Evaluation of three selective chromogenic media, CHROMagar ESBL, CHROMagar CTX-M and CHROMagar KPC, for the detection of Klebsiella pneumoniae producing OXA-48 carbapenemase Michael Hornsey, 1 Lynette Phee, 1,2 Neil Woodford, 1,3 Jane Turton, 3 Daniele Meunier, 3 Claire Thomas, 4 David W Wareham 1,2 1 Antimicrobial Research Group, Centre for Immunology and Infectious Disease, Blizard Institute, Barts & The London School of Medicine and Dentistry, Queen Mary University London, London, UK 2 Division of Infection, Barts Health NHS Trust, London, UK 3 Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Health Protection Agency, Microbiology Services, Colindale, London, UK 4 Hammersmith Hospital NHS Trust, London, UK Correspondence to Dr David W Wareham, Antimicrobial Research Group, Centre for Immunology and Infectious Disease, Blizard Institute, 4 Newark Street, Whitechapel, London E1 2AT, UK; d.w.wareham@qmul.ac.uk Received 21 September 2012 Revised 29 October 2012 Accepted 5 November 2012 To cite: Hornsey M, Phee L, Woodford N, et al. J Clin Pathol Published Online First: [ please include Day Month Year] doi:10.1136/ jclinpath-2012-201234 ABSTRACT Three selective chromogenic culture media (CHROMagars ESBL, CTX-M and KPC) were evaluated for their ability to support the growth of nine Klebsiella pneumoniae isolates producing OXA-48 carbapenemase in combination with other β-lactamases. CHROMagar ESBL and CHROMagar KPC were the most sensitive media, supporting growth of all isolates with a detection limit as low as < 100 CFU/ml. Five isolates failed to grow on CHROMagar CTX-M, and five were recovered on CHROMagar KPC only at counts > 10 6 CFU/ml. Both CHROMagar ESBL and CHROMagar KPC may be useful for enhanced isolation of K pneumoniae producing OXA- 48-like carbapenemases. Carbapenemase producing Enterobacteriaceae (CPE) present a major threat to public health and are rapidly disseminating globally, 1 aided by the spread of successful bacterial clones, successful plasmids and international travel. The most preva- lent CPE include those which produce the class A carbapenemase KPC, the metallo-β-lactamases of the IMP, NDM and VIM families, and the class D OXA-48-like enzymes. As treatment options are limited, attention is increasingly focused on methods for the rapid detection of CPE that may direct effective infection prevention and control strategies. Some countries (eg, France) have intro- duced comprehensive screening of all ‘high risk’ patients for CPE on admission to hospital, 2 particu- larly those with a history of hospitalisation in foreign countries, and many others (UK Health Protection Agency, http://www.hpa.org.uk/webc/ HPAwebFile/HPAweb_C/1294740725984; US Centers for Disease Control, http://www.cdc.gov/ mmwr/preview/mmwrhtml/mm5810a4.htm) also advise that this is done. Strains of CPE producing OXA-48 carbapenem- ase raise several challenges. First, the gene is carried on plasmids (IncL/M) that have spread into multiple sequence types of Klebsiella pneumoniae, 3 which appear to be highly successful and capable of causing nosocomial outbreaks. 45 Second, detection of OXA-48 producers can be difficult unless there is co-carriage of additional β-lactamases, especially when using automated systems. 6 OXA-48 is an unusual enzyme as, unlike other carbapenemases, it is able to hydrolyse penicillins and carbapenems, but not cephalosporins. 7 This potential for a carbapenem-resistant but cephalosporin-susceptible phenotype may subvert standard laboratory detec- tion protocols and expert rules used to infer carba- penem resistance mechanisms. 8 In this study, we assessed the ability of three commercially available chromogenic agars to detect carbapenem-resistant K pneumoniae producing the OXA-48 enzyme. The nine K pneumoniae isolates used in this study were isolated from patients involved in an outbreak of carbapenem-resistant K pneumoniae in a London hospital renal unit. 9 Susceptibility testing was performed by the British Society for Antimicrobial Chemotherapy agar dilution method- ology and molecular typing was performed by pulsed-field gel electrophoresis (PFGE). 10 Molecular mechanisms of β-lactam resistance were determined using a series of multiplex PCRs for the detection of class A (TEM, SHV, CTX-M-1,2,9,8/25-like, VEB, PER, GES, KPC), D (OXA-1/4/30,48), plasmidic AmpC (ACC, FOX, MOX, DHA, CIT, EBC) and metallo-β-lactamases (IMP, VIM, NDM). 11 Genes encoding variants of TEM, SHV and CTX-M enzymes known to have ESBL activity were identified by sequencing follow- ing PCR amplification of the entire coding sequence. All of the isolates were resistant to ertapenem (MIC ≥2 mg/ml) and imipenem (MIC ≥4 mg/ml) according to the 2011 Clinical Laboratory Standards Institute breakpoints, but exhibited vari- able susceptibility to cephalosporins, aztreonam and meropenem (table 1). MICs of cloxacillin and piperacillin/tazobactam were >256 and >64 mg/ml, respectively, for all isolates tested. Each contained a bla OXA-48-like gene in combination with bla SHV (n=8), bla TEM -1 (n=3), bla CTX-M-15 (n=3) or bla OXA-1 (n=2) genes (table 1). Six carried bla SHV-11 (non-ESBL) and two had bla SHV-1 . No plasmidic AmpC or metallo-carbapenemase genes were detected. PFGE showed that the isolates belonged to three distinct strains; strain A lacked a coresident ESBL, whereas strains B and C both pro- duced a CTX-M-15 enzyme. The three selective media, CHROMagar ESBL, CHROMagar CTX-M and CHROMagar KPC, were obtained directly from the manufacturer (CHROMagar, Paris, France). All components were provided as dehydrated powders and media were prepared in-house according to the accompanying instructions; CHROMagar Orientation 12 was the base medium used for the preparation of CHROMagar ESBL and CHROMagar KPC plates, Hornsey M, et al. J Clin Pathol 2013;00:1–3. doi:10.1136/jclinpath-2012-201234 1 Short report JCP Online First, published on January 2, 2013 as 10.1136/jclinpath-2012-201234 Copyright Article author (or their employer) 2013. Produced by BMJ Publishing Group Ltd under licence. group.bmj.com on January 24, 2013 - Published by jcp.bmj.com Downloaded from