USOO9347888B2

(12) United States Patent (10) Patent No.: US 9,347,888 B2 Ghirardi et al. (45) Date of Patent: May 24, 2016

(54) DETECTION OF BACTERIA EXHIBITING A Wexler et al., “In Vitro Activities of Faropenem against 579 Strains of RESISTANCE TO Anaerobic Bacteria'. Antimicrobial Agents and Chemotherapy, Nov. 2002, vol. 46, No. 11, pp. 3669-3675.* (75) Inventors: Sandrine Ghirardi, Saint Genis les Naiemi et al., “Extended-spectrum beta-lactamases screening agar Ollieres (FR); John Perry, Newcastle with AmpC inhibition”, Eur J. Clin Microbiol Infect Dis (2009) upon Tyne (GB); Gilles Zambardi, 28:989-990. DOI 10.1007/s10096-009-0714-8. Chezeneuve (FR) Rodel et al., “In vitro activities of faropenem, , and against Borreliaburgdorferi s.l.. International Jour (73) Assignee: BIOMERIEUX, Marcy L’Etoile (FR) nal of Antimicrobial Agents 30 (2007) 83-86.* Pages etal; “Efflux Pump, the Masked Side of B-Lactam Resistance (*) Notice: Subject to any disclaimer, the term of this in Klebsiella pneumoniae Clinical Isolates.” PLoS ONE: Mar. 2009; patent is extended or adjusted under 35 vol. 4: Issue 3, e4817. U.S.C. 154(b) by 0 days. Samraetal; “Evaluation ofCHROMagar KPC for Rapid Detection of -Resistant Enterobacteriaceae.” Journal of Clinical (21) Appl. No.: 14/001565 Microbiology; Jul. 2008; vol. 46; No. 9: pp. 3110-3111. Nordmann et al; "How to Detect NDM-1 Producers.' Journal of (22) PCT Filed: Mar. 16, 2012 Clinical Microbiology; 2011; Dec. 2010; vol. 49; No. 2: pp. 718-721. Mushtaq et al., “Activity of faropenem against -resis (86). PCT No.: PCT/FR2O12/050556 tant Enterobacteriaceae.” Journal of Antimicrobial Chemotherapy; 2007: vol. 59: pp. 1025-1030. S371 (c)(1), Giske et al., “A Sensitive and specific phenotypic assay for detection (2), (4) Date: Aug. 26, 2013 of metallo-B-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks Supplemented with aminophenylboronic (87) PCT Pub. No.: WO2012/131216 acid, dipicolinic acid and cloxacilling” Clinical Microbiology and PCT Pub. Date: Oct. 4, 2012 Infection; Jun. 2010; vol. 17; No. 4; pp. 552-556. Malléa et al; "Inhibitors of efflux pump in resistant (65) Prior Publication Data Enterobacter aerogenes strains.” Biochemical and Biophysical Research Communications; 2002; vol. 293; pp. 1370-1373. US 2013/0344522-A1 Dec. 26, 2013 Orenga et al; "Enzymatic Substrates in microbiology,” Journal of Microbiological Methods: 2009; vol. 79: pp. 139-155. (30) Foreign Application Priority Data Panagea et al; “Evaluation of CHROMagarTM KPC for the detection of carbapenemase-producing Enterobacteriaceae in rectal Surveil Mar. 25, 2011 (FR) ...... 1152477 lance cultures;” International Journal of Antimicrobial Agents; 2010. May 22, 2012 Search Report issued in International Patent Applica (51) Int. Cl. tion No. PCT FR2012/050556. GOIN 2L/76 (2006.01) Translation of May 22, 2012 Written Opinion issued in International CI2N L/20 (2006.01) Patent Application No. PCT/FR2012/050556. CI2N I5/0 (2006.01) CI2O I/04 (2006.01) * cited by examiner (52) U.S. Cl. CPC ...... G0IN 21/763 (2013.01); C12N 1/20 (2013.01): CI2N 15/01 (2013.01); C12O I/04 Primary Examiner — Suzanne M Noakes (2013.01) (74) Attorney, Agent, or Firm — Oliff PLC (58) Field of Classification Search None (57) ABSTRACT See application file for complete search history. Disclosed is a process for detecting and/or identifying, in a (56) References Cited biological sample, bacteria exhibiting a resistance to carbap enems, including: a) contacting said sample with a reaction FOREIGN PATENT DOCUMENTS medium including at least one chromogenic agent and faro and/or ; b) incubating the whole so as to FR 2925 O70 A1 6, 2009 allow the bacteria to grow; and c) detecting the strains exhib WO WO 2010/010O83 A1 1, 2010 iting a resistance to carbapenems. The medium employed in OTHER PUBLICATIONS step a) also contains and/or a combination of clox acillin and PAbetaN. Walsh, F., “Doripenem: A new carbapenem antibiotic a review of comparative antimicrobial and bactericidal activities'. Therapeutics and Clinical Risk Management 2007:3(5) 789-794.* 21 Claims, No Drawings US 9,347,888 B2 1. 2 DETECTION OF BACTERIA EXHIBITINGA context, in particular with the emergence of new resistances RESISTANCE TO CARBAPENEMS such as NDM-1, and permits the screening of all of the resis tance mechanisms due to the production of all known types of The present invention relates to a detection and identifica carbapenemases. tion process Suitable for screening bacteria which are resis In this respect, the present invention relates to a process for tant to carbapenems. detecting and/or identifying, in a biological sample, bacteria The increase in the resistance to beta-lactam , exhibiting a resistance to carbapenems, comprising the steps Such as and , complicates the treat consisting in: ment of infections caused by strains of Gram-negative bacte a) contacting said sample with a reaction medium compris ria. These antibiotics are then replaced by other broad-spec 10 ing at least one chromogenic agent and faropenem and/ trum antimicrobials. Amongst these broad-spectrum or doripenem: antimicrobials, carbapenems have taken an important role, b) incubating the whole so as to allow the bacteria to grow; especially for treating hospitalised patients. Carbapenems act c) detecting the strains exhibiting a resistance to carbapen against the majority of Gram-positive and Gram-negative CS. aerobic bacteria, and on certain anaerobic bacteria. 15 The Applicant has shown that the addition of faropenem However, more and more strains resistant to carbapenems and/or doripenem into a chromogenic medium makes it pos are appearing in hospitalised patients. sible for the majority of carbapenemase-producing bacteria to The bacteria concerned are, non-exhaustively, Escherichia grow, whilst inhibiting a majority of strains which do not coli, Enterobacter cloacae, Enterobacter aerogenes, Citro produce them, such as wild strains, and strains which produce bacter sp., Klebsiella pneumoniae, Klebsiella Oxytoca, extended-spectrum beta-lactamases or high-level cepha , Providencia rettgeri, Pseudomo losporinases, for example. The tests have made it possible to nas putida, Stenotrophomonas maltophilia, Acinetobacter demonstrate a greater sensitivity and specificity than those of baumanii, Comamonas sp., Aeromonas sp., Morganella mor a chromogenic medium using meropenem. ganii, Enterococcus sp., Proteus mirabilis, Salmonella sen According to a first embodiment, the present invention fienberg, Serratia marcescens, Salmonella typhimurium, etc. 25 corresponds to a process for detecting and/or identifying, in a The reduced susceptibility to carbapenems can be due to: biological sample, bacteria exhibiting a resistance to carbap the expression of a gene which is resistant to beta-lactams: enems, comprising the steps consisting in: (i) hyperproduction of ampC beta-lactamases and/or a) contacting said sample with a culture medium compris (ii) ESBL (extended-spectrum beta-lactamase), ing at least one chromogenic Substrate, and at least faro combined with changes in the permeability of the 30 penem and/or doripenem: (impermeability resistance) and/or with the active efflux of b) incubating the whole so as to allow the bacteria to grow; the antibiotics (Pages et al., 2009; PloS ONE, 4 (3)); and/or c) detecting the strains exhibiting a resistance to carbapen the existence of enzymes which break down carbapenems, CS. called carbapenemases. According to a preferred embodiment of the invention, the The carbapenemase genes may be present in chromosomes 35 bacteria are NDM-1 bacteria. and/or in plasmids. Due to this presence in the form of plas Preferably, the carbapenem concentrations are between mids, these kinds of enzymatic resistance are capable of 0.05 and 32 mg/L. spreading to a great extent and, consequently, pose a major More preferably, the carbapenem concentrations are risk in epidemiological terms. between 2 and 32 mg/L for faropenem, and between 0.05 and The person skilled in the art has difficulty in easily detect 40 2 mg/L for doripenem. ing and/or identifying the strains of bacteria which are resis Advantageously, the medium employed in step a) also tant to carbapenems. comprises cloxacillin and/or a combination of cloxacillin and A method of characterising by using a chromogenic PAbetaN. These compounds provide an additional level of medium comprising meropenem and/or ertapenem was Sug selection and make it possible to distinguish the imperme gested in Application WO 2010/010083 and implemented in 45 ability resistances and other non-enzymatic resistances from the media CHROMagar R. KPC (Samra et al., 2008; J. Clin. the resistances by production of carbapenemase. Microbiol. 46 (9): 3110-31 11: CHROMagarTM, Paris, Biological sample, is to be understood to be a small part or France) and COLOREXTM KPC (BioMed Diagnostics Inc.). Small isolated quantity of an entity for analysis. This can be a This medium does not permit the detection of all of the clinical sample, human or animal, from a specimen of bio carbapenamase-producing strains, particularly the NDM-1 50 logical liquid, or a food sample, from any type of food or a strains which have appeared recently (Nordmann et al., 2011; sample from the food production or processing environment. J Clin Microbiol. 49(2): 718-721). In the current epidemio This sample can thus be liquid or solid. It is possible to cite in logical context, and particularly with the emergence of these a non-limiting manner, a clinical sample of whole blood, new NDM-1 resistances, there is still a need to improve, in serum, plasma, urines, faeces, specimens of nose, throat, skin, sensitivity and/or specificity, the screening of all of the 55 wounds, cerebrospinal fluid, a food sample of water, bever mechanisms of resistance to carbapenems. ages such as milk, fruit juices, yoghurt, meat, eggs, Veg Faropenem and doripenem are recently developed and etables, mayonnaise, cheese, fish, etc., a food sample from an tested carbapenems (Mushtaq et al., 2007: Journal of Antimi animal feed, such as in particular an animal meal sample, or a crobial Chemotherapy, 59: 1025-1030 Lee et al., 2011, sample for the control of a surface area or water body. This Microbial Drug Resistance). To the Applicant’s knowledge, 60 specimen can be used such as it is or, prior to the analysis, they have never been used to detect and/or identify, directly in undergo preparation by enrichment, dilution, extraction, con biological samples, bacterial Strains resistant to carbapenems. centration or purification, in accordance with methods known The Applicant has shown that it is possible to improve the to the person skilled in the art. detection of strains resistant to carbapenems, more particu Reaction medium, is to be understood to be a medium larly all of the strains which produce carbapenemases, includ 65 comprising all the elements necessary for the expression of ing KPC carbapenemases and NDM carbapenemases. This metabolism and/or for the growth of microorganisms. The meets the demands linked to the current epidemiological reaction medium can be solid, semi-solid or liquid. Solid US 9,347,888 B2 3 4 medium is understood to be a gelled medium, for example. the invention can also contain a pH indicator which is sensi Agar is the conventional gelling agent in microbiology for the tive to the pH variation induced by the consumption of the culturing of microorganisms, but it is possible to use gelatine, substrate and which reveals the metabolism of the target agarose, or other natural or artificial gel-forming Substances. microorganisms. Said pH indicator can be a chromophore or A number of preparations are commercially available, for a fluorophore. As examples of chromophores, mention can be instance Columbia agar, Trypcase-Soyagar, MacConkey agar made of bromocresol purple, bromothymol blue, neutral red, or more generally those described in the Handbook of Micro aniline blue and bromocresol blue. Fluorophores include for biological Media (CRC Press). example 4-methylumbelliferone, hydroxycoumarin deriva The reaction medium may comprise one or more elements tives or resorufin derivatives. in combination, such as amino acids, peptones, carbohy 10 According to the present invention, the chromogenic Sub drates, nucleotides, minerals, vitamins, etc. The medium may strate is preferably chosen from Indoxyl-based substrates also contain a dye. As an indication, possible dyes may be (3-Indoxyl, 5-Bromo-3-indoxyl, 5-Iodo-3-indoxyl, Evans blue, neutral red, sheep blood, horse blood, an opacifier 4-Chloro-3-indoxyl, 5-Bromo-4-chloro-3-indoxyl, Such as titanium oxide, nitroaniline, malachite green, brilliant 5-Bromo-6-chloro-3-indoxyl, 6-Bromo-3-indoxyl, green, one or more metabolic indicators, one or more meta 15 6-Chloro-3-indoxyl, 6-Fluoro-3-indoxyl, 5-Bromo-4- bolic regulators, etc. chloro-N-methyl-3-indoxyl, N-Methyl-3-indoxyl, AldolTM, The reaction medium may be a revealing medium or a culturing and revealing medium. In the first case, the cultur etc.); umbelliferone-based substrates (4-Methylumbellifer ing of the microorganisms is performed before seeding and, one, Cyclohexenoesculetin, etc.); Alizarin-based substrates; in the second case, the detection and/or identification medium p-Naphtholbenzein-based substrates; Nitrophenol-based also constitutes the culture medium. substrates (ortho-Nitrophenol, para-Nitrophenol, etc.); The person skilled in the art may also use a bi-plate, which Hydroxyquinoline-based substrates: Cathecol-based sub makes it possible to easily compare two media, comprising strates (Cathecol, Dihydroxyflavone, Hydroxyflavone, etc.); different substrates or different selective mixtures, onto Resorufin-based substrates; Chlorophenol Red-based sub which the same biological sample will have been deposited. 25 strates; Fluorescein-based Substrates; Aminophenol-based The reaction medium may comprise one or more selective Substrates (para-Aminophenol, Dichloro-aminophenol, etc.); agents. Selective agent is understood to be any compound Naphthol-based substrates (alpha-Naphthol, 2-Naphthol, capable of preventing or slowing the growth of a microorgan Naphthol-ASBI, etc.); Aminocoumarin-based substrates ism other than the target microorganism. Without being lim (7-Amino-4-methyl-coumarin, etc.); Naphthylamide-based iting, a concentration of between 0.01 mg/l and 5 g/l is par 30 Substrates: Acridine-based substrates (Amino-phenyl-acri ticularly suitable for the present invention. dine, etc.); Amino-phenoxazine-based substrates (Amino As a selective agent, mention can be made of antibiotics, antifungals, bile salts, crystal violet, basic fuchsine, brilliant benzophenoxazinone, Amino-pentyl-resorufin, etc.). green, etc. Antibiotics are to be understood to be any com As an indication, the enzymatic activities targeted by the pound capable of preventing or slowing the growth of a bac chromogenic Substrates can belong to the hydrolases group, terium. In particular, they belong to the beta-lactams, glyco 35 and preferably to theosidases, esterases or peptidases groups. peptides, aminosides, polypeptides, Sulfamides and As an indication, the substrates used for the detection of a quinolones groups. As an indication, it is in particular pos beta-glucuronidase activity can in particular be 4-Methylum sible to mention the antibiotics cloxacillin, , cef belliferyl-beta-glucuronide, 5-Bromo-4-chloro-3-indolyl Sulodin, , , to , , beta-glucuronide, 5-Bromo-6-chloro-3-indolyl-beta-glucu , , gentamicin, Trimethoprim, tobra 40 ronide, 6-Chloro-3-indolyl-beta-glucuronide, Alizarin-beta mycin, moxalactam, , D-, , glucuronide, Cyclohexenoesculetin-beta-glucuronide or salts , and quinolones such as nalidixic acid. thereof. Antifungals are to be understood to be any compound The substrates used for the detection of a beta-galactosi capable of preventing or slowing the growth of a yeast or a dase activity can in particular be 4-Methylumbelliferyl-beta mould. By way of indication, it is possible to mention in 45 galactoside, 5-Bromo-4-chloro-3-indolyl-beta-galactoside, particular amphotericin B, fluconazole, itraconazole, Vori 5-Bromo-6-chloro-3-indolyl-beta-galactoside, 6-Chloro-3- conazole and cycloheximide. indolyl-beta-galactoside, Alizarin-beta-galactoside, Cyclo Cloxacillin corresponds to an antibiotic in the hexenoesculetin-beta-galactoside or their salts. class. It is used in vitro to inhibit certain beta-lactamases The substrates used for the detection of a beta-glucosidase (Giske et al., 2010: Clin. Microbiol. Infect.; 17 (4): 552-6). 50 activity can in particular be 4-Methylumbelliferyl-beta-glu and are advantageously consid coside, 5-Bromo-4-chloro-3-indolyl-beta-glucoside, ered as equivalent to cloxacillin. Preferably, cloxacillin is 5-Bromo-4-chloro-3-indolyl-N-methyl-beta-glucoside, used at a concentration of between 25 and 300 mg/l. PABN or PAbetaN corresponds to phenylalanine-arginine 5-Bromo-6-chloro-3-indolyl-beta-glucoside, 6-Chloro-3-in beta-naphthylamide. This compound is known as an efflux dolyl-beta-glucoside, Alizarin-beta-glucoside, Cyclohex pump inhibitor, making it possible, for example, to reduce the 55 enoesculetin-beta-glucoside, Nitrophenyl-beta-glucoside, minimum inhibitory concentration (MIC) of chlorampheni Dichloroaminophenylglucoside or their salts. col for strains of Enterobacter aerogenes (Mallea et al., 2002: As an indication, the Substrates used to detect an esterase Biochemical and Biophysical Research Communications activity can in particular be the esters of Saturated or unsat 293: 1370-3). Preferably, PAbetaN is used at a concentration urated linear fatty acids, having between 6 and 14 carbons, of between 1 and 50 mg/l. 60 preferably between 7 and 9 carbons and of 4-Methylumbel Chromogenic substrate is to be understood to be a substrate liferone, 5-Bromo-4-chloro-3-indoxyl, 5-Bromo-6-chloro-3- making it possible to detect an enzymatic or metabolic activ indoxyl, 6-Chloro-3-indoxyl, 5-Bromo-3-indolyl or of ity of the target microorganisms by means of a directly or Alizarin or their salts. Preferably, they are chosen from 4-Me indirectly detectable signal. For direct detection, this sub thylumbelliferyl-octanoate, 5-Bromo-4-chloro-3-indoxyl strate can be linked to apartacting as a fluorescent or coloured 65 octanoate, 5-Bromo-6-chloro-3-indoxyl-octanoate, label (Orenga et al., 2009; J. Microbiol. Methods: 79(2): 139 6-Chloro-3-indoxyl-octanoate, 5-Bromo-3-indolyl-oc 55). For indirect detection, the reaction medium according to tanoate or Alizarin-octanoate. US 9,347,888 B2 5 6 The substrates used for the detection of a deaminase activ- in Table I, were tested on media containing different concen ity can in particular be L-Tryptophan, L-Phenylalanine, trations of faropenem in order to establish the sensitivity/ L-Tyrosine and L-Histidine. specificity of each formulation. The dishes were read after 24 The substrates used for the detection of a sulfatase activity hours of incubation at 37° C. can in particular be 4-Methylumbelliferyl-sulfate, 5-Bromo- 5 4-chloro-3-indoxyl-sulfate, 5-Bromo-6-chloro-3-indoxyl- TABLE I sulfate, 3-indoxyl-sulfate, phenolphthalein-disulfate or their salts. resistance mechanisms characterising the strains tested Preferably, the chromogenic substrate is chosen from: 5-Bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside 10 Number of strains (X-glucoside), 5-Bromo-6-chloro-3-indoxyl-beta-D-galac- hal topyranoside (Magenta beta-Gal), 6-Chloro-3-indoxyl-beta- ESBL or cephalosporinase (ESBL or Case) 15 Class A KPC carbapenemase (KPC) 34 D-glucuronide (Rose beta Gur), 5-Bromo-4-chloro-3-in- Class A non-KPC carbapenemase (Carba A) 8 doxyl-N-methyl-beta-D-glucopyranoside (Green A beta Class B carbapenemase (Carba B) 10 Glu), Methyl-beta-D-glucopyranoside (methyl beta D gluco- 15 Class D carbapenemase (Oxa) 6 side) and L-Tryptophan. Impermeability resistance (IR) 6 Incubate is to be understood to mean raising to and holding at, for between 1 and 48 hours, preferably between 4 and 24 hours, more preferably between 16 and 24 hours, an appro- The medium used is a conventional agar medium compris priate temperature, generally of between 20 and 50° C., pref 20 ing at least one chromogenic Substrate, and Supplemented erably between 30 and 40°C. with faropenem at the following concentrations:

Medium 1 Medium 2 Medium 3 Medium 4 Medium 5 Medium 6 Medium 7 Medium 8 Faropenem (mg/L) O O.S 1 2 4 8 16 32

Detect is to be understood to mean discerning, with the 2. Test naked eye or using an optical apparatus, the existence of a The media are divided into 120x120 square dishes. growth of the target bacteria. Advantageously, when the The seeding is performed from 24-h pre-cultures at 37°C. medium employed contains a chromogenic substrate, the on trypcase Soy agar. detection can also make it possible to identify the target For each strain, a 0.5 McF suspension in physiological bacteria. The detection takes place using an optical apparatus water is produced and then diluted 1/100. for the fluorescent substrates, or with the naked eye or using Each suspension is spot-seeded (1 to 2 ul) on each medium an optical apparatus for the coloured substrates. with the aid of a multi-point inoculator according to the Agar Enzymatic resistance to carbapenems is to be understood Dilution (AD) method. to mean, as indicated Supra, the resistance to carbapenem Readings are performed after 24 hours of incubation at 37° antibiotics due to the expression, by the target bacteria, of C. carbapenemases. 40 For interpretation, the following cases are considered as The most frequently encountered bacteria which are resis- corresponding to growth inhibition: tant to carbapenems are, as indicated Supra: Escherichia coli, absence of bacterial growth Enterobacter cloacae, Enterobacter aerogenes, Citrobacter number of coloniess3 sp., Klebsiella pneumoniae, Klebsiella Oxytoca, Pseudomo- The presence of 4 colonies or more is considered to be nas aeruginosa, Providencia rettgeri, Pseudomonas putida, 45 positive growth. Stenotrophomonas maltophilia, Acinetobacter baumanii, 3. Results and Interpretation Comamonas sp., Aeromonas sp., Morganella morganii, Detection sensitivity and specificity are indicated in Table Enterococcus sp., Proteus mirabilis, Salmonella Senftenberg, II. Serratia marcescens, Salmonella typhimurium, etc. Table II: detection sensitivity/specificity of the carbapen The present invention also relates to a culture medium for so emase-producing Strains on a medium containing 16 mg/L of detecting and/or identifying bacteria exhibiting a resistance faropenem. to carbapenems, said culture medium corresponding to a basic culture medium, further comprising at least one chro- TABLE II mogenic Substrate and at least faropenem and/or doripenem. detection sensitivity specificity of the carbapenemase-producing The examples developed below aim to facilitate the under- 55 Strains on a medium containing 10 mg/L of farOpenen. standing of the invention. They are given by way of explana tion and are not intended to limit the scope of the invention. Inoculum: 0.5 McF Inoculum: 0.5 McF diluted /100 EXAMPLES Detection sensitivity 53.58 53.58 60 91.4% 91.4% Example 1 Detection specificity 1421 1S,21 66.7% 71.4% Test of Faropenem on Pure Strains 1. Media and Microorganisms Faropenem, used for example at 16 mg/L, makes it possible Seventy-nine strains of Gram-negative bacteria, of which 65 to clearly distinguish between the carbapenemase-producing 69 are enterobacteria and 10 non-enterobacteria (non-fer- bacteria and the other types of resistance. The MIC of the menting bacteria), whose resistance characteristics are set out totality of KPC strains is always >32, whatever the inoculum US 9,347,888 B2 7 8 tested. All of the KPC strains develop on the medium com- supra (0.065 mg/L of doripenem) and on the COLOREXTM prising faropenem at 16 mg/L. KPC medium. This latter medium is provided by BioMed Diagnostics Inc. and corresponds to the CHROMagarTM KPC Example 2 ready-to-use medium. 5 Doripenem Test on Pure Strains TABLEV 1. Media and Microorganisms One hundred strains of Gram-negative bacteria, of which 98 are enterobacteria and 2 non-enterobacteria (non-ferment resistance mechanisms characterising the strains tested ing bacteria), whose resistance characteristics are set out in 10 Table III, were tested on media containing different concen Number of strains trations of faropenem in order to establish the sensitivity and - Y - ESBL 42 the specificity of each formulation. A medium containing Cephalosporinase AmpC (29), others (4 33 0.125 mg/L meropenem was also tested. The chromogenic 8. pC (29), others (4) medium UriSelect(R) (Biorad) is used as a growth control. The 15 Class A KPC carbapenemase 6 dishes were read after 24 h and 48 h of incubation at 37° C. Class B carbapenemase (Carba B) 19

TABLE III 2. Test resistance mechanisms characterising the strains tested 2O Each Strain is spot-seeded on each medium, using 1 LL Number of strains (containing around 10 microorganisms) of a diluted 0.5McF Suspension. ESBL 42 Cephalosporinase: AmpC (30), others (4) 34 Readings are performed after 24 and 48 hours of incuba Class A KPC carbapenemase (KPC) 5 tion at 37° C. Class B carbapenemase (Carba B) 19 25 3. Results and Interpretation The medium used is a conventional agar medium compris- TABLE VI ing at least one chromogenic Substrate, and Supplemented with carbapenem at the following concentrations: 30 percentage of strains which develop on the media Medium B Medium B (0.065 mg/L (0.065 mg/L Medium Medium Medium Medium Medium COLOREX COLOREX doripenem) doripenem) A. B C D E KPC24 KPC 48 24h 48 h. Doripenem (mg/L) O O.06S O.12S O.25 O 35 % carba- 8O 8O 88 92 Meropenem (mg/L) O O O O O.12S penemase producing strains 2.Test - - - - % other 5 5 10.7 10.7 The media are divided into 90 mm dishes. strains Each Strain is spot-seeded on each medium, using 1 uL 40 (containing around 10 microorganisms) of a diluted 0.5McF Suspension. The medium containing 0.065 mg/L of doripenem permits Readings are performed after 24 and 48 hours of incuba- the growth of 92% of the carbapenemase-producing Entero tion at 37° C. bacteria tested, against only 80% on the commercial medium, 3. Results and Interpretation whatever the incubation time. The medium containing dorip The results are brought together in Table IV. enem is therefore Superior to the commercial medium. TABLE IV percentage of strains of each resistance category which develop on the different media.

Medium E Medium B Medium C Medium D 0.125 mg/L UriSelect (R) 0.065 mg/L 0.125 mg/L 0.25 mg/L meropenem (Biorad) Medium A doripenem doripenem doripenem (mg/l) Carbapenemases 100 1OO 83 50 33 50 Others 100 98.7 8 5 4 5

Example 3 60 Example 4 Comparison of the Medium Containing Doripenem with a Tests on Clinical Specimens Commercially Available Medium 1. Media and Specimens Two hundred swabs (with transport medium) loaded with 1. Media and Microorganisms bacterial populations isolated from stools were seeded on One hundred enterobacteria strains, of which 25 produce 65 medium B indicated supra (0.065 mg/L of doripenem), on a carbapenemases and of which the resistance characteristics medium similar to medium 7 indicated Supra (16 mg/L of are set out in Table V, were tested on medium B described faropenem) and on the COLOREXTM KPC medium. US 9,347,888 B2 9 10 2. Test Example 5 0.5 mL of sterile water are added to the tubes containing the swab and the transport medium. The tubes are vortexed. Test of Stability of the Different Carbapenems in Agar 10 ul of the obtained suspension are seeded in 4 quadrants Medium on each medium. Readings of the media are performed after 5 Agar media modelled on those indicated Supra were pre 24 hours of incubation at 37° C. pared and kept at a temperature of between 2 and 8°C. for up 3. Results and Interpretation to 9 weeks (63 days). ESBL and AmpC strains, and carbapenemases from class TABLE VII 10 B, IR or KPC, were seeded on these different media on D0, results from screening carbapenemase-producing strains D14, D42 and D63. from bacterial populations isolated from Stools. These tests have made it possible to show that doripenem and faropenem exhibit a stability in agar medium which is All Medium B Medium 7 close to that of ertapenem, and greater than that of imipenem media (0.065 mg/L Colorex (16 mg/L mixed doripenem) KPC faropenem) 15 and of meropenem. No growth 141 1S6 145 Positive Specimens Example 6 Metallo-carbapenemase Impact of cloxacillin and/or PAbetaN on the Minimum E. coi 30 30 12 30 E. cloacae 22 22 22 17 Inhibitory Concentrations (MICs) of the strains which are Citrobacter sp. 7 3 2 6 impermeability-resistant (IR) or exhibit another non-enzy K. pneumoniae 3 O 2 2 matic resistance mechanism, or are carbapenemase-produc Prettgeri 2 O 2 O ing, in the presence of faropenem. Total CPE: 64 55 40 55 1. Media and Microorganisms Sensitivity (%) 85.9 62.5 85.9 25 Seventy-seven strains of Gram-negative bacteria, of which S. maltophilia 4 4 3 4 A. battmannii 3 2 3 O 71 are enterobacteria and 6 non-enterobacteria (non-ferment Ps putida 1 O 1 O ing bacteria), whose resistance characteristics are set out in Comamonas aquatic 1 O 1 O Table VIII, were tested on media containing different concen Aeromonas sp. 1 O 1 O trations of faropenem and cloxacillin, with or without Negative Specimens 30 PAbetaN, in order to establish the sensitivity and the speci Metallo-carbapenemase ficity of each formulation. The tested media are described in E. coi 13 13 2 9 Table IX. A. battmannii 4 3 3 1 Paeruginosa 1 1 1 O TABLE VIII M. morganii 5 5 1 1 35 Total false positives: 33 28 16 15 mechanisms of resistance to antibiotics (Only Gram-negatives) characterising the strains tested. Enterococci is sp. 13 11 O 13 Number of strains ICarbapenemase-producing enterobacteria 40 ESBL (7), Cephalosporinase AmpC (9) 16 Class A KPC carbapenemase (KPC) 10 The 64 strains of Carbapenemase-producing Enterobacte Class A non-KPC carbapenemase (Class A) 5 ria (CPE), of the NDM-1 type, were isolated from 37 stool Class B NDM-1 carbapenemase (NDM) 15 specimens. Medium 7 comprising 16 mg/L of faropenem Class B non-NDM carbapenemase (Class B) 6 Class DOXA carbapenemase (OXA) 6 exhibits the best performances in terms of sensitivity (86%) Impermeability resistance (IR) 19 and specificity (15 Gram-false positives). Medium B com 45 prising 0.065 mg/L of doripenem exhibits an equally high TABLE IX composition of the tested media Media C 1 S 9 2 6 10 3 7 11 4 8 12 13 16 19 14 17 20 15 18 21 Chromogenic base 47 g/L PABN (mg/L) O 25 Cloxacillin (mg/L) O 50 100 200 50 100 2OO Faropenem (mg/L) O 8 16 32 8 16 32 8 16 32 8 16 32 8 16 32 8 16 32 8 16 32 sensitivity, but a slightly lower specificity (28 Gram-false 2. Test positives). The COLOREXTMKPC medium makes it possible to The chromogenic media are divided into 120x120 square to detect only 62.5% of the CPE, for a specificity/selectivity dishes. equivalent to that of the medium containing faropenem. The seeding is performed from 24-h pre-cultures at 37°C. In conclusion, the method according to the invention of on trypcase Soyagar. For each strain, a 0.5 McFSuspension in detecting carbapenem-resistant bacteria, demonstrates physiological water is produced. Each Suspension is spot through examples 1, 2, 3 and 4, the improvement which it 65 seeded (1 to 2 LL) on each medium with the aid of a multi brings to the state of the art, both on laboratory strains and on pointinoculator according to the Agar Dilution (AD) method. clinical specimens. Readings are performed after 24 hours of incubation at 37°C. US 9,347,888 B2 11 12 3. Results These results also confirm the better inhibition of the IR The results are brought together in Table X. strains in the presence of cloxacillin+PAbetaN. This effect is An absence of bacterial growth or a number of colonies visible for the majority of the cloxacillin and faropenem which is less than or equal to 3 are considered as correspond concentrations tested, but is very distinctly marked in the ing to growth inhibition. The presence of 4 colonies or more 5 presence of 200 mg/L of cloxacillin (whatever the faropenem is considered to be positive growth. concentration). TABLE X impact of cloxacillin and/or of PAbetaN on the growth (number of strains developing) of the strains which produce carbapenemases or which are resistant to beta-lactans due to another resistance mechanism, in the presence of faropenen Media C 1 S 9 2 6 10 3 7 11 4 8 12 13 16 19 14 17 20 15 18 21 PABN (mg/L) O 25 Cloxacillin (mg/L) O 50 1OO 2OO 50 1OO 200 Faropenem (mg/L) O 8 16 32 8 16 32 8 16 32 8 16 32 8 16 32 8 16 32 8 16 32 ESBL 7 1 1 O 1 O O 1 O O 1 O O 3 1 O 1 O O 1 O O AmpC 9 3 2 2 2 2 2 2 2 2 2 2 1 4 3 2 3 2 2 2 2 O IR 19 16 1S 10 14 9 4 11 6 2 11 6 1 11 1 O S 10 S 1 7 1 1

Total non- 3S 20 18 12 17 11 6 14 8 4 14 8. 2 18 14 7 14 7 3 10 3 1 carbapenemases Class A 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Class B 6 4 3 3 4 4 3 3 3 3 3 3 3 S 3 2 4 2 2 4 2 2 KPC 10 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 10 NDM 1S 12 8 8 12 8 6 12 9 7 12 8 7 14 12 8 12 12 6 10 9 6 OXA 6 4 4. 2 4 4 1 4 3 2 4 4 1 S 4 O S 4 O S 4 O

Total 42 35 30 28 35 31 25 34 30 27 34 30 26 39 34 2S 36 33 23 34 30 23 carbapenemases

4. Interpretation TABLE XI sensitivity and specificity of media containing faropenem in the presence of cloxacillin, combined or not with PAbetaN, for strains which produce carbapenemases or which are resistant to beta-lactams due to another resistance mechanism.

Media C 1 5 9 2 6 10 3 7 11 4 8 12 13 eve 19 14 17 20 15 18 21 PABN (mg/L) O 25 Cloxacillin (mg/L) O 50 100 2OO 50 1OO 2OO Faropenem (mg/L) O 8 16 32 8 16 32 8 16 32 8 16 32 8 16 32 8 16 32 8 16 32 Sensitivity (%) 1OO 83 71 67 83 74 6O 81 71 64 81 71 62 93 81 60 86 79 SS 81 71 SS Specificity (%) O 43 49 66 S1 69 83 60 77 89 60 77 94 49 60 80 60 8O 91 71 91 97

4a. Impact of Cloxacillin and PAbetaN on IR Strains TABLE XII Effects of cloxacillin, associated or not with PAbetaN, on the growth of the 19 IR strains tested. PAbetaN (mg/L) O 25 Cloxacillin (mg/L) O 50 1OO 2OO 50 1OO 200 Faropenem (mg/L) O 8 16 32 8 16 32 8 16 32 8 16 32 8 16 32 8 16 32 8 16 32 No. IR developing 19 16 1S 10 14 9 4 11 6 2 11 6 1 11 1 O S 10 S 1 7 1 1 (19)

55 Faropenem alone has little impact on these strains, up to a 4b. Impact of Cloxacillin and PAbetaN on the Carbapen concentration of 16 mg/L. An inhibition of the IR strains is emase-Producing Strains observed from the moment when 50 mg/L of cloxacillin is The results obtained in presence of faropenem confirm that added, and is even more marked for the highest concentra the cloxacillin alone has little or no impact on the growth of 60 the carbapenemase-producing strains (at the 3 concentrations tions of faropenem tested (16 and 32 mg/L). This inhibitory of faropenem tested). They also confirm that associating effect increases with the concentration of cloxacillin (for PAbetaN with the cloxacillin makes it possible to increase the example, for a concentration of 16 mg/L of faropenem, the MIC of certain strains to carbapenems so long as the faro addition of 50 mg/L or 200 mg/L cloxacillin respectively penem concentration remains less than 32 mg/L. Thus, the permits the inhibition of 6 and 9 additional IR strains). A 65 detection sensitivity in the presence of 8 or 16 mg/L of faro combined effect of the concentration of faropenem and of that penem associated with 50 or 100 mg/L of cloxacillin is of cloxacillin on the IR strains is observed. greater in the presence of PAbetaN. For 200 mg/L of clox US 9,347,888 B2 13 14 acillin and 8 or 16 mg/L of faropenem, the addition of 7. The process of claim 1, wherein the culture medium is a PAbetan does not change the detection sensitivity. solid culture medium. 5. Conclusion 8. The process of claim 1, wherein the culture medium is a These results confirm the inhibitory role of cloxacillin on liquid culture medium. the strains which are impermeability-resistant, in the pres 5 9. The process of claim 1, wherein the culture medium ence of a carbapenem. These results also confirm optimum further comprises cloxacillin, dicloxacillin, or flucloxacillin. inhibition of the IR strains when cloxacillin is associated with 10. The process of claim 3, wherein the culture medium PAbetaN. The addition of cloxacillin and PAbetaN thus further comprises cloxacillin at a concentration from 25 to makes it possible to very greatly increase the specificity of the 300 mg/L. medium. 10 The addition of cloxacillin (at the 3 concentrations tested) 11. The process of claim 1, wherein the culture medium and of PAbetaN (25 mg/L) into the medium does not alter the further comprises cloxacillin and phenylalanine-arginine detection of the carbapenemase-producing strains in the pres beta-naphthylamide (PAbetaN). ence of 8 or 16 mg/L of faropenem. It is confirmed that the 12. The process of claim 3, wherein the culture medium addition of PAbetan tends to improve their detection at faro 15 further comprises cloxacillin at a concentration from 25 to penem concentrations equal to 8 or 16 mg/L, associated with 300 mg/L, and phenylalanine-arginine-beta-naphthylamide 50 or 100 mg/L cloxacillin. (PAbetan) at a concentration from 1 to 50 mg/L. In the presence of faropenem, the results observed with 13. The process of claim 1, wherein the culture medium ertapenem (E-test) are therefore confirmed, namely a better comprises faropenem at a concentration from 8 to 32 mg/L. inhibition of the IR strains by addition of cloxacillin, and an 14. The process of claim 3, wherein the culture medium improvement of the detection of the carbapenemase-produc further comprises doripenemata concentration from 0.065 to ing strains in the presence of PAbetaN. 0.25 mg/L. The invention claimed is: 15. The process of claim 1, wherein the culture medium 1. A process for detecting whether at least one carbapenem further comprises doripenem. resistant bacterial strain is present in a biological sample, 25 16. A process for detecting whether at least one carbap comprising: enem-resistant bacterial strain is present in a biological contacting the biological sample with a culture medium to sample, comprising: obtain an inoculated culture medium, the culture contacting the biological sample with a culture medium to medium comprising (i) at least one chromogenic sub obtain an inoculated culture medium, the culture strate, and (ii); 30 medium comprising (i) at least one chromogenic sub incubating the inoculated culture medium to culture the strate, (ii) at least one of faropenem or doripenem, (iii) at carbapenem-resistant bacterial strain if present in the least one of cloxacillin, dicloxacillin, or flucloxacillin, biological sample; and and (iv) phenylalanine-arginine-beta-naphthylamide detecting whether the carbapenem-resistant bacterial (PAbetaN); strain is present on or in the culture medium. 35 incubating the inoculated culture medium to culture the 2. The process of claim 1, wherein the carbapenem-resis carbapenem-resistant bacterial strain if present in the tant bacterial strain is an NDM-1 bacterial strain. biological sample; and 3. The process of claim 1, wherein the culture medium detecting whether the carbapenem-resistant bacterial comprises faropenem at a concentration from 2 to 32 mg/L. strain is present on or in the culture medium. 4. The process of claim 3, wherein the culture medium 40 17. The process of claim 16, wherein the culture medium further comprises doripenem at a concentration from 0.05 to comprises cloxacillin. 2 mg/L. 18. The process of claim 16, wherein the culture medium 5. The process of claim 1, wherein the chromogenic sub comprises at least one of faropenem or doripenem at a con Strate detects glucuronidase, glucosidase, galactosidase, centration from 0.05 to 32 mg/L. esterase, sulfatase, or deaminase activity. 45 19. The process of claim 18, wherein the culture medium 6. The process of claim 1, wherein the chromogenic sub comprises cloxacillinata concentration from 25 to 300 mg/L. Strate is selected from the group consisting of 5 Bromo-4 and PAbetan at a concentration from 1 to 50 mg/L. chloro-3 indoxyl-beta D glucopyranoside (X-glucoside), 5 20. The process of claim 16, wherein the culture medium Bromo-6 chloro-3 indoxyl-beta D galactopyranoside (Ma comprises faropenem at a concentration from 2 to 32 mg/L. genta beta-Gal), 6 Chloro-3 indoxyl-beta D glucuronide 50 21. The process of claim 16, wherein the culture medium (Rosebeta Gur), 5 Bromo-4 chloro-3 indoxyl-N methyl-beta comprises doripenem at a concentration from 0.05 to 2 mg/L. D glucopyranoside (Green Abeta Glu), and L Tryptophan.