ITFI20130203A1 - METHOD FOR THE ASSESSMENT OF SYNERGY BETWEEN ANTIBIOTIC PRODUCTS, AND ITS RELATED PRODUCT KIT FOR THE IMPLEMENTATION OF THIS METHOD - Google Patents

Description

TITLE

METHOD FOR EVALUATING THE SYNERGY BETWEEN PRODUCTS

ANTIBIOTICS, AND RELATED KIT OF PRODUCTS

FOR THE IMPLEMENTATION OF THIS METHOD

Field of the invention

The present invention relates to the field of clinical microbiology, and more particularly, it has as its object a method and a kit of products for the implementation of this method which allow the synergy of antibiotic products used to be evaluated quickly and reliably. in association with each other, against bacterial strains of Enterobacteriaceae not sensitive to carbapenems.

State of the art

The bacterial strains belonging to the Enterobacteriaceae family that are not sensitive to carbapenems (or CNS) are Gram-negative bacteria that are particularly difficult to treat, since they are clinically resistant even to the class of antibiotics that goes under the name of "carbapenems", antibiotics active against many bacteria, both Gram-positive and Gram-negative, considered the broadest spectrum antibiotic products, effective even in cases of severe and drug-resistant infections. It is therefore evident that infections caused by bacteria that prove resistant to treatment with these antibiotics are of particular concern.

Among the bacterial strains that have become resistant to carbapenems there are some strains of Enterobacteria, the spread of which represents a serious public health problem (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, in fact, shows resistance to most of the antibiotics commonly used in clinical practice for the treatment of serious infections by Gram negative bacteria (see for example Rapp R. P. et al. Pharmacotherapy, (2012) 32 (5), 399–407). The problem reaches its greatest relevance in Klebsiella pneumoniae bacterial isolates, which can produce enzymes capable of hydrolyzing carbapenems and accumulating resistance determinants that can be transferred to different classes of antibiotics (Nordmann P. et al.

Emerging Infectious Diseases, (2011) 17 (10), 1791–1798). The possible residual therapeutic options are represented by colistin, tigecycline and gentamicin, and by the associations of these drugs with each other and with carbapenems, for example imipenem and meropenem, or with rifampicin (Livermore D.M. et al. International Journal of Antimicrobial Agents (2011) 37 (5), 415–419).

Although belonging to a relatively small number of highly diffusible intra-hospital clones, especially Sequence Types 258, 512 and 101, the resistance phenotype of these K. pneumoniae is not identical for all isolates and potentially possible combinations of drugs. effective are not constant (Woodford N. et al. FEMS Microbiology Reviews, (2011) 35 (5), 736–755). This variability makes it necessary to perform sensitivity tests, using the broth-dilution technique on each bacterial isolate from severe 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 shown that the survival rate is significantly higher in patients treated with two or three drugs than in those treated with monotherapy (Tumbarello M. et al. (2012) Clinical Infectious Diseases: an Official Publication of the Infectious Diseases Society of America, 55 (7), 943–950).

To identify potentially active antibiotic associations on a given multi-resistant isolate, and thus optimize the therapy, antibiotic synergy assays have been developed which, up to now, are mainly performed with two different techniques, the so-called "time kill assay." assay "and the" checkerboard "assay 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 ones currently existing to offer guarantees of reproducibility and reliability, nevertheless allow to test, in a single assay, only the association between only two molecules. These methods therefore have an objective limitation due to the fact that they cannot be used to test for example the synergy between three antibiotics which, as mentioned above, in some cases have proved necessary for the treatment of certain resistant infections. The major limitation of these methods is represented by the laboriousness of the procedure, which has hampered its entry into the daily practice of the clinical microbiology laboratory, despite having been introduced for a considerable number of years now. The application of the two methods in fact requires specific technical skills and a long execution time, so that the two assays are only practicable by large specialized laboratories. Both known methods provide for the synergy test between two molecules for a wide range of concentrations (generally between 0.06 mg / L and 128 mg / L). This potential, although relevant from a scientific point of view, has no clinical utility, since the highest concentrations are not achievable in human serum after administration of a standard dose of the molecules tested, while the results provided by the lowest concentrations are often redundant. and not very informative for therapeutic purposes. To complete the methods, it generally takes 24 hours, which can reach 48 hours for the "time-kill" method and, as a starting material, a pure culture of the bacterial isolate in question is needed. It can be estimated that in order to have a complete evaluation of the possible synergies on a single isolate, it is necessary to perform about 10 individual time kill assays or checkerboards, each of which evaluates only the association between only two drugs. A complete evaluation would therefore require 10 96-well plates of checkerboards each of which tests only one association.

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, such as the method called "Cross Etest" which, although simple, suffer from poor reproducibility and standardization, being subject to interpretation by the operator. (Bonapace C. R. (2000) Diagnostic Microbiology and Infectious Disease, 48, 43–50).

From the above it is therefore evident that the problem of developing a reliable method for the study of the synergy between antibiotics still remains unsolved, which does not however present the practical limits characteristic of the methods available today, and in particular allows to carry out tests of synergy even between more than two antibiotic products.

Summary of the Invention

The Applicant has now found a new method for the evaluation of the synergy between antibiotics against bacterial strains of Enterobacteriaceae not sensitive to carbapenems, which also allows to test the synergy between three antibiotic products, hitherto impracticable, and also combines reliability of some of the known methods described above, a much greater practicality of application and shorter times.

Thanks to these characteristics, the method of the invention is easily implemented in the daily laboratory routine and provides, with a considerable saving of material and time compared to traditional methods, a large number of information currently not obtainable with known methods, or obtainable. only in much longer times and with much more complex procedures.

The object of the present invention is therefore a method for evaluating the synergy between antibiotics against bacterial strains of Enterobacteriaceae not sensitive to carbapenems, the essential characteristics of which are defined in the first of the appended claims. Further important characteristics of the present method are defined in the attached dependent claims.

A further object of the invention is a kit of products for the implementation of the method of the invention, the essential characteristics of which are defined in claim 6 attached hereto.

Other features and advantages of the invention will become clearer from the detailed description which follows.

Brief description of the figures

Figure 1 represents a schematic illustration of the distribution of the antibiotic products tested in a container with 96 separate compartments, in which single antibiotics are distributed, and their combinations of two and three products in certain concentrations, in three distinct sectors: in sector 1 the single antibiotics are found, in sector 2 the associations of two antibiotics and in sector 3 the associations 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 separate compartments at least equal to the number of antibiotic concentrations to be tested, and preferably in microdilution plates, which can also be completely similar to those already in use in clinical microbiology laboratories. , for example for performing the checkerboard technique.

In contrast to the checkerboard technique, however, where the entire microdilution plate is used to test a single association between antibiotic products in a high number of concentrations, in the present method the same plate is used to test a large number of associations between antibiotic products in a small number of concentrations, appropriately selected to obtain significant results for the purpose of the method, that is to evaluate the sensitivity and synergy of antibiotic associations.

Thanks to this, the method of the invention is able, with a single plate, to provide information both on the associations of two and on the associations of three antibiotics, while also maintaining a space to test the individual products and thus obtain useful data of comparison on the behavior of the tested products towards a certain bacterial isolate. With the traditional checkerboard technique, the same information obtainable with the present method would require a use of materials and times approximately 10 times higher than that envisaged according to the present invention.

However, this method is very fast, faster than even the simplest traditional techniques, such as 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 the advantages linked to the speed of this method are even greater when compared to other traditional techniques, such as the time-kill assay, which requires significantly longer execution times, as well as extremely more laborious operating modes, within the reach of laboratories only. specialized.

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 plate with microdilution wells, the bacterial isolate belonging to the Enterobacteriaceae family not sensitive to carbapenems is distributed, in against which antibiotic associations are to be tested; the plate therefore allows you to determine the minimum inhibitory concentration values (hereinafter "MIC" from the English Minimal Inhibitory Concentration) of the individual antibiotics, the synergy of the associations of two antibiotics, and the associations of three antibiotics.

Within the scope of the present invention, the antibiotic products subject to examination with the present method are selected from among the antibiotics most commonly used in the treatment of severe infections from carbapenemase-producing Enterobacteriaceae strains, and in particular the antibiotic products are selected from colistin, gentamicin, rifampicin, tigecycline, imipenem, meropenem, and their two- and three-product combinations. These associations are chosen on the basis of the mechanisms of action and tolerability of the molecules, taking into account the complementarity of the mechanisms of action and the possible effects in terms of toxicity caused by the simultaneous administration of several antibiotics.

For example, in the combinations of two or three products, meropenem and imipenem, belonging to the same family of carbapenems, are never tested together, but always in association 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 similar products always chosen according to the same criteria mentioned above, for example one or more of polymyxin B, amikacin and doripenem can be used as an alternative to replace colistin, gentamicin and meropenem respectively, possibly varying the values of the concentrations tested where necessary.

The known concentrations of each antibiotic product and their associations selected for the implementation of this method are chosen by taking only the concentrations that are actually achievable in the plasma of a patient following the administration of the antibiotic; the maximum achievable concentration values are known for any antibiotic product that can be used in the present method.

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

Table 1

Antibiotic Dosage Cmax (mg / L)

475 mg / day (in 2-3 doses) 5-7.5 colistin

5.1 mg / kg every 24 hours 4-6 gentamicin

500 mg every 6 hours 40 imipenem

1000 mg every 8 hours 49 meropenem

600 mg / day 4-32 rifampicin

100 mg loading dose followed by 50 mg every 12 hours 0.63 tigecycline

200 mg loading dose followed by 100 mg every 12 hours 1.25 tigecycline

For the antibiotic products and the respective combinations selected, in the context of this method, the ranges or concentration values to be used were therefore chosen, after determining the MIC values for the antibiotic products colistin, gentamicin, imipenem, meropenem, rifampimycin , and tigecycline, and identified their potentially active associations for a large number of clinical isolates of Klebsiella pneumoniae, producing Klebsiella pneumoniae carbapenemase (KPC), as described in detail later in the experimental part. This preliminary study made it possible to discard low relevant concentrations, identifying the concentration ranges to be tested for each antibiotic product or combination of products, reported in the following Tables 2-4:

Table 2

<Single antibiotics> Concentrations tested (µg / ml) colistin 0.25-16

tigecycline 0.125-8

gentamicin 0.25-16

rifampicin 16-128

imipenem 4-128

meropenem 8-128

Table 3

<Combination of two antibiotics> Concentrations tested (µg / ml) colistin 0.5-4

rifampicin 4

tigecycline 0.25-1

colistin 0.5-4

gentamicin 1-2

meropenem 1-8

rifampicin 4

tigecycline 1-0.125

imipenem 1-8 gentamicin 1

meropenem 1-8

tigecycline 0.25-1

meropenem 1-8

colistin 2-4

imipenem 1-8

tigecycline 0.25

tigecycline 0.5

gentamicin 0.5-4

Table 4

<Combination of three antibiotics> Concentrations tested (µg / ml) 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 associations of two of them and the associations of three are each tested in three different sectors in which the container or plate is ideally divided, also placing on the same row, in abscissa or ordinate, the different concentrations to be tested for the same product in descending order of dilution.

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 therefore ideally divided into three sectors: in sector 1 known, scalar concentrations of the single antibiotics are distributed. the synergy of the respective associations is to be tested, ie colistin (or polymyxin B), gentamicin (or amikacin), imipenem, meropenem (or doripenem), rifampimycin and tigecycline; in sector 2 of the same container or plate, known concentrations of the associations of two antibiotics whose synergy is to be tested are instead distributed; while in sector 3 known concentrations of the combinations of three antibiotics are distributed, the synergy of which is to be tested against the bacterial isolate under examination. For each antibiotic alone or in combination, significant concentrations are identified to be tested: for the single antibiotics and the combinations of two, values within the ranges indicated above in Tables 2 and 3 are selected, taking the lower limit value of the range and increasing for example, doubling up to the maximum value, while for the combinations of three antibiotics the single concentration values indicated in Table 4, corresponding to the sensitivity limit of the product, are taken. In a 96-well microdilution plate, the method for evaluating the synergy between the combinations of the 6 antibiotic products indicated above, will for example be carried out with a sector 1 comprising 36 wells, in which each single antibiotic is tested in a sector 2 comprising 52 wells with known concentrations of the 9 associations of two selected antibiotics, and a sector 3 comprising 8 wells with known concentrations of the 8 different associations of selected antibiotics; antibiotic concentrations are shown above in Tables 2-4. Figure 1 schematically illustrates the distribution of single or combined antibiotic products, indicated with an abbreviation of the name (for example COL = colistin, TIGE = tigecycline, IMI = 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 obtained, 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). Preferred is the Mueller Hinton 'cation adjusted' culture medium, freshly prepared no more than 24 hours ago. In the compartments or wells already containing the desired concentrations of antibiotics, the bacterial inoculum under test is added in order to obtain a solution containing about 5x10 <5> CFU / ml. After about 16-18 hours of incubation at about 35 ° C ± 2 ° C, the minimum bacterial growth inhibiting concentration (MIC) is then measured for the individual antibiotics, corresponding to the lowest antibiotic concentration in which no growth is visible. bacterial, visually or by spectrophotometry, while the fractional inhibitory concentration value (hereinafter FIC, from the English Fractional Inhibitory Concentration) is determined for the associations, which takes into account the MIC values of each antibiotic alone and in association. For a combination of two antibiotics A and B, the FIC fractional inhibitory concentration value can be calculated with the following formula (Bonapace C.R. (2000) Diagnostic Microbiology and Infectious Disease, 48, 43-50):

FIC = FICA + FICB

where FICA = MICA in combination / MICA and FICB = MICB in combination / MICB. For a combination of three antibiotics, a non-growth or growth information of the bacterial strain is visually detected, on the basis of which it is established whether there is, respectively, synergy or non-synergy between the antibiotics tested by comparison with the MICs of the individual antibiotics .

This method can be implemented by automating one or more stages, for example by using a program for the automatic interpretation of the MIC data detected and the calculation of the FIC values, in order to evaluate the synergy between two antibiotics in combination starting from the relative MIC data of the individual products and their association, as described above. For example, the program can be based on an Excel spreadsheet set up to automatically calculate the FIC data having entered the detected MIC data in the same sheet.

The present invention also refers to a kit of products for implementing the method described above. This kit for determining the synergy between antibiotics in association 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 plate with wells for microdilution; antibiotic products already distributed inside the compartments or wells in freeze-dried form or in frozen form pre-diluted in the culture medium; and an illustrative leaflet describing the method of the invention that can be implemented with the kit. The kit according to the present invention may also include a suitable culture medium in a separate container, for example a bottle, for possible addition at the time of use.

The method of the invention represents a significant progress in the field of antibiotic sensitivity and synergy tests against Enterobacteriaceae CNS strains; it allows to test, with a single microdilution plate and in just 16-18 hours, the MICs of the most relevant antibiotic products for the treatment of infections caused by these bacterial strains, and of a large number of their associations, both at two and three products, with a considerable saving of time, work and materials. Unlike the techniques known up to now, the method of the invention is easily implemented in the daily routine of any laboratory, providing a large number of information of clinical relevance, currently not obtainable except in a very long time and with a much greater use of resources.

The information obtained is also clinically useful for identifying an effective therapy against bacterial isolates that have a resistance profile to multiple antibiotic classes. In addition to the clinical utility point of view for the rapid identification of an effective and "personalized" antibiotic therapy, the method and the kit of the invention are also useful as a research tool to collect an enormous amount of data on the synergies between antibiotics, data that until now were not available precisely due to the lack of a simple and economical method, applicable on a large scale, but at the same time reliable and reproducible, such as that of the invention.

In the context of the present invention, the expression "bacterial strains of Enterobacteriaceae not sensitive to carbapenems" means any strain of an Enterobacterial that is not sensitive, or having reduced sensitivity, to carbapenems; for example chosen from Klebsiella spp., Escherichia coli, Enterobacter spp. and Proteus mirabilis; preferably a Klebsiella pneumoniae strain producing Klebsiella pneumoniae Carbapenemasi (KPC) is meant.

EXPERIMENTAL PART

The concentration ranges to be used for the selected antibiotic products colistin, gentamicin, imipenem, meropenem, rifampicin, and tigecycline were chosen and their potentially active associations with the respective concentration values to be used were identified, having determined the MIC values for a large number of Klebsiella pneumoniae producing Klebsiella pneumoniae (KPC) clinical isolates, 204 Klebsiella pneumoniae producing Klebsiella pneumoniae (KPC) clinical isolates, belonging to all major circulating clones, previously characterized (Giani T. (2013 ) Eurosurveillance, Volume 18, Issue 22, May 30). Thus, the concentration values and associations reported above in Tables 2-4 were found.

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

39 analyzes were then performed on as many 96-well microdilution plates, in which the individual six antibiotics and their combinations of two and three products were distributed in the positions and concentrations shown in Figure 1. The antibiotic dilutions were made using freshly prepared Mueller Hinton “cation adjusted” culture medium according to 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. The bacterial inoculum under test was added to each well in order to obtain a solution containing about 5x10 <5> CFU / ml. After about 16 hours of incubation at about 35 ° C, the MIC was measured for the single antibiotics, and for the combinations the FIC value calculated as explained above. The interpretation of the MIC values was carried out according to the EUCAST criteria (guidelines of the European Committee on Antimicrobial Susceptibility Testing), by visual determination of the MIC, checking the first well, in increasing order of concentration, in which there is no it is a clear sign of bacterial growth.

Out of the total of 39 tests performed, 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, since 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) on 162 tested combinations. The associations for which the greatest number of synergy cases was found were colistin-rifampicin (16 synergy cases) and colistin-tigecycline (14 synergy cases). In sector 3 of the plate, the association of the three antibiotics tested was found to be active in 294 cases out of 312 associations tested. Among the combinations of three antibiotics that have consistently been most active are meropenemtigecycline-gentamicin and meropenem-colistin-rifampicin. In this case, a non-growth or growth information of the bacterial strain is visually detected, on the basis of which it is established whether there is, respectively, synergy or non-synergy between the antibiotics tested.

In a limited number of cases, no synergy was found between the combination products, being the MIC for the individual products equal to or lower than the MIC for their combination.

Each determination was carried out in duplicate, obtaining almost identical values as proof of the high reproducibility of the method.

The present invention has been described up to now with reference to a preferred embodiment. It is to be understood that there may be other embodiments that pertain to the same inventive core, as defined by the scope of the claims set out below.

Claims (12)

CLAIMS 1. A method for the evaluation of the synergy between antibiotics against bacterial strains of Enterobacteriaceae not sensitive to carbapenems, comprising the following stages: to. identification of associations of two or three antibiotics potentially active towards said bacterial strains, and of respective significant concentration values to be used for said antibiotics alone or in combination with each other, chosen within the concentration ranges actually achievable in a patient's plasma following the administration of said antibiotics; b. distribution of said antibiotics, alone and in combinations of two and three antibiotics, in a container having separate compartments in a number at least equal to the number of identified concentrations, so as to achieve in each compartment one of said concentration values by dilution in a suitable culture medium; c. addition of a bacterial inoculum selected from said strains in each of said compartments and incubation for 16-18 hours; d. determination of the MIC values of the single antibiotics and their associations to two antibiotics, and calculation of the FIC values for said associations, and evaluation of the synergy between the two antibiotics based on the calculated FIC value; And. evaluation of the synergy between three antibiotics on the basis of growth inhibition in the presence of said three antibiotics tested, compared to the MIC values of the same three antibiotics taken individually. 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 Klebsiella pneumoniae strains producing Klebsiella pneumoniae Carbapenemase (KPC). The method of claim 1 wherein said container is a 96-well microdilution plate. 5. The method according to claims 1-4, wherein the identified antibiotics whose synergy is evaluated are colistin, gentamicin, imipenem, meropenem, rifampicin and tigecycline, and the significant concentrations to be used for the distribution of the antibiotic alone are identified, within the following intervals, doubling starting from the lower limit of the interval: <Antibiotics> Significant concentrations (µg / ml) colistin 0.25-16 tigecycline 0.125-8 gentamicin 0.25-16 rifampicin 16-128 imipenem 4-128 meropenem 8-128 The method according to claim 5, wherein the associations of two and three antibiotics whose synergy is to be evaluated are those indicated below together with respective significant concentration values to be used or at intervals of concentrations, in which the values to be used are identified by doubling starting from the lower limit of the range: Combination of two antibiotics Significant concentrations (µg / ml) colistin 0.5-4 rifampicin 4 tigecycline 0.25-1 colistin 0.5-4 gentamicin 1-2 meropenem 1-8 rifampicin 4 tigecycline 1-0.125 imipenem 1-8 gentamicin 1 meropenem 1-8 tigecycline 0.25-1 meropenem 1-8 colistin 2-4 imipenem 1-8 tigecycline 0.25 tigecycline 0.5 gentamicin 0.5-4 <Combination of three antibiotics> Significant concentrations (µg / ml) 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 7. A kit of products for the evaluation of the synergy between antibiotics against bacterial strains of Enterobacteriaceae not sensitive to carbapenems, comprising: a container having a plurality of separate compartments, at least as many as the concentrations of antibiotics for which one wishes to evaluate the synergy; antibiotic products distributed within said compartments in freeze-dried form or in frozen form pre-diluted in a suitable culture medium; and a leaflet describing the method as defined in claims 1-6. The kit according to claim 7, further comprising a separate container with a suitable culture medium. The kit according to claim 7, wherein said bacterial strains are strains of Klebsiella spp., Escherichia coli, Enterobacter spp. and Proteus mirabilis. The kit according to claim 7, wherein said bacterial strains are Klebsiella pneumoniae strains producing Klebsiella pneumoniae Carbapenemase (KPC). The kit of claim 7 wherein said container is a 96-well microdilution plate. The kit according to claims 7-11, comprising the antibiotics and their associations as defined in claims 5 and 6.