Method for testing sensitivity of antibacterial agent

Abstract

The present document relates to a calorimetric method for determining the antimicrobial sensitivity of microorganisms in a sample, such as a clinical tissue sample. According to the method, a microbial sample is exposed to an antibacterial agent when the sample has reached a specific metabolic activity and differences in calorimetric signal (s) are compared relative to the sample incubated in the absence of the antibacterial agent to determine whether the microorganism (s) in the sample are sensitive or insensitive to the antibacterial agent (s).

Description

Technical Field

[0001] This document relates to a calorimetric method for determining the antimicrobial susceptibility of microorganisms in samples such as clinical tissue samples.

[0002] background

[0003] The purpose of antimicrobial susceptibility testing is to confirm susceptibility to selected empirical antimicrobial agents or to detect resistance in single bacterial isolates, which will alter the selected empirical therapy to a targeted therapy based on a specific antimicrobial profile.

[0004] There are many methods for determining antimicrobial susceptibility, such as broth microdilution, disk diffusion, gradient diffusion, and semi-automated and automated equipment (such as the VITEK®2 instrument, BD Phoenix, and Qlinea ASTar devices). All of these standard tests are adjusted to the correct ratio of microorganism count to antimicrobial agent to give reproducible results.

[0005] For assessing susceptibility, two main test definitions exist: i) quantitative minimum inhibitory concentration determination (MIC) and ii) relating MIC to qualitative breakpoint determination (BP).

[0006] The MIC is the lowest concentration of an antimicrobial drug (such as an antifungal, antibiotic, bactericide, or bacteriostatic agent) that will inhibit visible growth of a microorganism after overnight incubation. The MIC can be determined on plates of solid growth medium (agar growth medium) or by broth dilution (liquid growth medium) after isolating a pure culture.

[0007] For example, to determine the MIC by the broth dilution method, the same dose of bacteria is plated in wells of liquid growth medium containing decreasing drug concentrations. The minimum inhibitory concentration of an antibiotic lies between the concentration in the last well that showed no growth and the next lower dose that allowed growth. The broth dilution test involves preparing two-fold dilutions of the antibiotic (e.g., 1, 2, 4, 8, and 16 µg / mL) in liquid growth medium dispensed into test tubes. The tubes containing the antibiotic are inoculated with 1-5 × 10 5 A standardized bacterial suspension of 100 CFU / mL was prepared and incubated overnight at 35°C. The precision of this method is considered to be plus or minus one dilution, which is largely due to the practice of manually preparing serial dilutions of antibiotics.

[0008] The Kirby-Bauer disc diffusion test is a standardized technique used to test fast-growing pathogens for quantitative determination of MICs. A standardized inoculum is spread onto the surface of an agar plate (150 mm diameter). The reproducibility of this test depends on the logarithmic growth phase of the microorganism. A filter paper disc impregnated with a standardized concentration of antimicrobial agent is placed on the surface, and after overnight incubation, the size of the inhibition zone around the disc is measured in millimeters.

[0009] Gradient diffusion Etests and semi-automated and automated equipment such as those described above can also be used to quantitatively determine MICs.

[0010] Clinical breakpoints provide an interpretation of antimicrobial susceptibility test results. Breakpoints can help determine whether an antimicrobial agent is likely to be effective in treating microbial infections. The establishment of breakpoints requires integrating knowledge of the wild-type MIC distribution, assessment of the pharmacokinetics / pharmacodynamics of the antimicrobial agent, and studies of the clinical outcomes of infections with antimicrobial use. Depending on the test method, breakpoints can be expressed as MICs (in mg / liter or μg / ml) or disk diffusion zone diameters (in mm). Generally speaking, all susceptibility testing methods require breakpoints, also known as interpretative criteria, to allow for the interpretation of test results as sensitive, intermediately sensitive, or nonsusceptible to the microorganism. Clinical breakpoints are those concentrations (MICs) that distinguish isolates with a high probability of treatment success from those with a greater likelihood of treatment failure. These breakpoints are derived from prospective human clinical studies that compare results to the MIC of the infecting pathogen. A third use of the term "breakpoint" refers to antimicrobial concentrations calculated based on knowledge of pharmacodynamic parameters and the extent to which these parameters predict in vivo efficacy. These are pharmacokinetic / pharmacodynamic breakpoints, in which data generated in animal models are extrapolated to humans using mathematical or statistical techniques.

[0011] Isothermal microcalorimetry (IMC) is a measurement technique used for real-time monitoring and dynamic analysis of chemical, physical, and biological processes. IMC determines the onset, rate, extent, and energetic characteristics of such processes in samples within closed systems over time periods of minutes or days, measured in µW. In isothermal calorimetry, heat release is measured at a constant set temperature. The term "micro" is applied to smaller systems with heat release in the microwatt range.

[0012] For isothermal (micro)calorimetry measurements, the sample to be measured is placed in a sealed container at a constant temperature. Heat flow changes due to chemical and biological processes are specific to the type of system being studied. For example, any given isolate of a bacterial or fungal species under specific metabolic conditions will produce a plot of heat flow over time, known as a thermogram. This heat flow can be tracked in real time, a technique known as real-time isothermal calorimetry. For monitoring microbial systems such as fungi or bacteria, these specific thermograms can be used to infer the metabolic activity of the microorganisms present in the sample, and in some cases, even the species of microorganisms present.

[0013] Even though calorimetry has been used to study microorganisms, the technique is hampered by problems in obtaining high-quality calorimetric data and / or by difficulties in interpreting calorimetric data.

[0014] Therefore, there is a need for faster and / or more reliable methods for determining the microbial susceptibility of microorganisms.

[0015] Overview

[0016] It is therefore an object of the present invention to overcome or at least alleviate one or more of the problems described herein.

[0017] Thus, the present document relates to a calorimetric method for antimicrobial susceptibility testing of microbial samples, said method comprising or consisting of the following steps:

[0018] a) incubating a sample that may contain one or more microorganisms in an inoculated medium and tracking metabolic activity by measuring one or more calorimetric signals of the incubated sample;

[0019] b) when the metabolic rate has an R value of ≥ 0.98 for at least about 20 minutes, or when the metabolic activity of the incubated sample of step a) reaches an inflection point or within 2 hours after reaching said inflection point, reloading one or more aliquots of the incubated sample of step a) onto:

[0020] i) inoculated medium not supplemented with an antimicrobial agent; and

[0021] ii) the same inoculum medium as in step b) i), but wherein the inoculum medium is supplemented with an antimicrobial agent;

[0022] c) incubating the sample of steps b)i) and b)ii and measuring one or more calorimetric signals in the sample; and

[0023] d) determining whether the one or more microorganisms are sensitive or insensitive to the antimicrobial agent by comparing the one or more calorimetric signals obtained in step c) from the sample(s) supplemented with the antimicrobial agent with the one or more calorimetric signals obtained in step c) from the sample(s) not supplemented with the antimicrobial agent.

[0024] The calorimetric methods of the present document can be performed without first isolating the one or more microorganisms from the microbial sample.

[0025] One or two or more aliquots with different total metabolic activities may be reloaded in steps b)i) and b)ii), respectively, and the calorimetric signal(s) obtained from the aliquot(s) according to step b)ii) are used to determine the sensitivity or insensitivity in step d), the aliquot(s) according to step b)ii) corresponding to the aliquot(s) in which the metabolic activity (activity) in the sample(s) according to step b)i) at that time after reloading is below the detection limit of one or more calorimetric signals, and wherein the metabolic activity (activity) in the same aliquot(s) is detected within about 6 hours, such as 15 minutes to 3 hours (i.e., the detection time), after said reloading in step b)i).

[0026] The sample may be a clinical sample from a subject, an environmental sample, a food or feed sample, and / or a purified microbial sample.

[0027] The calorimetric method of the present document may further comprise the step of using the one or more calorimetric signals obtained in said step c) from the sample(s) not supplemented with the antimicrobial agent to determine the Gram state, genus and / or species of one or more microorganisms in said microbial sample.

[0028] In step b) ii), a combination of two or more different types of antimicrobial agents may be used in the same inoculation medium. Alternatively or additionally, two or more types of inoculation media may be used in step a) and / or step b). The two or more inoculation media in step b) ii) may contain different concentrations of one or more antimicrobial agents. The inoculation medium or media of step a) and / or step b) may include one or more adjuvants, such as adjuvants that enhance and / or increase the activity of the antimicrobial agents. Examples of inoculation media that may be used include, but are not limited to, Mueller Hinton broth, thioglycolate broth, mannitol salts, modified Sabouraud broth, and / or Scheduler broth.

[0029] The analysis of the calorimetric signals in steps b), c), and / or d) can be performed using a database that correlates at least one calorimetric signal with a plurality of microorganisms. Furthermore, the database that correlates at least one calorimetric signal with a plurality of microorganisms can be used to determine the Gram status, genus, and / or species of one or more microorganisms in the microbial sample.

[0030] The calorimetric signal determined can be, for example, a detection time, a metabolic rate, a maximum metabolic rate, an area under the curve, an area under the curve before the maximum metabolic rate, a heat flow, and / or a ratio between any of these signals. Thus, the calorimetric signal can also be represented as a calorimetric feature.

[0031] The incubation in step c) is generally performed for a period of about 1 hour to about 48 hours from the time the one or more calorimetric signals are detected, although longer and shorter incubation periods may be applied depending on the metabolic activity of the sample to be analyzed.

[0032] This document also relates to a system for performing the calorimetric method of this document. The system comprises a calorimeter and software for performing the analysis according to steps a), b), c), and / or d) of the method and / or for determining the Gram status, genus, and / or species of one or more microorganisms in the microbial sample.

[0033] This document also discloses a kit for performing the calorimetric method of this document, the kit comprising:

[0034] i) at least one inoculum medium that does not contain an antimicrobial agent;

[0035] ii) inoculation medium as in i), but supplemented with an antimicrobial agent;

[0036] iii) one or more databases establishing a correlation between at least one calorimetric parameter and a plurality of microorganisms;

[0037] iv) an optional software component that includes functionality for determining the Gram status, genus, and / or species of one or more microorganisms; and

[0038] v) a software component comprising functionality for determining whether the microorganism(s) are susceptible or insensitive to the antimicrobial agent.

[0039] The present document also relates to a computer program comprising computer program code which, if executed on a processor, is adapted to implement the calorimetric method for antimicrobial susceptibility testing of the present document. The present document also relates to a computer program product comprising a computer-readable storage medium having the computer program.

[0040] This document also relates to a computer-implemented method performed by a control unit, the method comprising:

[0041] receiving first measurement data indicative of the calorimetric signal (i.e., calorimetric signature) of step a), wherein the calorimetric signal (i.e., calorimetric signature) of step a) is indicative of metabolic activity of the one or more samples comprising the one or more microorganisms in the inoculated culture medium, and

[0042] Determining that the metabolic rate has an R value of ≥ 0.98 for at least about 20 minutes, or the metabolic activity of the sample incubated in step a) has reached an inflection point,

[0043] indicating to a system user that reloading of one or more aliquots of the incubated sample of step a) should be performed 2 hours after reaching said inflection point,

[0044] receiving second measurement data indicative of the calorimetric signal (ie the calorimetric signature) of step c),

[0045] By comparing the second measurement data of the one or more samples supplemented with the antimicrobial agent with the second measurement data of the sample(s) not supplemented with the antimicrobial agent, the one or more microorganisms are classified as being sensitive or not sensitive to the antimicrobial agent. Such a method can be used to analyze the calorimetric signal of the calorimetric method of the present document.

[0046] This document also relates to a control unit, said control unit (CU) comprising:

[0047] processor,

[0048] and a memory containing instructions executable by the processor, thereby operating the control unit to perform the computer-implemented method described herein.

[0049] Other features and advantages of the invention will be apparent from the following detailed description, the drawings, the examples, and from the claims.

[0050] definition

[0051] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0052] The term "comprising" includes the term "consisting of" unless it is clear from the context that this is not intended.

[0053] The terms "antimicrobial," "antimicrobial agent," "antimicrobial substance," and the like refer to substances that are active against microorganisms in the form of bacteria and / or fungi. Thus, an antimicrobial agent has antimicrobial or antifungal activity that kills, prevents, or reduces the growth and / or metabolic activity of bacteria and / or fungi. Examples of antimicrobial agents include, but are not limited to, antibiotics and antifungal agents.

[0054] "Microbial sample" and the like herein means a sample that may contain microorganisms in the form of bacteria and / or fungi.

[0055] "Calorie" and the like refer to the process of measuring the amount of heat released or absorbed during a chemical reaction, such as during the incubation of microorganisms in an inoculum. The temporal heat change in a sample (J / s versus time) can be recorded in a thermogram. Calorimetry according to this document is performed as known to those skilled in the art. Briefly, to determine the calorimetric signal of a microbial sample, the sample is inoculated into a vial containing an inoculum, which is then placed in a temperature-controlled chamber of a calorimetric apparatus for incubation. According to this document, a calorimetric signal can also be expressed as a calorimetric characteristic. For example, a calorimetric signal measured in J / s (W), which reflects the metabolic activity of the microorganism(s) that may be present in the sample, can be continuously tracked and analyzed and presented in a so-called thermogram. Other non-limiting examples of useful calorimetric signals include detection time, metabolic rate, maximum metabolic rate, area under the curve, area under the curve before maximum metabolic rate, heat flow, and / or ratios between any of these signals.

[0056] "Metabolic activity" refers to the chemical reactions within a cell that convert cellular food / fuel into cellular building blocks or energy. Thus, "metabolic activity" refers to all biochemical reactions that occur during the absorption and utilization of inorganic or organic compounds required for cellular growth and maintenance. In the context of this document, the terms "metabolic activity" and "metabolic vitality" are used interchangeably. Metabolic rate refers to metabolic activity over time, i.e., heat / time, expressed, for example, in J / s, also known as heat flow. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Inoculum calibration experiment. Thermogram of Staphylococcus epidermidis (S. epidermidis) in Mueller-Hinton broth indicates the metabolic inflection point (black dot) and arrows defining the ideal sampling time range. Open circles A (70 minutes before the inflection point), B (1 hour after the inflection point), and C (4 hours after the inflection point) represent the sampling times for the experiment. The black dashed line represents the region where the metabolic rate increases linearly for >30 minutes and the R value is ≥0.98.

[0059] Figure 2. Antimicrobial susceptibility testing using Staphylococcus epidermidis starting at different metabolic activity levels. A) Inoculum obtained 70 minutes before the inflection point; B) Inoculum obtained 1 hour after the inflection point; C) Inoculum obtained 4 hours after the inflection point. Continuous black line = thermogram of sample incubated in Mueller-Hinton broth; dashed black line = thermogram of sample incubated in Mueller-Hinton broth supplemented with 16 mg / L CTX.

[0060] Figure 3 Inoculum calibration experiment. Thermogram of Staphylococcus aureus (S. aureus) in Mueller-Hinton broth indicates the metabolic inflection point (black dot) and arrows defining the ideal sampling time range. Open circles A (90 minutes before the inflection point), B (1 hour after the inflection point), and C (3 hours after the inflection point) represent the sampling times for the experiment. The black dashed line represents the region where the metabolic rate increases linearly for >30 minutes and the R value is ≥0.98.

[0061] Figure 4. Antimicrobial susceptibility testing using Staphylococcus aureus starting at different metabolic activities. A) Inoculum obtained 90 minutes before the inflection point; B) Inoculum obtained 1 hour after the inflection point; C) Inoculum obtained 3 hours after the inflection point. Continuous black line = thermogram of sample incubated in Mueller Hinton broth; dashed black line = thermogram of sample incubated in Mueller Hinton broth supplemented with 16 mg / L CTX.

[0062] Figure 5 Inoculum calibration experiment. Thermogram of CIP-sensitive P. aeruginosa in Mueller-Hinton broth indicates the metabolic inflection point (black dot) and arrows defining the ideal sampling time range after the inflection point. Open circles A (1 hour before the inflection point), B (1 hour after the inflection point), and C (5 hours after the inflection point) represent the sampling times for the experiment. The black dashed line represents the region where the metabolic rate increases linearly for >30 minutes and the R value is ≥0.98.

[0063] Figure 6. Antimicrobial susceptibility testing using Pseudomonas aeruginosa starting at different metabolic activity levels. A) Inoculum obtained 1 hour before the inflection point; B) Inoculum obtained 1 hour after the inflection point; C) Inoculum obtained 5 hours after the inflection point. Continuous black line = thermogram of sample incubated in Mueller-Hinton broth; dashed black line = thermogram of sample incubated in Mueller-Hinton broth supplemented with 2 mg / L CIP.

[0064] Figure 7Inoculum calibration experiment. Thermogram of CIP-resistant Pseudomonas aeruginosa in Mueller-Hinton broth indicates the metabolic inflection point (black dot) and arrows defining the ideal sampling time range. Open circles A (50 minutes before the inflection point), B (1 hour after the inflection point), and C (5 hours after the inflection point) represent the sampling times for the experiment. The black dashed line represents the region where the metabolic rate increases linearly for >30 minutes and the R value is ≥0.98.

[0065] Figure 8. Antimicrobial susceptibility testing using Pseudomonas aeruginosa starting at different metabolic activity levels. A) Inoculum obtained 50 minutes before the inflection point; B) Inoculum obtained 1 hour after the inflection point; C) Inoculum obtained 5 hours after the inflection point. Continuous black line = thermogram of sample incubated in Mueller Hinton broth; dashed black line = thermogram of sample incubated in Mueller Hinton broth supplemented with 2 mg / L CIP.

[0066] Figure 9. Effects of different metabolic sampling percentages on antimicrobial susceptibility testing using susceptible clinical isolates of Staphylococcus epidermidis starting with the same metabolic activity. A) 30% inoculum obtained at the inflection point; B) 20% inoculum obtained at the inflection point; C) 10% inoculum obtained at the inflection point. Continuous black line = thermogram of samples incubated in Mueller Hinton broth; dashed black line = thermogram of samples incubated in Mueller Hinton broth supplemented with 16 mg / L CTX.

[0067] Details

[0068] According to this document, antimicrobial susceptibility testing (AST) can be performed using calorimetric methods (e.g., isothermal (micro)calorimetry) using the metabolic activity (i.e., metabolic activity) of microorganisms and antimicrobial substances. The action of the antimicrobial substance will result in changes in the thermogram, which can be used to deduce the antimicrobial efficacy of the antimicrobial substance.

[0069] As disclosed herein, calorimetry is used to calibrate the effects of antimicrobial agents on viability (as determined by measuring metabolic activity) directly on living cells, which are in their optimal metabolic conditions for antimicrobial susceptibility testing. This can be accomplished directly on patient samples, whereas compendial biomass-based assays cannot be directly applied to patient samples. Furthermore, the metabolic viability (metabolic activity) assay used in accordance with this document avoids problems associated with biomass-based assays and the inability to separate mixtures of dead and live cells, which obscures the correct ratio of viability to antimicrobial molecules, leading to impaired sensitivity readouts.

[0070] For example, the time of the detectable calorimetric signal (time to detection, TTD), the amount of energy released, and the kinetics of the metabolic activity of the microorganism are proportional to the sensitivity to the antimicrobial substance.

[0071] Therefore, this document provides an improved method for antimicrobial susceptibility testing. Knowing the antimicrobial susceptibility of microorganisms in a microbial sample is important in several situations, particularly in patients with microbial infections, where rapid and accurate initiation of treatment for the microbial infection is crucial to the patient's outcome. Currently available methods for antimicrobial susceptibility testing are hampered by being too slow, as they can take one or more days to determine the appropriate antimicrobial to use. In clinical situations, this can be detrimental to the patient.

[0072] Therefore, this document relates to an improved method for antimicrobial susceptibility testing of samples, such as clinical samples, based on calorimetry. Due to the use of calorimetry, the test avoids the need for microbial growth, resulting in faster results, as only the metabolic activity of the microbial cells is required. Furthermore, due to the use of calorimetry, the microbial sample to be analyzed for the presence of the microorganisms and their susceptibility to antimicrobial agents does not have to be purified from the patient sample and can be used without the initial step of isolating the microorganisms. This again results in faster results, but also improves the safety of the method, as potentially harmful microorganisms can be handled more safely due to fewer sample preparation steps.

[0073] Using methods based on microbial growth, antimicrobial susceptibility testing can only be performed after a sufficient number of microbial cells has been reached. In contrast, in the present method, actual growth of microbial cells is not required; only metabolic activity of the microbial cells is essential. Microbial cells can be metabolically active without accompanying growth, and increases in metabolic activity often precede growth. Therefore, using the methods of this document, much faster and more accurate antimicrobial susceptibility results can be obtained.

[0074] The method of this document is a calorimetric method for testing the susceptibility of microbial samples to antimicrobial agents, comprising or consisting of the following steps:

[0075] a) incubating a sample (possibly) comprising one or more microorganisms (i.e., a microbial sample) in an inoculation medium and tracking metabolic activity by measuring one or more calorimetric signals of the incubated sample;

[0076] b) when the metabolic rate has an R value of ≥ 0.98 for at least about 20 minutes, or when the metabolic activity of the incubated sample of step a) reaches an inflection point or within 2 hours of reaching said inflection point, reloading one or more aliquots of the incubated sample of step a) onto:

[0077] i) inoculated medium not supplemented with an antimicrobial agent; and

[0078] ii) the same inoculum medium as in step b) i), but wherein the inoculum medium is supplemented with an antimicrobial agent;

[0079] c) incubating the sample of steps b)i) and b)ii and measuring one or more calorimetric signals of said sample; and

[0080] d) determining whether the one or more microorganisms are sensitive or insensitive to the antimicrobial agent by comparing the one or more calorimetric signals obtained in step c) from the sample(s) (b)ii)) supplemented with the antimicrobial agent with the one or more calorimetric signals obtained in step c) from the sample(s) (b)i)) not supplemented with the antimicrobial agent.

[0081] By calorimetry, the heat flow in a sample can be measured, which reflects the metabolic activity of the microbial cells in the sample. In the method of this document, the metabolic activity of the incubated sample is measured by calorimetry. Higher metabolic activity leads to higher heat release. Any calorimetric signal related to metabolic activity can be used in the method of this document. An example of a calorimetric signal that can be used according to this document is the maximum metabolic rate, which is the measured value of the slope of the curve when the metabolic activity reaches the maximum difference over time. Another example is the maximum metabolic activity, which is the measured value of the maximum achievable curve height for a given organism. Another typical calorimetric signal useful in the method of this document is the detection time, which confirms the detection limit, at which the presence of metabolic activity can be distinguished from the measurement baseline. Heat flow is defined as the measurement signal in the isothermal calorimetric method, where the heat flow is expressed in J / s. Data can be compared between experiments or samples by using the ratio of the measured values ​​or signals obtained between different treatment conditions. In the context of this document, the terms calorimetric "signal" and calorimetric "signature" are used interchangeably.

[0082] To determine the inflection point, the metabolic rate (i.e., heat flow) is preferably used as the calorimetric signal. To determine which aliquots to analyze after reloading, the detection time of the calorimetric signal is used, i.e., the time at which the calorimetric signal, typically heat flow, is first detected.

[0083] Typically, the microbial sample is homogenized or suspended in a liquid, such as phosphate-buffered saline, physiological saline, or a typical bacterial growth medium, to break the sample into smaller pieces and release the microorganisms prior to incubation in step a). However, as mentioned above, in contrast to current methods for antimicrobial susceptibility testing, it is not necessary to isolate the microorganisms or quantify the number of microorganisms in the sample prior to testing. Furthermore, since the sample does not have to be purified, there is no risk of losing microorganisms present in the original sample, which would then remain undetected during the antimicrobial susceptibility testing process.

[0084] In the first step of the method, step a), a microbial sample is incubated in an inoculum. The metabolic activity of the microbial sample is then measured calorimetrically, and one or more calorimetric signals are determined after measuring the metabolic activity of the sample. Since only viable cells are metabolically active, the calorimetric readout is based solely on viable cells. On the other hand, prior art antimicrobial susceptibility testing methods based on counting cell numbers (e.g., by optical density) also read dead and / or inactive cells. This is an advantage of the present method, as the results are based only on the viable portion of the microbial population. As explained elsewhere herein, any suitable calorimetric signal can be used for calorimetric determination of metabolic activity.

[0085] When the metabolic rate has an R value of ≥ 0.98 for at least about 20 minutes, or when the metabolic activity of the incubated sample of step a) reaches an inflection point or within 2 hours, such as about 1 hour after reaching the inflection point, one or more aliquots of the incubated sample of step a) are reloaded (step b)) on:

[0086] i) inoculated medium not supplemented with an antimicrobial agent; and

[0087] ii) the same inoculum medium as in step i), but wherein the inoculum medium is supplemented with an antimicrobial agent;

[0088] Therefore, according to the present method, metabolic activity is tracked over time to determine when the metabolic rate reaches an R value ≥ 0.98. Reloading in step b) should be performed at the inflection point or within two hours after reaching the inflection point when the R value of the metabolic rate has been ≥ 0.98 for at least about 20 minutes, preferably at least 30 minutes. This ensures that the microbial population is sufficiently homogeneous in terms of metabolic activity and that the sample is in a state qualitatively suitable for AST. The inflection point of metabolic activity as defined herein represents the inflection point at which the metabolic rate increases from a linear increase of R ≥ 0.98 to a point at which it drops below R < 0.98. The linear increase in metabolic activity, i.e., when R ≥ 0.98, should preferably continue for a period of time and then drop to below R < 0.98 when the inflection point is determined. Typically, the linear increase in metabolic activity should continue for at least 10-30 minutes and then drop to R < 0.98. Preferably, the linear increase continues for at least 20 minutes or at least 30 minutes. It is found that the time point at or near the inflection point or the time point at which the R value of the metabolic rate has been ≥ 0.98 for a period of at least about 20 minutes, preferably at least 30 minutes, is optimal for proceeding to the reloading step b), wherein the incubated microorganism sample is transferred to a medium with and without an antimicrobial agent for antimicrobial susceptibility testing. The inflection point can be determined from the thermogram by identifying the point at which the increase in metabolic activity over time is highest, e.g., see Figure 1. Generally, the best results are obtained if the incubated sample of step a) is reloaded at the time of the inflection point or within two hours after reaching the inflection point, such as within one hour or two hours after the inflection point. However, good results can also be obtained if the incubated sample is reloaded when the R value is ≥0.98 and has been maintained for a period of at least about 20 minutes, preferably at least 30 minutes before reloading. It is preferred to reload at a time close to the inflection point. However, depending on the microbial species in the microbial sample, it is still possible to reload at a time point more than two hours after reaching the inflection point. As shown in the experimental part, if the time of reloading the sample is too early or too late relative to the inflection point, the microbial cells are not in a metabolic state that qualitatively allows reliable results to be obtained in the antimicrobial susceptibility test. Therefore, it was found that performing the reloading step at the inflection point or within a limited time thereafter is beneficial for giving reliable results in the calorimetric antimicrobial susceptibility test. Therefore, in the method of the present document, a microbial sample is first inoculated into an inoculation medium and metabolic activity is tracked until the R value of the metabolic rate is ≥0.98 for a period of at least about 20 minutes, preferably at least 30 minutes, or until an inflection point is reached. An aliquot of the incubated sample is then reloaded (within a maximum of two hours after reaching the inflection point) into a fresh inoculation medium with or without an antimicrobial agent. It has been found that pre-incubating the microbial sample and reloading it near the inflection point in the present method is beneficial for obtaining reliable results in antimicrobial susceptibility testing. Reloading is important for the present method when the R value of the metabolic rate is ≥0.98 for a period of at least about 20 minutes, preferably at least 30 minutes, or at the inflection point or within a time thereafter as defined herein, because at this time point the microbial cells are in a state that qualitatively allows reliable results to be obtained in AST.

[0089] Furthermore, the present inventors have discovered that an appropriate ratio of metabolic activity to the amount of antimicrobial molecules to be used for antimicrobial susceptibility testing (i.e., metabolic activity / antimicrobial molecules) is crucial for obtaining the best and most reliable results in antimicrobial susceptibility calorimetric methods. It was discovered that if the total metabolic activity reloaded in step b) ii) is too low relative to the amount of antimicrobial molecules used, the results may be interpreted as indicating that the microorganism(s) in the microbial sample are susceptible to the antimicrobial, even though this is not the case (false negatives—a significant error rate). Conversely, if the total metabolic activity reloaded in step b) ii) is too high relative to the amount of antimicrobial molecules used, the results may be interpreted as indicating that the microorganism(s) are resistant, even though this is not the case (false positives—a significant error rate). Therefore, it was discovered that using the correct ratio of total metabolic activity to the amount of antimicrobial molecules in the reloading step b) is crucial for calorimetric methods to provide reliable results in antimicrobial susceptibility testing. Therefore, reloading the appropriate amount of metabolic activity is crucial for this method to provide the most reliable results.

[0090] For process efficiency, it may be preferable to reload only one aliquot in step b). However, a method to ensure the correct ratio of total metabolic activity to amount of antimicrobial agent molecules is used, whereby in step b), aliquots containing different amounts of the incubated sample from step a) (i.e., different amounts of total metabolic activity) are reloaded in inoculation medium with and without antimicrobial agent, respectively.

[0091] Then, in step c), the metabolic activity of the reloaded sample(s) with and without the antimicrobial agent is tracked by measuring one or more calorimetric signals. It has been found that when metabolic activity in the antimicrobial-free medium does not appear immediately after reloading, but rather appears about 6 hours after reloading, such as about 15 minutes to about 6 hours or about 15 minutes to about 3 hours after reloading, the inoculated sample(s) provide the most reliable results in antimicrobial susceptibility testing (i.e., samples in which metabolism, i.e., a calorimetric signal, is detected within about 6 hours, such as 15 minutes to 6 hours or about 15 minutes to 3 hours after reloading). Therefore, the aliquot(s) reloaded into the antibiotic-containing inoculated medium for antimicrobial susceptibility testing correspond to the aliquot(s) in the antimicrobial-free inoculated medium for which a calorimetric signal appears about 6 hours after reloading, such as about 15 minutes to about 6 hours or about 15 minutes to about 3 hours. If the total metabolic activity at reload is too low, the ratio of total metabolic activity to the amount of antimicrobial molecules in the reloaded sample may be too low, and the results of the antimicrobial susceptibility test may be interpreted as indicating that the microorganism is susceptible to the antimicrobial, despite the fact that it is not. If the total metabolic activity at reload is too high, the ratio of total metabolic activity to the amount of antimicrobial molecules in the reloaded sample may be too high, and the results of the antimicrobial susceptibility test may be interpreted as indicating that the microorganism is not susceptible to the antimicrobial, despite the fact that it is. As demonstrated in the experimental section, a calorimetric signal appearing earlier than 15 minutes after reloading increases the risk of a false-positive result (interpreted as insensitive), while a calorimetric signal appearing later than approximately 6 hours after reloading, and typically later than 3 hours after reloading, increases the risk of a false-negative result (interpreted as sensitive). Preferably, the aliquot used for antimicrobial susceptibility testing is an aliquot corresponding to reloading into inoculum medium without antimicrobial, in which the calorimetric signal is detected between approximately 15 minutes and approximately 3 hours after reloading. However, in some cases, a metabolic signal appearing up to 6 hours after reloading is acceptable, particularly when the microorganism(s) in the sample have low metabolic activity. Non-limiting examples of such microorganisms with low metabolic activity are Cutibacterium acnes, Mycobacterium tuberculosis, Helicobacter pylori and Campylobacter spp.

[0092] Although it may be preferred to reload only one aliquot for process efficiency, in some cases, to obtain the most reliable results of the methods of the present document, it may be preferred to reload two or more aliquots with different total metabolic activities in steps b)i) and b)ii), respectively, and the calorimetric signals (multiple) obtained from the aliquot(s) are used to determine sensitivity or insensitivity in step d), the aliquot(s) corresponding to the aliquot(s) in which the metabolic activity (metabolic activity) in the sample(s) of step b)i) is below the detection limit of one or more calorimetric signals at the time of reloading, and the metabolic activity (metabolic activity) in the same aliquot(s) is detected within about 6 hours, such as about 15 minutes to 6 hours or about 15 minutes to about 3 hours, after reloading in step b)i). Typically, several different aliquots of the incubated sample from step a) are transferred so that the sample reloaded in step b) achieves a minimum of about 0.1% to a maximum of 50% (such as about 1% to about 15%, such as 1% to about 10%, or about 5% to about 10%) of the metabolic activity of the incubated sample from step a) at the time of reloading. That is, if the metabolic activity of the incubated sample from step a) is X J / ml at the time of reloading, then after reloading, the reloaded sample from step b) will have a metabolic activity corresponding to about 0.1% to 50% of X J / ml. Preferably, one or at least two aliquots are transferred to achieve different starting metabolic activities in the reloaded samples in the inoculated medium with and without the antimicrobial agent, respectively. This ensures that the volume of the incubated sample of step a) will be sufficient to reload a sufficient number of aliquots of varying volumes to ensure that at least one aliquot meets the following criteria: metabolic activity is below the detection limit of one or more calorimetric signals at the time of reloading, but is detectable within about 6 hours, such as within 15 minutes to 3 hours, after reloading in step b) i).

[0093] In the methods of this document, when more than one aliquot is reloaded, the amount of antimicrobial agent used in step b) ii) is generally fixed, and the amount of reloaded sample is varied if necessary. However, it is of course also possible to reload step b) using a fixed amount of the incubated sample of step a), while inoculating this amount into an inoculation medium supplemented with different amounts of antimicrobial agent(s).

[0094] The microbial sample of this document test is the sample that may comprise one or more types of microorganisms.Microbial sample can be for example the microbial sample from clinical sample, environmental sample, food or feed sample and / or purification of experimenter.Usually, sample is the clinical sample from human or animal experimenter.Such clinical sample can be the sample of any body fluid or tissue, such as the sample of blood, urine, blood plasma, synovial fluid, cerebrospinal fluid, bone tissue, soft tissue, connective tissue, skin tissue, sputum, lavage fluid, blood plasma / serum, urine, peritoneal fluid, pericardial fluid and / or pleural fluid.

[0095] The temperature during the incubation step in this method will vary depending on the sample type. For example, environmental samples can be incubated at lower temperatures than clinical samples, depending on the type of microorganisms expected to be present in the sample. Clinical samples are typically incubated at a temperature of about 35°C to about 39°C, such as about 36°C to about 38°C, such as about 37°C, and for some microorganisms, temperatures as low as 25°C are necessary for correctly interpreting antimicrobial susceptibility tests. In addition, other growth conditions, such as whether the microorganisms are grown under aerobic or anaerobic conditions and the type of culture medium to be used must be adapted to the sample type and / or the type of microorganisms expected to be present in the sample.

[0096] Different microorganisms produce different specific calorimetric signals when incubated under the same conditions. In this way, the presence of microbial species (species) in a microbial sample can be determined by studying one or more calorimetric signals obtained under one or several different incubation conditions (e.g., incubation with different inoculation media, different temperatures, aerobic / anaerobic conditions, etc.).

[0097] To determine the identity of the microorganism(s) present in a microbial sample, the obtained calorimetric signal(s) are compared with calorimetric data (i.e., calorimetric signal(s)) previously obtained from incubating different microorganisms under the same conditions. Due to the differences in calorimetric signals between the different microorganisms, the identity of the microorganism(s) in the sample can be determined, such as whether they are Gram-positive or Gram-negative, fungi and / or bacteria, and / or the species of microorganism present. The level of specificity in determining identity will depend on the type of sample and the desired data to be acquired. In mixed samples, i.e., samples containing more than one type of microorganism, the obtained calorimetric signal will be a combined calorimetric signal of the various microorganisms present in the sample. When two or more microorganisms are present in the same sample, they may influence each other's metabolic activity, resulting in a calorimetric signal that is not simply a sum of the individual calorimetric signals obtained when the same microorganisms were incubated individually. Consequently, the combined calorimetric signal may be higher or lower than the calorimetric signal expected based on the calorimetric signal obtained when a single microbial sample of the same microorganism was incubated.

[0098] For example, based on the calorimetric signal(s) obtained from the sample reloaded in step b)i) in an inoculum medium without an antimicrobial agent, the Gram status, genus, and / or species of one or more microorganisms in the sample can be determined. It is also possible to determine whether the sample contains bacteria and / or fungi. Therefore, another advantage of this method is that it not only provides a result on whether the microorganism(s) are susceptible to the tested antimicrobial agent(s), but also provides data on the identity of the specific microorganism(s) present. This is particularly important for the analysis of clinical samples.

[0099] Based on the one or more calorimetric signals obtained in step c) from samples reloaded with inoculated medium with and without the antimicrobial, in step d), a determination is made as to whether the one or more microorganisms in the sample are susceptible or resistant to the antimicrobial by comparing the one or more calorimetric signals obtained in the respective incubated samples with and without the antimicrobial, typically for up to 48 hours from the time when metabolic activity in the positive control (i.e., from sample b)i) is detectable, depending on the antimicrobial used. This determination is made based on aliquots in which a calorimetric signal is detected within approximately 6 hours, such as 15 minutes to 6 hours or 15 minutes to 3 hours, after reloading according to step b)i), as described above. For a subset of microorganisms and antimicrobials, an incubation period of up to 2 weeks may be necessary to accurately interpret the AST. If the microorganism(s) in the microbial sample are susceptible to the antimicrobial being tested, this will result in differences in the calorimetric signal(s) being studied between samples incubated with and without the antimicrobial. By using calorimetry, not only can a "yes" or "no" answer be obtained regarding sensitivity, but information can also be obtained regarding the degree of sensitivity of the microorganism(s) by determining the gradual effect of the antimicrobial on the metabolic (i.e., calorimetric) signal(s). Determining the identity of the microorganisms in a microbiological sample can aid in the interpretation of the calorimetric signal(s) obtained from samples incubated in the presence of an antimicrobial to determine whether they are sensitive or insensitive to the antimicrobial.

[0100] One challenge in antimicrobial susceptibility testing of microbial samples is mixed samples—that is, samples containing more than one type of microorganism. In such samples, some microorganisms may be sensitive to a particular antimicrobial agent while others are not. The present method allows for the identification of the specific microorganisms present in the sample (e.g., Gram-positive / negative, species, fungus / bacteria), rather than simply determining whether the sample as a whole is susceptible to the antimicrobial agent. Instead, by comparing the calorimetric signal(s) from a sample incubated without an antimicrobial agent with the calorimetric signal(s) obtained from a sample incubated with an antimicrobial agent, it is possible to determine which microorganisms in the microbial sample are susceptible to each specific antimicrobial agent tested. In this way, treatment for a patient with a microbial infection, for example, can be tailored to that specific patient.

[0101] In the methods for antimicrobial susceptibility testing of the present document, analysis of calorimetric data and / or determination of the identity of (multiple) microorganisms can be performed by visual inspection or automatically using a database of calorimetric data covering different microorganisms. Thus, such a database can be used to establish a correlation between at least one calorimetric signal and multiple microorganisms, allowing them to be distinguished from each other. Software can be used to perform this analysis. The database can be obtained by incubating known microorganisms under known conditions and tracking their metabolic activity using calorimetry to obtain one or more calorimetric signals.

[0102] The antimicrobial agent used in the antimicrobial susceptibility test can be a single type of antimicrobial agent or a combination of two or more antimicrobial agents. The antimicrobial agent can be an antibiotic or an antifungal substance or a combination thereof. As known to those skilled in the art, the antimicrobial agent(s) used is selected based on the sample type and the microorganisms that can be expected to be present in the sample. In the methods of this document, 1 to 20 different antimicrobial agents are typically used, alone and / or in combination. Non-limiting examples of antibacterial agents that can be used are cefoxitin, rifampicin, fosfomycin, ciprofloxacin, amoxicillin, clavulanic acid, piperacillin, tazobactam, meropenem, imipenem, ceftriaxone, linezolid, cefotaxime, vancomycin, clindamycin, fosfomycin, vancomycin, gentamicin, methicillin, oxacillin, rifampicin, cefazolin, trimethoprim-sulfamethoxazole, levofloxacin, trimethoprim-sulfamethoxazole, colistin, trimethoprim-sulfamethoxazole, cefepime, gentamicin, mipinem, meropenem, methicillin, oxacillin. Typical antibacterial combinations to be used include, but are not limited to, ampicillin and sulbactam, amoxicillin and clavulanic acid, ceftazidime and avibactam, cefuroxime and tazobactam, and piperacillin and tazobactam. The same antimicrobial agent or the same combination of antimicrobial agents at different concentrations may also be used.

[0103] Thus, by preparing several vials of incubation medium containing the desired antimicrobial(s) for reloading step b) ii), susceptibility to 1) different concentrations of the same antimicrobial, 2) different antimicrobials, and / or 3) combinations of antimicrobials (same or different concentrations) can be tested in parallel.

[0104] The inoculation medium containing the antimicrobial agent may also be supplemented with one or more adjuvants, such as adjuvants that enhance and / or increase the activity of the antimicrobial agent. Non-limiting examples of such adjuvants are iron and beta-lactam inhibitors.

[0105] In the method of the present document, two or more inoculation media can be used in both step a) and / or reloading step b). In this way, the inoculation media can be adapted and / or optimized for specific microorganisms that may be present in the microbial sample by incubating the sample in different vials containing different inoculation media. For example, an inoculation medium for the growth of aerobic microorganisms and an inoculation medium for the growth of anaerobic microorganisms can be used. As is known to those skilled in the art and mentioned above, the growth conditions must also be adjusted depending on whether aerobic or anaerobic microorganisms are suspected to be present in the sample. Non-limiting examples of culture media suitable for use in the method of the present document are Mueller Hinton broth, thioglycolate broth, mannitol salts, modified Sabouraud broth and / or Schaedler broth.

[0106] In step c), the time for which the sample must be incubated with or without the antimicrobial agent will depend on the type of microbial sample and the microorganism(s) present. Typically, the incubation in step c) is carried out for a period of about 1 hour to about 48 hours, starting from the time of detection (i.e., the time point at which the selected calorimetric signal appears in the sample incubated without inoculation with the antimicrobial agent).

[0107] This document also relates to a system for performing the calorimetric method disclosed herein for antimicrobial susceptibility testing.Such a system includes a calorimeter and software for performing analysis of the calorimetric signal.

[0108] The present invention also relates to a kit for performing the calorimetric method of antimicrobial susceptibility testing of the present document. Such a kit comprises:

[0109] i) at least one inoculum medium that does not contain an antimicrobial agent;

[0110] ii) inoculation medium as in i), but supplemented with an antimicrobial agent;

[0111] iii) one or more databases that establish a correlation between at least one calorimetric signal and a plurality of microorganisms;

[0112] iv) an optional software component that includes functionality for determining the Gram status, genus, and / or species of one or more microorganisms; and

[0113] v) a software component comprising functionality for determining whether said microorganism is susceptible or insensitive to said at least one antimicrobial agent.

[0114] Alternatively, instead of an inoculation medium without an antimicrobial agent and supplemented with an antimicrobial agent, the kit may comprise an inoculation medium and a separately provided antimicrobial agent, which is to be mixed in the inoculation medium to provide an inoculation medium supplemented with an antimicrobial agent.

[0115] Detailed information regarding suitable inoculation media and antimicrobial agents to be used in such kits is given elsewhere herein.

[0116] The kit may also include a software component that determines whether the ratio of metabolic activity to the number of antimicrobial molecules is suitable for performing an antimicrobial susceptibility test.

[0117] This document also relates to a computer-implemented method performed by a control unit, the method comprising:

[0118] receiving first measurement data indicative of the calorimetric signal of step a), wherein the calorimetric signal of step a) is indicative of metabolic activity of one or more samples comprising one or more microorganisms in an inoculated medium, and,

[0119] determining that the metabolic rate has an R value of ≥ 0.98 for at least about 20 minutes, preferably at least 30 minutes, or the metabolic activity of the incubated sample of step a) has reached an inflection point,

[0120] indicating to a system user that reloading of one or more aliquots of said incubated sample of step a) should be performed 2 hours after reaching said inflection point,

[0121] receiving second measurement data indicative of the calorimetric signal of step c),

[0122] By comparing the second measurement data of the one or more samples supplemented with the antimicrobial agent with the second measurement data of the sample(s) not supplemented with the antimicrobial agent, the one or more microorganisms are classified as being sensitive or not sensitive to the antimicrobial agent. Such a method can be used to analyze the calorimetric signal of the calorimetric method herein.

[0123] This document also relates to a control unit, said control unit (CU) comprising:

[0124] processor,

[0125] and a memory containing instructions executable by the processor, thereby operating the control unit to perform the computer-implemented method described herein.

[0126] The present document also relates to a computer program comprising computer program code which, if executed on a processor, is adapted to implement the calorimetric method for antimicrobial susceptibility testing of the present document. The present document also relates to a computer program product comprising a computer-readable storage medium having the computer program.

[0127] This document also discloses a calorimetric method for antimicrobial susceptibility testing of microbial samples, comprising or consisting of the following steps:

[0128] a) incubating a sample which (possibly) contains one or more microorganisms in an inoculation medium and determining the metabolic activity of the incubated sample by calorimetry;

[0129] b) determining the ratio of the metabolic activity of the incubated sample of step a) to the amount of antimicrobial agent molecules to be used for the antimicrobial test, and:

[0130] i) if the ratio of metabolic activity to the amount of antimicrobial molecules is suitable for performing an antimicrobial susceptibility test, performing an antimicrobial susceptibility test according to steps c)-d), and

[0131] ii) if the ratio of metabolic activity to the amount of antimicrobial molecules is not suitable for performing antimicrobial susceptibility testing, adjusting the incubated sample and / or the amount of antimicrobial molecules of step a) to obtain a suitable ratio of metabolic activity to the amount of antimicrobial molecules for performing antimicrobial susceptibility testing before performing antimicrobial susceptibility testing according to steps c) to d);

[0132] c) Incubation:

[0133] i) incubating an aliquot of the incubated sample of step a) or an aliquot of the conditioned sample of step b) ii) in an inoculum medium not supplemented with an antimicrobial agent and determining one or more calorimetric signals to determine the Gram state, genus and / or species of one or more microorganisms in the sample; and

[0134] ii) incubating an aliquot of the incubated sample of step a) or an aliquot of the conditioned sample of step b)ii) in the same inoculated medium as in step c)i) but supplemented with an amount of a molecular antimicrobial agent determined according to step b)i) or b)ii) and measuring the same calorimetric signal(s) as in step c)i); and

[0135] d) determining whether the one or more microorganisms are sensitive or insensitive to the antimicrobial agent by comparing the one or more calorimetric signals obtained in step c)i) with the calorimetric signal(s) obtained in step c)ii);

[0136] wherein the method is performed without first isolating the one or more microorganisms from the sample.

[0137] This document also discloses a calorimetric method for antimicrobial susceptibility testing of microbial samples, the method comprising or consisting of the following steps:

[0138] a) provide a microbial sample and an inoculum medium without an antimicrobial agent and the same inoculum medium with an antimicrobial agent;

[0139] b) Incubation

[0140] i) incubating an aliquot of said microbial sample in said inoculation medium without an antimicrobial agent; and

[0141] ii) incubating an aliquot of said microbial sample in said inoculation medium containing an antimicrobial agent;

[0142] c) determining the Gram state, genus and / or species of one or more microorganisms in the microbial sample using one or more calorimetric signals obtained from the microbial sample inoculated according to step b)i); and

[0143] d) using one or more calorimetric signals obtained from the microbial sample inoculated according to steps b)i) and b)ii) to:

[0144] i) determining whether the sample contains an appropriate ratio of metabolic activity to the amount of antimicrobial molecules for antimicrobial susceptibility testing;

[0145] ii) determining whether the microorganisms in the microbial sample are susceptible or insensitive to the antimicrobial agent by comparing the calorimetric signal(s) obtained from an inoculum without the antimicrobial agent to the same calorimetric signal(s) obtained from an inoculum containing the antimicrobial agent, when the sample contains an appropriate ratio of metabolic activity to the amount of antimicrobial agent molecules for antimicrobial susceptibility testing as determined in step d)i);

[0146] The method is performed without first isolating the microorganisms from the sample.

[0147] The present invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0148] Experimental part

[0149] Four homogenized clinical tissue biopsies from orthopedic surgery (Karolinska Hospital, Sweden) were used to determine the influence of qualitative and quantitative metabolic activity and the amount of antimicrobial molecules on obtaining accurate AST results.

[0150] Example 1: Treatment and pre-incubation of microbial samples

[0151] Four clinical tissue biopsies from the Department of Orthopedics (Karolinska Hospital, Sweden) were homogenized using a bullet blender BB50-DX (next advance) at the following settings: stainless steel UFO beads (3.5 mm diameter) at speed 4 for 12 minutes, and the biopsies were dissolved in 5 ml of liquid in a 50-ml tube. One biopsy was known to be infected with a strain of Staphylococcus epidermidis susceptible to cefotaxime (CTX); another was infected with Staphylococcus aureus resistant to CTX; a third was known to be infected with Pseudomonas aeruginosa susceptible to ciprofloxacin (CIP); and a fourth was infected with Pseudomonas aeruginosa resistant to CIP. Fifty microliters of tissue sample was added to Mueller Hinton broth (Sigma-Aldrich, Darmstadt, Germany) in sterile plastic cannulas dispensed into microcalorimetry vials (Symcel AB, Solna, Sweden). The microcalorimetry vials were sealed and introduced into a calScreener (Symcel AB, Solna, Sweden). Both samples were incubated at 37°C for 18 h, and the heat generated by each sample was measured.

[0152] Example 2: Qualitative Effect of Metabolic Activity on AST

[0153] To investigate the potential impact of metabolic rate on the accuracy of AST, bacteria with different initial metabolic rates were exposed to the same antibiotic concentration.

[0154] For qualitative assessment, all biopsies were incubated as described above. Then, tissue samples containing Staphylococci at different metabolic rates were taken: A = before the inflection point, B = within 2 hours after the inflection point, and C = 3-4 hours after the inflection point and reloaded into 300 µl of Mueller-Hinton broth (Sigma-Aldrich, Darmstadt, Germany) supplemented with 16 mg / L CTX or 2 mg / L CIP in sterile plastic cannulas in microcalorimetry vials. Aliquots of incubated biopsies (AC) with different metabolic activities per volume were adjusted to achieve the same metabolic activity in all reloaded samples, allowing the impact of metabolic quality to be assessed independently of the fixed metabolic amount between samples.

[0155] A tissue sample containing bacteria was used to inoculate another 300 µl of antibiotic-free Mueller Hinton broth as a positive metabolic control, with the same metabolic rate as described above (AC). The microcalorimetry vials were sealed and introduced into a calScreener (Symcel AB, Solna, Sweden). Heat generated by each sample was measured at 37°C for 18 hours.

[0156] Tissue samples containing P. aeruginosa were sampled at three different metabolic rates: A = before the inflection point, B = within 2 hours after the inflection point, and C = within 5 hours after the inflection point.

[0157] result

[0158] The inflection point represents the turning point in metabolic rate, where the metabolic rate increases linearly for >30 minutes with an R ≥ 0.98 and then drops below R < 0.98. This change represents the point at which the microorganism changes from the highest metabolic rate and has been found to be the optimal qualitative sampling point for reloading microbial samples. Using these experimental setup conditions, the inflection point for Staphylococcus epidermidis was determined to be 3 hours and 25 minutes (see Figure 1 ), the inflection point for Staphylococcus aureus is 3 hours and 30 minutes (see Figure 3 For Pseudomonas aeruginosa, the inflection point was determined to be 2 hours and 30 minutes in the case of a sensitive strain ( Figure 5 ), the inflection point for the drug-resistant strain was determined to be 2 hours and 15 minutes ( Figure 7 ).

[0159] Staphylococcus epidermidis (susceptible to CTX)

[0160] For qualitative evaluation (see Figure 2), in the case of S. epidermidis inoculum A, obtained 70 minutes before the inflection point, showed no metabolic activity upon exposure to CTX, indicating susceptibility to CTX. This is a correct result, as the S. epidermidis used in this experiment is sensitive to CTX. Inoculum B, obtained 1 hour after the inflection point, also showed no metabolic activity, indicating susceptibility to CTX, which is a correct result. Inoculum C, obtained 4 hours after the inflection point, showed metabolic activity upon exposure to CTX, indicating a lack of susceptibility to the antibiotic, leading to a false-positive result (non-susceptible). Therefore, this experiment demonstrates that if the reloading step is performed too late relative to the inflection point, the microorganism's metabolic activity is not at the correct stage to provide a reliable AST result.

[0161] Staphylococcus aureus (resistant to CTX)

[0162] In the case of S. aureus (see Figure 4), inoculum A, obtained before the inflection point, showed no metabolic activity, indicating susceptibility to CTX. Since the S. aureus strain used in this experiment is resistant to CTX, this is a false-negative result (susceptible). On the other hand, inocula B and C, obtained just after and 3 hours after the inflection point, showed metabolic activity, indicating resistance to CTX—a correct result. Therefore, this experiment demonstrates that if the reloading step is performed too early relative to the inflection point, the microorganism's metabolic activity is not at the correct stage to provide a reliable AST result.

[0163] Pseudomonas aeruginosa (CIP sensitive)

[0164] For the evaluation of Pseudomonas aeruginosa (see Figure 6), inoculum A, taken 1 hour before the inflection point, did not show any metabolic activity when exposed to CIP, indicating that Pseudomonas aeruginosa is sensitive to CIP. As expected, the no-antibiotic control showed metabolic activity within the first 3 hours of incubation. Inoculum B, taken 1 hour after the inflection point, also did not show any metabolic activity, confirming the sensitivity of Pseudomonas aeruginosa to CIP. The no-antibiotic control also showed metabolic activity within the first 3 hours of incubation. Inoculum C, taken 5 hours after the inflection point, showed metabolic activity when exposed to CIP, giving a false positive result (not sensitive). In addition, metabolic activity in the no-antibiotic control began before 15 minutes of incubation, indicating that the inoculum size at this time was too high. As seen in the case of Staphylococcus epidermidis, which is sensitive to CTX, the metabolic activity of Pseudomonas aeruginosa is not at the right stage to obtain reliable AST results.

[0165] Pseudomonas aeruginosa (Resistant to CIP)

[0166] For the second P. aeruginosa strain (see Figure 8), inoculum A, obtained 1 hour before the inflection point, showed no metabolic activity, indicating susceptibility to ciprofloxacin. Because the P. aeruginosa strain used in this experiment is resistant to ciprofloxacin, this is a false-negative result (susceptible). Inocula B and C showed metabolic activity in the presence of CIP, identifying the strain as resistant. However, inoculum C showed metabolic activity before 15 minutes of incubation, indicating that this time point is not suitable for reliable testing.

[0167] As these four examples illustrate, obtaining an inoculum too long after the inflection point has been reached can give a false-negative result (susceptible) even if the inoculated strain is susceptible to the tested antimicrobial (see Figure 2, Inoculum C). On the other hand, obtaining an inoculum before the inflection point can also give a false-negative result, even if the inoculated strain is resistant (see Figure 4, Inoculum A). The P. aeruginosa example illustrates the importance of performing susceptibility testing under the correct metabolic activity, which, as demonstrated here, is at or within a certain time period after the inflection point (Figures 6 and 8).

[0168] Example 3: Quantitative Effects of Metabolic Activity

[0169] Furthermore, to investigate the potential impact of the total amount of metabolic activity used for reloading during the reloading step (i.e., different metabolic activities per volume unit after reloading) on ​​the AST results, aliquots with different total metabolic activities (i.e., aliquots of different volumes) were reloaded at the inflection point (all samples had the same metabolic rate) and exposed to the same antibiotic concentrations as in the qualitative evaluation of Example 2. Dilutions of the aliquots produced different percentages of total metabolic activity in the newly incubated samples, corresponding to 30% to 10% of the metabolic activity per volume unit of the sample from which the aliquots were reloaded ( Figure 9A -C).

[0170] Therefore, for quantitative assessment, different percentages of total metabolic activity were taken from the incubated microbial samples of Example 1 at the inflection point, so that all samples were reloaded at the same metabolic rate: A) 30% of the total metabolic activity taken from the tissue sample containing bacteria at the inflection point; B) 20% of the total metabolic activity taken from the tissue sample containing bacteria at the inflection point; and C) 10% of the total metabolic activity taken. Aliquots were added to 300 μl of Mueller-Hinton broth supplemented with 16 mg / L CTX (Sigma-Aldrich, Darmstadt, Germany) in sterile plastic cannulas in microcalorimetric vials. Another 300 μl of antibiotic-free Mueller-Hinton broth served as a positive metabolic control, with the same percentage of total metabolic activity as the antibiotic-exposed tissue sample containing bacteria. The microcalorimetric vials were sealed and introduced into a calScreener (Symcel AB, Solna, Sweden). Heat generated by each sample was measured at 37°C for 18 hours.

[0171] result

[0172] Sample A) (reloaded with 30% total metabolic activity) showed metabolic activity upon exposure to CTX, indicating antibiotic resistance and yielding a false-positive result. Sample B) (reloaded with 20% total metabolic activity) showed metabolic activity upon exposure to CTX, indicating antibiotic resistance and also yielding a false-positive result. In both instances, metabolic activity began at the start of incubation, indicating unreliable results. Sample C) (reloaded with 10% total metabolic activity) showed no metabolic activity upon exposure to CTX, indicating antibiotic susceptibility and yielding a correct negative result. Metabolic activity in the antibiotic-free sample began after 2 hours of incubation.

[0173] Therefore, this example illustrates the importance of reloading the appropriate amount of metabolic activity given the amount of antimicrobial molecule used for the metabolic test, i.e. the ratio of metabolic activity to the amount of antimicrobial molecule after reloading must be correct to obtain reliable AST results. The example of Pseudomonas aeruginosa further illustrates this point.

[0174] in conclusion

[0175] The above examples illustrate the correlation between the metabolic inflection point used to determine the optimal qualitative metabolic activity sampling time range and the optimal quantitative shift in total metabolic activity required to achieve accurate antimicrobial susceptibility readings. Examples using susceptible S. epidermidis and P. aeruginosa clinical isolates, as well as resistant S. aureus clinical isolates and P. aeruginosa-infected orthopedic tissue biopsies, illustrate the importance of both qualitative and quantitative metabolic parameters.

[0176] The conclusion is that the optimal time to reload the microbial sample after the first incubation step is at the inflection point or within a maximum of two hours after the inflection point. Therefore, the inflection point of metabolic activity represents the transition from a linear increase in metabolic rate, i.e., R ≥ 0.98, to a decrease below R < 0.98. When determining the inflection point, the linear increase in metabolic activity, i.e., R ≥ 0.98, should preferably continue for a period of time before decreasing below R < 0.98. Typically, the linear increase in metabolic activity should continue for at least 10-30 minutes, preferably at least 20 or 30 minutes, before decreasing to R < 0.98. The results in Figures 2, 4, 6, and 8 illustrate the importance of sampling at the correct qualitative metabolic state, which impacts the accuracy of sensitivity readings. Reloading samples before the inflection point increases the risk of false-negative reads for resistant isolates, while the opposite is true within a time window > 3 hours after the inflection point, with an increased risk of false-positive reads for sensitive isolates. To ensure this does not occur, it is important that the metabolic activity of the microorganisms is homogeneous and in the correct qualitative state upon reloading. Reloading is preferably performed within two hours of the inflection point. However, if the metabolic rate has been R ≥ 0.98 for at least 20 minutes, preferably 30 minutes, before reloading, this state can also be achieved before the inflection point. Therefore, when the metabolic rate has been R ≥ 0.98 for more than 20 minutes, the microbial sample can be reloaded because the metabolic mass of the microorganisms in the sample at this time is equivalent to the metabolic mass at the inflection point or within two hours after the inflection point.

[0177] Figure 9 illustrates the importance of quantifying the correct total metabolic activity at the inflection point to avoid false negative or false positive results ( Figure 5False positive results are described in ). Samples used for AST determination after reloading are considered to be samples in which the reloaded aliquot gives a metabolic signal detected within 15 minutes to 3 hours after reloading. If the metabolic signal (i.e., calorimetric signal) is detected earlier than 30 minutes after reloading, there is a risk of a false positive result (interpreted as insensitive), and if the metabolic signal is first detected later than 3 hours after reloading, there is a risk of a false negative result (interpreted as sensitive). Therefore, reloaded samples in which a metabolic signal appears before or after the time range of 15 minutes to 3 hours (i.e., detection time) are generally not used for AST, although in some cases the appearance of a metabolic signal up to 6 hours after reloading is also acceptable, particularly when the (multiple) microorganisms in the sample have low metabolic activity. Non-limiting examples of such microorganisms with low metabolic activity are Propionibacterium acnes, Mycobacterium tuberculosis, Helicobacter pylori, and Campylobacter.

[0178] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0179] Unless expressly stated to the contrary, each preferred feature described herein may be used in combination with any and all other preferred features described herein.

Claims

1. A calorimetric method for antimicrobial susceptibility testing of microbial samples, comprising the following steps: a) incubating a sample that may contain one or more microorganisms in an inoculated medium and tracking metabolic activity by measuring one or more calorimetric signals of the incubated sample; b) when the metabolic rate has an R value of ≥ 0.98 for at least about 20 minutes, or when the metabolic activity of the incubated sample of step a) reaches an inflection point or within 2 hours after reaching said inflection point, reloading one or more aliquots of the incubated sample of step a) onto: i) inoculated medium not supplemented with an antimicrobial agent; and ii) the same inoculum medium as in step b) i), but wherein the inoculum medium is supplemented with an antimicrobial agent; c) incubating the sample of steps b)i) and b)ii, and measuring one or more calorimetric signals in the sample; as well as d) determining whether the one or more microorganisms are sensitive or insensitive to the antimicrobial agent by comparing the one or more calorimetric signals obtained in step c) from the sample(s) supplemented with the antimicrobial agent with the one or more calorimetric signals obtained in step c) from the sample(s) not supplemented with the antimicrobial agent.

2. The calorimetric method of claim 1, wherein the method is performed without first isolating the one or more microorganisms from the microbial sample.

3. The calorimetric method according to claim 1 , wherein two or more aliquots having different total metabolic activities are reloaded in steps b)i) and b)ii), respectively, and the calorimetric signals (multiple) obtained from the aliquots are used to determine the sensitivity or insensitivity in step d), said aliquots corresponding to aliquots in which the metabolic activity in the sample(s) of step b)i) at the time of reloading was below the detection limit of one or more calorimetric signals, and the metabolic activity in the same aliquots was detected within about 6 hours, such as 15 minutes to 3 hours, after said reloading in step b)i).

4. The calorimetric method according to any one of the preceding claims, wherein the sample is a clinical sample from a subject, an environmental sample, a food or feed sample and / or a purified microbial sample.

5. The calorimetric method according to any one of the preceding claims, wherein the method comprises the further step of using the one or more calorimetric signals obtained in step c) from the sample(s) not supplemented with an antimicrobial agent to determine the Gram state, genus and / or species of one or more microorganisms in the microbial sample.

6. The calorimetric method according to any one of the preceding claims, wherein in step b) ii) a combination of two or more different types of antimicrobial agents is used in the same inoculation medium.

7. The calorimetric method according to any one of the preceding claims, wherein two or more types of inoculation media are used in step a) and / or step b).

8. The calorimetric method according to any one of the preceding claims, wherein at least two inoculation media containing different concentrations of one or more antimicrobial agents are used in step b) ii).

9. The calorimetric method according to any one of the preceding claims, wherein steps b), c) and / or d) are performed using a database establishing a correlation between at least one calorimetric signal and a plurality of microorganisms.

10. The calorimetric method according to claim 5 or 9, wherein a database establishing a correlation between at least one calorimetric signal and a plurality of microorganisms is used to determine the Gram state, genus and / or species of one or more microorganisms in the microbial sample.

11. The calorimetric method according to any one of the preceding claims, wherein the inoculation medium or media of step a) and / or step b) further comprises one or more adjuvants, such as adjuvants that enhance and / or increase the activity of the antimicrobial agent.

12. The calorimetric method according to any one of the preceding claims, wherein the calorimetric signal is a detection time, a metabolic rate, a maximum metabolic rate, an area under the curve, an area under the curve before the maximum metabolic rate, a heat flow and / or a ratio between any of these signals.

13. The calorimetric method according to any one of the preceding claims, wherein the incubation in step c) is performed for a period of about 1 hour to about 48 hours from the time the one or more calorimetric signals are detected.

14. The calorimetric method according to any one of the preceding claims, wherein the inoculation medium is Mueller Hinton broth, thioglycolate broth, mannitol salts, modified Sabouraud broth and / or Scheduler broth.

15. A system for performing the calorimetric method according to any of the preceding claims, comprising a calorimeter and software for performing the analysis according to steps a, b), c) and / or d) of the method and / or for determining the Gram state, genus and / or species of one or more microorganisms according to claim 5 in the microbial sample.

16. A kit for performing the calorimetric method according to any one of claims 1 to 14, comprising: i) at least one inoculum medium that does not contain an antimicrobial agent; ii) inoculation medium as in i), but supplemented with an antimicrobial agent; iii) one or more databases establishing a correlation between at least one calorimetric parameter and a plurality of microorganisms; iv) an optional software component that includes functionality for determining the Gram status, genus, and / or species of one or more microorganisms; and v) a software component comprising functionality for determining whether the microorganism(s) are susceptible or insensitive to the antimicrobial agent.

17. A computer program comprising computer program code adapted to carry out the method according to any one of claims 1 to 14 if executed on a processor.

18. A computer program product comprising a computer-readable storage medium having the computer program according to claim 17.

19. A computer-implemented method performed by a control unit, the method comprising: receiving first measurement data indicative of the calorimetric signal of step a), wherein the calorimetric signal of step a) is indicative of metabolic activity of one or more samples comprising one or more microorganisms in an inoculated medium, and, Determine that the metabolic rate R value is ≥ 0.98 for at least about 20 minutes, or the metabolic activity of the incubated sample in step a) has reached an inflection point, indicating to a system user that reloading of one or more aliquots of said incubated sample of step a) should be performed 2 hours after reaching said inflection point, receiving second measurement data indicative of the calorimetric signal of step c), Classifying one or more microorganisms as sensitive or insensitive to the antimicrobial agent by comparing second measurement data of one or more samples supplemented with the antimicrobial agent with second measurement data of (multiple) samples not supplemented with the antimicrobial agent, wherein the calorimetric signal is optionally obtained by performing the calorimetric method of any one of claims 1-14.

20. A control unit, comprising: processor, and a memory containing instructions executable by the processor to operate the control unit to perform the computer-implemented method of claim 19.

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