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

Abstract

This provides an accurate method for detecting carbapenemase production. [Solution] A method for determining the presence of zero, one or more Ambler class carbapenemases expressed by intestinal bacteria, comprising the steps of: providing a sample containing the intestinal bacteria; applying the intestinal bacteria in the test sample to a plurality of at least four test compositions over a duration, each of which comprises a growth medium and an antibiotic, and at least one of the test compositions further comprising at least one carbapenemase inhibitor; and determining the presence of zero, one or more Ambler class carbapenemases expressed by the intestinal bacteria by detecting the presence or inhibition of growth of the intestinal bacteria in each of the test compositions after the duration.

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 present 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 Art

[0002] Carbapenemase-producing Gram-negative bacteria are a major and serious global public health threat because there are few options available as next-generation antibiotics for use against these pathogens. Pharmaceutical companies are currently targeting a number of new antibiotics in their pipelines, but none have a broad spectrum of activity against all of the types (classes) of carbapenemase enzymes that these bacteria can acquire.

[0003] Accurate detection of carbapenemase production and discrimination of β-lactamase classes are important for decisions regarding antimicrobial therapy, epidemiology and infection control measures.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

[0005] One embodiment is a method for determining the presence of zero, one or more Ambler class carbapenemases expressed by Enterobacteriaceae, comprising the steps of: providing a sample containing Enterobacteriaceae; applying the Enterobacteriaceae in the test sample to a plurality of at least four test compositions over a duration, each of which comprises 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 Enterobacteriaceae by detecting the presence or inhibition of growth of Enterobacteriaceae in each of the plurality of at least four test compositions after the duration. In any embodiment disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed 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 comprises, or is essentially derived from, 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 comprises, or is essentially derived from, 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 comprises, or is essentially derived from, a second concentration of DOR.In any embodiment disclosed herein, the method may include the step of determining that one or more Ambler class carbapenemases expressed by Enterobacteria are class D by detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprises a first concentration of TEM and an Ambler class B carbapenemase inhibitor, or is essentially composed of such growth. In any embodiment disclosed herein, the method may include the step of determining that one or more Ambler class carbapenemases expressed by Enterobacteriaceae are class A by detecting the following: inhibition of growth in a first test composition, wherein the antibiotics and inhibitors comprise or are essentially comprised of a first concentration of TEM and an Ambler class B carbapenemase inhibitor; presence of growth in a second test composition, wherein the antibiotics and inhibitors comprise or are essentially comprised 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 antibiotics and inhibitors comprise or are essentially comprised of a first concentration of MEM, an Ambler class C carbapenemase inhibitor and an Ambler class A carbapenemase inhibitor.

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

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

[0008] In any embodiment disclosed herein, the method may include the step of determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by Enterobacteriaceae by detecting the presence of growth inhibition in a first test composition, wherein the antibiotics and inhibitors comprise or are essentially comprised of TEM, an Ambler class B carbapenemase inhibitor, and a first concentration of TEM, an Ambler class B carbapenemase inhibitor; the presence of growth in a second test composition, wherein the antibiotics and inhibitors comprise or are essentially comprised of TEM, 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 antibiotics and inhibitors comprise or are essentially comprised 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, or is essentially composed of, a third concentration of MEM and an Ambler class C carbapenemase inhibitor.In any of the embodiments disclosed herein, the method comprises inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprises a first concentration of TEM and an Ambler class B carbapenemase inhibitor, comprising or essentially comprising the inhibition of growth, and the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprises a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, comprising or essentially comprising the presence of growth, and the presence of growth in a third test composition among a plurality of at least four test compositions, wherein the antibiotic and inhibitor comprises a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, comprising the presence of growth, and the presence of growth in a third test composition among a plurality of at least four test compositions, wherein the The biomaterials and inhibitors include a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor, and the presence of growth consisting of or essentially derived from them, as well as inhibition of growth in a fifth test composition, wherein the antibiotics and inhibitors include a third concentration of MEM and an Ambler class C carbapenemase inhibitor, and the inhibition of growth consisting of or essentially derived from them, thereby determining that no answer is obtained regarding the identification of one or more Ambler class carbapenemases expressed by enterobacteria.In any of the embodiments disclosed herein, the method comprises inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprises a first concentration of TEM and an Ambler class B carbapenemase inhibitor, comprising or essentially comprising the inhibition of growth, and the presence of growth in a second test composition, wherein the antibiotic and inhibitor comprises a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, comprising or essentially comprising the presence of growth, and the presence of growth in a third test composition, wherein the antibiotic The antibiotics and inhibitors may include the step of determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by Enterobacteria by detecting the presence of growth consisting of or essentially derived from a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor, as well as the presence of growth in a fifth test composition.

[0009] One embodiment is a method for determining the presence of zero, one or more Ambler class carbapenemases expressed by Enterobacteriaceae, comprising the steps of: providing a sample containing Enterobacteriaceae; applying the Enterobacteriaceae in the test sample to a plurality of at least four test compositions over a duration, each of which comprises 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 Enterobacteriaceae by detecting the presence or inhibition of growth of Enterobacteriaceae in each of the plurality of at least four test compositions after the duration. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed 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, or is essentially derived from, 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, or is essentially derived from, 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, or is essentially derived from, a second concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler A carbapenemase inhibitor.In any embodiment disclosed herein, the method may include the step of determining that one or more Ambler class carbapenemases expressed by Enterobacteriaceae are class A by detecting an inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprises a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor, comprising or essentially consisting of these, and an inhibition of growth in a second test composition, wherein the antibiotic and inhibitor comprises a second concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor, comprising or essentially consisting of these. In any embodiment disclosed herein, the method may include the step of determining that one or more Ambler class carbapenemases expressed by Enterobacteriaceae are class B by detecting the presence of growth in a first test composition, wherein the antibiotics and inhibitors comprise or are essentially comprised of MEM at a first concentration, 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 antibiotics and inhibitors comprise or are essentially comprised of MEM at a second concentration, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor.In any embodiment disclosed herein, the method may include the step of determining that one or more Ambler class carbapenemases expressed by Enterobacteriaceae are class A by detecting the presence of growth in a first test composition, wherein the antibiotics and inhibitors comprise a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor, and detecting the inhibition of growth in a third test composition, wherein the antibiotics and inhibitors comprise a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor, and detecting the inhibition of growth comprised of or comprising these. In any embodiment disclosed herein, the method may include the step of determining that one or more Ambler class carbapenemases expressed by Enterobacteriaceae are class D by detecting the presence of growth in a first test composition, wherein the antibiotics and inhibitors comprise or are essentially composed 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 antibiotics and inhibitors comprise or are essentially composed 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 antibiotics and inhibitors comprise or are essentially composed of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

[0010] In any embodiment disclosed herein, the method may include the step of 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, wherein the antibiotics and inhibitors comprise 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 antibiotics and inhibitors comprise 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 fourth test composition, wherein the antibiotics and inhibitors comprise a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. In any embodiment disclosed herein, the method may include the step of determining that no answer is obtained regarding the identification of one or more Ambler class carbapenemases expressed by Enterobacteriaceae by detecting the presence of growth in a first test composition, wherein the antibiotics and inhibitors comprise 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 antibiotics and inhibitors comprise 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 fourth test composition, wherein the antibiotics and inhibitors comprise a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor, or are composed of or essentially derived from them.

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

[0012] In any embodiment disclosed herein, the method may further encompass a method for determining the presence of zero, one or more Ambler class carbapenemases expressed by non-fermenting bacteria, the method comprising the steps of: providing a sample containing non-fermenting bacteria; applying the non-fermenting bacteria in the test sample to a plurality of at least three test compositions over a duration, 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 more Ambler class carbapenemases expressed by 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 duration.

[0013] One embodiment is a method for determining the presence of zero, one or more Ambler class carbapenemases expressed by non-fermenting bacteria, comprising the steps of: providing a sample containing non-fermenting bacteria; applying the non-fermenting bacteria in the test sample to a plurality of at least three test compositions over a duration, 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 further one or more Ambler class carbapenemases expressed by 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 duration. In any of the embodiments disclosed herein, the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed 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, or is essentially derived from, 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, or is essentially derived from, 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 one or more Ambler class carbapenemases expressed by non-fermenting bacteria as class B by detecting inhibition of growth consisting of, or essentially composed of, a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, wherein the antibiotics and inhibitors comprise 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 non-fermenting bacteria as class D by detecting the presence of growth in a first test composition, wherein the antibiotics and inhibitors comprise or are essentially composed of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor at a third concentration, and the inhibition of growth in a second test composition, wherein the antibiotics and inhibitors comprise or are essentially composed of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a fourth concentration.In any of the embodiments disclosed herein, the method may include determining one or more Ambler class carbapenemases expressed by non-fermenting bacteria as class A by detecting the presence of growth in a first test composition, wherein the antibiotics and inhibitors comprise or are essentially composed 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 antibiotics and inhibitors comprise or are essentially composed of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a fourth concentration; and inhibition of growth in the third test composition, wherein the antibiotics and inhibitors comprise or are essentially composed of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a fifth concentration. In any embodiment disclosed herein, the method may include the step of determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by non-fermenting bacteria by detecting the presence of growth in a first test composition, wherein the antibiotics and inhibitors comprise or are essentially derived from 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 antibiotics and inhibitors comprise or are essentially derived from 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 third test composition, wherein the antibiotics and inhibitors comprise or are essentially derived from 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 antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially derived from, 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, or is essentially derived from, 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, or is essentially derived from, a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor. In any of the embodiments disclosed herein, the method is the presence of growth in a first test composition, wherein the antibiotics and inhibitors comprise, or are essentially composed of, a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor. and The step may include determining whether one or more Ambler class carbapenemases expressed by non-fermenting bacteria are of class A, B, or D by detecting the presence of growth in a second test composition, the presence of growth consisting of or essentially derived from antibiotics and inhibitors comprising a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

[0014] In any embodiment disclosed herein, the method may include the step of determining that one or more Ambler class carbapenemases expressed by non-fermenting bacteria are class D by detecting the presence of growth in a first test composition, wherein the antibiotics and inhibitors comprise or are essentially derived from a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the inhibition of growth in a second test composition, wherein the antibiotics and inhibitors comprise or are essentially derived from a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. In any embodiment disclosed herein, the method may include the step of determining that one or more Ambler class carbapenemases expressed by non-fermenting bacteria are class B by detecting the presence of growth in a first test composition, wherein the antibiotics and inhibitors comprise or are essentially comprised 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 the third test composition, wherein the antibiotics and inhibitors comprise or are essentially comprised of a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor.In any embodiment disclosed herein, the method may include a step of determining that no answer is obtained regarding the identification of one or more Ambler class carbapenemases expressed by non-fermenting bacteria by detecting an inhibition of growth in a first test composition comprising, or essentially comprising, an antibiotic and inhibitor comprising a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and an inhibition of growth in a third test composition comprising, or essentially comprising, a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor. In any embodiment disclosed herein, the method may include the step of determining whether one or more Ambler class carbapenemases expressed by non-fermenting bacteria are of class A, B, or D by detecting an inhibition of growth in a first test composition comprising, or essentially comprising, an antibiotic and inhibitor comprising a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and an inhibition of growth in a third test composition comprising, or essentially comprising, a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor.

[0015] In any of the embodiments disclosed herein, the method may include the step of applying non-fermenting bacteria in a test sample to a plurality of at least four test compositions over a period of time, wherein the antibiotics and carbapenemase inhibitors in at least one test composition consist of or are essentially composed 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 includes the presence of growth in a first test composition, wherein the antibiotics and inhibitors consist of or are essentially composed 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 antibiotics and inhibitors consist of, or are essentially composed of, a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor. The steps may include determining whether one or more Ambler class carbapenemases expressed by non-fermenting bacteria are of class A, B, or D by detecting the presence of growth consisting of or essentially derived from the following, as well as growth in a fourth test composition, wherein the antibiotics and inhibitors include a fifth concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.In any embodiment disclosed herein, the method may include the step of determining that one or more Ambler class carbapenemases expressed by non-fermenting bacteria are class A by detecting the presence of growth in a first test composition, wherein the antibiotics and inhibitors comprise or are essentially composed 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 antibiotics and inhibitors comprise or are essentially composed of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a fourth concentration; and inhibition of growth in the fourth test composition, wherein the antibiotics and inhibitors comprise or are essentially composed of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor at a fifth concentration.

[0016] In any embodiment disclosed herein, the method may further include a step of determining whether the bacteria in the sample are intestinal, non-fermentable, 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 approximately 0.1 μg / ml to approximately 400 μg / ml, approximately 0.5 μg / ml to approximately 3 μg / ml, or approximately 1 μg / ml. In any of the embodiments disclosed herein, the fourth concentration of DOR is approximately 0.2 μg / ml to approximately 40 μg / ml, approximately 0.5 μg / ml to approximately 4 μg / ml, or approximately 2 μg / ml. In any of the embodiments disclosed herein, the fifth concentration of DOR is approximately 0.03125 μg / ml to approximately 80 μg / ml, approximately 2 μg / ml to approximately 24 μg / ml, or approximately 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 approximately 0.015625 μg / ml to approximately 0.125 μg / ml, approximately 0.003 μg / ml to approximately 0.3 μg / ml, approximately 0.0075 μg / ml to approximately 0.12 μg / ml, approximately 0.01 μg / ml to approximately 0.12 μg / ml, or approximately 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 includes a compound selected from the group consisting of AVI, clavulanic acid, boronic acid, tazobactam, sulbactam, bavolbactam (RPX-7009), and BLI-489. In any of the embodiments disclosed herein, the Ambler class B carbapenemase inhibitor is a metal chelating agent. In any of the embodiments disclosed herein, the Ambler class B carbapenemase inhibitor includes 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 includes 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 includes a compound selected from the group consisting of babolbactam (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 includes a compound selected from the group consisting of BLI, AVI, clavulanic acid, boronic acid, tazobactam, sulbactam, babolbactam, and (RPX-7009).

[0019] In any of the embodiments disclosed herein, the Ambler class B carbapenemase inhibitor combined with a first concentration of TEM and / or a first concentration of DOR comprises, consists of, or is essentially composed of EDTA. In any of the embodiments disclosed herein, the Ambler class C carbapenemase inhibitor comprises, consists of, or is essentially composed of CLOX. In any of the embodiments disclosed herein, the Ambler class A carbapenemase inhibitor combined with a first concentration of MEM and / or a second concentration of MEM comprises, consists of, or is essentially composed of RPX. In any of the embodiments disclosed herein, the Ambler class B carbapenemase inhibitor combined with a first concentration of MEM and / or a third concentration of DOR comprises, consists of, or is essentially composed of DPA. In any of the embodiments disclosed herein, the Ambler class D carbapenemase inhibitor combined with a first concentration of DOR and / or a fifth concentration of DOR comprises, consists of, or is essentially composed of AVI. In any of the embodiments disclosed herein, the Ambler class D carbapenemase inhibitor combined with a fourth concentration of MEM comprises, consists of, or is essentially composed of, a 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 approximately 0.2 μg / ml to approximately 320 μg / ml, approximately 1.5 μg / ml to approximately 40 μg / ml, or approximately 8 μg / ml. In any of the embodiments disclosed herein, the concentration of DPA is approximately 0.018 mg / ml to approximately 1.8 mg / ml, approximately 0.07 mg / ml to approximately 0.73 mg / ml, or approximately 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 enterobacteria growth is about 6 hours to about 8 hours. In any of the embodiments disclosed herein, the duration for detecting the presence or inhibition of enterobacteria growth is about 7 hours. In any of the embodiments disclosed herein, the duration for detecting the presence or inhibition of non-fermenting bacteria growth is about 8 hours to about 11 hours. In any of the embodiments disclosed herein, the duration for detecting the presence or inhibition of non-fermenting bacteria growth is about 10 hours. In any of the embodiments disclosed herein, enterobacteria include 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 include bacteria selected from the group consisting of Pseudomonas aeruginosa and Acinetobacter baumanii complex.

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

[0022] One embodiment is a system for performing any of the methods of the embodiments disclosed herein, the system comprising a plurality of compartments, each of the plurality of compartments comprising a plurality of compartments containing a test composition according to any of the methods of the preceding claims, means for providing a sample comprising gut bacteria, non-fermenting bacteria, or both to the plurality of compartments, an instrument for obtaining a first signal from the plurality of compartments supplied with gut bacteria, non-fermenting bacteria, or both, an incubator for incubating the plurality of compartments supplied with gut bacteria, non-fermenting bacteria, or both over a period of time, an instrument for obtaining a second signal from the plurality of compartments comprising gut bacteria, non-fermenting bacteria, or both, a detector for detecting the presence or inhibition of growth in the plurality of compartments supplied with gut bacteria, non-fermenting bacteria, or both by comparing the first signal and the second signal, a computer for generating an output of the results from the detector, and an analyzer for interpreting the output of the results. In any of the embodiments disclosed herein, the plurality of compartments comprise compartments selected from the group consisting of wells, plates, and tubes. In any of the embodiments disclosed herein, the system comprises a BD Phoenix panel and / or system.

[0023] One embodiment is a kit for identifying one or more unclassified classes of carbapenemases expressed by gut bacteria 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 methods 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 the method of any one of claims 1-90, and wherein at least one of 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 composition comprises, consists of, or consists essentially of a test composition selected from the test compositions disclosed in Boxes 1-14. In any of the embodiments disclosed herein, the test composition comprises, consists of, or consists essentially of the test compositions disclosed in Boxes 1-5. 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, 8, 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 10, 11, 12, and 13. In any of the embodiments disclosed herein, the test composition comprises, consists of, or consists essentially of the test compositions disclosed in Boxes 10, 11, 12, 13, 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, 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 Description of the Drawings

[0025] [Figure 1] It is a diagram showing a box plot of TEM GAM data regarding Enterobacteria expressing any of Class A, Class B, or Class D carbapenemases. [Figure 2]This figure shows a box plot of TEM GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 3] This figure shows a box plot of TEM / CLOX / EDTA GAM data for intestinal bacteria expressing either class A, class B, or class D carbapenemases. [Figure 4] This figure shows a box plot of TEM / CLOX / EDTA GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 5] This figure shows a box plot of TEM / CLOX / EDTA GAM data for intestinal bacteria expressing either class A, class B, or class D carbapenemases. [Figure 6] This figure shows a box plot of TEM / CLOX / EDTA GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 7] This figure shows a box plot of MEM / CLOX GAM data for intestinal bacteria expressing either class A, class B, or class D carbapenemases. [Figure 8] This figure shows a box plot of MEM / CLOX GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 9] This figure shows a box plot of MEM / CLOX / DPA GAM data for intestinal bacteria expressing either class A, class B, or class D carbapenemases. [Figure 10] This figure shows a box plot of MEM / CLOX / DPA GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 11]This figure shows a box plot of MEM / CLOX / RPX GAM data for intestinal bacteria expressing either class A, class B, or class D carbapenemases. [Figure 12] This figure shows a box plot of MEM / CLOX / RPX GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 13] This figure shows a box plot of DOR / CLOX / AVI GAM data for intestinal bacteria expressing either class A, class B, or class D carbapenemases. [Figure 14] This figure shows a box plot of DOR / CLOX / AVI GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 15] This figure shows a box plot of DOR / CLOX / EDTA GAM data for intestinal bacteria expressing either class A, class B, or class D carbapenemases. [Figure 16] This figure shows a box plot of DOR / CLOX / EDTA GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 17] This figure shows a box plot of DOR GAM data for intestinal bacteria expressing either class A, class B, or class D carbapenemases. [Figure 18] This figure shows a box plot of DOR GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 19] This figure shows a box plot of DOR / CLOX GAM data for intestinal bacteria expressing either class A, class B, or class D carbapenemases. [Figure 20]This figure shows a box plot of DOR / CLOX GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 21] This figure shows a box plot of DOR / CLOX / DPA GAM data for intestinal bacteria expressing either class A, class B, or class D carbapenemases. [Figure 22] This figure shows a box plot of DOR / CLOX / DPA GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 23] This figure shows a box plot of MEM / CLOX / BLI GAM data for intestinal bacteria expressing either class A, class B, or class D carbapenemases. [Figure 24] This figure shows a box plot of MEM / CLOX / BLI GAM data for non-fermenting Gram-negative bacilli expressing either class A, class B, or class D carbapenemases. [Figure 25] This figure shows a flowchart of an embodiment of the algorithm related to Gram-negative enteric bacteria. [Figure 26] This figure shows a flowchart of an embodiment of the algorithm related to Gram-negative enteric bacteria. [Figure 27] This figure shows a flowchart of an embodiment of the algorithm related to Gram-negative enteric bacteria. [Figure 28] This diagram shows a flowchart of an embodiment of the algorithm for non-fermenting Gram-negative bacilli. [Figure 29] This diagram shows a flowchart of an embodiment of the algorithm for non-fermenting Gram-negative bacilli. [Figure 30] This diagram shows a flowchart of an embodiment of the algorithm for non-fermenting Gram-negative bacilli. [Figure 31]This figure shows box plots of MEM GAM data and MEM / CLOX GAM data for intestinal bacteria expressing class C carbapenemase. [Figure 32] This figure shows a flowchart of an embodiment of an algorithm for classifying Enterobacteriaceae into Class A, B, or D. [Figure 33] This diagram shows a flowchart of an embodiment of an algorithm for classifying nonfermenter strains into class B or D. [Figure 34] This figure shows a flowchart of an embodiment of an algorithm for classifying Enterobacteriaceae into Class A, B, or D. [Figure 35] This figure shows a flowchart of an embodiment of an algorithm for classifying non-fermenting strains into classes A, B, or D. [Figure 36] This diagram shows a flowchart of an embodiment of the algorithm relating to Enterobacteriaceae and non-fermenting strains. [Figure 37] This diagram shows a flowchart of an embodiment of an algorithm for the classification of Enterobacteriaceae and non-fermenting strains, as well as Enterobacteriaceae. [Figure 38] This figure shows a flowchart of an embodiment of an algorithm for classifying Enterobacteriaceae and non-fermenting strains, as well as Enterobacteriaceae and non-fermenting strains. [Figure 39] This figure shows a flowchart of an embodiment of an algorithm for classifying Enterobacteriaceae and non-fermenting strains. [Modes for carrying out the invention]

[0026] A global public health crisis is unfolding, with increasing antibiotic resistance and a declining supply of antibiotics leading to a growing number of patients infected with Gram-negative bacteria that are completely or nearly completely antibiotic-resistant. Carbapenemase-producing organisms (CPOs) are not only a threat to the treatment of bacterial infections, but are also becoming a driving force in the emergence of untreated pathogens, posing a threat to the use of antibiotics to protect patients undergoing cancer chemotherapy, transplant surgery, heart surgery, joint replacement, and even postpartum women.

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

[0028] Most laboratories currently use inaccurate carbapenemase phenotyping tests that require overnight incubation. A minority use more accurate but cumbersome phenotyping tests, or more expensive PCR-based tests with several unresolved accuracy issues. Current phenotyping 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 available bioMerieux Rapidec® Carba NP test is a manual, independent test that detects carbapenemases but does not classify them. Therefore, there is a therapeutic need to classify carbapenemases into molecular groups.

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

[0031] In some embodiments, this detection test incorporates one or more antibiotics and optionally one or more inhibitors in a test that enables more accurate and rapid identification of one or more Ambler class carbapenemases expressed by bacteria. One or more antibiotics inhibit the growth of Gram-negative bacteria in the sample. However, if the Gram-negative bacteria are resistant to one or more antibiotics in order to express one or more Ambler class carbapenemases, one or more inhibitors may be included to enable more accurate and rapid identification of one or more Ambler class carbapenemases expressed by bacteria. In some embodiments, the identification test involves exposing the sample to only one antibiotic or combination of antibiotics with or without an inhibitor. In other embodiments, the test may involve exposing a portion of the sample to multiple different antibiotics with or without an inhibitor in multiple wells so that the sample is tested against one or more antibiotics or combinations of antibiotics with or without an inhibitor in a single test. These multiple combinations are typically tested in parallel, with each combination placed in a separate well so that a portion of the sample is simultaneously exposed to all combinations. However, it is also possible to test by successively exposing a portion of the sample to various combinations. As described herein, multiple wells containing specific antibiotics with or without inhibitors can be tested depending on a given sample (e.g., two or three repetitions of a particular combination of antibiotic and inhibitor).

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

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

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

[0035] In some embodiments, the detection test may incorporate a combination of one or more antibiotics and one or more inhibitors to enable 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 enables 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 enables more accurate and rapid identification of the Ambler class D carbapenemase enzyme.

[0037] The various detection tests provided herein can be combined with automated detection systems that utilize one or more algorithms for phenotypic detection of bacterial carbapenemase expression and, optionally, for the Ambler classification of bacterial carbapenemase expression.

[0038] (BD Phoenix panel and system) Systems for microbiological diagnostic testing and for microbial identification (ID) and antimicrobial agent sensitivity testing (AST) are described in patents (see, for example, Patent Documents 1, 2, 3, 4, and 5) and published applications (see, for example, Patent Document 6), which are incorporated herein in their entirety. These references disclose panels and systems referred to as the BD Phoenix Gram-Negative Identification (ID) / Antimicrobial Sensitivity Test (AST) Panels and Systems (BD Phoenix Panels and Systems) for ID / AST of microorganisms and their sensitivity to one or more antibiotics. The BD Phoenix Panels and Systems are suitable for various AST determination methods. Immediately before adding the microbial sample to be tested by the instrument, for example, alamarBlue®, a redox-buffered redox indicator, is added to and mixed with the AST inoculum. Visible and UV light sources are used to obtain readings corresponding to red, green, blue, and fluorescence wavelengths of light (see, for example, Patent Document 6, which are incorporated herein in their entirety).

[0039] The BD Phoenix panel and system comprises a substrate having multiple test wells adapted to receive specific combinations of bacteria and reagents (e.g., antibiotics with or without inhibitors) suspended in a liquid culture medium. The bacterial response to the specific reagent combination in the panel is measured by installing a panel instrument system that includes multiple light sources (e.g., visible light sources and UV sources) emitting different wavelengths (e.g., red, green, blue, and fluorescence wavelengths). This instrument system can receive the panel and perform microbial ID / AST based on detection by colorimetric and / or fluorescence measurements (see, for example, Patent Document 6, which is entirely incorporated herein). Based on the results of the BD Phoenix panel and system, the sensitivity of the microorganism (e.g., Gram-negative bacteria) to the antibiotic is determined. In addition, the BD Phoenix panel and system can distinguish between enterobacteria and non-fermentable bacteria.

[0040] Those skilled in the art will likely find it easy to understand the cutting edge technologies by considering the aforementioned patents and applications. Those skilled in the art will also likely recognize the improvements that the novel detection tests, including the compositions, methods, systems, and kits disclosed herein, offer beyond existing cutting edge technologies such as BD Phoenix panels and systems.

[0041] [BD Phoenix (trademark) CPO Detect (Phoenix (trademark) CPO Detect)] A novel BD Phoenix® CPO Detect, also known as Phoenix® CPO Detect, is provided herein, which includes a detection test that can be extended on BD Phoenix panels and systems by incorporating CPO detection. The CPO Detect provides a rapid, highly sensitive, specific algorithm-based automated detection test for the detection and identification of bacteria expressing one or more classes of carbapenemases.

[0042] The BD Phoenix® CPO Detect detection test extends 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 the specific identification of classes of carbapenemases 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 a Gram-negative bacterium is resistant to one or more antibiotics to express one or more Ambler class carbapenemases, one or more inhibitors are used to identify one or more Ambler class carbapenemases. For more accurate and rapid identification of Ambler class carbapenemases, one or more antibiotics and one or more inhibitors are used in a test that typically involves a sample being tested in multiple wells containing different combinations of antibiotics with / 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 positive isolates 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 the initial detection-based positive / negative result for carbapenemase detection, the “sensitivity” of the test or “sensitivity” of detection is defined as the percentage of CPO detected in the positive / negative phase of the test.

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

[0047] In the context of classifying positive isolates according to the molecular class of carbapenemase as used herein, a classification result is considered good if it is either an accurate classification or a positive result with an unknown type. Detection of carbapenemases without classification is important and highly beneficial for patient management. Accurate classification of carbapenemases enhances the value of the result. If the carbapenemase belongs to class A, ceftazidime / avibactam is a potential therapeutic candidate. Detection of class B carbapenemases indicates a contraindication to ceftazidime / avibactam therapy because class B CPOs are inherently resistant to this drug. Regarding infections with class D carbapenemase-producing strains, there is currently no clear indication for ceftazidime / avibactam therapy. Accurate negative results are also a good outcome for guiding patient management and infection control.

[0048] Misclassifying carbapenemases as class B or class D is considered unhelpful but relatively harmless. This misclassification does not diminish the value of carbapenemase detection, but it may delay consideration of ceftazidime / avibactam therapy until sensitivity results become available. Similarly, a "no answer" result is unhelpful in that it provides neither benefit nor harm.

[0049] One possible consequence is the misclassification of a class B carbapenemase as a class A carbapenemase. This could lead to patients receiving ineffective ceftazidime / avibactam therapy. False negative results are also considered potentially harmful because the consequences of CPO not being detected could be ineffective therapy and / or failure to implement infection control measures.

[0050] Non-limiting examples of bacteria, antibiotics, inhibitors, and detection reagents are provided herein. Non-limiting examples of concentration ranges for these antibiotics and inhibitors are also provided. However, it is expected that those skilled in the art will readily recognize that the detection tests can be adapted to be performed with other bacteria, antibiotics, inhibitors, and detection reagents, and furthermore, with other concentration ranges for these antibiotics and inhibitors.

[0051] (antibiotics) Non-exclusive examples of antibiotics include temocillin (TEM), doripenem (DOR), or meropenem (MEM). TEM (6-beta-(2-carboxy-2-thien-3-ylacetamide)-6-alpha-methoxypenicillane disodium) is a carboxypenicillin stable against the hydrolysis of chromosomal and plasmid β-lactamases, including extended-spectrum β-lactamases (ESBL) and AmpC-type β-lactamases. TEM is currently used in Belgium and the United Kingdom for the treatment of multidrug-resistant Enterobacteriaceae.

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

[0053] MEM is a broadband injectable antibiotic used to treat a wide variety of infections. It is a β-lactam and belongs to the carbapenem subgroup. 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 β-lactam antibiotics, it inhibits the synthesis of bacterial cell walls.

[0054] In some embodiments, the MEM concentration range in the detection tests provided herein is 0.0039 μg / ml to 128 μg / ml or approximately thereto. In some embodiments, the MEM concentration range in the detection tests provided herein is 0.0078 μg / ml to 64 μg / ml or approximately thereto (Figures 7-12, 23 and 24). In some embodiments, the MEM concentration range in the detection tests provided herein is 0.0156 μg / ml to 64 μg / ml or approximately thereto (Figure 31). Other concentration ranges are approximately 0.016 μg / ml to approximately 1 μg / ml, and in some embodiments, have a concentration of approximately 0.0625 μg / ml.

[0055] DOR is a broadband injectable antibiotic. It is a beta-lactam and belongs to the carbapenem subgroup. DOR can be used for bacterial infections, such as complex abdominal infections, hospital-acquired pneumonia, and urinary tract complications, including kidney infections associated with sepsis. DOR reduces the process of cell wall growth, thereby ultimately leading to the complete elimination of the infectious bacteria.

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

[0057] Other non-exclusive examples of antimicrobial agents include CLOX, EDTA, and RPX7009, avibactam, BLI-489, and DPA.

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

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

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

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

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

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

[0064] (Ambler's carbapenemase) Carbapenemases are β-lactamase enzymes with diverse hydrolytic activities. Carbapenemases can hydrolyze penicillin, cephalosporins, monobactams, and carbapenems. The rapid spread of these enzymes in clinically important bacteria, such as Enterobacteriaceae and non-fermenting bacteria, including Acinetobacter and Pseudomonas species, poses a significant threat to public health.

[0065] Carbapenemases belong to two main families. These two main families are distinguished by their hydrolysis mechanism at the active site (either zinc or serine). Classification based on amino acid homology (Ambler classification) has resulted in four main 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 susceptible to mechanism-based inhibitors. Mechanism-based inhibition is an irreversible form of enzyme inhibition that occurs when, during a "normal" catalytic reaction, the enzyme binds to a substrate analog and forms an irreversible complex with it via covalent bonding. 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's class B carbapenemases contain metallic zinc in their active site. Bacteria expressing Ambler's class B carbapenemases are susceptible to chelating agents that bind to and remove zinc (metallic ions) from the active site of the class B carbapenemase. Non-exclusive 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] Similar to Ambler class A carbapenemase, Ambler class C β-lactamase contains the amino acid serine in its active site. However, Ambler class C β-lactamase does not hydrolyze carbapenems. Overexpression of Ambler class C β-lactamase in bacteria does not make them insensitive to carbapenems, therefore this is not a carbapenemase. Nevertheless, carbapenem resistance may arise if other mutations are present, including loss of porins in the outer membrane or activation of efflux pumps. Overexpression of Ambler class C β-lactamase in bacteria makes bacteria insensitive to broadband cephalosporins.

[0069] Bacteria expressing Ambler class C β-lactamase (referred to herein, for convenience, as class C carbapenemase) may be susceptible to carbapenems, but they may still be insensitive to carbapenems through other mechanisms. To detect class A, B, and D carbapenemases, bacteria expressing class C carbapenemase must be selectively made susceptible in phenotypic studies; otherwise, false positives may occur.

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

[0071] In some cases, more than one class of carbapenemase may be produced by the organism. For example, in some embodiments, two, three, or four classes of carbapenemase may be 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 ESBL, SHV-5, SHV-5-like, SHV-12, SHV-12-like, SHV-18, TEM ESBL, OXA-45), AmpCs (including overproduction strains) (e.g., plasmid-mediated AmpCs, 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), broadband β-lactamases, and porin mutants.

[0073] (Carbapenemase inhibitors and discriminant factors) Non-restrictive classes of carbapenemase inhibitors include mechanism-based inhibitors, chelators, and β-lactam antibiotics.

[0074] Non-exclusive examples of mechanism-based inhibitors include, but are not limited to, β-lactamase inhibitors, as well as boronic acid-based inhibitors such as bavolbactam (RPX7009), BLI-489, CLOX, clavulanate, tazobactam, or avibactam.

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

[0076] Bacteria expressing Ambler class A carbapenemase are susceptible to TEM at lower concentrations, for example, in the range of approximately 6 μg / ml to approximately 12 μg / ml (Figure 1). On the other hand, bacteria expressing class D typically exhibit elevated MICs to TEM and are therefore susceptible at considerably higher concentrations of TEM, for example, above approximately 128 μg / ml (Figure 1).

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

[0078] Non-specific 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), susceptible to EDTA at 280 μg / ml (Figures 5 and 6), and susceptible to DPA at 180 μg / ml (Figures 9, 10, 21, and 22).

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

[0081] (BD Phoenix® CPO Detect Test) The BD Phoenix® CPO Detect detection test can be used as a qualitative in vitro diagnostic test to phenotypically detect carbapenemase expression in bacteria. In addition to providing detection of bacteria expressing carbapenemase, it further distinguishes the type of carbapenemase enzyme into Ambler class A, class B, or class D. With the BD Phoenix® CPO Detect detection test, clinical trial laboratories are expected to be able to identify Ambler class carbapenemase expression by bacteria, in addition to testing all Gram-negative bacteria isolated from patient samples for isolate identification and antibiotic susceptibility.

[0082] In some embodiments, non-limiting examples of samples may include one or more of the following: blood, urine, feces, sputum, saliva, etc. Samples are collected from a human, one or more companion animals, or one or more commercially important animals. In some embodiments, a human, one or more companion animals, or one or more commercially important animals may have a bacterial infection. The bacterial infection may be due to enterobacteria or non-fermenting bacteria. In some embodiments, the bacteria may be other than enterobacteria or non-fermenting bacteria.

[0083] Non-specific examples of intestinal bacteria include Klebsiella pneumoniae, Escherichia coli, and Enterobacter aerogenes.

[0084] Non-fermentative bacteria include non-specific examples such as Pseudomonas aeruginosa and Acinetobacter baumanni complex strains.

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

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

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

[0088] Figures 1–24 and 31 illustrate non-limiting embodiments of detection tests for enterobacteria and non-fermenting bacteria using various concentration ranges of one or more antibiotics and one or more inhibitors. These figures illustrate the concentrations of antibiotics tested along the x-axis (in μg / ml) (not all concentrations are tested in any figure), and the amount of bacterial growth in the sample along the y-axis. Boxes illustrate the median, interquartile range, non-outlier minimum and maximum values, and asterisks represent single outliers. Each figure has panels for bacteria producing class A, class B, and class D, plus a panel for non-carbapenemase-producing bacteria (NEGs). 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 enterobacteria 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 bacteria. At a concentration of 24 μg / ml, almost all class A enterobacteria strains tested are inhibited by TEM, while class D remains unaffected until the concentration reaches 192 μg / ml. Almost all NEG enterobacteria are susceptible to the lowest concentrations of TEM. Class B enterobacteria tested begin to show susceptibility at 48 μg / ml, as evidenced by the decrease in the mean growth line, and at 384 μg / ml, the growth of most class B enterobacteria is inhibited. Figures 1–24 and 31 demonstrate the results of numerous combinations of antibiotics with and without inhibitors that can be used to identify class A, B, D, and NEG enterobacteria and non-fermenting bacteria.

[0090] In some embodiments, the detection test may include multiple wells containing enteric bacteria and TEM with concentrations ranging from approximately 12 μg / ml to approximately 512 μg / ml (Figure 1).

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

[0092] In some embodiments, the detection test may include multiple wells containing enterobacteria, 0.1 mg / ml of CLOX, 250 μg / ml of EDTA, and TEM in a concentration range of approximately 12 μg / ml to approximately 512 μg / ml (Figure 3).

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

[0094] In some embodiments, the detection test may include multiple wells containing enterobacteria, 0.1 mg / ml of CLOX, 280 μg / ml of EDTA, and TEM in a concentration range of approximately 12 μg / ml to approximately 512 μg / ml (Figure 5).

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

[0096] In some embodiments, the detection test may include multiple wells containing enteric bacteria, 0.1 mg / ml of CLOX, and MEM in a concentration range from approximately 0.0078 μg / ml to approximately 64 μg / ml (Figure 7).

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

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

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

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

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

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

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

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

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

[0106] In some embodiments, the detection test may include multiple wells containing enteric bacteria and DOR in a concentration range from approximately 0.0156 μg / ml to approximately 64 μg / ml (Figure 17).

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

[0108] In some embodiments, the detection test may include multiple wells containing enteric bacteria, 0.1 mg / ml of CLOX, and DOR in a concentration range from about 0.0156 μg / ml to about 64 μg / ml (Figure 19).

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

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

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

[0112] In some embodiments, the detection test may include multiple wells containing enterobacteria, 0.1 mg / ml of CLOX, 5 μg / ml of BLI, and MEM in a concentration range from approximately 0.0078 μg / ml to approximately 64 μg / ml (Figure 23).

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

[0114] In some embodiments, the detection test may include multiple wells containing enteric bacteria, 0.1 mg / ml of CLOX, and MEM in a concentration range from approximately 0.0156 μg / ml to approximately 64 μg / ml (Figure 31).

[0115] In detection tests, a determination is made as to whether a bacterium is susceptible to one or more antibiotics provided herein, based on whether or not the bacterium grows in the presence of a specific concentration of the antibiotic. If the bacterium is insufficient to one or more antibiotics, the identification of one or more classes of carbapenemase expressed by the bacterium that confer insufficiency to this antibiotic is achieved by using one or more carbapenemase inhibitors or discriminants provided herein.

[0116] This invention provides non-limiting examples (Examples 1 to 4) of detection tests for identifying the expression of one or more Ambler class carbapenemases by intestinal or non-fermenting bacteria. Whether a sample contains intestinal or non-fermenting bacteria can be determined by methods known in the art, such as biochemical tests including spot oxidase tests, MALDI-TOF, and the Phoenix ID system. The concentrations of antibiotics and inhibitors disclosed in the following examples are illustrative and non-limiting. Other concentrations or ranges of acceptable concentrations are disclosed in the disclosure of the invention, including the drawings.

[0117] In some embodiments, the BD Phoenix® CPO Detect includes wells (or optionally several 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 wells contain one of the combinations disclosed in Table 0.1 below, comprising one or more antibiotics, with or without one or more carbapenemase inhibitors.

[0118] [Table 1]

[0119] In any of the combinations 1 to 10 provided in Table 0.1, the concentration of CLOX is 2.5 μg / ml to 40,000 μg / ml or approximately therein, or the concentration of CLOX is 20 μg / ml to 500 μg / ml or approximately therein. In some embodiments, the concentration of CLOX is 20 μg / ml to 150 μg / ml or approximately therein, the concentration of CLOX is 150 μg / ml to 250 μg / ml or approximately therein, the concentration of CLOX is 250 μg / ml to 350 μg / ml or approximately therein, or the concentration of CLOX is 350 μg / ml to 500 μg / ml or approximately therein, and in some embodiments, the concentration is 100 μg / ml or approximately therein. In some embodiments, the concentration of CLOX is 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 the range defined by any two of the above values.

[0120] In any of the combinations 1 to 10 provided in Table 0.1, the concentration of AVI is 0.5 μg / ml to 20 μg / ml or approximately therein. In some embodiments, the concentration of AVI is 0.5 μg / ml to 5 μg / ml or approximately therein, the concentration of AVI is 5 μg / ml to 10 μg / ml or approximately therein, the concentration of AVI is 10 μg / ml to 15 μg / ml or approximately therein, or the concentration of AVI is 15 μg / ml to 10 μg / ml or approximately therein, and in some embodiments, the concentration is 4 μg / ml or approximately therein. In some embodiments, the concentration of AVI is 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 approximately within that range, or within the range defined by any two of the above values.

[0121] In any of the combinations 1 to 10 provided in Table 0.1, the concentration of BLI-489 is 1 μg / ml to 25 μg / ml or approximately therein. In some embodiments, the concentration of BLI-489 is 1 μg / ml to 5 μg / ml or approximately therein, the concentration of BLI-489 is 5 μg / ml to 10 μg / ml or approximately therein, the concentration of BLI-489 is 10 μg / ml to 17.5 μg / ml or approximately therein, or the concentration of BLI-489 is 17.5 μg / ml to 25 μg / ml or approximately therein, and in some embodiments, the concentration is 5 μg / ml or approximately therein. In some embodiments, the concentration of BLI-489 is 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 approximately within that range, or within the range defined by any two of the above values.

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

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

[0124] In any of the combinations 1 to 10 provided in Table 0.1, the concentration of RPX7009 is 1.5 μg / ml to 40 μg / ml or approximately therein. In some embodiments, the concentration of RPX7009 is 1.5 μg / ml to 3 μg / ml or approximately therein, the concentration of RPX7009 is 3 μg / ml to 15 μg / ml or approximately therein, the concentration of RPX7009 is 15 μg / ml to 25 μg / ml or approximately therein, or the concentration of RPX7009 is about 25 μg / ml to 40 μg / ml or approximately therein, and in some embodiments, the concentration is 8 μg / ml or approximately therein. In some embodiments, the concentration range of RPX7009 is 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 approximately within that range, or within the range defined by any two of the above values.

[0125] In combination 1, the concentration of DOR is 0.0625 μg / ml to 0.25 μg / ml or approximately within that range. In some embodiments, the concentration of DOR is 0.0125 μg / ml to 1.25 μg / ml or approximately within that range, the concentration of DOR is 0.0625 μg / ml to 0.0825 μg / ml or approximately within that range, the concentration of DOR is 0.0825 μg / ml to 0.125 μg / ml or approximately within that range, the concentration of DOR is 0.125 μg / ml to 0.175 μg / ml or approximately within that range, or the concentration of DOR is 0.175 μg / ml to 0.25 μg / ml or approximately within that range. In some embodiments, the concentration of DOR is 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 approximately in that range, or within the range defined by any two of the above values.

[0126] In combination 2, the concentration of DOR is 0.5 μg / ml to 4 μg / ml or approximately within that range. In some embodiments, the concentration of DOR is 0.1 μg / ml to 40 μg / ml or approximately within that range, the concentration of DOR is 0.1 μg / ml to 10 μg / ml or approximately within that range, the concentration of DOR is 0.2 μg / ml to 20 μg / ml or approximately within that range, the concentration of DOR is 0.5 μg / ml to 4 μg / ml or approximately within that range, the concentration of DOR is 0.5 μg / ml to 1 μg / ml or approximately within that range, the concentration of DOR is 1 μg / ml to 2 μg / ml or approximately within that range, the concentration of DOR is 2 μg / ml to 3 μg / ml or approximately within that range, or the concentration of DOR is 3 μg / ml to 4 μg / ml or approximately within that range. In some embodiments, the concentration of DOR is 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 the range defined by any two of the above values.

[0127] In combination 3, the concentration of DOR is between 0.03125 μg / ml and 16 μg / ml, or between 0.02 μg / ml and 600 μg / ml or approximately therein. In some embodiments, the concentration of DOR is between 0.03126 μg / ml and 1 μg / ml or approximately therein, the concentration of DOR is between 1 μg / ml and 4 μg / ml or approximately therein, the concentration of DOR is between 4 μg / ml and 8 μg / ml or approximately therein, or the concentration of DOR is between 8 μg / ml and 16 μg / ml or approximately therein. In some embodiments, the concentration of DOR is 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 approximately within that range, or within the range defined by any two of the above values. In some embodiments, the concentration of DOR is 0.006 μg / ml to 0.6 μg / ml or approximately within that range. In some embodiments, the concentration of DOR is between 0.03125 μg / ml and 0.0625 μg / ml or approximately therein, between 0.015 μg / ml and 0.24 μg / ml or approximately therein, between 0.0625 μg / ml and 0.0775 μg / ml or approximately therein, between 0.0775 μg / ml and 0.1 μg / ml or approximately therein, or between 0.1 μg / ml and 0.125 μg / ml or approximately therein. In some embodiments, the concentration of DOR is 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 approximately within that range, or within the range defined by any two of the above values.

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

[0129] In combination 5, the concentration of DOR is 0.03125 μg / ml to 0.125 μg / ml or approximately within that range. In some embodiments, the concentration of DOR is 0.03125 μg / ml to 0.0625 μg / ml or approximately within that range, the concentration of DOR is 0.0625 μg / ml to 0.0775 μg / ml or approximately within that range, the concentration of DOR is 0.0775 μg / ml to 0.1 μg / ml or approximately within that range, or the concentration of DOR is 0.1 μg / ml to 0.125 μg / ml or approximately within that range. In some embodiments, the concentration of DOR is 0.006 μg / ml to 0.6 μg / ml or approximately within that range. In some embodiments, the concentration of DOR is 0.015 μg / ml to 0.24 μg / ml or approximately within that range. In some embodiments, the concentration of DOR is 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 approximately within that range, or within the range defined by any two of the above values.

[0130] In combination 6, the concentration of MEM is 0.03125 μg / ml to 1 μg / ml or approximately within that range. In some embodiments, the concentration of MEM is 0.0125 μg / ml to 5 μg / ml or approximately within that range, the concentration of MEM is 0.03125 μg / ml to 0.0625 μg / ml or approximately within that range, the concentration of MEM is 0.0625 μg / ml to 0.125 μg / ml or approximately within that range, the concentration of MEM is 0.125 μg / ml to 0.5 μg / ml or approximately within that range, the concentration of MEM is 0.125 μg / ml to 2 μg / ml or approximately within that range, or the concentration of MEM is 0.5 μg / ml to 1 μg / ml or approximately within that range. In some embodiments, the concentration of MEM is 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 the range defined by any two of the above values. In some embodiments, the concentration of MEM is 0.006 μg / ml to 0.6 μg / ml or within the range. In some embodiments, the concentration of MEM is between 0.03125 μg / ml and 0.0625 μg / ml or approximately therein, between 0.015 μg / ml and 0.24 μg / ml or approximately therein, between 0.0625 μg / ml and 0.0775 μg / ml or approximately therein, between 0.0775 μg / ml and 0.1 μg / ml or approximately therein, or between 0.1 μg / ml and 0.125 μg / ml or approximately therein.In some embodiments, the concentration of MEM is 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 approximately within that range, or within the range defined by any two of the above values.

[0131] In combination 7, the concentration of MEM is 2 μg / ml to 8 μg / ml or approximately within that range. In some embodiments, the concentration of MEM is 0.4 μg / ml to 40 μg / ml or approximately within that range, the concentration of MEM is 1 μg / ml to 16 μg / ml or approximately within that range, the concentration of MEM is 2 μg / ml to 4 μg / ml or approximately within that range, the concentration of MEM is 4 μg / ml to 6 μg / ml or approximately within that range, the concentration of MEM is 6 μg / ml to 7.5 μg / ml or approximately within that range, or the concentration of MEM is 7.5 μg / ml to 8 μg / ml or approximately within that range. In some embodiments, the concentration of MEM is 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 approximately within that range, or within the range defined by any two of the above values.

[0132] In combination 8, the concentration of MEM is 0.03125 μg / ml to 0.125 μg / ml or approximately within that range. In some embodiments, the concentration of MEM is 0.03125 μg / ml to 0.0625 μg / ml or approximately within that range, the concentration of MEM is 0.0625 μg / ml to 0.0775 μg / ml or approximately within that range, the concentration of MEM is 0.0775 μg / ml to 0.1 μg / ml or approximately within that range, or the concentration of MEM is 0.1 μg / ml to 0.125 μg / ml or approximately within that range. In some embodiments, the concentration of MEM is 0.006 μg / ml to 0.6 μg / ml or approximately within that range. In some embodiments, the concentration of MEM is 0.015 μg / ml to 0.24 μg / ml or approximately within that range. In some embodiments, the concentration of MEM is 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 approximately within that range, or within the range defined by any two of the above values.

[0133] In combination 9, the concentration of MEM is between 0.015625 μg / ml and 0.125 μg / ml or approximately therein. In some embodiments, the concentration of MEM is between 0.03125 μg / ml and 0.0625 μg / ml or approximately therein, the concentration of MEM is between 0.0625 μg / ml and 0.0775 μg / ml or approximately therein, the concentration of MEM is between 0.0775 μg / ml and 0.1 μg / ml or approximately therein, or the concentration of MEM is between 0.1 μg / ml and 0.125 μg / ml or approximately therein. In some embodiments, the concentration of MEM is 0.006 μg / ml to 0.6 μg / ml or approximately therein, the concentration of MEM is 0.015 μg / ml to 0.24 μg / ml or approximately therein, the concentration of MEM is 0.003 μg / ml to 0.3 μg / ml or approximately therein, or the concentration of MEM is 0.0075 μg / ml to 0.12 μg / ml or approximately therein. In some embodiments, the concentration of MEM is 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 approximately within that range, or within the range defined by any two of the above values.

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

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

[0136] For example, in some embodiments, Examples 10.1 to 10.4, each “box” in the algorithm represents a test site (e.g., a well, or optionally an average of several identical wells) of a detection test provided herein, which includes 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, and the CPO detection and / or classification wells of the algorithm for enterobacteria can be reordered and / or combined with the CPO detection and / or classification wells of the algorithm for non-fermenting bacteria to achieve detection of CPO enterobacteria, CPO non-fermenting bacteria, or both, and / or Ambler classification of enterobacteria, non-fermenting bacteria, or both.

[0137] In some embodiments, the wells contain one of a combination of one or more antibiotics, with or without one or more carbapenemase inhibitors as disclosed in Table 0.1. In some embodiments, the test comprises at least two wells, one well for the detection of CPO enterobacteria and the other well 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 the detection of CPO enterobacteria, one well is for the detection of CPO non-fermenting bacteria, and four wells are for Ambler classification of carbapenemases produced by enterobacteria (e.g., Figure 37). Thus, in some embodiments, at least four wells enable Ambler classification of carbapenemases produced by enterobacteria (e.g., Figure 37). In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by enterobacteria is two to five. In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by enterobacteria is two, three, four, five or more.

[0139] In some embodiments, the test comprises at least nine wells, where one well is for the detection of CPO enterobacteria, one well is for the detection of CPO non-fermenting bacteria, four wells are for the Ambler classification of carbapenemases produced by enterobacteria, and three wells are for the Ambler classification of carbapenemases produced by non-fermenting bacteria (e.g., Figure 38). Thus, in some embodiments, at least four wells enable the Ambler classification of carbapenemases produced by enterobacteria, at least three wells enable the Ambler classification of carbapenemases produced by non-fermenting bacteria, and at least seven wells enable the Ambler classification of carbapenemases produced by both enterobacteria and non-fermenting bacteria (e.g., Figure 38). In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by enterobacteria is 2 to 5, and the number of wells used for Ambler classification of carbapenemases produced by non-fermenting bacteria is 2 to 4. In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by enterobacteria is 2, 3, 4, 5 or more, and the number of wells used for Ambler classification of carbapenemases produced by non-fermenting bacteria is 2, 3, 4, 5 or more. In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by enterobacteria and non-fermenting bacteria is 4 to 9. In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by enterobacteria and non-fermenting 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 the detection of CPO enterobacteria, one well is for the detection of CPO non-fermenting bacteria, and three wells are for Ambler classification of carbapenemases produced by non-fermenting bacteria (e.g., Figure 39). Thus, in some embodiments, at least three wells enable Ambler classification of carbapenemases produced by non-fermenting bacteria (e.g., Figure 39). In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by non-fermenting bacteria is two to four. In some embodiments, the number of wells used for Ambler classification of carbapenemases produced by non-fermenting bacteria is two, three, four or more.

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

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

[0143] If intestinal bacteria grow in the presence of 0.06 μg / ml MEM and 0.1 mg / ml CLOX, in the presence of 0.06 mg / ml MEM, 0.1 mg / ml CLOX, and 0.18 mg / ml DPA, and do not grow in the presence of 0.06 mg / ml MEM, 0.1 mg / ml CLOX, and 8 μg / ml RPX, then the detection test indicates the expression of class A carbapenemase by these bacteria.

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

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

[0146] (Example 2) (Identification of Class B carbapenemase expression) (intestinal bacteria) If intestinal bacteria proliferate in the presence of 0.06 μg / ml MEM and 0.1 mg / ml CLOX, but do not proliferate in the presence of 0.06 μg / ml MEM, 0.1 mg / ml CLOX, and 0.18 mg / ml DPA, and proliferate in the presence of 0.03 μg / ml MEM, 0.1 mg / ml CLOX, and 8 μg / ml RPX, then the detection test indicates the expression of class B carbapenemase by these bacteria.

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

[0148] (Non-fermenting bacteria) If non-fermenting bacteria grow in the presence of 1 μg / ml DOR and 0.1 mg / ml CLOX, but do 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 the expression of class B carbapenemase by these bacteria. Growth in 2 μg / ml DOR and 0.1 mg / ml CLOX indicates the expression of class B.

[0149] If non-fermenting bacteria grow in the presence of 1 μg / ml DOR and 0.1 mg / ml CLOX, but do not grow in 1 μg / ml DOR, 0.1 mg / ml CLOX, and 0.18 mg / ml DPA, the detection test indicates the expression of class B carbapenemase by these bacteria. Growth in 2 μg / ml DOR and 0.1 mg / ml CLOX indicates the expression of class B.

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

[0151] When intestinal bacteria proliferate in the presence of 0.06 μg / ml MEM and 0.1 mg / ml CLOX, and also in the presence of 64 μg / ml TEM and 0.25 mg / ml EDTA, the detection test indicates the expression of class D carbapenemase by these bacteria.

[0152] If intestinal bacteria grow in the presence of 0.06 μg / ml MEM and 0.1 mg / ml CLOX, but do not grow in the presence of 64 μg / ml TEM and 0.25 mg / ml EDTA, or in the presence of 0.06 μg / ml DOR, 0.1 mg / ml CLOX, 0.25 mg / ml EDTA, or 0.125 μg / ml DOR, then the detection test indicates the expression of class D carbapenemase by this bacterium.

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

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

[0155] (Example 4) (Identification of Class A, B, or D carbapenemase expression) (intestinal bacteria) If an intestinal bacterium 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, grows in the presence of 0.06 μg / ml MEM, 0.1 mg / ml CLOX and 8 μg / ml RPX, and grows in the presence of 0.5 μg / ml MEM and 0.1 mg / ml CLOX, then the detection test indicates the expression of class A, B, and / or D carbapenemases by this bacterium.

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

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

[0158] (Duration of incubation) The detection test is performed over 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 outcome of the detection test—whether growth occurred or not—is obtained within the time frame defined by the incubation period.

[0159] Conventional assays (e.g., plate-based assays) require at least 16 hours to about 24 hours or more for the 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 that range. In some embodiments, the duration of the detection test may be in the range of about 5 hours to about 10 hours. In some embodiments, the duration of the detection test for enterobacteria is in the range of about 6 hours to about 8 hours. In some embodiments, the duration of the detection test for enterobacteria is in the range of about 5 hours to about 7 hours. In some embodiments, the duration of the detection test for non-fermentable bacteria is in the range of about 8 hours to about 11 hours. In some embodiments, the duration of the detection test for non-fermentable bacteria is in the range of about 7 hours to about 14 hours.

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

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

[0162] The detection test in each of the multiple wells is redox reaction-based, and one or more detection reagents in the wells enable redox reaction-based detection of the absence or presence of growth. In some embodiments, the detection test in each of the multiple wells for detecting growth is based on turbidity in each of the multiple wells. In some embodiments, the detection test in each of the multiple wells for detecting growth is based on a combination of redox reaction and turbidity in each of the multiple wells.

[0163] Redox reactions are well known in the art and include chemical reactions in which the oxidation state of an atom is changed. Redox reactions involve the transfer of electrons between two or more chemical species. When one or more electrons are transferred from one chemical species, that species is oxidized, and when one or more electrons are transferred to another chemical species, that 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 types of redox reactions. In some embodiments, redox reactions may be based on changes in pH, color, etc.

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

[0166] In some embodiments, the outcome of the detection test is that one or more bacteria grow in multiple wells. In some embodiments, the outcome of the detection test is that one or more bacteria do not grow in the sample in multiple wells.

[0167] The assays disclosed to date 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-fermenting bacteria. In some embodiments, the detection test can be used for both enterobacteria and non-fermenting bacteria.

[0168] In some embodiments, the detection test can be modified to automate the process to enable rapid identification and differentiation of different carbapenemase classes. In 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 data from one or more detectors to query results in the wells. The algorithms take approximately 1 to 10 minutes to process the data. Based on the presence or absence of bacterial growth in the wells, the algorithms provide an output of "growth present" or "growth absent" in the wells. In some embodiments, the time frame for the entire algorithm is approximately 6 to 12 hours. In some embodiments, the time frame for the entire algorithm is approximately 5 to 7 hours.

[0170] Although this disclosure describes tests performed entirely in a “well,” it is expected that those skilled in the art will recognize that numerous test sites are suitable for the tests disclosed herein, and therefore the “well” is not limiting. For example, microtiter plates, cuvettes, test tubes, or any other suitable structure known in the art may be used.

[0171] Examples 6 (Figure 25) to 10 (Figure 30) provide non-limiting examples of the algorithm. Each "box" in the algorithm represents an assay well of the detection test provided herein.

[0172] (BD Phoenix (trademark) CPO Detect Algorithm) In some embodiments, one or more algorithms are provided that enable rapid, automated identification of carbapenemase-expressing bacteria, along with identification of the Ambler class of carbapenemases. Integrating these algorithms into an automated platform results in a high level 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 detection tests obtained using CPO Detect. For example, the computer queries the results of detection tests obtained in multiple wells and provides an output based on the results from the queried detection tests (e.g., growth present or growth absent) as defined by the algorithm. The detection test results provided to the system are either growth positive (G) or growth absent (NG) in one or more wells of the detection test within a given time frame. Based on the results provided for the queried wells (growth present or growth absent), the system proceeds to the next query as defined by 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] The following provides non-limiting examples of the algorithm (Examples 5 to 10). Each “box” in the algorithm represents a well (or, optionally, an average of several identical wells) of a detection test provided herein, 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. For example, Box 1 in Figure 25 represents a well of a detection test containing a combination of input samples comprising one or more enterobacteria, one or more detection reagents, 0.06 μg / ml MEM, and 0.1 mg / ml CLOX. As will be discussed, the determination of whether a sample contains enterobacteria or non-fermentable 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 has been put through the test. In the examples of algorithms shown herein, the determination of whether a sample contains enteric or non-fermentable bacteria is made either before or after the steps of the algorithm shown. In some embodiments, the algorithms shown herein can be attempted without prior determination of whether a sample contains enteric or non-fermentable bacteria. If the test is not suitable to provide determined CPO detection and / or classification of the type of bacteria (e.g., non-fermentable or enteric) after the test has been attempted, 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 carbapenemases. Box 1 represents a well containing an input sample combination containing one or more Enterobacteriaceae, 0.06 μg / ml of MEM, 0.1 mg / ml of CLOX, and one or more detection reagents. As shown in Figure 25 with respect to the 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 carbapenemases in the sample. If the result of the test in Box 1 is No Growth (NG), the reported output result is negative, meaning the sample does not contain Enterobacteriaceae that produce class A, B, or D carbapenemases. As discussed herein, the reporting of results depends on determining whether the bacteria being tested are intestinal, either before or after the test is attempted, to ensure that an appropriate algorithm is used depending on the type of bacteria present.

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

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

[0178] (Example 7) (Identification of carbapenemase classes related to intestinal bacteria) Figure 27 illustrates a flowchart of an embodiment of an algorithm for determining whether enterobacteria in a sample produce carbapenemase, and if so, which class. As shown in Figure 27, the system is expected to query the results of the test in box 1 for the enterobacteria, and if growth is reported, it will proceed to query the results of the test in box 6. Box 6 represents a well containing an input sample combination including one or more enterobacteria, 64 μg / ml TEM, 0.25 mg / ml EDTA, and one or more detection reagents. If the system is expected to query the results of the test in box 6 and no growth is reported, it will proceed to query the results of the test in box 7. Box 7 represents a well containing an input sample combination including one or more enterobacteria, 0.06 μg / ml DOR, 0.1 mg / ml CLOX, 0.25 mg / ml EDTA, and one or more detection reagents. If the system is expected to query the results of the test in box 7 and growth is reported, it will proceed to query the results of the test in box 3. If the algorithm queries for the results of the test in box 3 and reports no growth, it is expected to terminate the query and output a result indicating that the sample contains Enterobacteria expressing class A carbapenemase. If the system queries for the results of the test in box 6 and reports no growth, it is expected to proceed to query for the results of the test in box 7. If the algorithm queries for the results of the test in box 7 and reports no growth, it is expected to proceed to query for the results of the test in box 9. Box 9 represents a well containing a combination of input samples containing one or more Enterobacteria, 0.125 μg / ml DOR, and one or more detection reagents. If the system queries for the results of the test in box 9 and reports growth, it is expected to terminate the query and output a result indicating that the sample contains Enterobacteria expressing class B carbapenemase.The system is expected to query the results of the test in box 6, and if growth is reported, it will terminate the query and output a result indicating that the sample contains Enterobacteria expressing class D carbapenemase. The system is expected to query the results of the test in box 9, and if no growth is reported, it will terminate the query and output a result indicating that the sample contains Enterobacteria expressing class D carbapenemase. The system is expected to query the results of the test in box 3, and if growth is reported, it will proceed to query the results of the test in box 8. Box 8 represents a well containing a combination of input samples containing one or more Enterobacteria, 0.5 μg / ml MEM, 0.1 mg / ml CLOX, and one or more detection reagents. The system is expected to query the results of the test in box 8, and if growth is reported, it will terminate the query and output a result indicating that the sample contains Enterobacteria expressing one or more class A, B, or D carbapenemases. The system is expected to query the results of the Box 8 test, and if no growth is reported, it will terminate the query and output a result indicating that it was unable to determine which class of carbapenemase the enterobacteria express.

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

[0180] (Example 8) Figure 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 carbapenemases. Box 10 represents a well containing an input sample combination comprising one or more enterobacteria, 1 μg / ml of DOR, 0.1 mg / ml of CLOX, and one or more detection reagents. As shown in Figure 28 with respect to 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 enterobacteria that produce class A, B, or D carbapenemases in the sample. If the result of the test in Box 10 is no growth (NG), the reported output result is negative, meaning the sample does not contain non-fermenting bacteria that produce class A, B, or D carbapenemases. As discussed herein, the reporting of results depends on determining whether the bacteria being tested are non-fermentative, either before or after the test is attempted, to ensure that an appropriate algorithm is used depending on the type of bacteria present.

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

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

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

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

[0185] (Example 10.1) (Detection of carbapenemases in Enterobacteriaceae and non-fermenting strains) Figure 36 shows a flowchart of an embodiment of the algorithm for detecting CPO in Enterobacteriaceae and non-fermenting strains. For Enterobacteria, as shown in Figure 36, the system can query the results of the detection test in box 1. If the test result in box 1 is growth, the system reports a positive output result indicating the presence of Enterobacteria producing class A, B, or D carbapenemases in the sample. If the test result in box 1 is no growth (inhibition), the reported output result is negative, meaning the sample does not contain Enterobacteria producing class A, B, or D carbapenemases. For non-fermenting bacteria, as shown in Figure 36, the system can query the results of the detection test in box 10. If the test result in box 10 is growth, the system reports a positive output result indicating the presence of non-fermenting bacteria producing class A, B, or D carbapenemases in the sample. If the test result in Box 10 is no growth (inhibition), the reported output result is negative, meaning the sample does not contain non-fermenting bacteria that produce class A, B, or D carbapenemases.

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

[0187] (Example 10.2) (Detection of carbapenemases in Enterobacteriaceae and non-fermenting strains, and Ambler classification of Enterobacteriaceae) Figure 37 shows a flowchart of an embodiment of the algorithm for CPO detection of Enterobacteriaceae and non-fermenting strains and classification of Enterobacteriaceae. The algorithm in Figure 37 is based on the algorithm in Figure 36. For Enterobacteriaceae, as shown in Figure 37, the system can query the results 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 producing 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, meaning the sample does not contain Enterobacteriaceae producing class A, B, or D carbapenemase. For non-fermenting bacteria, as shown in Figure 37, the system can query the results 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 producing class A, B, or D carbapenemase in the sample. If the test result in Box 10 is no growth (inhibition), the reported output result is negative, meaning the sample does not contain non-fermenting bacteria that produce class A, B, or D carbapenemases.

[0188] The algorithm in Figure 37 further enables the determination of the class of carbapenemase produced by enterobacteria. As shown in Figure 37 with respect to enterobacteria, 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 6. If the system queries box 6 and growth is reported, it is expected to terminate the query and output a result indicating that the sample contains enterobacteria expressing class D carbapenemase. If the system queries the results of the test in box 6 and no growth (inhibition) is reported, it is expected to proceed to query the results 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 results 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 a result indicating that the sample contains enterobacteria expressing class D carbapenemase. If the system queries box 7 and growth is reported, it is expected to query the results 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 a result indicating that the sample contains Enterobacteria expressing class A carbapenemase. If the system queries box 9 and reports growth, it is expected to terminate the query and output a result indicating that the sample contains Enterobacteria expressing class B carbapenemase. If the system queries box 3 and reports growth, it is expected to terminate the query and output a result indicating that it was unable to determine which class of carbapenemase the Enterobacteria expresses.

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

[0190] (Example 10.3) (Detection of carbapenemases and Ambler classification in Enterobacteriaceae and non-fermenting strains) Figure 38 shows a flowchart of an embodiment of an algorithm for detecting CPO in Enterobacteriaceae and non-fermenting strains, and for classifying Enterobacteriaceae and non-fermenting 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 enables the determination of the class of carbapenemase produced by non-fermenting bacteria. As shown in Figure 38 for Intestinal Bacteria, if the system queries the results of the test in box 10 and reports growth, it is expected to proceed to query the results of the test in box 11. If the system queries box 11 and reports no growth (inhibition), it is expected to terminate the query and output a result indicating that the sample contains non-fermenting bacteria expressing class B carbapenemase. If the system queries the results of the test in box 11 and reports growth, it is expected to proceed to query the results of the test in box 12. If the system queries box 12 and reports no growth (inhibition), it is expected to terminate the query and output a result indicating that the sample contains non-fermenting bacteria expressing class D carbapenemase. If the system queries box 12 and reports growth, it is expected to proceed to query the results of the test in box 14. If the system queries box 14 and reports no growth (inhibition), it is expected to terminate the query and output a result indicating that the sample contains non-fermenting bacteria expressing class A carbapenemase. If the system queries box 14 and reports growth, it is expected to terminate the query and output a result indicating that it was unable to determine which class of carbapenemase the non-fermenting bacteria express.

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

[0193] (Example 10.4) (Detection of carbapenemases in Enterobacteriaceae and non-fermenting strains, and Ambler classification of non-fermenting strains) Figure 39 shows a flowchart of an embodiment of the algorithm for detecting CPO in Enterobacteriaceae and non-fermenting strains, and for classifying non-fermenting strains. The algorithm in Figure 39 is the same as in Figure 38, except that it does not include some of the algorithm for classifying Enterobacteriaceae.

[0194] (Example 11) (Comparison between the BD Phoenix® CPO Detect trial and the bioMerieux Rapidec® Carba NP trial) The data in this embodiment relate to a study designed to assess the performance of 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 reduce operator time and expedite carbapenemase reporting by providing both CPO detection and carbapenemase classification. The comparative test was the bioMerieux Rapidec® Carba NP test, a currently commercially available independent test that detects carbapenemases but does not classify them. Thus, the BD Phoenix® CPO Detect IUO panel and the bioMerieux Rapidec® Carba NP test were compared in terms of accuracy and impact on workflow.

[0195] (Example 11.1) Method The studies were conducted at BD Life Sciences, Sparks, MD laboratories by BD research staff providing laboratory and computing support. GKID Inc. prepared inoculum for both studies and interpreted all bioMerieux Rapidec® Carba NP studies. BD staff were not involved in any aspect of the bioMerieux Rapidec® Carba NP studies. Both studies were blinded and performed according to manufacturer recommendations. Inoculum were prepared from cells grown overnight on BD blood agar plates adjacent to imipenem disks, which were used to enhance retention of the carbapenemase-encoding plasmid in unstable isolates.

[0196] The bioMerieux Rapidec® Carba NP test was sometimes difficult to interpret. The manufacturer's definition of a positive test is a "significant color variation" between the test well and the control well. This definition was problematic because it did not provide a boundary between significant and non-significant color variations. For example, when using E. cloacae 0164 (IMI class A carbapenemase), E. coli 0104 (KPC class A carbapenemase), E. coli 0058 (ESBL), and Klebsiella pneumoniae G1673 (CMY-2 plasmid-mediated AmpC), which were expected to yield positive tests, no significant color variation was observed. For this reason, borderline results were interpreted as both positive (interpretation 1) and negative (interpretation 2). This provides two sets of results for the bioMerieux Rapidec® Carba NP.

[0197] (Example 11.2) Isolated strain In addition to 294 isolates, three quality control strains were tested. The tested isolates consisted of 236 isolates from the Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii that produced a single carbapenemase, seven isolates producing 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 type, plus the group of resistance mechanisms). Table 1.3 provides the symbol for the mechanisms in Tables 1.1-1.3.

[0198] The isolated strain was obtained from the following:

[0199] FDA / CDC Challenge Panel on 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 in both tests.

[0203] Klebsiella pneumoniae ATCC700603 (negative) - Used for both tests.

[0204] Used exclusively for E. coli ATCC BAA2452 (positive, NDM-1)-BD Phoenix™ CPO detection.

[0205] These were not conventional clinical isolates. They were selected to provide an extreme test of diagnostic capability. The reference standard was prior characterization by molecular, phenotypic, and biochemical testing. There were 110 strains producing class A carbapenemases, including KPC, NMC-A, IMI, and SME enzymes; 91 strains producing class B carbapenemases (metallo-β-lactamases), including NDM, GIM, SPM, IMP, and VIM enzymes; 35 strains producing class D carbapenemases, including OXA-23, 40, 48, 58, 72, 181, and 232; and 7 isolates producing two carbapenemases. 51 negative controls (35 AmpC and 16 other non-AmpC) produced ESBL, AmpC (including overproduction strains), K1, broadband β-lactamase, and porin mutants.

[0206] [Table 2]

[0207] [Table 3-1]

[0208] [Table 3-2]

[0209] [Table 4]

[0210] (Example 11.3) Sensitivity for detection of all carbapenemases Given the extreme diagnostic difficulties in some of the test isolates, the results obtained in this manner for both BD Phoenix® CPO Detect and bioMerieux Rapidec® Carba NP were outstanding in terms of overall sensitivity for the detection of all types of carbapenemases.

[0211] BD Phoenix® CPO Detect achieved a sensitivity of 97.1% (236 out of 243 CPOs were detected). 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 carbapenemase molecular classes The sensitivity of both BD Phoenix® CPO Detect and bioMerieux Rapidec® Carba NP detection was very good in terms of each molecular class of carbapenemase. BD Phoenix® CPO Detect interrupted the panel for one isolate of Pseudomonas aeruginosa G15303. Conventional clinical laboratories expect to immediately perform replicate testing on this isolate and obtain results.

[0213] (Sensitivity for detecting class A carbapenemases) BD Phoenix® CPO Detect achieved a sensitivity of 97.3% (107 out of 110 isolates) for detecting class A carbapenemase-producing strains. bioMerieux Rapidec® Carba NP achieved a sensitivity of 100.0% (110 out of 110 isolates) using interpretation 1 and a sensitivity of 98.2% (108 out of 110 isolates) using interpretation 2. The detection of 97.3% of class A-producing strains 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) producing strains resulted in false negatives.

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

[0216] KPC-4-producing Klebsiella pneumoniae G1511-KPC-4 is a weakly active enzyme. Because the MICs were clearly elevated (ertapenem > 1; imipenem 4; meropenem 2 μg / ml), the isolate is not expected to be unfairly reported as carbapenem-sensitive. This isolate is extremely difficult to identify as CPO for most phenotypic studies.

[0217] K. oxytoca 0147 - This isolate possessed high, off-scale carbapenem MICs (MICs) of ertapenem > 1; imipenem > 8; and meropenem > 8 μg / ml, suggesting it may not have been unfairly reported as being susceptible to carbapenems. The reason why its potential carbapenemase production was not recognized remains unclear.

[0218] These three isolates are not very common types of CPO, as there may be limited data available at present to generate robust algorithms. Two of them are expected to be reported as clearly carbapenem-insensitive and therefore not candidates for carbapenem therapy.

[0219] (Sensitivity for detecting class B carbapenemases) BD Phoenix® CPO Detect achieved a sensitivity of 95.6% (87 out of 91 isolates) for detecting class B-producing strains. bioMerieux Rapidec® Carba NP achieved a sensitivity of 98.9% (90 out of 91 isolates) using interpretations 1 and 2.

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

[0221] The four isolated strains 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 abnormal phenotype of this organism, particularly the low but elevated MEM MIC, may have contributed to the false-negative test.

[0223] VIM-producing Pseudomonas aeruginosa G15557 and VIM-2-producing Pseudomonas aeruginosa: These had identical phenotypes, namely ERT > 1; IMP > 8; MEM 4 μg / ml. This is a frequently encountered phenotype associated with reduced OprD porin production. This may have contributed to false-negative tests.

[0224] The fourth isolate was an IMP-27-producing Proteus mirabilis. The MICs for carbapenems with ERT > 1; IMP 8; MEM > 8 μg / ml were distinctly different from the species' inherently reduced susceptibility to imipenems, but not for other carbapenems. The irregular phenotype is likely what led to suspicion of carbapenemase production, although IMP-27 is a carbapenemase that is extremely difficult to detect by phenotypic testing.

[0225] (Sensitivity for detecting class D carbapenemases) Both BD Phoenix® CPO Detect and bioMerieux Rapidec® Carba NP demonstrated excellent detection of class D carbapenemase production by Acinetobacter species and Enterobacteriaceae.

[0226] BD Phoenix® CPO Detect achieved 100% detection sensitivity (35 out of 35 isolated strains) for class D-producing strains. This is particularly unprecedented, given that class D production presents one of the most challenging diagnostic issues, as class D carbapenemases exhibit only weak activity and are extremely difficult or nearly impossible to detect in some current tests.

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

[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 isolate contributed to the lower-than-usual specificity. BD Phoenix® CPO Detect yielded a specificity of 68.6% (35 out of 51 isolates). bioMerieux Rapidec® Carba NP yielded a specificity of 60.8% (31 out of 51 isolates) using interpretation 1 and a specificity of 78.4% (40 out of 51 isolates) using interpretation 2.

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

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

[0232] Table 2 lists the isolates that produce false-positive results, their resistance mechanisms, and their carbapenem MICs. False-positive results due to high levels of AmpC production are problematic for many carbapenemase detection tests. High levels of AmpC production itself does not explain false-positive results when using the BD Phoenix® CPO Detect test. This test correctly yielded negative calls for E. coli G1634 and G1700, which are very high-level AmpC-producing strains. This tends to exclude AmpC production as an explanation for false-positive results in other AmpC-producing strains. Similarly, it is unlikely that 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 that hydrolyzes carbapenems. Another possible explanation is inhibition by chelating agents in class B carbapenemase detection tests. This can lead to false positive results in testing for ESBL or AmpC-producing strains regarding class B carbapenemase production.

[0233] The identification of the isolates listed in Table 2 provides further support for the possibility of porin mutations as an explanation. Fifteen of the 17 isolates were Klebsiella pneumoniae, Escherichia coli, and Enterobacter species. 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. Escherichia coli and Enterobacter species also have a relatively high tendency to undergo porin mutations. In Table 2, all isolates have elevated out-of-scale MICs for at least one carbapenem, and most have out-of-scale MICs for all carbapenems. Confirmation of porin mutations is usually not attempted because it is redundant, costly, and technically difficult. Distinguishing between porin mutants and carbapenemase-producing strains is best achieved by tests that detect the presence or absence of carbapenem hydrolysis.

[0234] False positive results for P. mirabilis G1745 can, in some cases, be corrected using software editing. This isolate is typical of a species that has a characteristically higher MIC for imipenem than for ertapenem and meropenem. The elevated MIC for imipenem alone in this isolate is unlikely to be caused by 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 investigation. Four BD Phoenix® CPO detection algorithms were analyzed for their ability to classify types of carbapenemase production.

[0237] The bioMerieux Rapidec® Carba NP trial is unable to classify carbapenemases. Currently, the BD Phoenix® CPO Detect's ability to classify carbapenemases is unparalleled. There are no standards for evaluating this type of trial. In fact, any correct classification of carbapenemases may have clinical benefit.

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

[0239] Algorithms 1 to 4 (Figures 26, 27, 29, 30, and 32 to 35) are illustrative and non-limiting. The antibiotic concentrations provided by Algorithms 1 to 4 (Figures 26, 27, 29, 30, and 32 to 35) were within the concentration ranges of the antibiotics disclosed in Table 2.1. In Algorithms 1 to 4 (Figures 26, 27, 29, 30, and 32 to 35) and Table 2.1, the concentration of CLOX was 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 (Figures 26, 27, 29, 30, and 32-35) operate as previously described in Figures 25-30. For example, each “box” in Algorithms 1-4 (e.g., Box 1 in Figure 32) represents a well (or, optionally, an average of several identical wells) of a detection test provided herein, containing an input sample with or without one or more carbapenemase inhibitors, one or more bacteria, one or more detection reagents, and one or more antibiotics. The detection test results provided to the system are either positive growth (G) or no growth (NG) in one or more wells of the detection test within a given time frame. Based on the results provided for the queried well (growth or no growth), 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.

[0242] Table 3 summarizes the algorithm results for all isolates except for seven dual carbapenemase-producing strains. On the left side of Table 3, we list the total of good results, i.e., correct classification, correct negative results, unclassified carbapenemases (type unclassified), and correct detections regardless of whether they were classified or unclassified (i.e., assigned to the column "A, B, D or type unknown" / type unclassified).

[0243] [Table 7]

[0244] All algorithms functioned well, identifying at least 80% of class A-producing CPOs. Algorithms 2 and 4 were slightly more accurate than algorithms 1 and 3 (81.2%), correctly classifying 91 out of 110 class A-producing strains (82.7%). The incidence of misclassification was very low, and clinically important, the isolates misclassified as class A-producing were not class B-producing. Only one isolate, OXA-40-producing A. baumannii G1734, produced false-positive class A results 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, also produced false-positive class A results using algorithms 1 and 3. The high level of accuracy in identifying class A-producing CPOs indicates that the availability of ceftazidime / avibactam therapy meets an important but currently unmet clinical need.

[0245] All algorithms correctly classified 63 out of 91 (69.2%) of class B-producing strains. This is useful in identifying cases where ceftazidime / avibactam should not be used. This can, consequently, save lives by preventing patients from receiving ineffective ceftazidime / avibactam therapy. The consequence of false-positive class B classification is that it may delay the initiation of effective ceftazidime / avibactam therapy or alternative anti-CPO therapy. Generally, these are expected to be non-life-threatening consequences, only applicable until additional testing (e.g., molecular) is performed. Algorithms 2 and 4 showed 13 false-positive results, which was more than double the number of false-positives in algorithms 1 and 3. Taken together, the performance of BD Phoenix® CPO Detect in identifying class B-producing strains offers considerable potential for clinical benefit and minimal risk to patients.

[0246] Algorithm 2 correctly classified most class D-producing strains, i.e., 31 out of 35 isolates (88.6%). Of all carbapenemases, these are the most difficult to detect, let alone classify. The performance of all algorithms on class D-producing strains was excellent. False positive class D calls can lead to unnecessary separations. Algorithms 2 and 4 each had 4 inaccurate calls, but they outperformed algorithms 1 and 3, which each produced 20 false positive calls. The "no answer" results of algorithms 3 and 4 are also unhelpful in that they provide neither benefit nor harm.

[0247] All algorithms correctly reported 33 (64.7%) of the 51 carbapenemase-negative isolates as negative. Given that the diagnostic difficulty in conventional clinical performance should be considerably lower than in this study, the percentage of correct negative results should be significantly higher.

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

[0249] Tables 4.1 and 4.2 show the carbapenemase-producing and non-carbapenemase-producing isolates that resulted in inaccurate classifications, as well as the MICs of their carbapenems, respectively. Given the prospect of highly elusive CPOs in this study, the false negative rate of 7 out of 244 CPOs tested (2.9%) is not surprising. While carbapenemase detection testing is not perfect, it is desirable to reduce the false negative rate to 1%. Apart from these 7 false negative results by each algorithm, other inaccurate classifications are of minimal potential to cause harm.

[0250]

Table 8

[0251]

Table 9

[0252] (Example 11.7) Performance of the algorithm for non-fermenting strains against Enterobacteriaceae The algorithm described in Example 11.6 was tested for its ability to classify non-fermenting strains producing a single carbapenemase and Enterobacteriaceae. Non-fermenting strains, such as Pseudomonas aeruginosa and A. baumannii, may be an unexpected reservoir of class A and class B carbapenemases, and A. baumannii also has an endogenous class D carbapenemase and may acquire other transmissible class D carbapenemases. Accurate detection of carbapenemases produced by non-fermenting strains is an important but technically difficult task. This is because other mechanisms of carbapenem resistance can result in the same phenotype as carbapenemases.

[0253] As shown in Table 5.1 and Table 5.2, the classification of carbapenemases in Enterobacteriaceae was able to achieve a higher level of accuracy than in the case of non-fermenting strains. However, the comparison was not ideal because there were differences between the various biological groups in terms of their large number of isolates and the type of β-lactamase production. This is reflected in the fact that the numbers obtained for class A-producing strains (105 in the case of Enterobacteriaceae compared to 5 in the case of non-fermenting strains), negative control isolates (51 in the case of Enterobacteriaceae compared to 0 in the case of non-fermenting strains), and the total number of CPOs (185 in the case of Enterobacteriaceae compared to 51 in the case of non-fermenting strains) were very different.

[0254]

Table 10

[0255] [Table 11]

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

[0257] (Example 11.7) Algorithmic performance of an isolated strain producing two carbapenemases The seven isolated strains that produced two carbapenemases were as follows: A. Baumann 0063: OXA-23 + OXA-40 A. Baumann 0083: OXA-23+NDM Klebsiella pneumoniae 0068:OXA-181+NDM Klebsiella pneumoniae 0153:OXA-232+NDM Klebsiella pneumoniae G15406:OXA-181+NDM E. croakae G6809: KPC-18+VIM-1 E. croacae G6810: KPC-18 + VIM-1.

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

[0259] [Table 12]

[0260] Each isolate was correctly reported as carbapenemase-positive in the positive / negative phase of the test. Algorithms 1, 2, and 4 assigned all isolates to either one of the molecular classes or to the category of carbapenemase-positive, type unknown. 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 where the algorithm assigned 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. baumanni 0063, which produced two class D carbapenemases, it was correct for both carbapenemases. There were not enough isolates to analyze the classification trends regarding dual-carbapenemase-producing strains, but it appeared to be a possible choice for assigning carbapenemases 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 furthermore, this test did not require operator involvement after loading the panel into the instrument, thus eliminating waiting time. 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 a 30-minute incubation period, and 2 minutes 24 seconds for a negative test after 30 minutes, thus requiring additional processing and incubation. Table 7 provides a summary of the workflow analysis.

[0263] [Table 13]

[0264] (Example 11.9) Outlook / Summary / Conclusion This example presents 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). BD Phoenix® CPO Detect is an innovative test integrated with a susceptibility panel for detecting and classifying carbapenemases. The bioMerieux Rapidec® Carba NP test is a standalone carbapenemase detection test. A selection of 294 study isolates from Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumanni presented significant diagnostic challenges. These have previously been characterized by molecular, phenotypic, and biochemical analyses regarding β-lactamase production type. Both tests were blinded and performed according to manufacturer recommendations.

[0265] This study presented a very challenging evaluation of the carbapenemase detection capabilities of the BD Phoenix® CPO Detect and bioMerieux Rapidec® Carba NP tests. Both tests demonstrated very high sensitivity. The 100% detection of class D-producing CPO by the BD Phoenix® CPO Detect is excellent and should be recognized as a remarkable achievement, as these are among the most difficult carbapenemases to detect. The extremely challenging nature of carbapenemase-negative isolates contributed to lower specificity than usual. In typical clinical use, it is rare to encounter the isolate type that caused the false-positive test, and this specificity should be significantly higher.

[0266] 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 the test, both tests demonstrated high sensitivity for carbapenemase detection (97.1% for BD Phoenix® CPO Detect and 97.1% to 98.8% for bioMerieux Rapidec® Carba NP test). Both tests exhibited lower-than-usual specificity in this study due to the extremely difficult nature of carbapenemase-negative isolates.

[0267] BD Phoenix® CPO Detect is the first automated test capable of detecting carbapenemases and can be integrated into conventional susceptibility testing. This represents a major technological advancement as it avoids the individual-dependent decision of whether or not a carbapenemase detection test should be set up. The test can also assign carbapenemases to different molecular classes. In current studies, BD Phoenix® CPO Detect demonstrated a high ability to detect and differentiate between CPOs producing class A carbapenemases and CPOs producing class B carbapenemases. This diagnostic characteristic is clinically important for determining the appropriateness of ceftazidime / avibactam as a potential therapeutic option. Three of the four survey algorithms correctly classified more than 90% of carbapenemases as either A, B, D, or type-unknown positive carbapenemases, with algorithm 2 showing slightly better performance.

[0268] During the classification phase of the trial, the BD Phoenix® CPO Detect correctly classified more than 90% of carbapenemases as either class A, B, D, or type-unknown positive carbapenemases. This demonstrated a high ability to detect and differentiate between CPOs producing class A carbapenemases and those producing class B carbapenemases, a diagnostic feature that is clinically important for determining the appropriateness of ceftazidime / avibactam as a potential treatment option. The bioMerieux Rapidec® Carba NP trial lacked the ability to classify carbapenemases. Overall, the BD Phoenix® CPO Detect is a completely novel type of phenotypic test with a range of capabilities unmatched by any currently marketed test. This symbolizes a remarkable advance in meeting a critical clinical need.

[0269] The production of multiple carbapenemases is currently rare, and its detection presents a significant diagnostic and therapeutic challenge. Current research is hampered by the extremely limited number of available isolates of this type. Until suitable trials become available, it is crucial for current trials 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 class B carbapenemase-producing strains, thereby correctly indicating a contraindication to ceftazidime / avibactam therapy. Two A. baumannii isolates producing both class B and class D carbapenemases were both classified as class D-producing strains. This result also prevented patients from receiving inappropriate ceftazidime / avibactam therapy.

[0270] (Example 12) Multicenter evaluation of the BD Phoenix® CPO Detect test in the BD Phoenix® automated microbiology system for the detection and classification 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 baumanni.

[0271] A total of 1034 new frozen isolates, including 722 Enterobacteriaceae and 312 non-fermenting strains (Pseudomonas aeruginosa and Acinetobacter baumanni), were evaluated for carbapenemase production using the BD Phoenix® CPO Detect test at three medical facilities. Isolates were evaluated in parallel using a modified carbapenemase inactivation method (mCIM) and meropenem and ertapenem MIC as reference methods. The Ambler classification (class A, B, or D) of the carbapenemases was determined by multiplex PCR performed by BD. The agreement of positive and negative percentages (PPA and NPA, respectively) between the CPO Detect results and the reference method results was assessed. Disagreements were duplicated and repeated using the BD Phoenix system and appropriate reference methods. The data is shown in Table 8.1 (for Enterobacteriaceae), Table 8.2 (for non-fermenting strains), and Table 8.3 (combinations of Enterobacteriaceae and non-fermenting strains).

[0272] [Table 14]

[0273] [Table 15]

[0274] [Table 16]

[0275] These results indicate that, for Enterobacteriaceae, the PPA (when the reference system detects carbapenemase and %Phoenix detects carbapenemase), 96.0% NPA (when the reference system does not detect carbapenemase and %Phoenix does not detect carbapenemase), and 98.2% classification accuracy (when both Phoenix and the reference system are positive, provide classification, and %Phoenix is ​​correct) were observed. For non-fermenting strains, the PPA (when the reference system detects carbapenemase and %Phoenix detects carbapenemase), 95.0% NPA (when the reference system does not detect carbapenemase and %Phoenix does not detect carbapenemase), and 96.3% classification accuracy (when both Phoenix and the reference system are positive, provide classification, and %Phoenix is ​​correct) were observed. The combined results showed a PPA of 98.3% (when the reference system detects carbapenemase and %Phoenix detects carbapenemase), a NPA of 95.7% (when the reference system does not detect carbapenemase and %Phoenix does not detect carbapenemase), and a classification accuracy of 97.7% (when both Phoenix and the reference system are positive, provide classification, and %Phoenix is ​​correct).

[0276] Results are provided for 1034 specification-compliant clinical isolates tested and analyzed for carbapenemase detection by CPO Detect. After inconsistent analysis, PPA and NPA in Enterobacteriaceae were 99.4% and 96.0%, respectively. Sixteen false positives (2.2%) and two false negatives (0.3%) were observed. For non-fermenting strains, PPA and NPA were 96.0% and 95.0%, respectively, with eight false positive results (2.6%) and six false negative results (1.9%). Among the specification-compliant isolates tested, the results of 385 CPO Detect strains were compared to multiplex PCR for carbapenemase classification. The overall class precision was 98.2% (272 / 277) for Enterobacteriaceae and 96.3% (104 / 108) for non-fermenting strains.

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

[0278] (abbreviation) CLOX (cloxacillin)

[0279] EDTA (Ethylenediaminetetraacetic acid)

[0280] DPA Dipicolinic Acid

[0281] RPX Bavolbactam (RPX-7009)

[0282] AVI Avibactam

[0283] 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 lowest inhibitory concentration.

[0291] As used herein, GAM refers to a generalized additive model, which is the conversion of instrument readings to measured values.

[0292] Where used herein, chapter headings are for systematization purposes only and should not be construed as limiting the subject matter described herein. All references and similar materials cited herein, including but not limited to patents, patent applications, articles, books, professional texts, and web pages on the Internet, are expressly incorporated by reference in their entirety for any purpose. Where the definitions of terms in the incorporated references differ from those provided in the teachings of the present invention, the definitions provided in the teachings of the present invention shall prevail. It is acknowledged that there is an implicit "about" before the temperature, concentration, time, etc., discussed in the teachings of the present invention, and that minor, non-substantial deviations may fall within the scope of the teachings of the present invention as described herein.

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

[0294] When used in the specification and claims, the singular forms “a,” “an,” and “the” encompass plural subjects unless the context clearly indicates otherwise.

[0295] As used herein, “approximately” means that a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length differs from the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference by a range of 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 understand that the 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 also within the scope of the invention and are expected to be readily apparent to those skilled in the art based on this disclosure. Furthermore, it is expected that various combinations or subcombinations of specific features and aspects of the embodiments can be created and still be included within the scope of the invention. It will be understood that the various features and aspects of the disclosed embodiments can be combined or substituted for each other to form various styles 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] However, since various changes and modifications within the essence and scope of the present invention will be obvious to those skilled in the art, please understand that this detailed description illustrates embodiments of the present invention, but is provided for illustrative purposes only.

[0298] The terms used in the descriptions herein are not intended to be construed in any restrictive or limiting manner. Rather, the terms are simply used in conjunction with the detailed descriptions of embodiments of the systems, methods, and related components. Furthermore, embodiments may include a number of novel features, none of which are considered to be the sole cause of the desired properties or essential for carrying out the invention as described herein.

Claims

1. A method for determining the presence of zero, one or more Ambler class carbapenemases expressed by intestinal bacteria, The steps include providing a sample containing the aforementioned intestinal bacteria, A step of applying the intestinal bacteria in a test sample to a plurality of at least four test compositions over a 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. The steps include determining the presence of zero, one or more Ambler class carbapenemases expressed by the intestinal bacteria by detecting the presence or inhibition of the growth of the intestinal bacteria in each of the plurality of at least four test compositions after the duration, and A method that includes this.

2. The method according to claim 1, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises or is essentially composed of a first concentration of TEM and an Ambler class B carbapenemase inhibitor.

3. The method according to claim 1 or 2, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed of, a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor.

4. The method according to any one of claims 1 to 3, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed of, a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler A carbapenemase inhibitor.

5. The method according to any one of claims 1 to 4, wherein the antibiotic in at least one test composition comprises or is essentially composed of a second concentration of DOR.

6. A method comprising the step of determining that one or more Ambler class carbapenemases expressed by enterobacteria are of class D by detecting the presence of growth in a first test composition, wherein the antibiotic and inhibitor comprise, or are essentially comprised of, a first concentration of TEM and an Ambler class B carbapenemase inhibitor.

7. The method according to any one of claims 1 to 6, comprising the step of determining that one or more Ambler class carbapenemases expressed by intestinal bacteria are of class A by detecting the following: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise a first concentration of TEM and an Ambler class B carbapenemase inhibitor, comprising or essentially comprising these, The presence of growth in the second test composition, wherein the antibiotic and inhibitor comprises a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. Inhibition of growth in a third test composition, wherein the antibiotic and inhibitor comprise, or are essentially composed of, a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor.

8. The method according to any one of claims 1 to 7, comprising the step of determining that one or more Ambler class carbapenemases expressed by intestinal bacteria are of class B by detecting the following: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprises, or essentially comprises, 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, or essentially comprises, 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 the antibiotic comprises, or is essentially composed of, a second concentration of DOR.

9. The method according to any one of claims 1 to 8, comprising the step of determining that one or more Ambler class carbapenemases expressed by intestinal bacteria are of class D by detecting the following: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise a first concentration of TEM and an Ambler class B carbapenemase inhibitor, comprising or essentially comprising these, Inhibition of growth in a second test composition, wherein the antibiotic and inhibitor comprises, or essentially comprises, 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, or is essentially composed of, a second concentration of DOR.

10. The method according to any one of claims 1 to 9, comprising the step of determining the presence of one or more carbapenemases of the Ambler class expressed by intestinal bacteria (wherein the Ambler class is not identified) by detecting the following: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise a first concentration of TEM and an Ambler class B carbapenemase inhibitor, comprising or essentially comprising these, The presence of growth in the second test composition, wherein the antibiotic and inhibitor comprises a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in a third test composition among a plurality of at least four test compositions, wherein the antibiotic and inhibitor comprises, consists of, or essentially comprises MEM at a first concentration as the antibiotic, and further comprises an Ambler class C carbapenemase inhibitor and an Ambler class A carbapenemase inhibitor.

11. The method according to any one of claims 1 to 10, comprising the step of determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by intestinal bacteria by detecting the following: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise, or consist of, a first concentration of TEM and an Ambler class B carbapenemase inhibitor, or are essentially derived from them. The presence of growth in the second test composition, wherein the antibiotic and inhibitor comprises a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in a third test composition, wherein the antibiotic and inhibitor comprises, or is essentially composed of, a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor.

12. The method according to any one of claims 1 to 11, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises or is essentially composed of a third concentration of MEM and an Ambler class C carbapenemase inhibitor.

13. The method according to any one of claims 1 to 12, comprising the step of determining that no answer can be obtained regarding the identification of one or more Ambler class carbapenemases expressed by intestinal bacteria by detecting the following: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise a first concentration of TEM and an Ambler class B carbapenemase inhibitor, comprising or essentially comprising these, The presence of growth in the second test composition, wherein the antibiotic and inhibitor comprises a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in a third test composition among a plurality of at least four test compositions, wherein the antibiotic and inhibitor comprises a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. Inhibition of growth in a fifth test composition, wherein the antibiotic and inhibitor comprise a third concentration of MEM and an Ambler class C carbapenemase inhibitor, comprising or essentially comprising these.

14. The method according to any one of claims 1 to 13, comprising the step of determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by intestinal bacteria by detecting the following: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprise a first concentration of TEM and an Ambler class B carbapenemase inhibitor, comprising or essentially comprising these, The presence of growth in the second test composition, wherein the antibiotic and inhibitor comprises a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in a third test composition, wherein the antibiotic and inhibitor comprises a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in a fifth test composition, wherein the antibiotic and inhibitor comprise a third concentration of MEM and an Ambler class C carbapenemase inhibitor, and the presence of growth consisting of or essentially derived from them.

15. The method according to claim 1, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed of, a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor.

16. The method according to any one of claims 1 and 15, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed of, a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler A carbapenemase inhibitor.

17. The method according to any one of claims 1 and 15 to 16, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed of, a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

18. The method according to any one of claims 1 and 15-17, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed of, a second concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler A carbapenemase inhibitor.

19. The method according to any one of claims 1 and 15 to 18, comprising the step of determining that one or more Ambler class carbapenemases expressed by intestinal bacteria are of class A by detecting the following: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprises, or essentially consists 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 or are essentially composed of a second concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor.

20. The method according to any one of claims 1 and 15 to 19, comprising the step of determining that one or more Ambler class carbapenemases expressed by intestinal bacteria are of class B by detecting the following: Inhibition of growth in a first test composition, wherein the antibiotic and inhibitor comprises, or essentially consists 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 a second concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor, and the presence of growth consisting of or essentially comprising them.

21. The method according to any one of claims 1 and 15 to 20, comprising the step of determining that one or more Ambler class carbapenemases expressed by intestinal bacteria are of class A by detecting the following: The presence of growth in the first test composition, wherein the antibiotic and inhibitor comprises a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor, and the presence of growth consisting of or essentially derived from them, and Inhibition of growth in a third test composition, wherein the antibiotic and inhibitor comprise, or are essentially composed of, a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor.

22. The method according to any one of claims 1 and 15 to 21, comprising the step of determining that one or more Ambler class carbapenemases expressed by intestinal bacteria are of class D by detecting the following: The presence of growth in the first test composition, wherein the antibiotic and inhibitor comprises a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in a third test composition, wherein the antibiotic and inhibitor comprises a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. Inhibition of growth in a fourth test composition, wherein the antibiotic and inhibitor comprises, or is essentially composed of, a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

23. The method according to any one of claims 1 and 15 to 22, comprising the step of determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by intestinal bacteria by detecting the following: The presence of growth in the first test composition, wherein the antibiotic and inhibitor comprises a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in a third test composition, wherein the antibiotic and inhibitor comprises a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in a fourth test composition, wherein the antibiotic and inhibitor comprises, or is essentially composed of, a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

24. The method according to any one of claims 1 and 15 to 22, comprising the step of determining that no answer can be obtained with respect to identifying one or more Ambler class carbapenemases expressed by intestinal bacteria by detecting the following: The presence of growth in the first test composition, wherein the antibiotic and inhibitor comprises a first concentration of MEM, an Ambler class B carbapenemase inhibitor, and an Ambler class C carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in a third test composition, wherein the antibiotic and inhibitor comprises a first concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class A carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in a fourth test composition, wherein the antibiotic and inhibitor comprises, or is essentially composed of, a first concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

25. The method according to any one of claims 1 to 24, comprising the step of applying the intestinal bacteria in a test sample to a plurality of at least five test compositions over a period of time, wherein the antibiotic and carbapenemase inhibitor in at least one test composition consist of or are essentially composed of a first concentration of MEM and an Ambler class C carbapenemase inhibitor.

26. A method according to any one of claims 1 and 25, comprising the step of determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by intestinal bacteria by detecting the presence of growth in a first test composition, The method comprises or essentially consists of the antibiotic and inhibitor, comprising a first concentration of MEM and an Ambler class C carbapenemase inhibitor.

27. A method according to any one of claims 1 and 25, comprising the step of determining the absence of one or more Ambler class A, B, or D carbapenemases expressed by intestinal bacteria by detecting inhibition of growth in a first test composition, The method comprises or essentially consists of the antibiotic and inhibitor, comprising a first concentration of MEM and an Ambler class C carbapenemase inhibitor.

28. A method according to any one of claims 1 to 27, further comprising a method for identifying zero, one or more Ambler class carbapenemases expressed by non-fermenting bacteria, wherein the method is: A step of providing a sample containing non-fermenting bacteria, The steps include applying the non-fermenting bacteria in the test sample to a test composition containing a growth medium, an antibiotic, and a carbapenemase inhibitor over a sustained period of time, The steps include determining the presence of zero, one or more Ambler class carbapenemases expressed by non-fermenting bacteria by detecting the presence or inhibition of growth of the non-fermenting bacteria in the test composition after the aforementioned duration, and A method that includes this.

29. The method according to claim 28, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises or is essentially composed of a third concentration of DOR and an Ambler class C carbapenemase inhibitor.

30. The method according to claim 28 or 29, comprising the step of determining the presence of one or more Ambler class A, B, or D carbapenemases expressed by non-fermenting bacteria by detecting the presence of growth in a test composition, The method comprises or is essentially composed of the antibiotic and inhibitor, comprising a third concentration of DOR and an Ambler class C carbapenemase inhibitor.

31. The method according to claim 28 or 29, comprising the step of determining the absence of one or more Ambler class A, B, or D carbapenemases expressed by non-fermenting bacteria by detecting inhibition of growth in a test composition, The aforementioned antibiotics and inhibitors include a third concentration of DOR and an Ambler class C carbapenemase inhibitor, comprising or essentially comprising them, for the inhibition of growth.

32. A method according to any one of claims 1 to 31, further comprising a method for determining the presence of zero, one or more Ambler class carbapenemases expressed by non-fermenting bacteria, wherein the method is A step of providing a sample containing non-fermenting bacteria, A step of applying the non-fermenting bacteria in a test sample to a plurality of at least three test compositions over a 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. The steps include determining the presence of zero, one, or 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 duration, and A method that includes this.

33. A method for determining the presence of zero, one or more Ambler class carbapenemases expressed by non-fermenting bacteria, The steps include providing a sample containing the non-fermenting bacteria, A step of applying the non-fermenting bacteria in the test sample to a plurality of at least three test compositions over a 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. The steps include determining the presence of zero, one, or 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 duration, and A method that includes this.

34. The method according to claim 32 or 33, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed of, a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor.

35. The method according to any one of claims 32 to 34, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed of, a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

36. The method according to any one of claims 32 to 35, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises or is essentially composed of a fifth concentration of DOR, and an Ambler class C carbapenemase inhibitor and an Ambler class D carbapenemase inhibitor.

37. A method according to any one of claims 32 to 36, comprising determining one or more Ambler class carbapenemases expressed by non-fermenting bacteria as class B by detecting inhibition of growth in a first test composition, The antibiotics and inhibitors include a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, or a method comprising or essentially comprising them.

38. The method according to any one of claims 32 to 37, comprising determining one or more Ambler class carbapenemases expressed by non-fermenting bacteria as class D by detecting the presence of growth in the first test composition by detecting the following: The aforementioned antibiotics and inhibitors include a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth consisting of or essentially derived from them. Inhibition of growth in a second test composition, wherein the antibiotic and inhibitor comprise, or are essentially composed of, a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

39. The method according to any one of claims 32 to 38, comprising determining that one or more carbapenemases of the Ambler class expressed by non-fermenting bacteria are class A by detecting the following: The presence of growth in the first test composition, wherein the antibiotic and inhibitor comprises a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in the second test composition, wherein the antibiotic and inhibitor comprises a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. Inhibition of growth in a third test composition, wherein the antibiotic and inhibitor comprises, or is essentially composed of, a fifth concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

40. The method according to any one of claims 32 to 39, which determines the presence of one or more Ambler class A, B, or D carbapenemases expressed by non-fermenting bacteria by detecting the following: The presence of growth in the first test composition, wherein the antibiotic and inhibitor comprises a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in the second test composition, wherein the antibiotic and inhibitor comprises a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in a third test composition, wherein the antibiotic and inhibitor comprises, or is essentially composed of, a fifth concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

41. The method according to claim 32 or 33, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed of, a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor.

42. The method according to any one of claims 32-33 and 41, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed of, a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

43. The method according to any one of claims 32-33 and 41-42, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises or is essentially composed of a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor.

44. The method according to any one of claims 32-33 and 41-43, comprising the step of determining whether one or more Ambler class carbapenemases expressed by non-fermenting bacteria are of class A, B, or D by detecting the following: The presence of growth in the first test composition, wherein the antibiotic and inhibitor comprises a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in a second test composition, wherein the antibiotic and inhibitor comprises a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor, and the presence of growth consisting of or essentially consisting of these.

45. The method according to any one of claims 32-33 and 41-44, comprising the step of determining that one or more Ambler class carbapenemases expressed by non-fermenting bacteria are of class D by detecting the following: The presence of growth in the first test composition, wherein the antibiotic and inhibitor comprises a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. Inhibition of growth in a second test composition, wherein the antibiotic and inhibitor comprise, or are essentially composed of, a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

46. The method according to any one of claims 32-33 and 41-45, comprising the step of determining that one or more Ambler class carbapenemases expressed by non-fermenting bacteria are of class B by detecting the following: Inhibition of growth in the first test composition, wherein the antibiotic and inhibitor comprises, or essentially comprises, 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 comprise a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor, and the presence of growth consisting of or essentially comprising them.

47. The method according to any one of claims 32-33 and 41-46, comprising the step of determining that no answer has been obtained regarding the identification of one or more Ambler class carbapenemases expressed by non-fermenting bacteria by detecting the following: Inhibition of growth in the first test composition, wherein the antibiotic and inhibitor comprises, or essentially comprises, 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 comprise a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor, comprising or essentially comprising them.

48. The method according to any one of claims 32-33 and 41-47, comprising the step of determining whether one or more Ambler class carbapenemases expressed by non-fermenting bacteria are of class A, B, or D by detecting the following: Inhibition of growth in the first test composition, wherein the antibiotic and inhibitor comprises, or essentially comprises, 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 comprise a fourth concentration of DOR and an Ambler class C carbapenemase inhibitor, comprising or essentially comprising them.

49. The method according to any one of claims 32 to 48, comprising the step of applying the non-fermenting bacteria in a test sample to a plurality of at least four test compositions over a period of time, wherein the antibiotic and carbapenemase inhibitor in at least one test composition comprises, or is essentially composed of, a fifth concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

50. The method according to claim 49, comprising the step of determining whether one or more carbapenemases of the Ambler class expressed by non-fermenting bacteria are of class A, B, or D by detecting the following: The presence of growth in the first test composition, wherein the antibiotic and inhibitor comprises a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in the second test composition, wherein the antibiotic and inhibitor comprises a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in a fourth test composition, wherein the antibiotic and inhibitor comprises, or is essentially composed of, a fifth concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

51. The method according to claim 49 or 50, comprising the step of determining that one or more Ambler class carbapenemases expressed by non-fermenting bacteria are of class A by detecting the following: The presence of growth in the first test composition, wherein the antibiotic and inhibitor comprises a third concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class B carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. The presence of growth in the second test composition, wherein the antibiotic and inhibitor comprises a fourth concentration of MEM, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor, and the presence of growth consisting of or essentially composed of them. Inhibition of growth in a fourth test composition, wherein the antibiotic and inhibitor comprises, or is essentially composed of, a fifth concentration of DOR, an Ambler class C carbapenemase inhibitor, and an Ambler class D carbapenemase inhibitor.

52. The method according to any one of claims 1 to 51, further comprising the step of determining whether the bacteria in the sample are intestinal, non-fermentable, or both.

53. The method according to any one of claims 1 to 52, wherein 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.

54. The method according to any one of claims 1 to 53, wherein 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.

55. The method according to any one of claims 1 to 54, wherein 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.

56. The method according to any one of claims 1 to 55, wherein the third concentration of DOR is approximately 0.1 μg / ml to approximately 400 μg / ml, approximately 0.5 μg / ml to approximately 3 μg / ml, or approximately 1 μg / ml.

57. The method according to any one of claims 1 to 56, wherein 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.

58. The method according to any one of claims 1 to 57, wherein the fifth concentration of DOR is approximately 0.03125 μg / ml to approximately 80 μg / ml, approximately 2 μg / ml to approximately 24 μg / ml, or approximately 8 μg / ml.

59. The method according to any one of claims 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 according to any one of claims 1 to 59, wherein the second concentration of MEM is approximately 0.015625 μg / ml to approximately 0.125 μg / ml, approximately 0.003 μg / ml to approximately 0.3 μg / ml, approximately 0.0075 μg / ml to approximately 0.12 μg / ml, approximately 0.01 μg / ml to approximately 0.12 μg / ml, or approximately 0.03 μg / ml.

61. The method according to any one of claims 1 to 60, wherein 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.

62. The method according to any one of claims 1 to 61, wherein 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.

63. The method according to any one of claims 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, bavolbactam (RPX-7009), and BLI-489.

64. The method according to any one of claims 1 to 63, wherein the Ambler class B carbapenemase inhibitor is a metal chelating agent.

65. The method according to any one of claims 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 according to any one of claims 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 according to any one of claims 1 to 66, wherein the Ambler class A carbapenemase inhibitor comprises a compound selected from the group consisting of bavolbactam (RPX-7009), AVI, clavulanic acid, boronic acid, tazobactam, sulbactam, and BLI-489.

68. The method according to any one of claims 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, bavolbactam, and (RPX-7009).

69. The method according to any one of claims 1 to 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 is essentially composed of EDTA.

70. The method according to any one of claims 1 to 69, comprising, or essentially comprising CLOX, the Ambler class C carbapenemase inhibitor.

71. The method according to any one of claims 1 to 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 is essentially derived from RPX.

72. The method according to any one of claims 1 to 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 is essentially composed of DPA.

73. The method according to any one of claims 1 to 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 is essentially derived from AVI.

74. The method according to any one of claims 1 to 73, comprising or essentially comprising BLI, wherein the Ambler class D carbapenemase inhibitor combined with the fourth concentration of MEM comprises a first concentration.

75. The method according to any one of claims 1 to 74, wherein the concentration of EDTA is approximately 0.025 mg / ml to approximately 10 mg / ml, approximately 0.05 mg / ml to approximately 1.25 mg / ml, or approximately 0.25 mg / ml.

76. The method according to any one of claims 1 to 75, wherein the concentration of CLOX is approximately 0.0025 mg / ml to approximately 40 mg / ml, approximately 0.020 mg / ml to approximately 0.5 mg / ml, or approximately 0.1 mg / ml.

77. The method according to any one of claims 1 to 76, wherein the concentration of RPX is approximately 0.2 μg / ml to approximately 320 μg / ml, approximately 1.5 μg / ml to approximately 40 μg / ml, or approximately 8 μg / ml.

78. The method according to any one of claims 1 to 77, wherein the concentration of DPA is approximately 0.018 mg / ml to approximately 1.8 mg / ml, approximately 0.07 mg / ml to approximately 0.73 mg / ml, or approximately 0.178 mg / ml.

79. The method according to any one of claims 1 to 78, wherein the concentration of AVI is approximately 0.1 μg / ml to approximately 40 μg / ml, approximately 0.5 μg / ml to approximately 20 μg / ml, or approximately 4 μg / ml.

80. The method according to any one of claims 1 to 79, wherein the concentration of BLI is approximately 0.1 μg / ml to approximately 200 μg / ml, approximately 1 μg / ml to approximately 25 μg / ml, or approximately 5 μg / ml.

81. The method according to any one of claims 1 to 80, wherein 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.

82. The method according to any one of claims 1 to 81, wherein the duration for detecting the presence or inhibition of the growth of intestinal bacteria is about 6 hours to about 8 hours.

83. The method according to any one of claims 1 to 82, wherein the duration for detecting the presence or inhibition of the growth of intestinal bacteria is about 7 hours.

84. The method according to any one of claims 1 to 83, wherein the duration for detecting the presence or inhibition of growth of non-fermenting bacteria is about 8 hours to about 11 hours.

85. The method according to any one of claims 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 according to any one of claims 1 to 85, wherein the intestinal bacteria include bacteria selected from the group consisting of Klebsiella pneumoniae, Escherichia coli, and Enterobacter aerogenes.

87. The method according to any one of claims 1 to 86, wherein the non-fermenting bacteria are selected from the group consisting of Pseudomonas aeruginosa and Acinetobacter baumanni complex strains.

88. The method according to any one of claims 1 to 87, wherein the detection of the presence or inhibition of proliferation is not performed by imaging changes in cell morphology.

89. An automated system for performing the method described in any one of claims 1 to 88, A plurality of compartments, each of which contains the test composition described in any of claims 1 to 88, The plurality of compartments are provided with means for supplying samples containing intestinal bacteria, non-fermenting bacteria, or both. A device for obtaining a first signal from the plurality of compartments to which the aforementioned intestinal bacteria, non-fermenting bacteria, or both are supplied, An incubator for incubating the plurality of compartments supplied with the aforementioned intestinal bacteria, non-fermenting bacteria, or both, over a sustained period of time. A device for obtaining a second signal from the plurality of compartments containing intestinal bacteria, non-fermenting bacteria, or both, A detector for detecting the presence or inhibition of growth in the plurality of compartments supplied with the intestinal bacteria, non-fermenting bacteria, or both, by comparing the first signal and the second signal, A computer for generating the output of the results from the detector, An analyzer for interpreting the output of the above results and An automated system equipped with [the following features].

90. The automated system according to claim 89, wherein the plurality of compartments include compartments selected from the group consisting of wells, plates, and tubes.

91. A kit for identifying one or more Ambler class carbapenemases expressed by intestinal bacteria and / or non-fermenting bacteria, comprising a substrate or panel having a plurality of compartments, each of which comprises a test composition according to any one of claims 1 to 90.

92. The kit according to claim 91, wherein the substrate comprises at least three or at least four different test compositions.

93. The kit according to claim 91 or 92, wherein the kit comprises a second substrate having a plurality of compartments, each of which comprises a test composition according to the method of any one of claims 1 to 90, and at least one of the plurality of test compositions in the first substrate is different from the plurality of test compositions in the second substrate.

94. A method, system, or kit according to any one of claims 1 to 88, comprising a plurality of test compositions, wherein the test compositions comprise or are essentially comprised of test compositions selected from the test compositions disclosed in boxes 1 to 14.

95. The method, system, or kit according to claim 94, comprising, or essentially comprising, the test compositions disclosed in boxes 1 to 5.

96. The method, system, or kit according to claim 94, comprising, or essentially comprising, the test compositions disclosed in boxes 1, 6, 7, 3, 8, and 9.

97. The method, system, or kit according to claim 94, comprising, or essentially consisting of, the test compositions disclosed in boxes 10, 11, 12, and 13.

98. The method, system, or kit according to claim 94, comprising, or essentially comprising, the test compositions disclosed in boxes 10, 11, 12, 13, and 14.

99. The method, system, or kit according to claim 94, comprising, or essentially comprising, the test compositions disclosed in boxes 1, 6, 7, 3, and 9.

100. The method, system, or kit according to claim 94, comprising, or essentially comprising, the test compositions disclosed in boxes 1, 6, 7, 9, 3, and 10.

101. The method, system, or kit according to claim 94, comprising, or essentially comprising, the test compositions disclosed in boxes 1, 6, 7, 3, 9, 10, 11, 12, and 14.

102. The method, system, or kit according to claim 94, comprising, or essentially comprising, the test compositions disclosed in boxes 1, 10, 11, 12, and 14.

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