Methods for Antimicrobial Susceptibility Testing

Abstract

The present specification is directed to a calorimetric method for determining the antimicrobial susceptibility of microorganisms in a sample, such as a clinical tissue sample. According to the method, a microbial sample is exposed to an antimicrobial when the sample reaches a specific metabolic activity, and the difference in calorimetric signal is compared to a sample incubated without the antimicrobial to determine whether the microorganisms in the sample are susceptible or non-susceptible to the antimicrobial.

Description

[Technical Field]

[0001] The present specification is directed to calorimetric methods for determining antimicrobial susceptibility of microorganisms in a sample, for example, in a clinical tissue sample. [Background technology]

[0002] The purpose of antimicrobial susceptibility testing is to confirm susceptibility or detect resistance to selected empirical antimicrobial agents in individual bacterial isolates, which converts the selected empirical therapy into targeted therapy based on a specific antibiogram.

[0003] There are many methods for determining antimicrobial susceptibility, such as broth microdilution, disk diffusion assays, gradient diffusion, and semi-automated and automated instruments (such as the VITEK® 2 instrument, BD Phoenix, and Q linea ASTar instruments). All of these standard assays are calibrated to a precise ratio of microbial counts to antimicrobial substances to obtain reproducible results.

[0004] To assess susceptibility, there are two main test definitions: i) quantitative determination of the minimum inhibitory concentration (MIC), and ii) relating the MIC to a qualitative breakpoint determination (BP).

[0005] The MIC is the lowest concentration of an antimicrobial agent (e.g., an antifungal, antibiotic, bactericidal, or bacteriostatic agent) that inhibits visible growth of a microorganism after overnight incubation. The MIC can be determined on plates of solid growth media (agar growth media) or by the broth dilution method (liquid growth media) after a pure culture has been isolated.

[0006] For example, to identify the MIC via broth dilution, identical doses of bacteria are cultured in wells of liquid medium containing decreasing concentrations of the drug. The minimum inhibitory concentration of the antibiotic is between the concentration in the last well where bacteria did not grow and the next lowest dose where bacterial growth was possible. Broth dilution testing involves preparing two-fold dilutions of antibiotic (e.g., 1, 2, 4, 8, and 16 μg / mL) in liquid growth medium that are dispensed into test tubes. 1–5 × 10 5 A standardized bacterial suspension of 100 CFU / mL is inoculated and incubated overnight at 35°C. The accuracy of this method is considered to be plus or minus one dilution, primarily because the preparation of serial antibiotic dilutions is performed manually.

[0007] The Kirby-Bauer disk diffusion assay is a standardized technique for testing rapidly growing pathogens and quantitatively determining their MICs. A standardized inoculum is applied to the surface of an agar plate (150 mm diameter) with a cotton swab. The reproducibility of this assay depends on the logarithmic growth phase of the microorganism. A filter paper disk impregnated with a standardized concentration of antimicrobial agent is placed on the surface, and after overnight incubation, the size of the zone of inhibition around the disk is measured in millimeters.

[0008] Gradient diffusion Etest and semi-automated and automated instruments such as those described above can also be used for quantitative determination of MIC. Clinical breakpoints provide an interpretation of antimicrobial susceptibility testing results. Breakpoints can aid in determining whether an antimicrobial is potentially useful in treating a microbial infection. Setting breakpoints requires integrating knowledge of the wild-type distribution of MICs, evaluation of the antimicrobial's pharmacokinetics / pharmacodynamics, and studies of the clinical outcome of infection when the antimicrobial is used. Depending on the testing method, breakpoints are expressed either as MICs (in mg / liter or μg / ml) or as disk diffusion zone diameters (in mm). Typically, all susceptibility testing methods require breakpoints, also known as interpretive criteria, so that test results can be interpreted as indicating that the microorganism is susceptible, intermediate, or nonsusceptible. Clinical breakpoints refer to the concentration (MIC) that separates isolates likely to be treated successfully from those likely to be unsusceptible. These breakpoints are derived from prospective human clinical studies that compare outcomes with the MIC of the infectious pathogen. A third use of the term "breakpoint" refers to antimicrobial drug concentrations calculated from knowledge of pharmacodynamic parameters and their dimensionality that predict in vivo efficacy. These are pharmacokinetic / pharmacodynamic breakpoints where data obtained in animal models are extrapolated to humans using mathematical or statistical techniques.

[0009] Isothermal microcalorimetry (IMC) is a measurement technique for real-time monitoring and dynamic analysis of chemical, physical, and biological processes. Over minutes or days, IMC determines the initiation, rate, extent, and energetics of such processes for specimens in closed systems, measured in μW. Isothermal calorimetry measures heat release at a constant set temperature. The term "micro" applies to smaller systems, where heat release is in the microwatt range.

[0010] In isothermal (micro)calorimetry measurements, the sample to be measured is placed in a sealed container in a constant temperature environment. The changes in heat flow due to chemical and biological processes are specific to the type of system being studied; for example, any given bacterial or fungal isolate under specific metabolic conditions will produce a graph of heat flow over time called a thermogram. This heat flow can be tracked in real time, i.e., in real-time isothermal calorimetry. In monitoring microbial systems, such as fungal or bacterial systems, these specific thermograms can be used to derive the metabolic capacity of the microorganisms present in the sample, and in some cases, even the microbial species present.

[0011] Although calorimetry has been used in the study of microorganisms, the technique is limited by challenges in obtaining good quality calorimetry data and / or due to difficulties in interpreting the calorimetry data.

[0012] As a result, there is a need for faster and / or more reliable methods for determining the antimicrobial susceptibility of microorganisms. Summary of the Invention [Problem to be solved by the invention]

[0013] It is therefore an object of the present invention to overcome or at least mitigate one or more of the problems described herein. [Means for solving the problem]

[0014] The present specification therefore provides a calorimetric method for antimicrobial susceptibility testing of a microbial sample, comprising: a) incubating a sample potentially containing one or more microorganisms with an inoculation medium and tracking metabolic activity by determining one or more calorimetric signals of the incubated sample; b) when the metabolic rate R value is ≧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, one or more aliquots of said incubated sample of step a); i) inoculation medium without the addition of antimicrobial agents, and ii) an inoculation medium identical to step b) i), but supplemented with an antimicrobial agent; Step 2: Reload the c) incubating the samples of steps b)i) and b)ii) and determining one or more calorimetric signals in said samples; and d) determining whether the one or more microorganisms are susceptible or non-susceptible to the antimicrobial agent by comparing the one or more calorimetric signals obtained in step c) from the sample spiked with the antimicrobial agent with the one or more calorimetric signals obtained in step c) from the sample not spiked with the antimicrobial agent. The present invention is directed to a method comprising or consisting of:

[0015] The calorimetry methods herein can be performed without first isolating the one or more microorganisms from the microbial sample. One, two or more aliquots with different total metabolic activities may be reloaded in steps b)i) and b)ii), respectively, and the calorimetric signals obtained from the aliquots in step b)ii) corresponding to aliquots in which the metabolic capacity (activity) in the sample in step b)i) immediately after reloading is below the detection limit of one or more calorimetric signals and the metabolic capacity (activity) in the same aliquots is detected within about 6 hours after said reloading in step b)i), for example between 15 minutes and 3 hours (i.e., time to detection), are used to determine sensitivity or insensitivity in step d).

[0016] The sample may be a clinical sample from a subject, an environmental sample, a food or feed sample, and / or a purified microbial sample. The calorimetry methods herein may further comprise a step of determining the Gram stain status, genus, and / or species of one or more microorganisms in the microbial sample using the one or more calorimetry signals obtained in step c) from a sample to which no antimicrobial agent has been added.

[0017] A combination of two or more different types of antimicrobial agents may be used in the same inoculation medium in step b)ii). Alternatively, or in addition, two or more types of inoculation medium may be used in step a) and / or step b). The two or more inoculation media may contain different concentrations of one or more antimicrobial agents in step b)ii). One or more inoculation media in step a) and / or step b) may contain one or more adjuvants, such as adjuvants that enhance and / or potentiate the activity of the antimicrobial agents. Examples of inoculation media that may be used include, but are not limited to, Mueller-Hinton broth, thioglycollate broth, salt mannitol, modified Sabouraud broth, and / or Schuler's broth.

[0018] The analysis of the calorimetric signals in steps b), c), and / or d) can be performed using a database establishing a correlation between at least one calorimetric signal and a plurality of microorganisms. Further, the database establishing a correlation between at least one calorimetric signal and a plurality of microorganisms can be used to determine the Gram stain status, genus, and / or species of one or more microorganisms in the microbial sample.

[0019] The calorimetry signal determined can be, for example, time to detection, 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. The calorimetry signal can therefore also be described as a calorimetric feature.

[0020] The incubation in step c) is typically carried out for a period of about 1 hour to about 48 hours from the time of detection of said one or more calorimetric signals, although longer and shorter incubation times may also be applied depending on the metabolic activity of the sample to be analyzed.

[0021] The present specification is also directed to a system for carrying out the calorimetry method herein, the system 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 stain status, genus, and / or species of one or more microorganisms in the microbial sample.

[0022] The present disclosure also provides a kit for carrying out the calorimetry method of the present disclosure, comprising: i) at least one antimicrobial-free inoculation medium; ii) the inoculation medium of i) supplemented with an antimicrobial agent; iii) one or more databases establishing correlations between at least one calorimetric parameter and a plurality of microorganisms; iv) optionally, a software component including functionality for determining the Gram stain status, genus, and / or species of one or more microorganisms; and v) a software component that includes functionality for determining whether the microorganism is susceptible or non-susceptible to the antimicrobial agent; A kit is disclosed comprising:

[0023] The present specification is also directed to a computer program comprising computer program code, the computer program code being adapted, when executed on a processor, to implement the calorimetric method for antimicrobial susceptibility testing of the present specification. The present specification is also directed to a computer program product comprising a computer-readable storage medium, the computer-readable storage medium having the computer program thereon.

[0024] The present specification also provides a computer-implemented method performed by a control unit, comprising: receiving first measurement data indicative of a calorimetry signal (i.e., a calorimetry characteristic) of step a), wherein the calorimetry signal (i.e., a calorimetry characteristic) of step a) is indicative of metabolic activity in one or more samples comprising one or more microorganisms in an inoculation medium; and determining that the metabolic rate R value is ≧0.98 for at least about 20 minutes or that the metabolic activity of the incubated sample of step a) has reached an inflection point; indicating to a user of the system that reloading of one or more aliquots of the incubated sample of step a) should occur two hours after the inflection point is reached; receiving second measurement data indicative of the calorimetry signal (i.e., calorimetry signature) of step c; classifying the one or more microorganisms as susceptible or non-susceptible to the antimicrobial agent by comparing the second measurement data of the one or more samples to which the antimicrobial agent has been added with the second measurement data of the sample to which the antimicrobial agent has not been added. Such methods may be used to analyze the calorimetric signals of the calorimetry methods herein.

[0025] This specification also provides: processor, and memory wherein the memory has instructions executable by the processor, whereby the control unit operates to perform the computer-implemented methods described herein.

[0026] Other features and advantages of the present invention will become apparent from the following detailed description, drawings, examples, and claims. definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0027] The term "comprising" includes the term "consisting of," unless it is clear from the context that this is not intended. "Antimicrobial agent," "antimicrobial agent," "antimicrobial substance," and the like refer to substances active against microorganisms in the form of bacteria and / or fungi. Antimicrobial agents thus have antibacterial or antifungal activity, killing, arresting or reducing the growth and / or metabolic activity of bacteria and / or fungi. Examples of antimicrobial agents include, but are not limited to, antibiotics and antifungals.

[0028] By "microbial sample" and the like herein is meant a sample potentially containing microorganisms in the form of bacteria and / or fungi. "Calorimetry" and the like refer to the process of measuring the amount of heat released or absorbed during a chemical reaction, for example, during the incubation of microorganisms in an inoculated medium. The heat change over time in a sample (J / s vs. time) can be recorded in a thermogram. Calorimetry according to the present specification is performed as known to those skilled in the art. Briefly, in determining the calorimetric signal from a microbial sample, the sample is inoculated into a vial containing an inoculated medium, and the vial is then placed in a temperature-controlled chamber of a calorimetric device for incubation. According to the present specification, the calorimetric signal can also be described as a calorimetric characteristic. For example, the calorimetric signal, measured in J / s (W), which reflects the metabolic activity of microorganisms potentially present in the sample, can be continuously tracked and analyzed and represented in a so-called thermogram. Other non-limiting examples of calorimetry signals that can be used are, for example, time to detection, 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.

[0029] "Metabolic activity" refers to the chemical reactions within a cell that convert cellular food / fuel into cellular building blocks or energy. "Metabolic activity" therefore refers to all biochemical reactions occurring within a microorganism during the uptake and utilization of inorganic or organic compounds necessary for growth and maintenance of cellular processes. In the context of this specification, the terms metabolic activity and metabolic capacity can be used interchangeably. Metabolic rate is metabolic activity over time, i.e., heat / time expressed in J / s, also known as heat flow. [Brief explanation of the drawings]

[0030] [Figure 1]Figure 1. Inoculum calibration assay. Thermogram of S. epidermidis in Mueller-Hinton broth showing metabolic inflection points (black dots) and arrows delimiting ideal sampling time frames. Open circles A, 70 min before the inflection, B, 1 h after the inflection, and C, 4 h after the inflection, indicate sampling time points for the experiment. The dashed black line indicates the area of ​​linear metabolic rate increase over 30 min with an R value of ≥ 0.98. [Figure 2A] Antimicrobial susceptibility testing with S. epidermidis starting at different metabolic activities. A) Inoculum taken 70 minutes before the inflection point, B) Inoculum taken 1 hour after the inflection point, C) Inoculum taken 4 hours after the inflection point. The solid black line is the thermogram of the sample incubated in Mueller-Hinton broth, and the dashed black line is the thermogram of the sample incubated in Mueller-Hinton broth supplemented with 16 mg / L CTX. [Figure 2B] FIG. 1 shows antimicrobial susceptibility testing in S. epidermidis starting at different metabolic activities. [Figure 2C] FIG. 1 shows antimicrobial susceptibility testing in S. epidermidis starting at different metabolic activities. [Figure 3] Figure 1. Inoculum calibration assay. Thermogram of S. aureus in Mueller-Hinton broth showing metabolic inflection points (black dots) and arrows delimiting ideal sampling time frames. Open circles A, 90 min before the inflection point, B, 1 h after the inflection point, and C, 3 h after the inflection point indicate sampling time points for the experiment. The dashed black line indicates the area of ​​linear metabolic rate increase over 30 min with an R value of ≥ 0.98. [Figure 4A]Antimicrobial susceptibility testing with S. aureus starting at different metabolic activities. A) Inoculum taken 90 minutes before the inflection point, B) Inoculum taken 1 hour after the inflection point, C) Inoculum taken 3 hours after the inflection point. The solid black line is the thermogram of the sample incubated in Mueller-Hinton broth, and the dashed black line is the thermogram of the sample incubated in Mueller-Hinton broth supplemented with 16 mg / L CTX. [Figure 4B] FIG. 1 shows antimicrobial susceptibility testing in S. aureus starting at different metabolic activities. [Figure 4C] FIG. 1 shows antimicrobial susceptibility testing in S. aureus starting at different metabolic activities. [Figure 5] Figure 1. Inoculum calibration assay. Thermogram of P. aeruginosa susceptible to CIP in Mueller-Hinton broth showing metabolic inflection points (black dots) and arrows delimiting ideal sampling time frames after the inflection point. Open circles A, 1 hour before the inflection, B, 1 hour after the inflection, and C, 5 hours after the inflection indicate sampling time points for the experiment. The dashed black line indicates the area of ​​linear metabolic rate increase over 30 minutes with an R value of ≥ 0.98. [Figure 6A] Antimicrobial susceptibility testing with P. aeruginosa starting at different metabolic activities. A) Inoculum taken 1 hour before the inflection point, B) Inoculum taken 1 hour after the inflection point, C) Inoculum taken 5 hours after the inflection point. The solid black line is the thermogram of the sample incubated in Mueller-Hinton broth, and the dashed black line is the thermogram of the sample incubated in Mueller-Hinton broth supplemented with 2 mg / L CIP. [Figure 6B] FIG. 1 shows antimicrobial susceptibility testing with P. aeruginosa starting at different metabolic activities. [Figure 6C] FIG. 1 shows antimicrobial susceptibility testing with P. aeruginosa starting at different metabolic activities. [Figure 7] Figure 1. Inoculum calibration assay. Thermogram of P. aeruginosa resistant to CIP in Mueller-Hinton broth showing metabolic inflection points (black dots) and arrows delimiting ideal sampling time frames. Open circles A, 50 min before the inflection point, B, 1 h after the inflection point, and C, 5 h after the inflection point indicate sampling time points for the experiment. The dashed black line indicates the area of ​​linear metabolic rate increase over 30 min with an R value of ≥ 0.98. [Figure 8A] Antimicrobial susceptibility testing with P. aeruginosa starting at different metabolic activities. A) Inoculum taken 50 minutes before the inflection point, B) Inoculum taken 1 hour after the inflection point, and C) Inoculum taken 5 hours after the inflection point. The solid black line is the thermogram of the sample incubated in Mueller-Hinton broth, and the dashed black line is the thermogram of the sample incubated in Mueller-Hinton broth supplemented with 2 mg / L CIP. [Figure 8B] FIG. 1 shows antimicrobial susceptibility testing with P. aeruginosa starting at different metabolic activities. [Figure 8C] FIG. 1 shows antimicrobial susceptibility testing with P. aeruginosa starting at different metabolic activities. [Figure 9A] Figure 1 shows the effect of different metabolic sampling percentages starting at the same metabolic activity on antimicrobial susceptibility testing using a susceptible clinical isolate of S. epidermidis. A) 30% inoculum taken at the inflection point, B) 20% inoculum taken at the inflection point, C) 10% inoculum taken at the inflection point. The solid black line is the thermogram of the sample incubated in Mueller-Hinton broth, and the dashed black line is the thermogram of the sample incubated in Mueller-Hinton broth supplemented with 16 mg / L CTX. [Figure 9B] FIG. 1 shows the effect on antimicrobial susceptibility testing of different metabolic sampling percentages starting at the same metabolic activity using susceptible clinical isolates of S. epidermidis. [Figure 9C] FIG. 1 shows the effect on antimicrobial susceptibility testing of different metabolic sampling percentages starting at the same metabolic activity using susceptible clinical isolates of S. epidermidis. DETAILED DESCRIPTION OF THE INVENTION

[0031] According to the present specification, antimicrobial susceptibility testing (AST) can be performed by calorimetry (e.g., isothermal (micro)calorimetry) using the metabolic capacity (i.e., metabolic activity) of the microorganism and the antimicrobial agent. The effect of the antimicrobial agent produces a change in the thermogram, which can be used to derive the antimicrobial efficacy of the antimicrobial agent.

[0032] As disclosed herein, when using calorimetry, the impact of antimicrobials on viability (determined by determining metabolic activity) is measured directly on live cells at their metabolic optimum in antimicrobial susceptibility testing. While this can be done directly on patient samples in some settings, official biomass-based assays cannot be directly applied to patient samples. Furthermore, the determination of metabolic capacity (metabolic activity) used in accordance with the present invention avoids the challenges associated with biomass-based assays, which can lead to false susceptibility readouts, and the inability to separate the mixture of dead and live cells, which masks the true viability of antimicrobial molecules.

[0033] For example, the time to a detectable calorimetric signal (time to detection, TTD), the total amount of energy released, and the kinetic signals of metabolic activity of the microorganism are proportional to the susceptibility to antimicrobial agents.

[0034] The present specification therefore provides an improved antimicrobial susceptibility testing method. Knowledge of the antimicrobial susceptibility of microorganisms in a microbial sample is important in some situations, particularly in the context of microbial infection in patients, where rapid and accurate initiation of treatment for the microbial infection can be critical to patient outcome. Currently available antimicrobial susceptibility testing methods are limited by being very slow, as it can take one or several days before the appropriate antimicrobial can be determined. In a clinical setting, this can be detrimental to the patient.

[0035] The present disclosure is therefore directed to an improved method for testing samples, such as clinical samples, for antimicrobial susceptibility, based on calorimetry. The use of calorimetry avoids the need for testing to rely on microbial growth, thereby providing faster results because only the metabolic activity of microbial cells is required. Furthermore, the use of calorimetry means that microbial samples analyzed for the presence of microorganisms and their susceptibility to antimicrobial drugs do not need to be purified from patient samples and can be used without the initial step of isolating the microorganisms. This also provides faster results, but also improves the safety of the method, since potentially harmful microorganisms can be handled in a safer manner due to fewer sample preparation steps.

[0036] Using a method based on microbial growth, antimicrobial susceptibility testing can only be performed if a sufficient number of microbial cells are obtained. Conversely, the present method does not require the actual growth of microbial cells, but only the metabolic activity of microbial cells. Microbial cells can be metabolically active without simultaneous growth, and increased metabolic activity often occurs before growth. Therefore, the method herein can obtain much faster and more accurate results of antimicrobial susceptibility.

[0037] The method herein is a calorimetric method for antimicrobial susceptibility testing of a microbial sample, comprising: a) incubating a sample (i.e., a microbial sample) (potentially) containing one or more microorganisms in an inoculation medium and following metabolic activity by determining one or more calorimetric signals of the incubated sample; b) when the metabolic rate R value is ≧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 about 2 hours after reaching said inflection point, one or more aliquots of said incubated sample of step a); i) inoculation medium without the addition of antimicrobial agents, and ii) an inoculation medium identical to step b) i), but supplemented with an antimicrobial agent; Step 2: Reload the c) incubating the samples of steps b)i) and b)ii) and determining one or more calorimetric signals in said samples; and d) determining whether the one or more microorganisms are susceptible or non-susceptible to the antimicrobial agent by comparing one or more calorimetric signals obtained in step c) from sample (b) ii) spiked with the antimicrobial agent with one or more calorimetric signals obtained in step c) from sample (b) i) without the antimicrobial agent. A calorimetry method comprising or consisting of:

[0038] Calorimetry can measure heat flow in a sample, which reflects the metabolic activity of microbial cells in the sample. In the methods herein, the metabolic activity of the incubated sample is measured by calorimetry. The higher the metabolic activity, the more heat is released. Any calorimetry signal related to metabolic activity can be used in the methods herein. One example of a calorimetry signal that can be used in accordance with the present specification is maximum metabolic rate, which is a measurement of the slope of the curve where metabolic activity reaches its maximum difference over time. Another example is maximum metabolic activity, which is a measurement of the maximum achievable curve height for a given organism. Another typical calorimetry signal useful in the methods herein is time to detection, which identifies the lower detection limit at which the presence of metabolic activity can be distinguished from the baseline measurement. Heat flow is defined as the measured signal in isothermal calorimetry, where heat flow is expressed as J / s. Data can be compared between experiments or samples by using the ratio of measured values ​​or derived signals between different treatment conditions. The terms calorimetric "signal" and calorimetric "feature" may be used interchangeably in the context of this specification.

[0039] In determining the inflection point, metabolic rate (i.e., heat flow) is preferably used as the calorimetric signal. The time to detection of the calorimetric signal, i.e., the time point at which a calorimetric signal, typically heat flow, is first detected, is used to determine which aliquot to analyze after reloading.

[0040] Microbial samples are typically homogenized or suspended in a liquid such as phosphate-buffered saline, saline, or a typical bacterial growth medium to break the sample into smaller pieces and release the microorganisms prior to the incubation step (a). However, as noted above, in contrast to currently used antimicrobial susceptibility testing methods, it is not necessary to isolate the microorganisms or determine the number of microorganisms in the sample prior to testing. Furthermore, because sample purification is not required, there is no risk that microorganisms present in the original sample will be lost and therefore not be detected during antimicrobial susceptibility testing.

[0041] In the first step of the method, step a), a microbial sample is incubated in an inoculation medium. The metabolic activity of the microbial sample is then determined by calorimetry, where the metabolic activity of the sample is tracked by determining one or more calorimetric signals. Because only viable cells are metabolically active, the calorimetric readout is based solely on viable cells. Prior art antimicrobial susceptibility testing methods based on counting cell numbers (e.g., by optical density), on the other hand, also read out dead and / or inactive cells. This is an advantage of the present method, since the results are based solely on the viable portion of the microbial population. As described elsewhere herein, any suitable calorimetric signal can be used for the calorimetric determination of metabolic activity.

[0042] When the metabolic rate R value is ≧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 about 2 hours, e.g., within about 1 hour, after reaching said inflection point, one or more aliquots of the incubated sample of step a) are i) inoculation medium without the addition of antimicrobial agents, and ii) an inoculation medium identical to that in step i), but supplemented with an antimicrobial agent; (step b)).

[0043] According to the present method, metabolic activity is therefore tracked over time to identify the point at which the metabolic rate reaches an R value of ≥ 0.98. The reloading step (b) is performed over a period of at least about 20 minutes, preferably at least 30 minutes, when the metabolic rate has an R value of ≥ 0.98; at the inflection point; or within 2 hours after the inflection point is reached. This ensures that the microbial population is sufficiently homogeneous with respect to metabolic activity and that the sample is qualitatively suitable for performing AST. The inflection point of metabolic activity, as defined herein, refers to the inflection from a linear increase in metabolic rate R ≥ 0.98 to when it drops below R < 0.98. The linear increase in metabolic activity, i.e., when R ≥ 0.98, preferably continues for a period of time, after which it drops below R < 0.98, at which point the inflection point is determined. Typically, the linear increase in metabolic activity continues for at least 10-30 minutes, after which it drops below R < 0.98. Preferably, the linear increase continues for at least 20 minutes or at least 30 minutes. It has been found that the inflection point, or a time point near the inflection point, or when the metabolic rate has an R value of ≥ 0.98 for a period of at least about 20 minutes, preferably at least 30 minutes, is best to continue with the reload step b), in which the incubated microbial sample is transferred to media with and without antimicrobial agents 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 greatest. See, for example, FIG. 1. Typically, best results are obtained if the incubated sample of step a) is reloaded at the inflection point or within 2 hours after reaching the inflection point, e.g., within 1 or 2 hours after the inflection point. However, good results can also be obtained when reloading when the incubated sample has an R value of ≥ 0.98 and was at least about 20 minutes, preferably at least 30 minutes, before reloading. It may be preferable to reload at a time close to the inflection point, however, depending on the microbial species in the microbial sample, it may also be possible to reload more than 2 hours after the inflection point is reached.As shown in the experimental section, if the sample is reloaded too early or too late from the inflection point, the microbial cells are not in a qualitatively metabolic state that will yield reliable antimicrobial susceptibility testing results. Therefore, performing the reloading step at or within a limited time after the inflection point has been found to be advantageous for calorimetric antimicrobial susceptibility testing to yield reliable results. Accordingly, in the methods herein, the microbial sample is first inoculated into an inoculation medium, and metabolic activity is followed for a period of at least about 20 minutes, preferably at least 30 minutes, until the metabolic rate has an R value of ≥ 0.98 or until the inflection point is reached. An aliquot of the incubated sample is then reloaded into fresh inoculation medium with or without an antimicrobial (within a maximum of 2 hours after the inflection point is reached). Preincubating the microbial sample and reloading it around the inflection point has been found to be advantageous for this method to yield reliable results in antimicrobial susceptibility testing. Reloading for a period of at least about 20 minutes, preferably at least 30 minutes, when the metabolic rate has an R value of ≥ 0.98, or at the inflection point, or within the hours specified herein after the inflection point, is important to the method because the microbial cells at this time are in a qualitative state that will provide reliable AST results.

[0044] Furthermore, the inventors have found that the ratio of metabolic activity to the amount of antimicrobial molecule (i.e., metabolic activity / antibiotic molecule) suitable for use in antimicrobial susceptibility testing is important for obtaining the best and most reliable results in calorimetric methods for antimicrobial susceptibility. It has been found that if the total metabolic activity reloaded in step b) ii) is too low in terms of the amount of antimicrobial molecule used, the results may be interpreted as indicating that the microorganism in the microbial sample is susceptible to the antimicrobial (false negative—a very significant error rate). Conversely, if the total metabolic activity reloaded in step b) ii) is too high in terms of the amount of antimicrobial molecule used, the results may be interpreted as indicating that the microorganism is resistant to the antimicrobial (false positive—a significant error rate). It has been found that using the correct ratio of total metabolic activity to the amount of antimicrobial molecule in the reloading step b) is therefore important for calorimetric methods for antimicrobial susceptibility testing to produce reliable results. Therefore, reloading the appropriate amount of metabolic activity is important for the method to produce the most reliable results.

[0045] For procedural efficiency, it may be preferable to reload only one aliquot in step b), but one way to ensure that the correct ratio of total metabolic activity to amount of antimicrobial molecule is used is to reload aliquots with different amounts of the incubated sample from step a) (i.e., different total metabolic activity amounts) into inoculation medium with and without the antimicrobial, respectively, in step b).

[0046] The metabolic activity in the reloaded samples with and without antimicrobial agents is then tracked in step c) by determining one or more calorimetric signals. It has been found that samples inoculated with aliquots in which metabolic activity in the medium without antimicrobial agents does not appear immediately after reloading, but appears within about 6 hours, for example, between about 15 minutes and about 6 hours, or between about 15 minutes and about 3 hours after reloading, provide the most reliable results in antimicrobial susceptibility testing (i.e., reloaded samples in which the time to detection of metabolic or calorimetric signals is within about 6 hours, for example, between 15 minutes and about 6 hours, or between 15 minutes and about 3 hours). Therefore, the aliquots reloaded into the inoculated medium with antibiotics corresponding to the aliquots in the antimicrobial-free inoculated medium in which the calorimetric signals appear within about 6 hours, for example, between about 15 minutes and about 6 hours, or between about 15 minutes and about 3 hours after reloading, are used to determine antimicrobial susceptibility. If too little total metabolic activity is reloaded, the ratio of total metabolic activity to the amount of antimicrobial molecules in the reloaded sample will be too low, and the antimicrobial susceptibility test result may be interpreted as indicating that the microorganism is susceptible to the antimicrobial when it is not. If too much total metabolic activity is reloaded, the ratio of total metabolic activity to the amount of antimicrobial molecules in the reloaded sample will be too high, and the antimicrobial susceptibility test result may be interpreted as indicating that the microorganism is not susceptible to the antimicrobial when it is not. As demonstrated in the experimental section, a calorimetric signal appearing earlier than 15 minutes after reloading increased the risk of a false-positive result (interpreted as non-susceptible), and a calorimetric signal appearing later than about 6 hours, typically later than 3 hours, after reloading increased the risk of a false-negative result (interpreted as susceptible). Preferably, the aliquot used for antimicrobial susceptibility determination is the aliquot corresponding to the aliquot reloaded into inoculation medium without antimicrobial, in which a calorimetric signal is detected from about 15 minutes to about 3 hours after reloading.However, in some cases, metabolic signals appearing up to 6 hours after reloading may be acceptable, especially if the metabolic activity of the microorganisms in the sample is low. Non-limiting examples of such low metabolic activity microorganisms include Cutibacterium acnes, Mycobacterium tuberculosis, Helicobacter pylori, and Campylobacter spp.

[0047] Although reloading only one aliquot is preferred for procedural efficiency, in some cases, in order to obtain the most reliable results of the methods herein, it may be preferable to reload two or more aliquots with different total metabolic activities in each of steps b)i) and b)ii), and the calorimetric signals obtained from aliquots corresponding to those aliquots in which the metabolic capacity (metabolic activity) in the sample in step b)i) immediately after reloading is below the detection limit of one or more calorimetric signals and the metabolic capacity (metabolic activity) in the same aliquot is detected within about 6 hours, for example between about 15 minutes and 6 hours, or between about 15 minutes and about 3 hours after said reloading in step b)i), are used to determine sensitivity or insensitivity in step d). Typically, a pair of different aliquots of the incubated sample in step a) are transferred so that the reloaded sample in step b) has a metabolic activity of at least 0.1% to at most about 50% (e.g., about 1% to about 15%, e.g., 1% to about 10%, or about 5% to about 10%) of the incubated sample in step a) at the time of reloading. That is, if the metabolic activity in the incubated sample in step a) at the time of reloading is XJ / ml, then the reloaded sample in step b) will have a metabolic activity corresponding to about 0.1% to 50% of XJ / ml immediately after reloading. Preferably, one or at least two aliquots are transferred to the inoculation medium with and without the antimicrobial agent, respectively, so as to result in different starting metabolic activities in the reloaded sample. This ensures that the volume of the incubated sample in step a) is sufficient to reload a sufficient number of aliquots of different volumes, so that at least one aliquot meets the criterion that metabolic activity immediately after reloading is below the detection limit of one or more calorimetric signals but is detectable within about 6 hours, for example between 15 minutes and 3 hours, after reloading in step b)i).

[0048] In the methods herein, the amount of antimicrobial agent used in step b)ii) is typically constant if two or more aliquots are reloaded, with the amount of sample reloaded varying, if desired. However, it will be appreciated that it is also possible to use a constant amount of the incubated sample from step a) for the reloading step b) and instead inoculate this amount into an inoculation medium supplemented with different amounts of antimicrobial agent.

[0049] The microbial sample tested according to the present specification is a sample that potentially contains one or more types of microorganisms.The microbial sample can be, for example, a clinical sample from a subject, an environmental sample, a food or feed sample, and / or a purified microbial sample.Typically, the sample is a clinical sample obtained from a human subject or an animal subject.Such a clinical sample can be any body fluid or tissue sample, for example, blood, urine, plasma, synovial fluid, cerebrospinal fluid, bone tissue, soft tissue, connective tissue, skin tissue, sputum, lavage fluid, plasma / serum, urine, peritoneal fluid, pericardial fluid, and / or pleural fluid sample.

[0050] The temperature during the incubation step of the method varies depending on the type of sample. For example, environmental samples may be incubated at a lower temperature than clinical samples, depending on the type of microorganism expected to be present in the sample. Clinical samples are typically incubated at a temperature of about 35°C to about 39°C, e.g., about 36°C to about 38°C, e.g., about 37°C, and for certain microorganisms, a temperature reduction to 25°C is necessary for correct interpretation of antimicrobial susceptibility testing. Additionally, other growth conditions, such as whether the microorganism grows aerobically or anaerobically and the type of medium used, must be adapted to the type of sample and / or the type of microorganism expected to be present in the sample.

[0051] Different microorganisms produce different specific calorimetric signals when incubated under identical conditions. Thus, it is possible to determine which microorganisms are present in a microbial sample by studying one or more calorimetric signals obtained under one or more different incubation conditions (e.g., incubation with different inoculation media, different temperatures, aerobic / anaerobic conditions, etc.).

[0052] To determine the identity of the microorganisms present in a microbial sample, the obtained calorimetric signal or signals are compared with the calorimetric data (i.e., calorimetric signals) previously obtained for different microorganisms incubated under the same conditions. Due to the difference in calorimetric signals between different microorganisms, it is possible to determine the identity of the microorganisms in the sample, for example, whether they are gram-positive or gram-negative, whether they are fungi and / or bacteria, and / or which microbial species are present. The level of specificity in determining identity depends on the type of sample and the desired data to be obtained in view of it. In a mixed sample, i.e., a sample containing two or more types of microorganisms, the obtained calorimetric signal is the combined calorimetric signal derived from each microorganism present in the sample. When two or more microorganisms are present in the same sample, they may affect each other's metabolic activity, and as a result, the calorimetric signal is not simply the sum of the calorimetric signals obtained when the same microorganism is incubated individually. The combined calorimetric signal may therefore be higher or lower than would be predicted based on the calorimetric signal obtained when incubating single microbial samples of the same microorganism.

[0053] For example, based on the calorimetric signal obtained from the sample reloaded in step b)i) into an inoculation medium without an antimicrobial agent, the Gram stain 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. This is therefore another advantage of the method, namely, not only can it provide a result on whether the microorganism is susceptible to the antimicrobial agent being tested, but it can also provide data on the identity of the specific microorganisms present. This is particularly important for the analysis of clinical samples.

[0054] Based on the one or more calorimetric signals obtained in step c) from the samples reloaded with the inoculation medium with and without the antimicrobial, in step d) it is determined whether the one or more microorganisms in the sample are susceptible or insusceptible to the antimicrobial by comparing the one or more calorimetric signals obtained from each incubated sample with and without the antimicrobial, typically for up to 48 hours, depending on the antimicrobial used, from the time metabolic activity becomes detectable in the positive control (i.e., the sample obtained from b)i). This determination is made based on aliquots in which a calorimetric signal is detected within about 6 hours, for example, between 15 minutes and 6 hours, or between 15 minutes and 3 hours, after reloading according to step b)i), as described above. For incubation of a subset of microorganisms with antimicrobials, up to two weeks may be required for correct interpretation of the AST. If the microorganisms in the microbial sample are susceptible to the antimicrobial being tested, this will result in a difference in the calorimetric signal being investigated between the sample incubated with the antimicrobial and the sample incubated without the antimicrobial. Calorimetry can be used to not only provide a yes or no answer regarding susceptibility, but also to obtain information regarding the degree of susceptibility of a microorganism by determining the gradual effect of an antimicrobial on the metabolic (i.e., calorimetry) signal. Determining the identity of the microorganisms in a microbial sample can facilitate interpreting the calorimetric signal obtained from a sample incubated in the presence of an antimicrobial to determine whether they are susceptible or non-susceptible to the antimicrobial.

[0055] One challenge with antimicrobial susceptibility testing of microbial samples is mixed samples, i.e., microbial samples containing two or more types of microorganisms. In such samples, some microorganisms may be susceptible to a particular antimicrobial, while others are not. The present method allows for the determination of not only whether the entire sample is likely to be susceptible to an antimicrobial, but also the identity (e.g., Gram-positive / negative, species, fungi / bacteria) of the specific microorganisms present in the sample. Rather, by comparing the calorimetric signal obtained from a sample incubated without an antimicrobial with the calorimetric signal obtained from a sample incubated with an antimicrobial, it is possible to demonstrate which microorganisms in the microbial sample are susceptible to each of the specific antimicrobials tested. In this way, treatment of a patient with, for example, a microbial infection, can be tailored to that particular patient.

[0056] The analysis of calorimetry data in the methods herein for antimicrobial susceptibility testing and / or determining the identity of microorganisms can be performed by visual inspection or automatically by using a database of calorimetry data for various microorganisms. Such a database can therefore be used to establish a correlation between at least one calorimetric signal and multiple microorganisms to distinguish them from each other. Software can be used for this analysis. The database can be obtained by incubating known microorganisms under known conditions, and tracking their metabolic activity by calorimetry to obtain one or more calorimetric signals.

[0057] The antimicrobial agents used in antimicrobial susceptibility testing can be a single type of antimicrobial agent or a combination of two or more antimicrobial agents. The antimicrobial agents can be antibiotics or antifungal agents, or a combination thereof. As known to those skilled in the art, the antimicrobial agents used are selected based on the type of sample and the microorganisms that may be expected to be present in the sample. Typically, 1 to 20 different antimicrobial agents are used in the method herein, alone and / or in combination. Non-limiting examples of antimicrobial agents that may be used are cefoxitin, rifampicin, fosfomycin, ciprofloxacin, amoxicillin, clavulanic acid, piperacillin, tazobactan, meropenem, imipenem, ceftriaxone, linezolic acid, cefotaxime, vancomycin, clindamycin, fosfomycin, vancomycin, gentamicin, methicillin, oxacillin, rifampicin, cefazolin, cotrimoxazole, levofloxacin, cotrimoxazole, colistin, cotrimoxazole, cefepime, gentamicin, mipinem, meropenem, methicillin, oxacillin. Typical combinations of antimicrobial agents that may be used include, but are not limited to, ampicillin and sulbactam, amoxicillin and clavulanic acid, ceftazidime and avibactam, ceftolozane and tazobactam, and piperacillin and tazobactam. Different concentrations of the same antimicrobial agent or the same combination of antimicrobial agents may also be used.

[0058] Thus, by preparing several vials of incubation medium with the desired antimicrobial for reloading step b)ii), it is possible to test in parallel for susceptibility to 1) the same antimicrobial at different concentrations, 2) different antimicrobials, and / or 3) combinations of antimicrobials (at the same or different concentrations).

[0059] One or more adjuvants can be added to the inoculation medium containing the antimicrobial agent, for example, adjuvants that enhance and / or potentiate the activity of the antimicrobial agent. Non-limiting examples of such adjuvants are iron and beta-lactam inhibitors.

[0060] In the methods herein, it is also possible to use two or more inoculation media in both step a) and / or reloading step b). In this manner, by incubating samples in different vials containing different inoculation media, the inoculation media can be adapted and / or optimized for the specific microorganisms potentially present in the microbial sample. For example, it is possible to use an inoculation medium for the growth of aerobic microorganisms and an inoculation medium for the growth of anaerobic microorganisms. As known to those skilled in the art and as described above, growth conditions must also be adapted depending on whether aerobic or anaerobic microorganisms are expected to be present in the sample. Non-limiting examples of media suitable for use in the methods herein are Mueller-Hinton broth, thioglycollate broth, salt mannitol, modified Sabouraud broth, and / or Schaedler broth.

[0061] The time for which the sample should be incubated with and without the antimicrobial agent in step c) depends on the type of microbial sample and the microorganisms present. Typically, the incubation in step c) is carried out for a period of about 1 hour to about 48 hours, counting from the time to detection (i.e., the time at which the selected calorimetric signal appears in the sample inoculated without the antimicrobial agent).

[0062] The present specification is also directed to systems for performing the calorimetric methods for antimicrobial susceptibility testing disclosed herein, such systems including a calorimeter and software for performing the analysis of the calorimetric signals.

[0063] The present specification is also directed to a kit for carrying out the calorimetric method for antimicrobial susceptibility testing herein. Such a kit comprises: i) at least one antimicrobial-free inoculation medium; ii) the inoculation medium of i) supplemented with an antimicrobial agent; iii) one or more databases establishing correlations between at least one calorimetric signal and a plurality of microorganisms; iv) optionally, a software component including functionality for determining the Gram stain status, genus, and / or species of one or more microorganisms; and v) a software component that includes functionality for determining whether the microorganism is susceptible or non-susceptible to the at least one antimicrobial agent; Includes:

[0064] Alternatively, instead of an antimicrobial-free inoculation medium and an antimicrobial-supplemented inoculation medium, the kit may include an inoculation medium and a separately provided antimicrobial that is mixed into the inoculation medium to provide an antimicrobial-supplemented inoculation medium.

[0065] Details regarding suitable inoculation media and antimicrobial agents for use in such kits are provided elsewhere herein. The kit may further include a software component that determines whether the ratio of metabolic activity to number of antimicrobial molecules is suitable for performing antimicrobial susceptibility testing.

[0066] The present specification also provides a computer-implemented method performed by a control unit, comprising: receiving first measurement data indicative of the calorimetric signal of step a) indicative of metabolic activity in one or more samples comprising one or more microorganisms in an inoculation medium; determining that the metabolic rate R value is ≧0.98 for at least about 20 minutes, preferably at least 30 minutes, or that the metabolic activity of the incubated sample of step a) has reached an inflection point; indicating to a user of the system that reloading of one or more aliquots of the incubated sample of step a) should occur two hours after the inflection point is reached; receiving second measurement data indicative of the calorimetric signal of step c; classifying the one or more microorganisms as susceptible or non-susceptible to the antimicrobial agent by comparing the second measurement data of the one or more samples to which the antimicrobial agent has been added with the second measurement data of the sample to which the antimicrobial agent has not been added. Such methods may be used to analyze the calorimetric signals of the calorimetry methods herein.

[0067] This specification also provides: processor, and memory wherein the memory has instructions executable by the processor, whereby the control unit operates to perform the computer-implemented methods described herein.

[0068] The present specification is also directed to a computer program comprising computer program code, the computer program code being adapted, when executed on a processor, to implement the calorimetric method for antimicrobial susceptibility testing of the present specification. The present specification is also directed to a computer program product comprising a computer-readable storage medium, the computer-readable storage medium having the computer program thereon.

[0069] Also disclosed herein is a calorimetric method for antimicrobial susceptibility testing of a microbial sample, comprising: a) incubating a sample (potentially) containing one or more microorganisms in an inoculation medium and determining the metabolic activity of the incubated sample by calorimetry; b) determining the ratio of metabolic activity of the incubated sample of step a) to the amount of antimicrobial molecule used in the antimicrobial test; i) if the ratio of metabolic activity to amount of antimicrobial molecule is suitable for performing antimicrobial susceptibility testing, proceeding with antimicrobial susceptibility testing according to steps c)-d); and ii) if the ratio of metabolic activity to amount of antimicrobial molecule is not suitable for performing antimicrobial susceptibility testing, adjusting the incubated sample and / or the amount of antimicrobial molecule of step a) to obtain a ratio of metabolic activity to amount of antimicrobial molecule that is suitable for performing antimicrobial susceptibility testing, and then proceeding with antimicrobial susceptibility testing according to steps c) to d); c) i) incubating an aliquot of the incubated sample of step a) or an aliquot of the conditioned sample of step b)ii) in an inoculation medium without the addition of an antimicrobial agent and determining one or more calorimetric signals to determine the Gram stain status, genus, and / or species of one or more microorganisms in said sample; and ii) incubating an aliquot of the incubated sample of step a) or an aliquot of the adjusted sample of step b)ii) in an inoculation medium identical to step c)i), but supplemented with an antimicrobial agent of the molecular weight determined according to step b)i) or b)ii), and determining one or more calorimetric signals identical to step c)i); and d) determining whether said one or more microorganisms are susceptible or non-susceptible to said antimicrobial agent by comparing the one or more calorimetric signals obtained in step c)i) with the calorimetric signals obtained in step c)ii); comprising or consisting of Also disclosed are calorimetric methods that are performed without first isolating said one or more microorganisms from said sample.

[0070] Also disclosed herein is a calorimetric method for antimicrobial susceptibility testing of a microbial sample, comprising: a) providing a microbial sample, and an inoculation medium without an antimicrobial agent and an identical inoculation medium with an antimicrobial agent; b) i) an aliquot of the microbial sample in the inoculation medium without an antimicrobial agent; and ii) an aliquot of said microbial sample in said inoculation medium with an antimicrobial agent; Incubating the c) determining the Gram stain status, 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 d) using one or more calorimetric signals obtained from the microbial sample inoculated according to steps b)i) and b)ii), i) determining whether the sample has a ratio of metabolic activity to quantity of antimicrobial molecule suitable for performing antimicrobial susceptibility testing; and ii) if the sample has a ratio of metabolic activity to amount of antimicrobial molecule as determined in step d)i) that is suitable for antimicrobial susceptibility testing, determining whether the microorganisms in the microbial sample are susceptible or non-susceptible to the antimicrobial by comparing the calorimetric signal obtained from an inoculated medium without the antimicrobial with the same calorimetric signal obtained from an inoculated medium with the antimicrobial. comprising or consisting of Calorimetric methods are also disclosed that are performed without first isolating the microorganisms from the sample.

[0071] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0072] Experimental Section Four homogenized clinical tissue biopsies obtained from the Orthopedic Surgery Department (Karolinska Hospital, Sweden) were used to determine the influence of qualitative and quantitative metabolic activity and amount of antimicrobial drug molecules on producing correct AST results.

[0073] Example 1 Microbiological sample processing and pre-incubation Four clinical tissue biopsies obtained from the Orthopedic Surgery Department (Karolinska Hospital, Sweden) were homogenized using a bullet blender BB50-DX (next advance) with the following settings: speed 4, stainless steel UFO beads (3.5 mm diameter), 12 min, and the biopsies were dissolved in 5 ml of liquid in a 50 ml test tube. One biopsy was known to be infected with a Staphylococcus epidermidis strain susceptible to cefotaxime (CTX). The other biopsy was infected with Staphylococcus aureus resistant to CTX. The third biopsy was known to be infected with Pseudomonas aeruginosa susceptible to ciprofloxacin (CIP). The fourth biopsy was infected with Pseudomonas aeruginosa resistant to CIP. From the tissue sample, 50 μl was added to Mueller-Hinton broth (Sigma-Aldrich, Darmstadt, Germany) that had been dispensed into a sterile plastic insert in a microcalorimetry vial (Symcel AB, Solna, Sweden). The microcalorimetry vial was sealed and placed in a calScreener (Symcel AB, Solna, Sweden). Two samples were incubated, and the heat produced by each sample was measured at 37°C for 18 hours.

[0074] Example 2 Qualitative influence of metabolic activity on AST To examine the potential effect of metabolic rate on AST accuracy, bacteria with different initial metabolic rates were exposed to identical antibiotic concentrations.

[0075] For qualitative assessment, all biopsies were incubated as described above. Staphylococci-containing tissue samples were then collected at different metabolic rates: A = before the inflection point, B = within 2 hours after the inflection point, and C = 3-4 hours after the inflection point. They were then 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 inserts within microcalorimetry vials. Aliquots (A-C) with different metabolic activities per volume of the incubated biopsies were adjusted to have the same metabolic activity across all reloaded samples, allowing the impact of metabolic quality to be assessed independently among samples with consistent metabolic activity.

[0076] Another 300 μl of Mueller-Hinton broth without antibiotics was inoculated with a tissue sample containing bacteria as a metabolic positive control with the same metabolic rates (A-C) as above. The microcalorimetry vials were sealed and placed in a calScreener (Symcel AB, Solna, Sweden). The heat produced by each sample was measured at 37°C for 18 hours.

[0077] 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. result The inflection point represents an inflection in metabolic rate, where the metabolic rate increases linearly for over 30 minutes with an R value of ≥ 0.98, followed by a drop to R < 0.98. This change represents the point of change from the microbial maximum metabolic rate and was found to be the optimal qualitative sampling point for reloading the microbial sample. In the experimental setup for S. epidermidis and S. aureus, the inflection point was determined to be 3 hours 25 minutes for S. epidermidis (see Figure 1) and 3 hours 30 minutes for S. aureus (see Figure 3). For P. aeruginosa, the inflection point was determined to be 2 hours 30 minutes for susceptible strains (Figure 5) and 2 hours 15 minutes for resistant strains (Figure 7).

[0078] S. epidermidis (susceptible to CTX) In the case of S. epidermidis, qualitative evaluation (see Figure 2) showed that inoculum A, collected 70 minutes before the inflection point, showed no metabolic activity when exposed to CTX, indicating susceptibility to CTX, which is correct because the S. epidermidis used in this experiment is susceptible to CTX. Inoculum B, collected 1 hour after the inflection point, also showed no metabolic activity, indicating susceptibility to CTX, which is correct. Inoculum C, collected 4 hours after the inflection point, showed metabolic activity when exposed to CTX, indicating a lack of susceptibility to the antibiotic, resulting in a false-positive result (non-susceptibility). This experiment therefore demonstrated that when the reload step is performed too late after the inflection point, the metabolic activity of the microorganism is not in the appropriate phase to produce reliable AST results.

[0079] S. aureus (resistant to CTX) In the case of S. aureus (see Figure 4), inoculum A, taken before the inflection point, showed no metabolic activity, indicating susceptibility to CTX. This is a false-negative result (susceptibility) because the S. aureus strain used in this experiment is resistant to CTX. Inocula B and C, taken immediately after the inflection point and 3 hours later, on the other hand, showed metabolic activity, indicating resistance to CTX, which is the correct result. This experiment therefore demonstrated that if the reloading step is performed too early after the inflection point, the metabolic activity of the microorganism is not in the appropriate phase to produce reliable AST results.

[0080] P. aeruginosa (susceptible to CIP) In the evaluation of P. 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 P. aeruginosa was susceptible to CIP. The no-antibiotic control showed metabolic activity within the first 3 hours of incubation, as expected. Inoculum B, taken 1 hour after the inflection point, also did not show any metabolic activity, confirming the susceptibility of P. aeruginosa to CIP. The no-antibiotic control again 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, resulting in a false-positive result (non-susceptibility). Furthermore, metabolic activity in the no-antibiotic control began before the 15-minute incubation, indicating an inoculum that was too high at this point. As seen in the case of S. epidermidis, which is susceptible to CTX, the metabolic activity of P. aeruginosa was not in the appropriate phase to obtain reliable AST results.

[0081] P. aeruginosa (resistant to CIP) For a second strain of P. aeruginosa (see Figure 8), inoculum A, taken 1 hour before the inflection point, showed no metabolic activity, indicating susceptibility to ciprofloxacin. This is a false-negative result (susceptible) because the P. aeruginosa strain used in this experiment is resistant to CIP. Inocula B and C showed metabolic activity in the presence of CIP, which identified these strains as resistant. However, inocula C developed metabolic activity before the 15-minute incubation, indicating that this time point was not suitable for reliable testing.

[0082] As shown in these four examples, inocula taken long after the inflection point is reached can produce false-negative results (susceptible) even if the inoculated strain is susceptible to the antimicrobial being tested (see Figure 2, inoculum C). On the other hand, inocula taken before the inflection point can produce false-negative results even if the inoculated strain is resistant (see Figure 4, inoculum A). The P. aeruginosa example illustrates the importance of performing susceptibility testing at the correct metabolic activity, which is either at the inflection point, as demonstrated herein, or within a specific period after the inflection point (Figures 6 and 8).

[0083] Example 3 Quantitative effects of metabolic activity To further investigate the potential impact of the total amount of metabolic activity used for reloading in the reloading step (i.e., different metabolic activities per volume unit after reloading) on ​​AST outcomes, aliquots with different total metabolic activities (i.e., different volumes) were reloaded at the inflection point (where all samples had the same metabolic rate) and exposed to the same antibiotic concentrations as in the qualitative assessment in Example 2. Dilution of the aliquots yielded different percentages of total metabolic activity per volume unit of sample, corresponding to 30% to 10% of the metabolic activity, which were then reloaded into newly incubated samples (Figures 9A-C).

[0084] For quantitative evaluation, different percentages of total metabolic activity of the incubated microbial samples from Example 1 were thus obtained at inflection points, whereby all samples were reloaded with the same metabolic rate: A) 30% of the total metabolic activity obtained at the inflection point from the tissue sample containing bacteria, B) 20% of the total metabolic activity obtained at the inflection point from the tissue sample containing bacteria, and C) 10% of the total metabolic activity obtained. Aliquots were added to 300 μl of Mueller-Hinton broth (Sigma-Aldrich, Darmstadt, Germany) supplemented with 16 mg / L CTX in a sterile plastic insert in a microcalorimetry vial. Another 300 μl of Mueller-Hinton broth without antibiotics but with the same percentage of total metabolic activity as the bacteria-containing tissue sample exposed to antibiotics was used as a metabolic positive control. The microcalorimetry vials were sealed and placed in a calScreener (Symcel AB, Solna, Sweden). The heat produced by each sample was measured at 37°C for 18 hours.

[0085] result Sample A) (reloaded with 30% of the total metabolic activity) showed metabolic activity when exposed to CTX, indicating resistance to the antibiotic, which produced a false-positive result. Sample B) reloaded with 20% of the total metabolic activity showed metabolic activity when exposed to CTX, indicating resistance to the antibiotic, which also produced a false-positive result. In both of these examples, metabolic activity began already at the start of incubation, which already indicated that the results were unreliable. Sample C) reloaded with 10% of the total metabolic activity showed no metabolic activity when exposed to CTX, indicating sensitivity to the antibiotic, which produced a correct negative result. Metabolic activity in the sample without antibiotics began after 2 hours of incubation.

[0086] This example therefore demonstrates the importance of reloading the appropriate amount of metabolic activity in terms of the amount of antimicrobial molecule used in metabolic testing, i.e., the ratio of metabolic activity to amount of antimicrobial molecule after reloading needs to be accurate to obtain reliable AST results, as further illustrated in the P. aeruginosa example.

[0087] conclusion The above example illustrates the correlation between metabolic inflection points and optimal quantitative transfer of total metabolic activity to identify the optimal qualitative metabolic activity sampling timeframe required to obtain an accurate antimicrobial susceptibility readout. Using the example of orthopedic tissue biopsies infected with susceptible clinical isolates of S. epidermidis and P. aeruginosa and resistant clinical isolates of S. aureus and P. aeruginosa, the importance of both qualitative and quantitative metabolic parameters is illustrated.

[0088] It was concluded that the optimal time to reload the microbial sample after the first incubation step is at the inflection point or within a maximum of 2 hours after the inflection point. The inflection point for metabolic activity, therefore, refers to the inflection point from a linear increase in metabolic rate, i.e., R ≥ 0.98, to a drop to R < 0.98. The linear increase in metabolic activity, i.e., R ≥ 0.98, preferably continues for some time before the drop to R < 0.98, which defines the inflection point. Typically, the linear increase in metabolic activity continues for at least 10 to 30 minutes, preferably at least 20 or 30 minutes, before the drop to R < 0.98. The importance of sampling at the correct qualitative metabolic state is illustrated with results in Figures 2, 4, 6, and 8, which have an impact on the accuracy of the sensitivity readout. Samples reloaded before the inflection point increase the risk of false-negative readings in resistant isolates, while a time window of more than 3 hours after the inflection point increases the risk of false-positive readings in susceptible isolates. To ensure this does not occur, it is important that the time point at which the microbial metabolic activity is reloaded is uniform and in the correct qualitative state. While reloading within 2 hours of the inflection point is preferred, this state can be reached even before the inflection point if the metabolic rate has an R of ≥ 0.98 for at least 20 minutes, preferably 30 minutes, before reloading. Therefore, it may be possible to reload a microbial sample when the metabolic rate has an R of ≥ 0.98 for more than 20 minutes, since the microorganisms in the sample have metabolic quality equivalent to that at or within 2 hours of the inflection point.

[0089] Figure 9 illustrates the importance of quantitatively transitioning the correct total metabolic activity at the inflection point to avoid false-negative or false-positive results (the impact of false-positive results is illustrated in Figure 5). It was concluded that samples used for post-reload AST determination are those in which the reloaded aliquot produces a metabolic signal that is detected between 15 minutes and 3 hours after reloading. If a metabolic signal (i.e., calorimetry signal) is detected earlier than 30 minutes after reloading, there is a risk of a false-positive result (interpreted as insensitive), whereas if a metabolic signal is first detected later than 3 hours after reloading, there is a risk of a false-negative result (interpreted as sensitive). Therefore, although a metabolic signal that appears up to 6 hours after reloading may be acceptable in some cases, especially if the microorganisms in the sample have low metabolic activity, reloaded samples in which a metabolic signal appears before or after the 15 minutes to 3 hour time frame (i.e., time to detection) are typically not used for AST. Non-limiting examples of such microorganisms with low metabolic activity are Cutibacterium acnes, Mycobacterium tuberculosis, Helicobacter pylori, and Campylobacter spp.

[0090] Although the present invention has been described in conjunction with its detailed description, it will be understood that the foregoing description is intended to illustrate, but not to limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0091] Unless expressly stated otherwise, each of the preferred features described herein can be used in combination with any and all of the other preferred features described herein.

Claims

1. 1. A calorimetric method for antimicrobial susceptibility testing of a microbial sample, comprising: a) incubating a sample potentially containing one or more microorganisms with an inoculation medium and following metabolic activity by determining one or more calorimetric signals of the incubated sample; b) when the metabolic rate R value is ≧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, one or more aliquots of said incubated sample of step a); i) inoculation medium without the addition of antimicrobial agents, and ii) an inoculation medium identical to step b) i), but with the addition of an antimicrobial agent; Step 2: Reload the c) incubating the samples of steps b)i) and b)ii) and determining one or more calorimetric signals in said samples; and d) determining whether the one or more microorganisms are susceptible or non-susceptible to the antimicrobial agent by comparing the one or more calorimetric signals obtained in step c) from the sample spiked with the antimicrobial agent with the one or more calorimetric signals obtained in step c) from the sample not spiked with the antimicrobial agent. A calorimetry method comprising:

2. 10. The calorimetry method of claim 1, performed without first isolating said one or more microorganisms from said microbial sample.

3. 3. The calorimetry method of claim 1 or 2, wherein two or more aliquots with different total metabolic activities are reloaded in steps b)i) and b)ii), respectively, and the calorimetry signals obtained from aliquots corresponding to those in which the metabolic activity in the sample in step b)i) immediately after reloading is below the detection limit of one or more calorimetry signals and the metabolic activity in the same aliquot is detected within about 6 hours, for example between 15 minutes and 3 hours, after the reloading in step b)i), are used to determine sensitivity or insensitivity in step d).

4. 4. The calorimetric method according to any one of claims 1 to 3, wherein the sample is a clinical sample of a subject, an environmental sample, a food or feed sample, and / or a purified microbial sample.

5. 5. The calorimetry method of claim 1, further comprising the step of determining the Gram stain status, genus, and / or species of one or more microorganisms in the microbial sample using the one or more calorimetric signals obtained in step c) from a sample to which no antimicrobial agent has been added.

6. 6. The calorimetry method according to any one of claims 1 to 5, wherein a combination of two or more different types of antimicrobial agents is used in the same inoculation medium in step b)ii).

7. 7. The calorimetry method according to any one of claims 1 to 6, wherein more than one type of inoculation medium is used in step a) and / or step b).

8. 8. The calorimetry method according to any one of claims 1 to 7, wherein at least two inoculation media containing different concentrations of one or more antimicrobial agents are used in step b)ii).

9. 9. The calorimetry method according to any one of claims 1 to 8, wherein steps b), c) and / or d) are carried out using a database that establishes correlations between at least one calorimetric signal and a plurality of microorganisms.

10. 10. The calorimetry method of 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 stain status, genus, and / or species of one or more microorganisms in the microbial sample.

11. 11. The calorimetry method according to any one of claims 1 to 10, wherein the one or more inoculation media of step a) and / or step b) further comprise one or more adjuvants, for example adjuvants that enhance and / or potentiate the activity of the antimicrobial agent.

12. 12. The calorimetry method of any one of claims 1 to 11, wherein the calorimetry signal is time to detection, metabolic rate, maximum metabolic rate, area under the curve, area under the curve before maximum metabolic rate, heat flow, and / or a ratio between any of these signals.

13. 13. The calorimetry method according to any one of claims 1 to 12, wherein the incubation in step c) is carried out for a period of about 1 hour to about 48 hours from the time until detection of said one or more calorimetric signals.

14. 14. The calorimetry method of any one of claims 1 to 13, wherein the inoculation medium is Mueller-Hinton broth culture medium, thioglycollate broth culture medium, salt mannitol culture medium, modified Sabouraud broth culture medium, and / or Schuller broth culture medium.

15. 15. A system for carrying out the calorimetry method of any one of claims 1 to 14, comprising a calorimeter and software for carrying out the analysis according to steps a), b), c), and / or d) of the method and / or for carrying out the determination of the Gram stain status, genus, and / or species of one or more microorganisms of claim 5 in the microbial sample.

16. A kit for carrying out the calorimetry method according to any one of claims 1 to 14, comprising: i) at least one antimicrobial-free inoculation medium; ii) the inoculation medium of i) to which an antimicrobial agent has been added; iii) one or more databases establishing correlations between at least one calorimetric parameter and a plurality of microorganisms; iv) optionally a software component that includes functionality for determining the Gram stain status, genus, and / or species of one or more microorganisms; and v) a software component that includes functionality for determining whether the microorganism is susceptible or non-susceptible to the antimicrobial agent; Includes a kit.

17. A computer program comprising computer program code, the computer program code being adapted to implement a method according to any one of claims 1 to 14 when the computer program code is run on a processor.

18. 20. A computer program product including a computer readable storage medium, the computer readable storage medium having the computer program of claim 17.

19. 1. A computer-implemented method performed by a control unit, the computer-implemented method comprising: receiving first measurement data indicative of the calorimetry signal of step a), wherein the calorimetry signal of step a) is indicative of metabolic activity in one or more samples comprising one or more microorganisms in an inoculation medium; and determining that the metabolic rate R value is ≧0.98 for at least about 20 minutes or that the metabolic activity of the incubated sample of step a) has reached an inflection point; indicating to a user of the system that reloading of one or more aliquots of the incubated sample of step a) should occur two hours after the inflection point is reached; receiving second measurement data indicative of the calorimetric signal of step c); classifying the one or more microorganisms as susceptible or non-susceptible to the antimicrobial agent by comparing the second measurement data of the one or more samples to which the antimicrobial agent has been added with the second measurement data of the sample to which the antimicrobial agent has not been added. wherein the calorimetric signal is optionally obtained by carrying out a calorimetry method according to any one of claims 1 to 14. Computer-implemented methods.

20. processor, and memory 20. A control unit comprising: said memory having instructions executable by said processor, whereby said control unit is operable to perform the computer-implemented method of claim 19.

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