and at least one of the at least three test compositions further comprising at least one carbapenemase inhibitor; determining the presence of zero, one, or an additional one or more Ambler-class carbapenemases expressed by the non-fermenting bacteria by detecting the presence or inhibition of growth of the non-fermenting bacteria in each of the plurality of at least three test compositions after the sustained period; A method comprising: 33. A method for determining the presence of zero, one or more Ambler-class carbapenemases expressed by a non-fermenting bacterium, comprising: providing a sample containing said non-fermenting bacteria; applying the non-fermenting bacteria in the test sample to a plurality of at least three test compositions over a sustained period of time, each of the plurality of at least three test compositions comprising a growth medium and an antibiotic, and at least one of the at least three test compositions further comprising at least one carbapenemase inhibitor; determining the presence of zero, one, or an additional one or more Ambler-class carbapenemases expressed by the non-fermenting bacteria by detecting the presence or inhibition of growth of the non-fermenting bacteria in each of the plurality of at least three test compositions after the sustained period; A method comprising: 34. The method of 32 or 33, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor. 35. The method of any one of 32-34, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 36. The method of any one of 32 to 35, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprise, consist of, or consist essentially of a fifth concentration of DOR, and an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 37. The method of any one of 32 to 36, comprising determining the one or more Ambler-class carbapenemases expressed by a non-fermenting bacterium as class B by detecting inhibition of growth in a first test composition, The method, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor. 38. The method of any one of 32 to 37, comprising determining the one or more Ambler-class carbapenemases expressed by a non-fermenting bacterium as class D by detecting the presence of growth in a first test composition by detecting: the presence of growth wherein said antibiotics and inhibitors comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; Inhibition of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 39. The method of any one of 32 to 38, comprising determining the one or more Ambler-class carbapenemases expressed by a non-fermenting bacterium as class A by detecting: the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor; Inhibition of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fifth concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 40. The method of any one of 32 to 39, wherein the presence of one or more Ambler class A, B, or D carbapenemases expressed by a non-fermenting bacterium is determined by detecting: the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor; The presence of growth in a third test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fifth concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 41. The method of 32 or 33, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor. 42. The method of any one of 32-33 and 41, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 43. The method of any one of 32-33 and 41-42, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor. 44. The method of any one of 32-33 and 41-43, comprising determining that the one or more Ambler class carbapenemases expressed by the non-fermenting bacterium are either class A, B, or D by detecting: the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; The presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 45. The method of any one of 32-33 and 41-44, comprising determining that the one or more Ambler-class carbapenemases expressed by the non-fermenting bacterium are class D by detecting: the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; Inhibition of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 46. ​​The method of any one of 32-33 and 41-45, comprising determining that the one or more Ambler-class carbapenemases expressed by the non-fermenting bacterium are class B by detecting: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprises, consists of, or consists essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; The presence of growth in a third test composition, wherein the antibiotic and inhibitor comprises, consists of, or consists essentially of a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor. 47. The method of any one of 32-33 and 41-46, comprising determining that there is no answer regarding identifying the one or more Ambler-class carbapenemases expressed by a non-fermenting bacterium by detecting: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprises, consists of, or consists essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; Inhibition of growth in a third test composition, wherein the antibiotic and inhibitor comprises, consists of, or consists essentially of a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor. 48. The method of any one of 32-33 and 41-47, comprising determining that the one or more Ambler class carbapenemases expressed by the non-fermenting bacterium are either class A, B, or D by detecting: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprises, consists of, or consists essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; Inhibition of growth in a third test composition, wherein the antibiotic and inhibitor comprises, consists of, or consists essentially of a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor. 49. The method of any one of 32-48, comprising applying the non-fermenting bacteria in a test sample to a plurality of at least four test compositions over a sustained period of time, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, consists of, or consists essentially of a fifth concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 50. The method of 49, comprising determining that the one or more Ambler class carbapenemases expressed by the non-fermenting bacterium are either class A, B, or D by detecting: the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor; The presence of growth in a fourth test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fifth concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 51. The method of 49 or 50, comprising determining that the one or more Ambler-class carbapenemases expressed by the non-fermenting bacterium are class A by detecting: the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor; the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor; Inhibition of growth in a fourth test composition, wherein the antibiotic and inhibitor comprise, consist of, or consist essentially of a fifth concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. 52. The method of any one of 1 to 51, further comprising determining whether the bacteria in the sample are enteric, non-fermentative, or both. 53. A method according to any one of 1 to 52, wherein the first concentration of TEM is from about 6 μg / ml to about 128 μg / ml, from about 32 μg / ml to about 128 μg / ml, from about 32 μg / ml to about 80 μg / ml, or about 64 μg / ml. 54. A method according to any one of 1 to 53, wherein the first concentration of DOR is from about 0.006 μg / ml to about 0.75 μg / ml, from about 0.03125 μg / ml to about 0.1 μg / ml, or from about 0.0625 μg / ml or about 0.06 μg / ml. 55. A method according to any one of 1 to 54, wherein the second concentration of DOR is from about 0.0125 μg / ml to about 2 μg / ml, 0.0625 μg / ml to about 0.25 μg / ml, or about 0.125 μg / ml. 56. A method according to any one of 1 to 55, wherein the third concentration of DOR is from about 0.1 μg / ml to about 400 μg / ml, from about 0.5 μg / ml to about 3 μg / ml, or about 1 μg / ml. 57. A method according to any one of 1 to 56, wherein the fourth concentration of DOR is from about 0.2 μg / ml to about 40 μg / ml, from about 0.5 μg / ml to about 4 μg / ml, or about 2 μg / ml. 58. A method according to any one of 1 to 57, wherein the fifth concentration of DOR is from about 0.03125 μg / ml to about 80 μg / ml, from about 2 μg / ml to about 24 μg / ml, or about 8 μg / ml. 59. The method of any one of 1 to 58, wherein the first concentration of MEM is 0.03125 μg / ml to 1 μg / ml, 0.03125 μg / ml to 0.125 μg / ml, 0.015625 μg / ml to 0.125 μg / ml, about 0.006 μg / ml to about 0.60 μg / ml, about 0.015 μg / ml to about 0.24 μg / ml, about 0.03 μg / ml to about 0.25 μg / ml, about 0.03 μg / ml to about 0.2 μg / ml, about 0.0625 μg / ml or about 0.060 μg / ml. 60. The method of any one of 1 to 59, wherein the second concentration of MEM is from about 0.015625 μg / ml to about 0.125 μg / ml, from about 0.003 μg / ml to about 0.3 μg / ml, from about 0.0075 μg / ml to about 0.12 μg / ml, from about 0.01 μg / ml to about 0.12 μg / ml, or about 0.03 μg / ml. 61. The method of any one of 1 to 60, wherein the third concentration of MEM is from about 0.0125 μg / ml to about 5 μg / ml, from about 0.125 μg / ml to about 1 μg / ml, or about 0.5 μg / ml. 62. The method of any one of 1 to 61, wherein the fourth concentration of MEM is from about 0.4 μg / ml to about 40 μg / ml, from about 1 μg / ml to about 16 μg / ml, from about 2 μg / ml to about 8 μg / ml, or about 4 μg / ml. 63. The method of any one of 1 to 62, wherein the Ambler class D carbapenemase inhibitor comprises a compound selected from the group consisting of AVI, clavulanic acid, boronic acid, tazobactam, sulbactam, vaborbactam (RPX-7009), and BLI-489. 64. The method of any one of 1 to 63, wherein the Ambler class B carbapenemase inhibitor is a metal chelator. 65. The method of any one of 1 to 64, wherein the Ambler class B carbapenemase inhibitor comprises a compound selected from the group consisting of EDTA, DPA, and deferoxamine. 66. The method of any one of 1 to 65, wherein the Ambler class C carbapenemase inhibitor comprises a compound selected from the group consisting of CLOX, dicloxacillin, and flucloxacillin. 67. The method of any one of 1 to 66, wherein the Ambler class A carbapenemase inhibitor comprises a compound selected from the group consisting of vaborbactam (RPX-7009), AVI, clavulanic acid, boronic acid, tazobactam, sulbactam, and BLI-489. 68. The method of any one of 1 to 67, wherein the Ambler class D carbapenemase inhibitor comprises a compound selected from the group consisting of BLI, AVI, clavulanic acid, boronic acid, tazobactam, sulbactam, vaborbactam and (RPX-7009). 69. The method of any one of 1-68, wherein the Ambler class B carbapenemase inhibitor in combination with the first concentration of TEM and / or the first concentration of DOR comprises, consists of, or consists essentially of EDTA. 70. The method of any one of 1-69, wherein the Ambler class C carbapenemase inhibitor comprises, consists of, or consists essentially of CLOX. 71. The method of any one of 1-70, wherein the Ambler class A carbapenemase inhibitor in combination with the first concentration of MEM and / or the second concentration of MEM comprises, consists of, or consists essentially of RPX. 72. The method of any one of 1-71, wherein the Ambler class B carbapenemase inhibitor in combination with the first concentration of MEM and / or the third concentration of DOR comprises, consists of, or consists essentially of DPA. 73. The method of any one of 1-72, wherein the Ambler class D carbapenemase inhibitor in combination with the first concentration of DOR and / or the fifth concentration of DOR comprises, consists of, or consists essentially of AVI. 74. The method of any one of 1-73, wherein the Ambler class D carbapenemase inhibitor in combination with the fourth concentration of MEM comprises, consists of, or consists essentially of a first concentration of BLI. 75. The method of any one of 1 to 74, wherein the concentration of EDTA is from about 0.025 mg / ml to about 10 mg / ml, from about 0.05 mg / ml to about 1.25 mg / ml, or about 0.25 mg / ml. 76. The method of any one of 1 to 75, wherein the concentration of CLOX is from about 0.0025 mg / ml to about 40 mg / ml, from about 0.020 mg / ml to about 0.5 mg / ml, or about 0.1 mg / ml. 77. The method of any one of 1 to 76, wherein the concentration of RPX is from about 0.2 μg / ml to about 320 μg / ml, from about 1.5 μg / ml to about 40 μg / ml, or about 8 μg / ml. 78. The method of any one of 1 to 77, wherein the concentration of DPA is from about 0.018 mg / ml to about 1.8 mg / ml, from about 0.07 mg / ml to about 0.73 mg / ml, or about 0.178 mg / ml. 79. The method of any one of 1 to 78, wherein the concentration of AVI is from about 0.1 μg / ml to about 40 μg / ml, from about 0.5 μg / ml to about 20 μg / ml, or about 4 μg / ml. 80. A method according to any one of 1 to 79, wherein the concentration of BLI is from about 0.1 μg / ml to about 200 μg / ml, from about 1 μg / ml to about 25 μg / ml, or about 5 μg / ml. 81. The method of any one of 1 to 80, wherein the duration for detecting the presence or inhibition of proliferation is less than about 24 hours, less than about 18 hours, less than about 16 hours, or less than about 14 hours. 82. The method of any one of 1 to 81, wherein the duration for detecting the presence or inhibition of enterobacterial growth is from about 6 hours to about 8 hours. 83. The method of any one of 1 to 82, wherein the duration for detecting the presence or inhibition of growth of enterobacteria is about 7 hours. 84. The method of any one of 1 to 83, wherein the duration for detecting the presence or inhibition of growth of non-fermenting bacteria is from about 8 hours to about 11 hours. 85. The method of any one of 1 to 84, wherein the duration for detecting the presence or inhibition of growth of non-fermenting bacteria is about 10 hours. 86. The method of any one of 1 to 85, wherein the enterobacteria include bacteria selected from the group consisting of Klebsiella pneumoniae, Escherichia coli, and Enterobacter aerogenes. 87. A method according to any one of 1 to 86, wherein the non-fermentative bacterium comprises a bacterium selected from the group consisting of Pseudomonas aeruginosa and Acinetobacter baumannii complex strains. 88. A method according to any one of 1 to 87, wherein detecting the presence or inhibition of proliferation is not carried out by imaging changes in cell morphology. 89. An automated system for carrying out the method according to any one of claims 1 to 88, comprising: a plurality of compartments, each of the plurality of compartments comprising a test composition according to any one of the methods of 1 to 88; a means for supplying a sample containing Enterobacteriaceae, non-fermenting bacteria, or both, to said plurality of compartments; an apparatus for obtaining a first signal from the plurality of compartments provided with the enterobacteria, the non-fermenting bacteria, or both; an incubator for incubating the plurality of compartments provided with the Enterobacteriaceae, non-fermenting bacteria, or both, for a sustained period of time; an instrument for obtaining a second signal from said plurality of compartments containing enterobacteria, non-fermenting bacteria, or both; a detector for detecting the presence or inhibition of growth in the plurality of compartments provided with the Enterobacteriaceae, non-fermenting bacteria, or both, by comparing the first signal and the second signal; a computer for generating an output of results from said detector; an analyzer for interpreting the output of said results; An automated system with 90. The automated system of 89, wherein the plurality of compartments comprises compartments selected from the group consisting of wells, plates, and tubes. 91. A kit for identifying one or more Ambler-class carbapenemases expressed by Enterobacteriaceae and / or non-fermenting bacteria, the kit comprising a substrate or panel having a plurality of compartments, each of the plurality of compartments comprising a test composition described in any one of 1 to 90. 92. The kit of 91, wherein the substrate comprises at least three, or at least four, different test compositions. 93. The kit of claim 91 or 92, wherein the kit includes a second substrate containing a plurality of compartments, each of the plurality of compartments containing a test composition according to the method of any one of 1 to 90, and wherein at least one test composition in the plurality of test compositions in the first substrate is different from the plurality of test compositions in the second substrate. 94. The method, system, or kit of any one of 1-88, comprising a plurality of test compositions, wherein the test compositions comprise, consist of, or consist essentially of a test composition selected from the test compositions disclosed in Boxes 1-14. 95. The method, system or kit according to 94, wherein said test composition comprises, consists of or consists essentially of the test compositions disclosed in Boxes 1-5. 96. The method, system or kit of 94, wherein said test composition comprises, consists of, or consists essentially of the test compositions disclosed in boxes 1, 6, 7, 3, 8 and 9. 97. The method, system or kit according to 94, wherein said test composition comprises, consists of or consists essentially of the test compositions disclosed in boxes 10, 11, 12 and 13. 98. The method, system or kit according to 94, wherein said test composition comprises, consists of or consists essentially of the test compositions disclosed in boxes 10, 11, 12, 13 and 14. 99. The method, system or kit of 94, wherein said test composition comprises, consists of, or consists essentially of the test compositions disclosed in Boxes 1, 6, 7, 3 and 9. 100. The method, system or kit of 94, wherein said test composition comprises, consists of, or consists essentially of the test compositions disclosed in boxes 1, 6, 7, 9, 3 and 10. 101. The method, system or kit according to 94, wherein said test composition comprises, consists of or consists essentially of the test compositions disclosed in boxes 1, 6, 7, 3, 9, 10, 11, 12 and 14. 102. The method, system or kit of 94, wherein the test composition comprises, consists of, or consists essentially of the test compositions disclosed in Boxes 1, 10, 11, 12 and 14. 。

Claims

1. 1. A method for identifying zero, one or more Ambler class carbapenemases expressed by a non-fermenting bacterium, the method comprising: providing a sample containing non-fermenting bacteria; applying the non-fermenting bacteria in a test sample to four test compositions for a sustained period of time, each of the four test compositions comprising a growth medium, an antibiotic, and at least one carbapenemase inhibitor; and identifying one or more Ambler classes by detecting the presence or inhibition of growth of said non-fermenting bacteria in each of at least two of said four test compositions after said duration; the antibiotic and inhibitor in the first test composition comprises or consists of a third concentration of DOR and an Ambler class C carbapenemase inhibitor; The antibiotic and inhibitor in the second test composition comprises or consists of a third concentration of DOR, and an Ambler class C carbapenemase inhibitor, an Ambler class B carbapenemase inhibitor; the antibiotic and inhibitor in the third test composition comprises or consists of a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor; The method, wherein the antibiotic and inhibitor in the fourth test composition comprise or consist of a fifth concentration of DOR, and an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

2. 10. The method of claim 1, comprising determining the one or more Ambler-class carbapenemases expressed by a non-fermenting bacterium as Class B by detecting: the presence of proliferation in the first test composition; and Inhibition of proliferation in said second test composition.

3. 10. The method of claim 1, comprising determining the one or more Ambler-class carbapenemases expressed by a non-fermenting bacterium as Class D by detecting: the presence of proliferation in said first test composition; the presence of proliferation in the second test composition; and Inhibition of proliferation in said third test composition.

4. 10. The method of claim 1, comprising determining the one or more Ambler-class carbapenemases expressed by a non-fermenting bacterium as class A by detecting: the presence of proliferation in said first test composition; the presence of proliferation in said second test composition; the presence of growth in said third test composition; and Inhibition of proliferation in said fourth test composition.

5. 10. The method of claim 1, comprising determining the presence of one or more Ambler class A, B or D carbapenemases expressed by a non-fermenting bacterium by detecting: the presence of proliferation in said first test composition; the presence of proliferation in said second test composition; the presence of growth in said third test composition; and The presence of proliferation in said fourth test composition.

6. 10. The method of claim 1, comprising determining the presence of one or more Ambler class A, B or D carbapenemases expressed by a non-fermenting bacterium by detecting: the presence of proliferation in said first test composition; the presence of proliferation in the second test composition; and The presence of growth in the third test composition.

7. The method of claim 1, further comprising: applying the non-fermenting bacteria in the test sample to a fifth test composition for a sustained period of time, the fifth test composition comprising a growth medium, an antibiotic, and at least one carbapenemase inhibitor; detecting the presence or inhibition of growth of said non-fermenting bacteria in said fifth test composition after said sustained period; The method, wherein the antibiotic and inhibitor in the fifth test composition comprises or consists of a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor.

8. 8. The method of claim 7, comprising determining that the one or more Ambler-class carbapenemases expressed by a non-fermenting bacterium are class B by detecting: the presence of proliferation in said first test composition; Inhibition of proliferation in the second test composition; and The presence of proliferation in said fifth test composition.

9. 8. The method of claim 7, comprising determining that the identification of one or more Ambler-class carbapenemases expressed by the non-fermenting bacterium is unanswerable by detecting: the presence of proliferation in said first test composition; Inhibition of proliferation in the second test composition; and Inhibition of proliferation in said fifth test composition.

10. 8. The method of claim 7, comprising determining the presence of one or more Ambler class A, B or D carbapenemases expressed by a non-fermenting bacterium by detecting: the presence of proliferation in said first test composition; Inhibition of proliferation in the second test composition; and Inhibition of proliferation in said fifth test composition.

11. 11. The method of any one of claims 1 to 10, further comprising determining whether the bacteria in the sample are enteric, non-fermentative, or both.

12. 12. The method of any one of claims 1 to 11, wherein the third concentration of DOR is from 0.5 μg / ml to 3 μg / ml.

13. 13. The method of any one of claims 1 to 12, wherein the third concentration of DOR is 1 μg / ml.

14. 14. The method of any one of claims 1 to 13, wherein the fourth concentration of DOR is from 0.5 μg / ml to 4 μg / ml.

15. 15. The method of any one of claims 1 to 14, wherein the fourth concentration of DOR is 2 μg / ml.

16. 16. The method of any one of claims 1 to 15, wherein the fifth concentration of DOR is from 2 μg / ml to 24 μg / ml.

17. 17. The method of any one of claims 1 to 16, wherein the fifth concentration of DOR is 8 μg / ml.

18. The method of any one of claims 1 to 17, wherein the fourth concentration of MEM is from 1 µg / ml to 16 µg / ml.

19. The method of any one of claims 1 to 18, wherein the fourth concentration of MEM is from 2 μg / ml to 8 μg / ml.

20. The method of any one of claims 1 to 19, wherein the fourth concentration of MEM is 4 µg / ml.

21. 21. The method of any one of claims 1 to 20, wherein the Ambler class D carbapenemase inhibitor comprises a compound selected from the group consisting of AVI, clavulanic acid, boronic acid, tazobactam, sulbactam, vaborbactam (RPX-7009), and BLI-489.

22. 22. The method of any one of claims 1 to 21, wherein the Ambler class B carbapenemase inhibitor is a metal chelator.

23. 23. The method of any one of claims 1 to 22, wherein the Ambler class B carbapenemase inhibitor comprises a compound selected from the group consisting of EDTA, DPA, and deferoxamine.

24. 24. The method of any one of claims 1 to 23, wherein the Ambler class C carbapenemase inhibitor comprises a compound selected from the group consisting of CLOX, dicloxacillin, and flucloxacillin.

25. 25. The method of any one of claims 1 to 24, wherein the Ambler class C carbapenemase inhibitor comprises, consists of, or consists of CLOX.

26. 26. The method of any one of claims 1-25, wherein the Ambler class B carbapenemase inhibitor in combination with the first concentration of MEM and / or the third concentration of DOR comprises or consists of DPA.

27. 27. The method of any one of claims 1-26, wherein the Ambler class D carbapenemase inhibitor in combination with the first concentration of DOR and / or the fifth concentration of DOR comprises or consists of an AVI.

28. 28. The method of any one of claims 1-27, wherein the Ambler class D carbapenemase inhibitor in combination with the fourth concentration of MEM comprises or consists of a first concentration of BLI.

29. 29. The method of any one of claims 1 to 28, wherein the concentration of CLOX is from 0.020 mg / ml to 0.5 mg / ml.

30. The method of any one of claims 1 to 29, wherein the concentration of CLOX is 0.1 mg / ml.

31. 31. The method of any one of claims 1 to 30, wherein the concentration of DPA is from 0.07 mg / ml to 0.73 mg / ml.

32. 32. The method of any one of claims 1 to 31, wherein the concentration of DPA is 0.

178.

33. 33. The method of any one of claims 1 to 32, wherein the concentration of AVI is from 0.5 μg / ml to about 20 μg / ml.

34. The method of any one of claims 1 to 33, wherein the concentration of AVI is 4 μg / ml.

35. The method of any one of claims 1 to 34, wherein the concentration of BLI is from 1 μg / ml to 25 μg / ml.

36. The method of any one of claims 1 to 35, wherein the concentration of BLI is 5 μg / ml.

37. 37. The method of any one of claims 1 to 36, wherein the duration for detecting the presence or inhibition of proliferation is less than 24 hours.

38. 38. The method of any one of claims 1 to 37, wherein the duration for detecting the presence or inhibition of non-fermenting bacterial growth is between 8 and 11 hours.

39. 39. The method of any one of claims 1 to 38, wherein the duration for detecting the presence or inhibition of non-fermenting bacterial growth is 10 hours.

40. 40. The method of any one of claims 1 to 39, wherein the non-fermenting bacteria comprises a bacterium selected from the group consisting of Pseudomonas aeruginosa and Acinetobacter baumannii complex.

41. The method of any one of claims 1 to 40, wherein detecting the presence or inhibition of proliferation is not performed by imaging changes in cell morphology.

42. An automated system for carrying out the method of any one of claims 1 to 41, comprising: a plurality of compartments, each of said plurality of compartments containing a test composition according to the method of any one of claims 1 to 41; a means for supplying a sample containing non-fermenting bacteria to said plurality of compartments; an apparatus for obtaining a first signal from the plurality of compartments provided with the non-fermenting bacteria; an incubator for incubating the plurality of compartments provided with the non-fermenting bacteria for a sustained period of time; an instrument for obtaining a second signal from said plurality of compartments containing non-fermenting bacteria; a detector for detecting the presence or inhibition of growth in the plurality of compartments provided with the non-fermenting bacteria by comparing the first signal and the second signal; a computer for generating an output of results from said detector; an analyzer for interpreting the output of said results; An automated system with

43. 43. The automated system of claim 42, wherein the plurality of compartments comprises compartments selected from the group consisting of wells, plates, and tubes.

44. A kit for identifying one or more Ambler class carbapenemases expressed by non-fermenting bacteria, comprising a substrate or panel having a plurality of compartments, each of said plurality of compartments containing a test composition according to a method described in any one of claims 1 to 41.

45. 45. The kit of claim 44, wherein the substrate comprises at least four different test compositions.

46. 45. The kit of claim 44, wherein the substrate comprises at least five different test compositions.

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