When Carbapenem-Hydrolyzing ß-Lactamase KPC. attributed to outer-membrane protein deficiency coupled with plasmid-mediated

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1 AAC Accepts, published online ahead of print on 18 July 2011 Antimicrob. Agents Chemother. doi: /aac Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Revised AAC Version 2 When Carbapenem-Hydrolyzing ß-Lactamase KPC meets Escherichia coli ST-131 in France Word count: 500 Carbapenem-resistance in E. coli isolates even though rare, can be attributed to outer-membrane protein deficiency coupled with plasmid-mediated ESBLs or class C ß-lactamases (2, 10, 11) or to carbapenemases. The latter can be either metallo-ß-lactamases (VIM, IMP and NDM) (3, 7, 12), oxacillinases (OXA-48) (1) or class A enzymes (mainly KPC) (9). KPC-positive E. coli isolates have been reported in the USA (6), in Israel (8), and in Brazil (5). For the first time, we described a bla KPC positive E. coli isolate ST131 in France. On May 2009, a 64-year-old bedridden woman was hospitalized in the Gerontology ward of the Lille University Hospital (France), for bedsores and malnutrition. During hospitalization, a carbapenem-resistant E. coli strain (designated LIL-1) was isolated from the patient s urine. E. coli LIL-1, which belonged to the sequence type (ST) 131 [according to Multi Locus Sequence Typing (14)] and to phylogenetic group B2, remained susceptible only to aminoglycosides, colistin, tigecycline and fosfomycin. β-lactam MICs, interpreted according to CLSI criteria (4) and shown in Table 1, revealed that E. coli LIL-1 was resistant to ertapenem, of intermediate susceptibility to imipenem 22 and susceptible to meropenem. The patient, who had not traveled recently, received imipenem three months before for urine infection due to ESBL- producing Enterobacter aerogenes. No other E. coli isolate with similar antibiotic 1

2 resistance pattern was recovered from hospitalized patients during this same period of time. PCR experiments from strain LIL-1 DNA, with primers for detection of Ambler class A, class D and class B ß-lactamase genes followed by DNA amplicon sequencing identified genes coding for the carbapenemase KPC-2, the narrow-spectrum TEM-1 and OXA-9, and the extended-spectrum CTX-M-9 ß- lactamases. Additionally, the bla KPC-2 gene was found to be associated with Tn4401a isoform. Plasmid content analysis of strain LIL-1 revealed three different sized plasmids. A ca. 75-kb plasmid, plil-1, was successfully transferred to E. coli TOP10 by electroporation and the transformant displayed a β-lactam resistance pattern consistent with the expression of KPC carbapenemase (Table 1). This plasmid, which also harbored bla TEM-1 and bla OXA-9 genes, belonged to the IncFiiA S incompatibility group. Up to now, KPC-type carbapenemases have largely disseminated worldwide among K. pneumoniae isolates, mostly belonging to the ST-258 clone, which has been associated with outbreaks of infection in hospitalized patients. On the contrary, other enterobacterial species expressing this carbapenemase, such as E. coli, have been rarely described. Here, we report an E. coli to ST-131 clone, the main clone responsible for CTX-M-15 diffusion in the community, harboring bla KPC-2 gene (13). No link of the infected patient with an endemic country such as the USA, Greece or Israel could be evidenced, as previously mentioned for KPC- 2-producing isolates identified in France. The presence of KPC-2 in a very successful and virulent E. coli clone, responsible for community-acquired infections in humans is of great concern in public health. Low MICs values of 2

3 49 50 carbapenems may make identification of a carbapenemase producer very difficult, which may lead to its under detection and silent spread in community settings ACKNOWLEDGMENTS We are Grateful to Dr Michel Simonet for proofreading this manuscript. This work was funded by INSERM, France, by a grant from the French Ministère de l'education Nationale et de la recherche (UPRES-EA3539), Université Paris XI, France, and by the European Community (7th Framework program FP7/ under grant agreement no ) REFERENCES 1. Carrer, A., L. Poirel, M. Yilmaz, O. A. Akan, C. Feriha, G.Cuzon, G. Matar, P. Honderlick, and P. Nordmann Spread of OXA-48-Encoding Plasmid in Turkey and Beyond. Antimicrob. Agents Chemother. 54: Chia, J. H., L. K. Siu, L. H. Su, H. S. Lin, A. J. Kuo, M. H. Lee, and T. L. Wu Emergence of carbapenem-resistant Escherichia coli in Taiwan: resistance due to combined CMY-2 production and porin deficiency. J Chemother. 21: Chihara S., K. Okuzumi, Y. Yamamoto, S. Oikawa, and A. Hishinuma First case of New Delhi metallo-beta-lactamase 1-producing Escherichia coli infection in Japan. Clin Infect Dis. 52: Clinical and Laboratory Standards Institute Performance standards for antimicrobial susceptibility testing; 20th informational supplement M100-S20. Clinical and Laboratory Standards Institute, Baltimore, MD. 5. D'Alincourt Carvalho-Assef, A. P., R. Souza Leão, R. Vianna da Silva, A. Guerra 3

4 Ferreira, L. Miyuki Seki, M. Dutra Asensi, and E. Andrade Marques Escherichia coli producing KPC-2 carbapenemase: first report in Brazil. Diagn Microbiol Infect Dis. 68 : Landman, D., C. Urban, M. Backer, P. Kelly, N. Shah, E. Babu, S. Bratu, and J. Quale Susceptibility Profiles, Molecular Epidemiology, and Detection of KPC-Producing Escherichia coli Isolates from the New York City Vicinity. J Clin Microbiol. 48: Miriagou, V., E. Tzelepi, D. Gianneli, and L. S. Tzouvelekis Escherichia coli with a self-transferable, multiresistant plasmid coding for metallo-betalactamase VIM-1. Antimicrob. Agents Chemother. 47: Navon-Venezia,S., I. Chmelnitsky, A. Leavitt, M. J. Schwaber, D. Schwartz, and Y. Carmeli Plasmid-Mediated Imipenem-Hydrolyzing Enzyme KPC-2 among Multiple Carbapenem-Resistant Escherichia coli Clones in Israel. Antimicrob. Agents Chemother. 50: Nordmann, P., G. Cuzon, and T. Naas The real threat of KPC carbapenemase-producing bacteria. Lancet Infect. Dis. 9: Oteo, J., A. Delgado-Iribarren, D. Vega, V. Bautista, M. C. Rodríguez, M. Velasco, J. M. Saavedra, M. Pérez-Vázquez, S. García-Cobos, L. Martínez- Martínez, and J. Campos Emergence of imipenem resistance in clinical Escherichia coli during therapy. Int J Antimicrob Agents. 32: Pavez, M., P. Neves, M. Dropa, M. H. Matté, R. S. Grinbaum, M. R. Elmor de Araújo, E. M. Mamizuka, and N. Lincopan Emergence of carbapenemresistant Escherichia coli producing CMY-2-type AmpC beta-lactamase in Brazil. J Med Microbiol. 57:

5 Peleg, A. Y., C. Franklin, J. M. Bell, and D. W. Spelman Dissemination of the metallo-beta-lactamase gene bla IMP-4 among gram-negative pathogens in a clinical setting in Australia. Clin. Infect. Dis. 41: Pitout, J. D., and K. B. Laupland Extended-spectrum beta-lactamaseproducing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis. 8: Wirth, T., D. Falush, R. Lan, F. Colles, P. Mensa, L. H. Wieler, H. Karch, P. R. Reeves, M. C. Maiden, H. Ochman, and M. Achtman Sex and virulence in Escherichia coli: an evolutionary perspective. Microbiol. 60: Thierry Naas* Gaelle Cuzon Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, Le Kremlin-Bicêtre, France Olivier Gaillot René Courcol Service de Microbiologie, Centre Hospitalier Régional Universitaire de Lille, Lille, France Patrice Nordmann Faculté de Médecine Paris-Sud, Université Paris XI, Le Kremlin- Bicêtre, France, INSERM U914. 5

6 *Phone: Fax:

7 TABLE. MICs of β-lactams for E. coli LIL-1, E. coli Top 10 recipient strain and E. coli transformant harboring plasmid plil-1 and E. coli Top 10 recipient strain. MIC (µg/ml) a β-lactam(s) E. coli E. coli Top10 E. coli Top10 LIL-1 plil-1 Amoxicillin >256 >256 2 Amoxicillin+CLA b 64 >256 2 Ticarcillin >256 >256 2 Ticarcillin+CLA 128 > Piperacillin 256 > Piperacillin+TZB c Cephalothin >32 >32 2 Cefoxitin > Cefotaxime Ceftazidime Cefepime Imipenem Meropenem Ertapenem

8 127 a MICs of β-lactams were determined by the E-test (biomérieux, Marcy l Etoile, France) b CLA, clavulanic acid at a fixed concentration of 2 µg/ml; c TZB, tazobactam at fixed concentration of 4 µg/ml. 131 Downloaded from on August 20, 2018 by guest 8