WO2015028983A1

WO2015028983A1 - Method for the evaluation of the synergy between antibiotics, and related kit of products for carrying out this method

Info

Publication number: WO2015028983A1
Application number: PCT/IB2014/064148
Authority: WO
Inventors: Fabio ARENA; Gian Maria Rossolini; Tommaso GIANI
Original assignee: Universita' Degli Studi Di Siena
Priority date: 2013-08-30
Filing date: 2014-08-29
Publication date: 2015-03-05
Also published as: ITFI20130203A1

Abstract

The invention refers to a method for the simultaneous evaluation, in a single assay, of the synergy of antibiotic products in multiple associations of two and three antibiotics, against bacterial strains of carbapenem-resistant Enterobacteriaceae, which is reliable and reproducible, and at the same time is fast; the invention also refers to a kit of products for carrying out the aforementioned method.

Description

TITLE

METHOD FOR THE EVALUATION OF THE SYNERGY BETWEEN ANTIBIOTICS, AND RELATED KIT OF PRODUCTS FOR CARRYING OUT THIS METHOD

Field of the invention

The present invention concerns the field of clinical microbiology, and more specifically, relates to a method and to a kit of products for carrying out this method that allow to quickly and reliably evaluate the synergy between several antibiotics, used in combination, against bacterial strains of Carbapenem-resistant Enterobacteriaceae.

State of the art

Bacterial strains belonging to the family of Carbapenem-resistant

Enterobacteriaceae (or CNS) are Gram-negative bacteria that are particularly difficult to treat, since they are indeed clinically resistant also to the class of antibiotics that are called "carbapenems", which are antibiotics active against many bacteria, both Gram- positive and Gram-negative bacteria, considered the antibiotics with the widest spectrum of action, also effective in the case of serious and drug-resistant infections. Therefore, it is clear that infections caused by bacteria, that are proved to be resistant to treatment with such antibiotics, are particularly worrying.

Among the bacterial strains that have become carbapenem-resistant are some strains of enterobacteria, whose spread represents a serious problem for public health (see for example Canton et al. "Rapid evolution and spread of carbapenemes among Enterobacteriaceae in Europe" Clinical Microbiology and Infection: the Official Publication of the European Society of Clinical Microbiology and Infectious Diseases, (2012) 18(5), 413-431). This type of bacterial isolates, indeed, shows resistance to most of the antibiotics commonly used in the clinical practice for treating serious infections from Gram-negative bacteria (see for example Rapp R.P. et al. Pharmacotherapy, (2012) 32(5), 399-407). The problem becomes most substantial in bacterial isolates of Klebsiella pneumoniae, which can produce enzymes capable of hydrolyzing the carbapenems and of accumulating transferable determinants of resistances to different classes of antibiotics (Nordmann P. et al. Emerging Infectious Diseases, (201 1) 77(10), 1791-1798). The possible remaining therapeutic options are represented by colistin, tigecycline and gentamicin, and by the associations of these drugs with each other and with carbapenems, such as imipenem and meropenem, and/or with rifampicin (Livermore D.M. et al. International Journal of Antimicrobial Agents (201 1) 37(5), 415-419).

Although they belong to a relatively small number of clones that are highly- transmissible intra-inter-hospitals, particularly Sequence Type 258, 512 and 101 , the resistance phenotype of these K. pneumoniae is not identical for all isolates and the possible associations of potentially effective drugs are not constant (Woodford N. et al. FEMS Microbiology Reviews, (201 1) 35(5), 736-755). This variability makes it necessary to perform susceptibility testing, through the broth dilution technique on each bacterial isolate coming from serious infections (CLSI, 2012, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Seventh Edition. M07-A9. Clinical and Laboratory Standards Institute. Wayne, PA., M07-A9). It has also been demonstrated that the survival rate is significantly higher in patients that are treated with two or three drugs than those treated with a single-drug therapy (Tumbarello M. et al. (2012) Clinical Infectious Diseases: an Official Publication of the Infectious Diseases Society of America, 55(7), 943-950).

In order to identify the antibiotic combinations potentially active on a given multi- resistant isolate, thus optimising the therapy, antibiotic synergy tests have been implemented that, up to now, are mainly carried out with two different methods, the so- called "time kill assay" test and the "checkerboard" test on microdilution plates (Appleman M. D. (2000) Antimicrobial Agents and Chemotherapy, 44{4), 1-6; Bonapace C. R. (2000) Diagnostic Microbiology and Infectious Disease, 48, 43-50; CLSI. (1999) M21-A Methodology for the Serum Bactericidal Test; Approved Guideline, 1-51). Both of these methods, which are the only current methods that offer guarantees of reproducibility and reliability, allow just the association of two molecules to be tested in a single test, however. Such methods therefore present an objective limitation due to the fact that they cannot be used to test, for example, the synergy among three antibiotics that, as stated above, in some cases have proven necessary for the treatment of certain resistant infections. The greatest limitation of these methods is represented by the laboriousness of the procedure, which has slowed down its introduction into the daily practice of clinical microbiology laboratories, even if these methods are available from a considerable number of years. The application of the two methods, indeed, requires specific technical capabilities and a long performance time, so that the two tests can only be carried out by large specialised laboratories. Both of the known methods foresee the synergy test between two molecules for a wide range of concentrations (in general comprised between 0.06 mg/L and 128 mg/L). This potentiality, although scientifically relevant, has no clinical use, since the higher concentrations cannot be reached in human serum after administration of a standard dose of the molecules tested, whereas the results provided by lower concentrations are often redundant and informative for therapeutic purposes. In order to complete the methods it generally takes 24 hours, or even 48 hours for the "time-kill" method, and a pure culture of the bacterial isolate in question is needed as starting material. It can be estimated that in order to have a complete evaluation of the possible synergies on a single isolate it is necessary to carry out about 10 single time kill assay or checkerboard tests, each of which only evaluates the association of two drugs only. For a complete evaluation, therefore, it would be necessary to have available 10 checkerboard 96-well plates each of which tests only one association.

Also Tascini et al. (2013) Antimicrobial Agents and Chemotherapy, 57(8), 3990- 3993, use a classical checkerboard method to test the activity of ten combinations of two antibiotics, in microtiter plates with 96 wells, and evaluate their synergic activity against bacterial isolates of colistin-resistant K.pneumoniae strains. In this method each plate is used for testing a single combination of two antibiotics in a range of concentrations, so that ten plates have to be used in order to evaluate the activity of ten different combinations of two antibiotics, and they come to the conclusion that the combination showing the higher synergic activity is the combination colistin-rifampicin.

Instead Jernigan M.G. et al. (2012) Antimicrobial Agents and Chemotherapy, 56(6), 3395-3398, use a "time-kill" method for testing four antibiotics, alone and in combinations of two, against different bacterial isolates of K.pneumoniae strains producing K.pneumoniae carbapenemase (KPC), thus obtaining information on their bactericidal effect, but not a quantitative measure of the synergic activity of the combinations of two antibiotics. Also in this case, moreover, in each test a single combination of two antibiotics may be tested in a range of different concentrations, and moreover for each test different assessments are required, carried out at different time intervals (0, 4, 8, 12 and 24 hours).

This complexity has the consequence that currently very few laboratories routinely perform synergy tests between antibiotics and even those that do often use alternative techniques, like for example the so-called "Etest cross-formation method" that, although simple, suffer from poor reproducibility and standardization, since they are subject to interpretation by the operator (Bonapace C. R. (2000) Diagnostic Microbiology and Infectious Disease, 48, 43-50).

From what outlined above it is thus clear that it remains unresolved the problem of devising a reliable method for studying the synergy of antibiotics, which does not have the practical limitations characteristic of the methods available up to now, and in particular which allows synergy tests to be carried out also on more than two antibiotics.

Summary of the invention

The Applicant has now found a novel method for the evaluation of the synergy between antibiotics against bacterial strains of Carbapenem-resistant Enterobacteriaceae, which also makes it possible to test the synergy among three antibiotics, not possible up to now, and also combines the reliability of some of the known methods described above with a much greater practicality of application and shorter times.

Thanks to these characteristics, the method of the invention can easily be carried out in the laboratory daily routine and provides, with a substantial saving of material and time compared to conventional methods, a great deal of information that is now unobtainable with the known methods, or obtainable only after a very long time and with extremely complex procedures.

Subject of the present invention is therefore a method for the simultaneous evaluation, in a single assay, of the synergy between antibiotics in multiple different combinations against bacterial strains of Carbapenem-resistant Enterobacteriaceae, whose essential characteristics are defined in the first of the claims attached hereto. Further important characteristics of the present method are defined in the attached dependent claims.

A further subject of the invention is a kit of products for carrying out the method of the invention, whose essential characteristics are defined in claim 6 attached hereto.

Further characteristics and advantages of the invention will become clearer from the following detailed description.

Brief description of the figures

Figure 1 represents a schematic illustration of the distribution of the antibiotics tested in a container with 96 separate compartments, in which single antibiotics are distributed, as well as their combinations of two and three products in certain concentrations, in three distinct sectors: in sector 1 the single antibiotics are dispensed, in sector 2 the combinations of two antibiotics and in sector 3 the combinations of three antibiotics. The products are identified with an abbreviation of the respective common name, accompanied by the indication of the concentration present in that compartment, expressed in μg/ml.

Detailed description of the invention

The method of the invention is carried out in suitable containers having a number of separated compartments at least equal to the number of antibiotics concentrations to be tested, and preferably in microdilution plates, which can also be totally analogous to those already in use in clinical microbiology laboratories, for instance to carry out the checkerboard technique.

Contrary to the checkerboard technique, however, where the entire microdilution plate is used to test a single combination of antibiotic products in a high number of concentrations, in the present method the same plate is used to test a large number of combinations of antibiotic products in a reduced number of concentrations, suitably selected to still obtain significant results for the purposes of the method, i.e. evaluating the sensitivity and the synergy of the antibiotic associations.

Thanks to this, the method of the invention is able, with a single plate, to provide information both on the combinations of two and on the combinations of three antibiotics, whilst still also maintaining a space to test the single products and thus obtain useful comparative data on the behaviour of the products tested against a certain bacterial isolate. With the conventional checkerboard technique the same information that can be obtained with the present method would require use of materials and times which are roughly 10-fold higher than those foreseen according to the present invention.

The present method is nevertheless very quick, even faster than the simplest conventional techniques, like the double Etest method, which provides a result in about 48 hours unlike the method of the invention, which can be completed in about 16-18 hours. And there are even greater advantages linked to the speed of the present method if compared to other conventional techniques, like for instance the time-kill assay, which requires much longer performance times, as well as extremely more laborious modes of operation, available only to specialised laboratories.

According to the invention in a container having a plurality of separate compartments, at least as many as the concentrations to be tested, for example in a microdilution plate with wells, the bacterial isolate belonging to the family of Carbapenem-resistant Enterobacteriaceae, against which it is wished to test antibiotic associations is distributed; the plate thus allows determining the Minimal Inhibitory Concentration (hereinafter referred to as "MIC") of the single antibiotics, the synergy of the combinations of two antibiotics, and the synergy of the combinations of three antibiotics.

In the present invention the antibiotics under examination with the present method are selected from among the antibiotics that are most commonly used in the treatment of serious infections from strains of Enterobacteriaceae producing carbapenemase, and in particular the antibiotics are selected among colistin, gentamicin, rifampicin, tigecycline, imipenem, meropenem, and their combinations of two and three products. Such combinations are selected based on the mechanisms of action and on the tolerability of the molecules, taking into account the complementarity of the mechanisms of action and of the possible effects in terms of toxicity caused by the simultaneous administration of many antibiotics.

For instance, in the combinations of two or three products, meropenem and imipenem, belonging to the same family of carbapenems, have never been tested together, but always in combination with other products not belonging to the same family, for example with rifampicin or with tigecycline, or with both. Some of the products indicated above can also be replaced by analogous products again selected according to the same criteria stated above. As an example, one or more from polymyxin B, amikacin and doxycycline can be used as an alternative to replace colistin, gentamicin and tigecycline, respectively, and doripenem or ertapenem may be used each as a replacement of imipenem or of meropenem, possibly varying the concentration values tested, where necessary.

The known concentrations of each antibiotic product and of their selected combinations to carry out the present method are selected taking only the concentrations that can actually be achieved in the patient's plasma following administration of the antibiotic; the maximum concentration values able to be reached are known for any antibiotic product that can possibly be used in the present method.

The following Table 1 shows the plasma levels that can be obtained during a therapy with standard doses of the antibiotics tested in the following experimental part, as shown for example by Mandell G.L. et al. (2010) in Mandell, Douglas, and Bennett's principles and practice of infectious diseases, Churchill Livingstone and da Ramirez et al. (2013) in Antimicrobial Agents and Chemotherapy, 57(4), 1756-1762:

Table 1

For the antibiotic products and their respective selected combinations, in the present method, the concentration ranges or values to be used were then selected, after having determined the MIC values for the antibiotics colistin, gentamicin, imipenem, meropenem, rifampimicin, and tigecycline, and their potentially active combinations have been identified for a substantial number of clinical isolates of Klebsiella pneumoniae, producing Klebsiella pneumoniae carbapenemase (KPC), as described in details in the following experimental part. Such a preliminary study allowed discarding concentrations of scarce relevance, and identifying the ranges of concentrations to be tested for each antibiotic product or combination of products, shown in the following Tables 2-4:

Combination of three Concentrations tested ^g/ml)

antibiotics

meropenem 8

rifampicin 4

tigecycline 0.5

tigecycline 1

rifampicin 4

gentamicin 1

meropenem 8

colistin 2

tigecycline 0.5

meropenem 8

tigecycline 0.5

gentamicin 1 imipenem 8

colistin 1

rifampicin 4

imipenem 8

colistin 2

tigecycline 0.5

rifampicin 4

tigecycline 0.5

colistin 2

meropenem 8

colistin 1

rifampicin 4

For greater convenience both in preparing the plate and in reading the results, the single antibiotics, the combinations of two of them and the combinations of three were each tested in three different sectors in which the container or the plate is ideally divided, also placing the different concentrations to be tested for a same product in decreasing order of dilution on the same row, in abscissas or in ordinate.

According to a particular embodiment of the invention the container with separate compartments, or the plate with wells, for carrying out the present method, is thus ideally divided into three sectors: in sector 1 known scalar concentrations are distributed for the single antibiotics for which the synergy of the respective combinations has to be tested, i.e. colistin (or polymyxin B), gentamicin (or amikacin), imipenem (or doripenem or ertapenem), meropenem (or doripenem or ertapenem), rifampimicin and tigecycline (or doxycycline); in sector 2 of the same container or plate, known concentrations are distributed for the combinations of two antibiotics whose synergy has to be tested; meanwhile, in sector 3 known concentrations are distributed for the combinations of three antibiotics, whose synergy against the bacterial isolate under examination has to be tested. For each antibiotic alone or in combination significant concentrations to be tested are identified: for the single antibiotics and their combinations of two, values within the ranges indicated above in Tables 2 and 3 are selected, taking the lower limit value and, for example, doubling up until the maximum value is reached, whereas for the combinations of three antibiotics the single concentration values indicated in Table 4 are taken, corresponding to the sensitivity limit of the product. In a 96-well microdilution plate the method for the evaluation of the synergy of the combinations of the 6 antibiotic products indicated above will be carried out for example with a sector 1 comprising 36 wells, in which each single antibiotic is tested, with a sector 2 comprising 52 wells with known concentrations of the 9 combinations of the two selected antibiotics, and with a sector 3 comprising 8 wells with known concentrations of the 8 different combinations of selected antibiotics; the concentrations of antibiotics are indicated above in Tables 2-4. Figure 1 schematically illustrates the distribution of the antibiotic products alone or in combination, indicated with an abbreviation of their names (for example COL= colistin, TIGE= tigecycline, I M l= Imipenem, and so on), followed by the respective concentration, expressed in μg/ml.

In the present method the antibiotic products are diluted in a suitable culture broth, until the desired concentration is reached, following standard procedures (CLSI, 2012, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Seventh Edition. M07-A9. Clinical and Laboratory Standards Institute, Wayne, PA., M07-A9). The preferred culture medium is Mueller Hinton "cation adjusted", freshly prepared, not more than 24 hours before. Into the compartments or wells already containing the desired concentrations of antibiotics, the bacterial inoculum under examination is added so as to obtain a solution containing about 5x10⁵ CFU/ml. After about 16-18 hours of incubation at about 35°C±2°C, for the single antibiotics the minimal inhibitory concentration of bacterial growth (MIC) is then measured, corresponding to the lowest concentration of antibiotic in which there is no visible bacterial growth, visually or by means of a spectrophotometer, whereas for the combinations the Fractional Inhibitory Concentration value (hereinafter referred to as "FIC") is determined, which takes into account the MIC values of each antibiotic alone and in combination. For a combination of two antibiotics A and B, the Fractional Inhibitory Concentration value FIC can be calculated with the following formula (Bonapace C.R. (2000) Diagnostic Microbiology and Infectious Disease, 48, 43-50):

in which FIC_A=MIC_A in association/MIC_A and FIC_B=MIC_B in association/MIC_B.

For a combination of three antibiotics, information of non-growth or of growth of the bacterial strain is visually detected; based on this information, it is established whether there is synergy or, respectively, lack of synergy, for the antibiotics tested by comparison with the MICs of the single antibiotics. The present method can be carried out by automating one or more steps, for example by using a program for automatically interpreting the MIC data detected and calculating the FIC values, in order to evaluate the synergy of two antibiotics in combination starting from the related MIC data of the single products and of their combination, as described above. The program can for example be based on an Excel spreadsheet that is so set as to automatically calculate the FIC data based on the detected MIC data inserted in the spreadsheet.

The present invention also refers to a kit of products for carrying out the method described above. Such a kit for determining the synergy of antibiotics in combination with each other against bacterial strains of Enterobacteriaceae CNS comprises a container having a plurality of separate compartments, at least as many as the concentrations of antibiotics to be tested, for example a microdilution plate with wells; the antibiotic products already distributed inside the compartments or wells in lyophilized form or in frozen form pre-diluted in the culture medium; and an information sheet that describes the method of the invention able to be carried out with the kit. The kit according to the present invention may also comprise a suitable culture medium in a separate container, for example a flask, for possible addition at the time of use.

The method of the invention represents a substantial progress in antibiotic sensitivity and synergy tests against strains of Enterobacteriaceae CNS; it allows indeed to test, with a single microdilution plate and in 16-18 hours only, the MICs of the most relevant antibiotic products for the treatment of the infections caused by these bacterial strains, and of a large number of their combinations, both with two and with three products, with a substantial saving of work time and of materials. Contrary to the methods known until today, the method of the invention can be easily introduced into the daily routine of any laboratories, providing a great deal of information of clinical relevance, currently not obtainable except by taking a long time and with much greater use of resources.

The information obtained is also clinically useful for identifying an effective therapy against bacterial isolates that have a profile of resistance to several antibiotic classes. Besides being clinically useful for quickly identifying an effective and "personalised" antibiotic therapy, the method and the kit of the invention are also useful as a research tool for collecting an enormous amount of data on the synergy of antibiotics, which were not available precisely until today due to the lack of a simple and cost-effective method, applicable on a large scale, and at the same reliable and reproducible, like that of the invention.

In the present invention, by the expression "bacterial strains of carbapenem- resistant Enterobacteriaceae" we mean any strain of Enterobacteria not sensitive, or having reduced sensitivity, to carbapenems; for example selected among Klebsiella spp., Escherichia coli, Enterobacter spp. and Proteus mirabilis; preferably we mean a strain of Enterobacteriaceae producing Klebsiella pneumoniae Carbapenemasi (KPC).

EXPERIMENTAL PART

The concentration ranges to be used for the selected antibiotic products colistin, gentamicin, imipenem, meropenem, rifampicin, and tigecycline and their potentially active combinations with the respective concentration values to be used were chosen, having determined the values of the MICs for a substantial number of clinical isolates of Klebsiella pneumoniae, producing Klebsiella pneumoniae carbapenemase (KPC), 204 clinical isolates of Klebsiella pneumoniae, producing Klebsiella pneumoniae Carbapenemasi (KPC), belonging to all of the greatest dines in circulation, previously characterised (Giani T. (2013) Eurosurveillance, Volume 18, Issue 22, May 30). The concentration values and the combinations shown above in Tables 2-4 were thus found.

Once the concentrations to be tested and the combinations of potentially active antibiotics were identified, the synergy of the same antibiotic products and of their combinations indicated above were then tested, using the method of the invention on a further 39 clinical isolates of Klebsiella pneumoniae producing carbapenemase, from invasive infections. A strain of Escherichia coli ATCC-25922 and one of Pseudomonas aeruginosa ATCC 27853 were used as control.

Then 39 analyses were carried out on as many 96-well microdilution plates, in which the single six antibiotics and their combinations of two and three products had been distributed in the positions and at the concentrations illustrated in Figure 1. The dilutions of the antibiotics were made using the culture medium Mueller Hinton "cation adjusted", freshly prepared, according to the standard procedures described for example in CLSI, 2012, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Seventh Edition. M07-A9. Clinical and Laboratory Standards Institute, Wayne, PA., M07-A9. Into each well the bacterial inoculum under examination was added so as to obtain a solution containing about 5x10⁵ CFU/ml. After about 16 hours of incubation at about 35°C, for the single antibiotics the MIC was measured, and for the combinations the FIC value was calculated as explained above. The interpretation of the MIC values was carried out according to EUCAST (guidelines of the European Committee on Antimicrobial Susceptibility Testing) criteria, by visually determining the MIC, by checking the first well, in increasing order of concentration, in which there is not a clear sign of bacterial growth.

Out of the total of the 39 tests carried out it was possible to identify a therapeutic indication in 36/39 cases (92%). In 21 cases the information was obtained from the reading of sector 1 of the plate, given that the isolate was simultaneously sensitive to colistin, tigecycline and gentamicin. In the remaining cases, from the interpretation of sector 2 of the plate, it was possible to detect at least one synergy in 18 isolates. In particular, it was possible to detect 24 synergies (FIC < 0.5) and 22 possible synergies (FIC 0.5<X<0.7) out of 162 combinations tested. The combinations for which the greatest number of cases of synergy was detected were colistin-rifampicin (16 cases of synergy) and colistin-tigecycline (14 cases of synergy). In sector 3 of the plate, the combination of the three antibiotics tested proved to be active in 294 cases out of 312 combinations tested. Among the combinations of three antibiotics that constantly proved more active there are meropenem-tigecycline-gentamicin and meropenem- colistin-rifampicin. In this case, information of non-growth or growth of the bacterial strain is detected visually, based on which it is established whether there is, respectively, synergy or lack of synergy of the antibiotics tested.

In a limited number of cases no synergy was detected between the products in combination, since the MIC for the single products was equal to or less than the MIC for their combination.

Each test was carried out in duplicate, obtaining substantially identical values, thus proving the high reproducibility of the method. The present invention has been described up to now with reference to a preferred embodiment. It should be understood that there may be other embodiments that derive from the same inventive core, as defined by the scope of protection of the claims given below.

Claims

A method for the simultaneous evaluation, in a single assay, of the synergy of antibiotics in multiple different combinations against bacterial strains of Carbapenem-resistant Enterobacteriaceae, comprising the following steps: a. distribution of said antibiotics, alone or in combinations of two and three antibiotics, in a multi-compartment container having a number of separated compartments at least equal to the number of significant concentration values for said antibiotics selected within a range of concentration values actually reachable in a patient's plasma following the administration thereof, so as to obtain in each compartment one of said concentration values by dilution in a suitable culture medium;

b. addition of a bacterial inoculum selected amongst said strains in each of said compartments, and incubation for 16-18 hours;

c. determination of the MIC values of the antibiotics alone and of their combination of two antibiotics, and calculation of the FIC values for said combinations, and evaluation of the synergy of the two antibiotics based on the calculated FIC value;

d. evaluation of the synergy of three antibiotics based on the inhibition of the bacterial growth in the presence of said three tested antibiotics in combination among each other, with respect to the MIC values of the same three antibiotics when taken alone.

The method according to claim 1 , wherein said bacterial strains are strains of Klebsiella spp., Escherichia coli, Enterobacter spp. and Proteus mirabilis.

The method according to claim 1 , wherein said bacterial strains are strains of Enterobacteriaceae producing Klebsiella pneumoniae Carbapenemase (KPC). The method according to claim 1 , wherein said container is a 96-well microdilution plate.

The method according to claims 1-4, wherein the antibiotics whose synergy is under evaluation, are colistin, gentamicin, imipenem, meropenem, rifampicin and tigecycline, and the significant concentration values to be used for the distribution of the antibiotic alone are selected, within the following ranges, by doubling starting from the lower limit of each range:

6. The method according to claim 5, wherein the combinations of two and three antibiotics, whose synergy is under evaluation, are indicated in the following table together with their respective significant concentration values or concentration ranges, wherein the values to be used are the single values indicated below or they are selected, within the ranges indicated below, by doubling starting from the lower limit of each range:

imipenem 8

colistin 1

rifampicin 4

imipenem 8

colistin 2

tigecycline 0,5

rifampicin 4

tigecycline 0,5

colistin 2

meropenem 8

colistin 1

rifampicin 4

7. The method according to claim 5 or claim 6, wherein imipenem or meropenem or both are replaced by doripenem or ertapenem.

8. The method according to claim 5, claim 6 or claim 7, wherein one or more among colistin, gentamicin and tigecycline are respectively replaced by polymyxin B, amikacin and doxycycline.

9. A kit of parts for the simultaneous evaluation, in a single assay, of the synergy of antibiotics in multiple different combinations against bacterial strains of Carbapenem-resistant Enterobacteriaceae, comprising: a multi-compartment container, having an amount of compartments at least equal to the amount of concentration values of the antibiotics whose synergy is under evaluation; single antibiotics and at least two combinations thereof, that are combinations of two and three antibiotics, distributed in said compartments in a freeze-dried or frozen form, pre-diluted in a suitable culture medium; and a leaflet which describes the method as defined in claims 1-8.

10. The kit according to claim 9, further comprising a separated container with a suitable culture medium.

1 1. The kit according to claim 9, wherein said bacterial strains are strains of Klebsiella spp., Escherichia coli, Enterobacter spp. and Proteus mirabilis.

12. The kit according to claim 9, wherein said bacterial strains are strains of Enterobacteriaceae producing Klebsiella pneumoniae Carbapenemase (KPC).

13. The kit according to claim 9, wherein said container is a 96-well microdilution plate.

14. The kit according to claims 9-13, comprising the antibiotics and the combinations thereof as defined in claims 5-8.