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 CARBAPENEMS 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, ertapenem, imipenem and meropenem 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 Carbapenem-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 cephalosporin-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 antibiotic 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 penem and/or doripenem; 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 cloxacillin 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 antibiotics, exhibiting a resistance to carbapenems, comprising the steps Such as penicillins and cephalosporins, 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 Pseudomonas aeruginosa, 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 cell wall 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