SE2350180A1 - Method for antimicrobial susceptibility testing - Google Patents

Abstract

ABSTRACT The present document is directed to a calorimetric method for determining the 5 antimicrobial susceptibility of microorganisms in a sample, such as a clinical tissue sample. According to the method, the microbial sample is exposed to an antimicrobial when the sample has reached a specific metabolic activity and the difference in calorimetric signal(s) is compared to a sample incubated without the antimicrobial to determine if the microorganism(s) in the sample are susceptible or 10 non-susceptible to the antimicrobial(s).

Description

108580 METHOD FOR ANTIMICROBIAL SUSCEPTIBILITY TESTING TECHNICAL FIELD The present document is directed to a calorimetric method for determining antimicrobial susceptibility of microorganisms in a sample, such as a clinical tissue sample.

BACKGROUND The performance of antimicrobial susceptibility testing is to confirm susceptibility to chosen empirical antimicrobial agents or to detect resistance in individual bacterial isolates that will alter the chosen empirical therapy to a targeted therapy based on the specific antibiogram.

There are a large number of antimicrobial susceptibility determination methods, for example broth microdilution, disk diffusion assays, gradient diffusion and semi- and automated devices (such as the VlTEK®2 instruments, BD Phoenix, Q linea ASTar devices). All standard assays are tuned to the correct ratio of the number of microorganisms to antimicrobial substance to give reproducible results.

For assessing susceptibility, two major test definitions exist i) a quantitative minimal inhibitory concentration determination (MIC) and ii) relating the MIC to a qualitative breakpoint determination (BP).

MIC is the lowest concentration of an antimicrobial (like an antifungal, antibiotic, bactericidal or bacteriostatic) drug that will inhibit the visible growth of a microorganism after overnight incubation. MlCs can be determined on plates of solid growth medium (agar growth media) or by broth dilution methods (liquid growth media) after a pure culture is isolated.

For example, to identify the MIC via broth dilution, identical doses of bacteria are cultured in wells of liquid media containing progressively lower concentrations of 108580 2 the drug. The minimum inhibitory concentration of the antibiotic is between the concentrations of the last well in which no bacteria grew and the next lower dose, which allowed bacterial growth. Broth dilution tests involve preparing two-fold dilutions of antibiotics (e.g., 1, 2, 4, 8, and 16 ug/mL) in a liquid growth medium dispensed in test tubes. The antibiotic-containing tubes are inoculated with a standardized bacterial suspension of 1-5><105CFU/mL and incubated overnight at 35°C. The precision of this method is considered to be plus or minus 1 two-fold concentration, due in large part to the practice of manually preparing serial dilutions of the antibiotics.

Disc diffusion assays, by the Kirby-Bauer method, is a standardized technique for testing rapidly growing pathogens for quantitatively determining the MIC. A standardized inoculum is swabbed onto the surface of an agar plate (150 mm in diameter). The reproducibility of this assay depends on the log growth phase of microorganisms. Filter paper disks impregnated with a standardized concentration of an antimicrobial agent are placed on the surface, and the size of the zone of inhibition around the disk is measured in mm after overnight incubation.

Gradient diffusion Etest and semi-and automated devices, such as the ones above, may also be used for quantitatively determining the MIC.

Clinical breakpoints give an interpretation of antimicrobial susceptibility testing results. Breakpoints can assist in determining if an antimicrobial is potentially useful in the treatment of a microbial infection. Breakpoint setting requires integration of knowledge of the wild-type distribution of MlCs, assessment of the pharmacokinetics/pharmacodynamics of the antimicrobial, and study of the clinical outcome of infections when the antimicrobial is used. Depending on the testing method, breakpoints are expressed as either a MIC (in mg/liter or ug/ml) or a Disk diffusion zone diameter (in mm). ln general, all susceptibility testing methods require breakpoints, also known as interpretive criteria, so that the results of the tests can be interpreted as the microorganism being susceptible, intermediate, or non-susceptible. Clinical breakpoints, which refer to those concentrations (MlCs) 108580 3 that separate isolate where there is a high Iikelihood of treatment success from those isolate where treatment is more likely to fail. These breakpoints are derived from prospective human clinical studies comparing outcomes with the MlCs of the infecting pathogen. The third use of the term "breakpoint" refers to antimicrobial concentrations calculated from knowledge of a pharmacodynamic parameter and the dimension of that parameter that predicts efficacy in vivo. These are the pharmacokinetic/pharmacodynamic breakpoints, where data that have been generated in an animal model are extrapolated to humans by using mathematical or statistical techniques. lsothermal microcalorimetry (IMC) is a measurement technology for real-time monitoring and dynamic analysis of chemical, physical and biological processes. Over a period of minutes or days, IMC determines the onset, rate, extent, and energetics of such processes for specimens in a closed system measured in uW. For isothermal calorimetry, the heat release at a constant set temperature is measured. The term 'micro' is applied for smaller systems where the heat release is in the micro-watt range.

For isothermal (micro)calorimetry measurements, the sample to be measured is placed in a sealed container in a constant temperature environment. Changes in heat flow due to chemical and biological processes are specific to the type of system studied, e.g., an isolate of any given bacterial or fungal species under specific metabolic conditions will give rise to a heat flow over time graph termed a thermogram. For monitoring of microbial, such as fungal or bacterial, systems, these specific thermograms can be used to derive the microbial species present and the metabolic viability of the microorganisms present in the sample.

SUMMARY An object of the present invention is thus to overcome or at least mitigate one or more of the problems described herein. 108580 4 The present document is therefore directed to a calorimetric method for antimicrobial susceptibility testing of a microbial sample, said method comprising or consisting of the steps of: a) incubating a sample potentially comprising one or more microorganisms in an inoculation medium and following the metabolic activity by determining one or more calorimetric signals of the incubated sample; b) when the metabolic activity of the incubated sample of step a) reaches the inflection point or within 2 hours after said inflection point is reached, reloading one or more aliquot(s) of said incubated sample of step a) in: i) an inoculation medium not supplemented with an antimicrobial; and ii) the same inoculation medium as in step b)i) but wherein said inoculation medium is supplemented with an antimicrobial; incubating the samples of step b)i) and b)ii and determining one or more calorimetric signal(s) in said samples; and d) determining if said one or more microorganisms is susceptible or non- susceptible to said antimicrobial by comparing the one or more calorimetric signal(s) obtained in step c) from the sample(s) supplemented with an antimicrobial to the one or more calorimetric signal(s) obtained in step c) from the sample(s) not supplemented with an antimicrobial.

The calorimetric method of the present document may be performed without first isolating said one or more microorganisms from said microbial sample.

Two or more aliquots with different total metabolic activity may be reloaded in step b)i) and b)ii), respectively, and the calorimetric signal(s) obtained from the aliquot(s) corresponding to the aliquot(s) wherein the metabolic viability in the sample(s) of step b)i) directly after reloading is below the detection limit of the one or more calorimetric signal(s) and the metabolic viability in the same aliquot(s) is detected between 15 min and 3 hours (i.e. time to detection) after said reloading in step b)i) is used for the determination of susceptibility or non-susceptibility in step d). 108580 The sample may be a clinical sample from a subject, an environmental sample, a food or feed sample, and/or a purified microbiological sample.

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

A combination of two or more different types of antimicrobials may used in the same inoculation medium in step b)ii). Alternatively or in addition, two or more types of inoculation media may be used in step a) and/or step b). The two or more inoculation media may contain different concentrations of one or more of an antimicrobial in step b)ii). The inoculation medium or inoculation media of step a) and/or step b) may comprise one or more adjuvants, such as an adjuvant potentiating and/or enhancing said antimicrobials activity. Non-limiting examples of inoculation media that may be used include, but are not limited to, Mueller Hinton broth, thioglycolate broth, salt mannitol, modified Sabouraud broth, and/or Scheduler broth.

The analysis of the calorimetric signal in step b), c) and/or d) may be performed using a database which establishes a correlation between at least one calorimetric signal and a plurality of microorganisms. Further, a database which establishes a correlation between at least one calorimetric signal and a plurality of microorganisms may be used to determine the Gram status, genus and/or species of one or more microorganisms in the microbial sample.

The calorimetric signal determined may for example be 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. 108580 6 The incubation in step c) is typically performed for a time period of from about 1 hour to about 48 hours from the time to detection of said one or more calorimetric signa|(s) even though longer and shorter incubation periods may also be applied depending on the metabolic activity of the sample to be analysed.

The present document is also directed to a system for performing the calorimetric method of the present document. The system comprises a calorimeter and a software for performing an analysis according to step a), b), c) and/or d) in said method and/or for performing a determination of the Gram status, genus and/or species of one or more microorganisms in said microbial sample.

The present document also discloses a kit for performing the calorimetric method of the present document, said kit comprising: i) at least one antimicrobial-free inoculation medium; ii) the inoculation medium of i) but supplemented with an antimicrobial; iii) one or more database(s) which establishes a correlation between at least one calorimetric parameter and a plurality of microorganisms; iv) optionally a software component comprising a function that determines the Gram status, genus and/or species of one or more microorganisms; and v) a software component comprising a function that determines if said microorganism(s) is susceptible or non-susceptible to said antimicrobial.

The present document is also directed to a computer program comprising computer program code, the computer program code being adapted, if executed on a processor, to implement the calorimetric method for antimicrobial susceptibility testing of the present document. The present document is also directed to a computer program product comprising a computer readable storage medium, the computer readable storage medium having this computer program.

Other features and advantages of the invention will be apparent from the following detailed description, drawings, examples, and from the claims. 108580 DEFINITIONS ln the context of the present document "a"/"an" refers to one or more.

By "antimicrobia|", "antimicrobial agent", "antimicrobial Substance" and the like is intended a substance that is active against microorganisms in the form of bacteria and/or fungi. An antimicrobial thus has an antibacterial or antifungal activity that kills, stops or impairs the growth and/or metabolic activity of bacteria and/or fungi. Examples of antimicrobial agents include, but are not limited to, antibiotics and antifungals.

By "microbial sample" and the like is herein intended a sample potentially comprising microorganisms in the form of bacteria and/or fungi.

"Calorimetry" and the like refers to a process of measuring the quantity of heat released or absorbed during a chemical reaction, such as during incubation of a microorganism in an inoculation medium. The heat changes in the sample over time [J/s vs. time] may be recorded in a thermogram. Calorimetry according to the present document is performed as known by the skilled person. ln short, for the determination of a calorimetric signal from a microbial sample, the sample is inoculated in a vial comprising an inoculation medium and the vial is then placed in the temperature-controlled chamber of the calorimetric device for incubation. The calorimetric signal measured in J/s(W), reflecting the metabolic activity of the microorganism(s) potentially present in the sample, can be continuously followed and analysed, presented in a so called thermogram.

"Metabolic activity" refers to the chemical reactions in a cell that convert cellular food/fuel into cellular building blocks or energy. 108580 BRIEF DESCRIPTION OF DRAWINGS Figure 1. lnoculum calibration assay. S.epidermidis thermogram in Mueller-Hinton broth indicating the metabolic inflection point (black dot) and the arrow delimiting the ideal metabolic viability sampling time frame. Open circles A, B and C show the sampling times for the experiment.

Figure 2. Antimicrobial susceptibility determination starting at different metabolic rates for the susceptible clinical S. epidermidis isolate. A) inoculum taken before the inflection point; B) inoculum taking 1 h after the inflection point; C) inoculum taken 4 h after the inflection point. Continuous black line = thermograms of the samples incubated in Mueller Hinton broth; dashed black line = thermograms of the samples incubated in Mueller Hinton broth supplemented with 16 mg/L of CTX.

Figure 3. lnoculum calibration assay. S. aureus thermogram in Mueller-Hinton broth indicating the metabolic inflection point (black dot) and the arrow delimiting the ideal sampling time frame. Open circles A, B and C show the sampling times for the experiment.

Figure 4. Antimicrobial susceptibility determination starting at different metabolic rates for the resistant clinical S. aureus isolate A) inoculum taken before the inflection point; B) inoculum taking 1 h after the inflection point; C) inoculum taken 3 h after the inflection point. Continuous black line =thermograms of the samples incubated in Mueller Hinton broth; dashed black line =thermograms of the samples incubated in Mueller Hinton broth supplemented with 16 mg/L of CTX.

Figure 5. Effect on antimicrobial susceptibility test starting with same metabolic activity at different metabolic sampling percentages with the susceptible clinical isolate of S. epidermidis. A) 30% inoculum taken at inflection point; B) 20% inoculum taken at inflection point C) 10% inoculum taken at inflection point. Continuous black line = thermograms of the samples incubated in Mueller Hinton broth; dashed black line = thermograms of the samples incubated in Mueller Hinton broth supplemented with 16 mg/L of CTX. 108580 DETAILED DESCRIPTION According to the present document, antimicrobial susceptibility testing (AST) can be performed by calorimetry (e.g. isothermal (micro)calorimetry), using the metabolic viability of the microorganisms and an antimicrobial substance. The effect of the antimicrobial substance will give rise to changes in a thermogram that can be used to derive the antimicrobial efficiency of the substance.

As disclosed herein, with calorimetry, the impact on the viability (as determined by determining the metabolic activity) of an antimicrobial is ca|ibrated directly against viable cells at their metabolic optimum for the antimicrobial susceptibility testing. This can be done in a setting directly on the patient sample whereas compendial biomass-based assays cannot be applied directly on the patient sample. Additionally, the metabolic viability determination avoids the problems associated with biomass-based assay and the inability to separate the mixture of dead and alive cells, masking the correct viable ratio to antimicrobial molecules, that can result in an impaired susceptibility readout.

The time to detectable signal, the total quantity of released energy, and the kinetic signals of microbial metabolic activity are proportional to the susceptibility against the antimicrobial substance.

The present document thus provides an improved method for antimicrobial susceptibility testing. Knowledge of the antimicrobial susceptibility of microorganisms in a microbial sample is important in several situations, in particular in the context of microbial infections in a patient, where quick and accurate onset of treatment against the microbial infection can be of crucial importance for the outcome for the patient. Currently available methods for antimicrobial susceptibility testing are hampered by being too slow as it may take one or several days before a decision on a suitable antimicrobial can be taken. ln a clinical situation, this can be detrimental for the patient. 108580 The present document is therefore directed to an improved method for antimicrobial susceptibility testing of a sample, such as a clinical sample, based on calorimetry. Due to the use of calorimetry, a mandatory dependency on growth of microorganisms for the testing is avoided, which leads to that a result is obtained faster since only metabolic activity of the microbial cells is necessary. Further, due to the use of calorimetry, the microbial sample to be analysed for the presence of microorganisms and their susceptibility to antimicrobials does not have to be purified from the patient sample but can be used without a first step of isolating the microorganisms. This again leads to that a result is obtained faster but also improves the safety of the method as potentially harmful microorganisms can be handled in a safer manner due to less sample preparation steps.

With methods based on growth of microorganisms, antimicrobial susceptibility testing can only be performed once a high enough number of microbial cells is achieved. ln contrast, in the present method, actual growth of the microbial cells is not necessary, but only metabolic activity of the microbial cells. Microbial cells can be metabolically active without concomitant growth and an increase in metabolic activity often precedes growth. Therefore, a much quicker and precise result of the antimicrobial susceptibility may be obtained with the method of the present document.

The method of the present document is a calorimetric method for antimicrobial susceptibility testing of a microbial sample comprising or consisting of the steps of: a) incubating a sample (potentially) comprising one or more microorganisms in an inoculation medium and following the metabolic activity by determining one or more calorimetric signals of the incubated sample; b) when the metabolic activity of the incubated sample of step a) reaches the inflection point or within about 2 hours after said inflection point is reached, reloading one or more aliquot(s) of said incubated sample of step a) in: i) an inoculation medium not supplemented with an antimicrobial; and ii) the same inoculation medium as in step b)i) but wherein said inoculation medium is supplemented with an antimicrobial; 108580 11 c) incubating the samples of step b)i) and b)ii and determining one or more calorimetric signa|(s) in said samples; and d) determining if said one or more microorganisms is susceptible or non- susceptible to said antimicrobial by comparing the one or more calorimetric signa|(s) obtained in step c) from the samp|e(s) supplemented with an antimicrobial to the one or more calorimetric signa|(s) obtained in step c) from the samp|e(s) not supplemented with an antimicrobial.

By calorimetry, the heat flow in a sample can be measured and this heat flow reflects the metabolic activity of microbial cells in the sample. ln the method of the present document, the metabolic activity of the incubated samples is measured by calorimetry. A higher metabolic activity results in a higher calorimetric signal. Any calorimetric signal related to metabolic activity may be used in the method of the present document. One example of a calorimetric signal that may be used in accordance with the present document is the maximum metabolic rate, which is a measurement of the slope of the curve where metabolic activity reaches the largest difference over time. Another example is the maximum metabolic activity, which is a measurement of the maximum achievable curve height for a given organism. Another typical calorimetric signal that is useful in the method of the present document is time to detection, wherein the lower limit of detection where the presence of metabolic activity is possible to distinguish from a measurement baseline is identified. Heat flow is defined as the measured signal in isothermal calorimetry where the heat flow is expressed as J/s. The data can be compared between experiments or samples by using a ratio of the measures or derived signals between different treatment conditions.

The microbial sample is typically homogenized or suspended in a liquid such as phosphate-buffered saline, saline, typical bacterial growth media in order to break up the sample into smaller pieces and release microorganisms before the incubation of step a). However, as mentioned above, as opposed to currently used methods for antimicrobial susceptibility testing, it is not necessary to isolate the microorganisms or to determine the number of microorganisms in the sample before the testing. Also, as no purification of the sample is necessary, there is no 108580 12 risk of losing microorganisms present in the original sample which then would not be detected during the antimicrobial susceptibility testing. ln the first step of the method, step a), the microbial sample is incubated in an inoculation medium. The metabolic activity of the microbial sample is then determined by calorimetry and the metabolic activity of the sample followed by determining one or more calorimetric signals. As only viable cells are metabolically active, the calorimetric read-out is only based on the viable cells. Prior art method for antimicrobial susceptibility testing based on counting the number of cells (e.g. by optical density) on the other hand read also on dead and/or inactive cells. This is one advantage with the present method, as the results are based only on the viable part of the microbial population. Any suitable calorimetric signal may be used for the calorimetric determination of metabolic activity as explained elsewhere herein.

When the metabolic activity of the incubated sample of step a) reaches the inflection point or within about 2 hours, such as within about 1 hour, after the inflection point is reached, one or more aliquot(s) of the incubated sample of step a) is reloaded in (step b)): i) an inoculation medium not supplemented with an antimicrobial; and ii) the same inoculation medium as in step i) but wherein said inoculation medium is supplemented with an antimicrobial.

According to the present method, the metabolic activity is thus followed over time to identify the inflection point for the maximum metabolic viability (i.e. where the increase in metabolic rate is the highest in the sample). The inflection point for metabolic viability as defined herein signifies the inflection from a linear increase in metabolic rate R 20.98 to when it is dropping below R <0.98. The inflection point is therefore when R is between 0.98 and 1. This time point, or a time point around this inflection point, was found to be the best for continuing with reloading step b) wherein the incubated microbial sample is transferred to a media with and without antimicrobials to perform the antimicrobial susceptibility testing. Typically, the best 108580 13 results are obtained if the incubated sample of step a) is reloaded at the time of the inflection point or within two hours after the inflection point is reached, such as within one or two hours after the inflection point. As is demonstrated in the experimental section, if the sample is reloaded too early or too late in view of the inflection point, the microbial cells are not in a qualitatively metabolic state that allows a reliable result of the antimicrobial susceptibility testing to be achieved. Performing the reloading step at the inflection point or within a limited time thereafter was therefore found to be crucial for the calorimetric antimicrobial susceptibility testing to give reliable results. Thus, in the method of the present document a microbial sample is first inoculated in an inoculation medium and the metabolic activity followed until the inflection point is reached and an aliquot of the incubated sample is thereafter reloaded (within maximum two hours after the inflection point is reached) in fresh inoculation medium with or without an antimicrobial. Pre-incubating the microbial sample and reloading it around the inflection point was found to be crucial in the present method to achieve reliable result in the antimicrobial susceptibility testing.

Further, the present inventors found that a suitable ratio of metabolic activity to amount of antimicrobial molecules to be used for the antimicrobial susceptibility testing (i.e. metabolic activity/antimicrobial molecule) was important to get the best and most reliable results in a calorimetric method for antimicrobial susceptibility. lt was found that if the total metabolic activity reloaded in step b)ii) is too low in view of the amount of antimicrobial molecules used, the results may be interpreted as that the microorganism(s) in the microbial sample are susceptible to the antimicrobial despite not actually being so (false positive - very major error rate). Conversely, if the total metabolic activity reloaded in step b)ii) is too high in view of the amount of antimicrobial molecules used, the results may be interpreted as that the microorganism(s) are resistant while not actually being this (false negative - major error rate). Using the right ratio of total metabolic activity to amount of antimicrobial molecules at the reloading step b) was therefore found to be important to for the calorimetric method for antimicrobial susceptibility testing to give reliable results. 108580 14 One way to ensure that the correct ratio of total metabolic activity to amount of antimicrobial molecules is used is to, in step b), reload aliquots with different amounts of the incubated sample of step a) (i.e. different total amounts of metabolic activity) in an inoculation medium with and without an antimicrobial, respectively. The metabolic activity in the reloaded samples with and without an antimicrobial are then followed by determining one or more calorimetric signals in step c). The sample(s) inoculated with an aliquot where the metabolic activity in the medium without an antimicrobial does not appear directly after reloading but within ca 15 min to 3 hours after reloading was found to give the most reliable results in the antimicrobial susceptibility testing (i.e. the reloaded samples where the time to detection of a metabolic, i.e. calorimetric, signal is 15 min to three hours). lf too little total metabolic activity is reloaded, the ratio of total metabolic activity to amount of antimicrobial molecules in the reloaded sample may be too low and the result of the antimicrobial susceptibility testing may be interpreted as that the microorganisms are susceptible to the antimicrobial despite not being so. lf too much total metabolic activity is reloaded, the ratio of total metabolic activity to amount of antimicrobial molecules in the reloaded sample may be too high and the result of the antimicrobial susceptibility testing interpreted as that the microorganisms are not susceptible to the antimicrobial despite being so. As is demonstrated in the experimental section, a calorimetric signal appearing earlier than 15 min after reloading increased the risk for false negative results (interpreted as non-susceptible) and calorimetric signal appearing later than 3 hours after reloading increased the risk of false positive results (interpreted as susceptible). ln order to get the most reliable results of the method of the present document, two or more aliquots with different total metabolic activity are reloaded in step b)i) and b)ii), respectively, and the calorimetric signal(s) obtained from the aliquot(s) corresponding to the aliquot(s) wherein the metabolic viability in the sample(s) of step b)i) directly after reloading is below the detection limit of the one or more calorimetric signal(s) and the metabolic viability in the same aliquot(s) is detected between 15 min and 3 hours after said reloading in step b)i) is used for the 108580 determination of susceptibility or non-susceptibility in step d). Typically a couple of different aliquots of the incubated sample of step a) are transferred so that from about minimum 0.1 % to maximum 50% (such as from about 1% to about 15%, such as from 1% to about 10% or from about 5% to about 10%) of the metabolic activity of the incubated sample of step a) is reached in the reloaded sample in step b) at the time of reloading. l.e., if the metabolic activity in the incubated sample of step a) at the time of reloading is X J/ml, then a metabolic activity corresponding to from about 0.1 % to 50% of X J/ml is present in the reloaded sample of step b) directly after reloading. Preferably, at least two aliquots are transferred to reach different metabolic starting activities in the reloaded sample in the inoculation medium with and without an antimicrobial, respectively. This ensures that the volume of the incubated sample of step a) will be enough to reload enough number of aliquots of different volume, to ensure that at least one aliquot fulfils the criteria of having a metabolic activity that is directly after reloading is below the detection limit of the one or more calorimetric signal(s) but that is detectable after between 15 min and 3 hours after reloading.

The microbial sample tested in accordance with the present document is a sample potentially comprising one or more types of microorganisms. The microbial sample may e.g. be a clinical sample from a subject, an environmental sample, a food or feed sample, and/or a purified microbiological sample. Typically, the sample is a clinical sample from a human or animal subject. Such a clinical sample may be a sample of any bodily fluid or tissue, such as blood, urine, plasma, synovial fluid, cerebrospinal fluid, bone tissue, soft tissue, connective tissue, skin tissue, sputum, lavage fluids, plasma/serum, urine, peritonea fluid, pericardial fluid, and/or pleural fluid.

The temperature during the incubation steps of the present method will differ depending on the type of sample. For example, an environmental sample may be incubated at a lower temperature than a clinical sample, depending on the types of microorganisms that are expected to be present in the sample. A clinical sample is typically incubated at a temperature of from about 35 °C, to about 39 °C, such as 108580 16 from about 36 °C, to about 38 °C, such as about 37 °C, for certain microorganisms down to 25 °C is necessary for correct antimicrobial susceptibility test interpretation. Further, other growth conditions, such as if the microorganisms are grown aerobically or anaerobically and the type of medium that is to be used, have to be adapted to the sample type and/or the type of microorganisms that are expected to be present in the sample.

Different microorganisms give different specific ca|orimetric signals when incubated under the same conditions. ln this way, it is possible to determine which microorganism(s) is/are present in a microbial sample by studying one or more ca|orimetric signals obtained under one or several different incubation conditions (such as incubation in different inoculation media, different temperatures, aerobic/anaerobic condition etc.).

For determination of identity of the microorganism(s) present in the microbial sample, one or more ca|orimetric signal(s) obtained is compared to previously obtained ca|orimetric data for different microorganisms incubated under the same conditions. Due to the differences in ca|orimetric signals between different microorganisms, it is possible to determine the identity of the microorganism(s) in the sample, such as if they are Gram positive or Gram negative, if it is fungi and/or bacteria, and/or what microbial species are present. The level of specificity in determination of the identity will depend on the type of sample and what data are desirable to obtain in view thereof. ln a mixed sample, i.e. a sample comprising more than one type of microorganism, the ca|orimetric signal obtained will be the combined signal from the respective microorganisms present in the sample.

For example, based on the ca|orimetric signal(s) obtained from the sample reloaded in the inoculation medium without an antimicrobial in step b)i), the Gram status, genus and/or species of one or more microorganisms in the sample may be determined. lt may also be possible to determine if the sample contains bacteria and/or fungi. This is thus another advantage with the present method, namely that it is possible to notjust obtain a result of whether or not the 108580 17 microorganism(s) is susceptible to the antimicrobia|(s) tested, but also to obtain data as to the identity of the specific microorganism(s) present. This is particularly important for the analysis of clinical samples.

Based on the one or more calorimetric signal(s) obtained in step c) from the sample reloaded in the inoculation medium with and without an antimicrobial, in step d) it is determined if said one or more microorganisms in the sample is/are susceptible or non-susceptible to the antimicrobial by comparing the one or more calorimetric signal(s) obtained in the respective incubated samples with and without antimicrobials for typically up to 48h from the time point when the metabolic activity in the positive control is detectable, depending on the antimicrobials used. This determination is made based on the aliquots where the calorimetric signal is detected between 15 min and 3 hours after the reloading according to step b)i) as described above. For a subset of microorganisms and antimicrobials incubations up to 2 weeks can be necessary for an accurate interpretation of the AST. lf the microorganism(s) in the microbial sample are susceptible to the antimicrobial tested, this will lead to a difference in the studied calorimetric signal(s) between the sample incubated with and without an antimicrobial. By using calorimetry, not only a yes or no answer as regards susceptibility is obtained but it may also be possible to obtain information regarding to what degree the microorganism(s) are susceptible, by determining the gradual impact of the antimicrobials on the metabolic (i.e. calorimetric) signal(s). Determining the identity of the microorganisms in the microbial sample may facilitate the interpretation of the calorimetric signal(s) obtained from sample incubated in the presence of an antimicrobial to determine if they are susceptible or non-susceptible to the antimicrobial.

One challenge with antimicrobial susceptibility testing of microbial samples are mixed samples, i.e. microbial samples comprising more than one type of microorganism. ln such a sample, some microorganisms may be susceptible to a specific antimicrobial while others are not. The present method allows the determination of the identity (e.g. Gram positive/negative, species, fungi/bacteria) 108580 18 of the specific microorganisms present in the sample and not just if the sample as a whole appears to be susceptible to an antimicrobial. Rather, by comparing the calorimetric signa|(s) from a sample incubated without an antimicrobial with the calorimetric signa|(s) obtained from a sample incubated with an antimicrobial, it is possible to establish which microorganisms in the microbial sample are susceptible to each specific antimicrobial tested. ln this way, the treatment of e.g. a patient with a microbial infection can be tailor-made for that specific patient.

The analysis of the calorimetric data in the method of the present document for antimicrobial susceptibility testing and/or the determination of the identity of the microorganism(s) may be performed by visual inspection or automatically by using a database of calorimetric data over different microorganisms. Such a database may thus be used to establish a correlation between at least one calorimetric signal and a plurality of microorganisms allowing them to be distinguished from each other. A software may be used for this analysis.

The antimicrobial used for the antimicrobial susceptibility testing may be a single type of antimicrobial or a combination of two or more antimicrobials. The antimicrobial may be an antibiotic or an antifungal substance or a combination thereof. The antimicrobial(s) used are chosen based on the sample type and what microorganisms that may be expected to be present in the sample, as is known by the person skilled in the art. Typically 1 to 20x different antimicrobials are used in the method of the present document, alone and/or in combination. Non-limiting examples of antimicrobials that may be used are cefoxitin, rifampicin, fosfomycin, cirpofloxacin, amoxicillin, clavulanic acid, piperacillin, tazobactan, meropenem, imipenem, ceftriaxon, linezolic acid, cefotaxime, vancomycin, clindamycin, fosfomycin, vancomycin, gentamicin, methicillin, oxacillin, rifampicin, cefazolin, cotrimoxazole, levofloxacin, cotrimoxazole, colistin, cotrimoxazole, cepefime, gentamicin, mipinem, meropenem, methicillin, oxacillin. Typical combinations of antimicrobials to be used include, but are not limited to, ampicillin and sulbactam, amoxicillin and clavulanic acid, ceftazidime and avibactam, ceftolozane and 108580 19 tazobactam and piperacillin and tazobactam. lt is also possible to use the same antimicrobial or same combination of antimicrobials but in different concentrations. Thus, it is possible to in parallel test the susceptibility to 1) different concentrations of the same antimicrobial, 2) different antimicrobials, and/or 3) combinations of antimicrobiais (in the same or different concentrations) by preparing several vials of incubation medium with the desired antimicrobial(s) for the reloading step b)ii). lt is also possible to supplement the inoculation medium comprising an antimicrobial with one or more adjuvants, such as an adjuvant potentiating and/or enhancing an antimicrobiais activity. Non-limiting examples of such adjuvants are iron and beta-lactam inhibitors. lt is also possible to use two or more inoculation media in the method of the present document, both in step a) and/or in reloading step b). ln this way, the inoculation medium may be adapted to and/or optimized for the specific microorganisms that potentially are present in the microbial sample by incubating a sample in different vials containing different inoculation media. For example, it is possible to use an inoculation medium for growth of aerobic microorganism and an inoculation medium for growth of anaerobic microorganisms. As is known to the person skilled in the art and as mentioned above, the growth conditions also have to be adapted depending on if it is suspected that aerobic or anaerobic microorganisms are present in the sample. Non-limiting examples of media that are suitable to use in the method of the present document are Mueller Hinton broth, thioglycolate broth, salt mannitol, modified Sabouraud broth, and/or Schaedler broth.

The time a sample has to be incubated with and without an antimicrobial in step c) will depend on the type of microbial sample and the microorganism(s) that are present. Typically, the incubation in step c) is performed for a time period of from about 1 hour to about 48 hours as counted from the time to detection (i.e. the time point when the chosen calorimetric signal appears in the sample inoculated without an antimicrobial). 108580 The present document is also directed to a system for performing the calorimetric method for antimicrobial susceptibility testing disclosed herein. Such a system comprises a calorimeter and a software for performing an analysis of a calorimetric signal.

The present document is also directed to a kit for performing the calorimetric method for antimicrobial susceptibility testing of the present document. Such a kit comprises: i) at least one antimicrobial free inoculation medium; ii) the inoculation medium of i) but supplemented with an antimicrobial; iii) one or more database(s) which establishes a correlation between at least one calorimetric signal and a plurality of microorganisms; iv) optionally a software component comprising a function that determines the Gram status, genus and/or species of one or more microorganisms; and v) a software component comprising a function that determines if said microorganism is susceptible or non-susceptible to said at least one antimicrobial.

Alternatively, instead of an antimicrobial-free and an antimicrobial supplemented inoculation medium, the kit may comprise an inoculation medium and a separately provided antimicrobial to be mixed in the inoculation medium to provide an antimicrobial supplemented inoculation medium.

The details regarding suitable inoculation media and antimicrobials to be used in such a kit are as given elsewhere herein.

The kit may further comprise a software component that determines if the ratio of metabolic activity to number of antimicrobial molecules is suitable for performing an antimicrobial susceptibility testing. 108580 21 The present document is also directed to a computer program comprising computer program code, the computer program code being adapted, if executed on a processor, to implement the calorimetric method for antimicrobial susceptibility testing of the present document. The present document is also directed to a computer program product comprising a computer readable storage medium, the computer readable storage medium having this computer program.

The present document also discloses a calorimetric method for antimicrobial susceptibility testing of a microbial sample comprising or consisting of the steps of: a) incubating a sample (potentially) comprising 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 an amount of molecules of an antimicrobial that is to be used for antimicrobial testing and: i) if the ratio of metabolic activity to amount of antimicrobial molecules is suitable for performing an antimicrobial susceptibility testing, proceeding with an antimicrobial susceptibility testing according to steps c)-d), and ii) if the ratio of metabolic activity to amount of antimicrobial molecules is not suitable for performing an antimicrobial susceptibility testing, adjusting said incubated sample of step a) and/or said amount of antimicrobial molecules to obtain a suitable ratio of metabolic activity to amount of antimicrobial molecules for performing an antimicrobial susceptibility testing before proceeding with an antimicrobial susceptibility testing according to steps c)-d); c) incubating: i) an aliquot of the incubated sample of step a) or an aliquot of the adjusted sample of step b)ii) in an inoculation medium not supplemented with an antimicrobial and determining one or more calorimetric signals to determine the Gram status, genus and/or species of one or more microorganisms in said sample; and 108580 d) 22 an aliquot of the incubated sample of step a) or an aliquot of the adjusted sample of step b)ii) in the same inoculation medium as in step c)i) but supp|emented with an antimicrobial in an amount of molecules as determined according to step b)i) or b)ii) and determining the same one or more calorimetric signals as in step c)i); and determining if said one or more microorganism is susceptible or non- susceptible to said antimicrobial by comparing the one or more calorimetric signal(s) obtained in step c)i) with the calorimetric signal(s) obtained in step C)ii); wherein said method is performed without first isolating said one or more microorganisms from said sample.

The present document also discloses a calorimetric method for antimicrobial susceptibility testing of a microbial sample, said method comprising or consisting of the steps of: a) b) C) d) providing a microbial sample and an inoculation medium without an antimicrobial and the same inoculation medium with an antimicrobial; incubaüng i) an aliquot of said microbial sample in said inoculation medium without an antimicrobial; and ii) an aliquot of said microbial sample in said inoculation medium with an antimicrobial; using one or more calorimetric signal(s) obtained from the microbial samples inoculated according to step b)i) to determine the Gram status, genus and/or species of one or more microorganisms in the microbial sample; and using one or more calorimetric signal(s) obtained from the microbial samples inoculated according to step b)i) and b)ii) to: i) determine if the sample contains a suitable ratio of metabolic activity to amount of antimicrobial molecules for performing an antimicrobial susceptibility testing; and 108580 23 ii) when the sample contains a suitable ratio of metabolic activity to amount of antimicrobial molecules for antimicrobial susceptibility testing as determined in step d)i), determine if a microorganism in said microbial sample is susceptible or non- susceptible to said antimicrobial by comparing the calorimetric signal(s) obtained from the inoculation medium without an antimicrobial with the same calorimetric signal(s) obtained from the inoculation medium with an antimicrobial; wherein said method is performed without first isolating microorganisms from said sample.

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

EXPERIMENTAL SECTION Two homogenized clinical tissue biopsies from orthopedic surgery (Karolinska Hospital, Sweden) were used to determine the effect of the qualitative and quantitative metabolic activity and amount of antimicrobial molecules that result in an accurate AST result.

Example 1: treatment and pre-incubation of microbial samples Two clinical tissue biopsies from orthopedic surgery (Karolinska Hospital, Sweden) were homogenized using a buiiet blender BBEO-DX (next advance) at the following settings, stainless Steei UFO Beads (35 mm in diameter) at speed 4, for 12 minutes with the biopsy ciisselveci in 5 mi of iiquid in a 50 ml tubeOne biopsy was known to be infected with a strain of Staphylococcus epidermidis susceptible to cefotaxime (CTX); the other biopsy was infected with Staphylococcus aureus resistant to CTX. From the tissue samples, 50 ul were added to Mueller Hinton broth (Sigma-Aldrich, Darmstadt, Germany) already dispensed in the sterile plastic insert in the microcalorimetry vial (Symcel AB, Solna, Sweden). The microcalorimetry vial was sealed and introduced in the calScreener (Symcel AB, 108580 24 Solna, Sweden). The two samples were incubated and heat produced by each sample was measured for 18 h at 37°C.

Example 2: Qualitative effect of metabolic activitv on AST ln order investigate the potential effect of metabolic rate on the accuracy of the AST bacteria with different initial metabolic rates were exposed to the same antibiotic concentration.

For the qualitative assessment, both biopsies were incubated as described above. Tissue sample containing bacteria were then taken at different metabolic rates: A = before the inflection point, B = within 2 h after the inflection point, C = 3 - 4 h after the inflection point and reloaded in 300 ul of Mueller Hinton broth supplemented with 16 mg/L of CTX (Sigma-Aldrich, Darmstadt, Germany) in sterile plastic inserts in the microcalorimetry vials. Aliquots with different metabolic activities per volume in the incubated biopsy (A-C) where adjusted to reach the same metabolic activity in all the reloaded samples, so that the effect of the metabolic quality could be assessed independently from the in between samples fixed metabolic quantity.

Another 300 ul of Mueller Hinton broth without antibiotic were inoculated with tissue sample containing bacteria as positive metabolic controls at the same metabolic rates as described above (A-C). The microcalorimetry vials were sealed and introduced in the calScreener (Symcel AB, Solna, Sweden). The heat produced by each sample was measured for 18 h at 37°C.

Results The inflection point for metabolic viability signifies the inflection from a linear increase in metabolic rate R 20.98 to when it is dropping below R <0.98. This change signifies the point of highest metabolic viability for the microorganism and was found to be the optimal qualitative sampling point for reloading of the 108580 microbial sample. The inflection point was determined to be at 3 h 25 min for S.epidermidis (see Fig. 1) and 3 h 30 min for S.aureus with these experimental setup conditions (see Fig. 3).

S. epidermis (susceptible to CTX) For the qualitative assessment (see Fig. 2), in the case of S. epidermidis inoculum A, taken 1 hour before the inflection point, shows no metabolic activity when exposed to CTX indicating susceptibility to CTX, which is a correct result as the S. epidermidis used in this experiment is susceptible to CTX. lnoculum B, taken 1 h after the inflection point, also shows no metabolic activity indicating susceptibility to CTX, which is the correct result. lnoculum C, taken 4 h after the inflection point shows metabolic activity when exposed to CTX, signaling lack of susceptibility to the antibiotic, resulting in a false negative result. ln this experiment it is therefore demonstrated that if the reloading step is performed too late in view of the inflection point, the metabolic activity of the microorganisms is not in the right phase to give a reliable AST result.

S. aureus (resistant to CTX) ln the case of S. aureus (see Fig. 4), inoculum A, taken before the inflection point, shows no metabolic activity indicating susceptibility to CTX. As the S. aureus strain used in this experiment is resistant to CTX, this is a false positive result. lnoculates B and C, taken just after the inflection point and 3 h after, on the other hand show metabolic activity indicating resistance towards CTX, which is a correct result. ln this experiment it is therefore demonstrated that if the reloading step is performed too early in view of the inflection point, the metabolic activity of the microorganisms is not in the right phase to give a reliable AST result.

As is demonstrated in this example, an inoculum taken too long after the inflection point is reached can give a false positive result if the inoculated strain is susceptible to the antimicrobial tested (see Fig. 2, inoculum C). On the other hand, 108580 26 an inoculum taken before the inflection point can give a false negative result if the inoculated strain is resistant (see Fig. 4, inoculum A). The examples for S. epidermidis and S. aureus in inoculums A and C, resulting in false negative or false positive results, highlights the importance of performing the susceptibility testing at the right metabolic activity, which as demonstrated herein is at the inflection point or within a certain time period thereafter.

Example 3: Quantitative effect of metabolic activitv Further, to investigate the potential effect of total amount of metabolic activity used for reloading in the reloading step (i.e. different metabolic activities per volume unit after reloading) on the outcome of the AST, aliquots with different total metabolic activities (i.e. aliquots of different volumes) were reloaded at the inflection point (were all samples had identical metabolic rate) and exposed to the same antibiotic concentration as in the qualitative assessment of Example 2. The dilution of the aliquots yielded different percentages of the total metabolic activity corresponding to 30-10% of the metabolic activity per volume unit of the sample that it was reloaded from, in the new incubated sample (Figs. 5 A-C).

For the quantitative assessment, different percentage of the total metabolic activity from the incubated microbial sample of Example 1 were thus taken at the inflection point, so that all samples were reloaded at the identical metabolic rate: A) 30% of total metabolic activity taken at inflection point from the tissue sample containing bacteria; B) 20% of total metabolic activity taken at inflection point from the tissue sample containing bacteria C) 10% of total metabolic activity taken. The aliquots were added to 300 ul of Mueller Hinton broth supplemented with 16 mg/L of CTX (Sigma-Aldrich, Darmstadt, Germany) in sterile plastic inserts in the microcalorimetry vials. Another 300 ul of Mueller Hinton broth without antibiotic with same percentages of total metabolic activity as for the antibiotic exposed tissue sample containing bacteria were used as positive metabolic controls. The microcalorimetry vials were sealed and introduced in the calScreener (Symcel AB, 108580 27 Solna, Sweden). The heat produced by each sample was measured for 18 h at 37°C.

Results Sampie A) (30% ot the totai metaboiic activity reioaded) shows metaboiic activity when exposed to GTX, indicating resistance to the antibiotic, conferring a taise negative resuit. Sampie B) reioaded at 20% ot total metaboiic activity shows metaboiic activity when exposed to GTX indicating resistance to the antibiotic, aiso conterring a faise negative resuit. Sample C) reioaded at 10% of total metabolic activity shows no metaboiic activity when exposed to (BTX indicating susceptibiiity to the antibiotic, conterring a correct positive result.

This example thus iilustrates the importance ot reioading a suitabie amount of metaboiic activity in view of the amount of antimicrobial moiecuies used tor the metaboiic testing, i.e. the ratio of metaboiic activity to amount of antimicrobiai moiecuies after reioading has to be correct to get a reiiable AST resuit.

Conclusion The above exampies iliustrate the correiation between the metaboiic infiection point tor the identification of the optimai quaiitative metaboiic viabiiity sampiing timeframe and the optimai quantitative transfer ot totai metaboiic activity that is required for achieving an accurate antimicrobiai susceptibiiity reading. Using the examples of a susceptibie clinicai isoiate of Sepidermidis and a resistant ciinicai isoiate of S. aureus intected orthopaedic tissue biopsies the importance of both the quaiitative and quantitative metaboiic parameters are iliustrated. it was concluded that the optimai time point for reioading the microbiai sampie after the first incubation step is at the intiection point or within maximum two hours after it. The intiection point is identified as the time frame where the metaboiic rate (R) is 20.98. The importance of sampling at the right quaiitativeiy metabolic state 108580 28 is iiiustrated with the results in Figs. 2 and 4 with the effect on acouracy of the susceptibility readings. Sampies are not optirnai before the inflection point, increasing the risk for faise positive readout for the resistant isoiates, with the reverse resuits in the time window > 3h after the inflection point, with an increased risk for false negative readout for susceptible isolates.

Fig. 5 iilustrates the importance of quahtitative transferrihg the right of totai metaboiic activity at the inflection point, to avoid faise negative or positive resuits (in Fig. 5, a false negative effect is iiiustrated). The sample to be used for the AST determination after reioading was conciuded to be the sampie where the reioaded aiiduot gives a rnetaboiic signai that is detected in between 15 min and 3 hours after reloading. if the metaboiic signai (i.e. caiorimetric signai) is detected eariier than 30 min after reloading, there is a risk for faise negative results (interpreted as non-susceptibie) and if the metaboiic signai is detected at first iater than 3 hours after reioading, there is a risk for faise positive resuits (interpreted as susceptibie). Thus, reioaded samples where the metaboiic signai appears (ie. time to detection) before or after a time frame of 15 min to three hours are not to be used for AST. lt is to be understood that while the invention has been described in conjunction with the detailed description thereof, 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.

Unless expressly described to the contrary, each of the preferred features described herein can be used in combination with any and all of the other herein described preferred features.

Claims (2)

1.Claims A calorimetric method for antimicrobial susceptibility testing of a microbial sample, said method comprising the steps of: a) incubating a sample potentially comprising one or more microorganisms in an inoculation medium and following the metabolic activity by determining one or more calorimetric signals of the incubated sample; b) when the metabolic activity of the incubated sample of step a) reaches the inflection point or within 2 hours after said inflection point is reached, reloading one or more aliquot(s) of said incubated sample of step a) in: i) an inoculation medium not supplemented with an antimicrobial; and ii) the same inoculation medium as in step b)i) but wherein said inoculation medium is supplemented with an antimicrobial; c) incubating the samples of step b)i) and b)ii and determining one or more calorimetric signal(s) in said samples; and d) determining if said one or more microorganisms is susceptible or non- susceptible to said antimicrobial by comparing the one or more calorimetric signal(s) obtained in step c) from the sample(s) supplemented with an antimicrobial to the one or more calorimetric signal(s) obtained in step c) from the sample(s) not supplemented with an antimicrobial. The calorimetric method of claim 1, wherein said method is performed without first isolating said one or more microorganisms from said microbial sample. The calorimetric method according to claim 1 or 2, wherein two or more aliquots with different total metabolic activity are reloaded in step b)i) and b)ii), respectively, and the calorimetric signal(s) obtained from the aliquot(s) corresponding to the aliquot(s) wherein the metabolic viability in the sample(s) of step b)i) directly after reloading is below the detection limit of the one or more calorimetric signal(s) and the metabolic viability in the same aliquot(s) is detected between 15 min and 3 hours after said reloading in step b)i) is used for the determination of susceptibility or non-susceptibility in step d). _ The calorimetric method according to any one of the preceding claims, wherein said sample is a clinical sample from a subject, an environmental sample, a food or feed sample, and/or a purified microbiological sample.The calorimetric method according to any one of the preceding claims, wherein said method comprises a further step of using said one or more calorimetric signals obtained in step c) from the samp|e(s) not supplemented with an antimicrobial to determine the Gram status, genus and/or species of one or more microorganisms in said microbial sample. The calorimetric method according to any one of the preceding claims, wherein a combination of two or more different types of antimicrobials are used in the same inocu|ation medium in step b)ii). The calorimetric method according to any one of the preceding claims, wherein two or more types of inocu|ation media are used in step a) and/or step b). The calorimetric method according to any one of the preceding claims, wherein at least two inocu|ation media containing different concentrations of one or more of an antimicrobial are used in step b)ii). The calorimetric method according to any one of the preceding claims, wherein step b), c) and/or d) is/are performed using a database which establishes a correlation between at least one calorimetric signal and a plurality of microorganisms. 10.The calorimetric method according to claim 5, wherein a database whichestablishes a correlation between at least one calorimetric signal and a plurality of microorganisms is used to determine the Gram status, genus and/or species of one or more microorganisms in said microbial sample. .The calorimetric method according to any one of the preceding claims, wherein the inocu|ation medium or inocu|ation media of step a) and/or step b) further comprises one or more adjuvants, such as an adjuvant potentiating and/or enhancing said antimicrobials activity. 12.The calorimetric method according to any one of the preceding claims, wherein said calorimetric 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.The calorimetric method according to any one of the preceding claims, wherein the incubation in step c) is performed for a time period of from about 1 hour toabout 48 hours from the time to detection of said one or more calorimetric signa|(s). 14.The calorimetric method according to any one of the preceding claims, wherein said inoculation medium is Mueller Hinton broth, thioglycolate broth, salt mannitol, modified Sabouraud broth, and/or Scheduler broth culture media. 15.A system for performing the calorimetric method according to any one of the preceding claims, said system comprising a ca|orimeter and a software for performing an analysis according to step a, b), c) and/or d) in said method and/or for performing a determination of the Gram status, genus and/or species of one or more microorganisms in said microbial sample. 16.A kit for performing the calorimetric method of any one of c|aims 1-14, said kit comprising: i) at least one antimicrobial-free inoculation medium; ii) the inoculation medium of i) but supplemented with an antimicrobial; iii) one or more database(s) which establishes a correlation between at least one calorimetric parameter and a plurality of microorganisms; iv) optionally a software component comprising a function that determines the Gram status, genus and/or species of one or more microorganisms; and v) a software component comprising a function that determines if said microorganism(s) is susceptible or non-susceptible to said antimicrobial. 17.A computer program comprising computer program code, the computer program code being adapted, if executed on a processor, to implement the method according to any one of the c|aims 1-14. 18. A computer program product comprising a computer readable storage medium, the computer readable storage medium having the computer program according to claim 17.