Resistance, Yonsei University College of Medicine, Seoul, Korea; and 2 Department of

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1 AAC Accepts, published online ahead of print on March 0 Antimicrob. Agents Chemother. doi:./aac.0- Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Title: Emergence of Escherichia coli sequence type ST carrying both the bla GES- and bla CTX-M-1 genes Running title: E. coli carrying the bla GES- gene Keywords: GES-, CTX-M-1, Escherichia coli, sequence type Authors: Juwon Kim 1, Seong Geun Hong, Il Kwon Bae 1, Ji Roung Kang 1, Seok Hoon Jeong 1*, Wookeun Lee, and Kyungwon Lee 1 Addresses: 1 Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea; and Department of Laboratory Medicine, CHA Bundang Medical Center, CHA University, Sungnam, Korea *Corresponding author: Seok Hoon Jeong. Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, -, 1 Shinchon-Dong, Seodaemun-Gu, Seoul, Republic of Korea Phone: +--- Fax: kscpjsh@yuhs.ac Downloaded from on July, 01 by guest 1 1

2 ABSTRACT Escherichia coli clinical isolate BD0 of ST recovered from a bed sore specimen exhibited a high level resistance to ceftazidime and cefotaxime but susceptibility to imipenem and meropenem. The isolate harbored two β-lactamase genes, the bla CTX-M-1 gene on a ~0-kbp plasmid carrying FIA and FIC replicons and the bla GES- gene on a class 1 integron in the chromosome. () MANUSCRIPT The class A carbapenemases, which have been identified in Gram-negative rods, involve chromosome-borne NMC-A and SME enzymes and plasmid-borne KPC, GES, and IMI enzymes (1). To date, 1 variants of GES-type extended-spectrum β-lactamases (ESBLs) have been identified ( but the carbapenem hydrolytic activity has been demonstrated in only some variants, including GES-, GES-, GES-, GES-, and GES-1, with a substitution at the Gly residue (,1). The bla GES- gene was first detected in a plasmid of Escherichia coli -0 from Greece in 00 (1). Then the detection of bla GES- gene in Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa has been reported from many parts of the world (1). E. coli clinical isolate BD0 was recovered from a bed sore sampled from a -yearold female who has taken home nursing care on May 1, 00. She had been hospitalized for days at a secondary-care hospital in Bundang, Korea for the treatment of chronic renal failure, type II diabetes mellitus, and hypertension. Multi-locus sequencing typing was performed on the isolate using the seven conserved housekeeping genes (adk, fumc, Downloaded from on July, 01 by guest

3 gyrb, icd, mdh, pura, and reca) following the protocols available at a website ( and the isolate was identified as sequence type (ST) ( ). Disk diffusion assay was performed (), and the isolate exhibited resistance to ampicillin, ampicillin-sulbactam, piperacillin-tazobactam, cefoxitin, aztreonam, ceftazidime, cefotaxime, cefepime, gentamicin, tobramycin, amikacin, and ciprofloxacin and was susceptible to imipenem, meropenem, and trimethoprim-sulfamethoxazole. Synergy was observed between amoxicillin-clavulanic acid (0/ µg) disk and ceftazidime (0 µg), cefotaxime (0 µg), cefepime (0 µg), and aztreonam (0 µg) disks (Becton Dickinson, Spark, MD) in double-disk synergy tests, indicating the production of ESBL (1). Agar dilution MIC testing on Muller-Hinton agar (Difco Laboratories, Detroit, MI) with an inoculum of CFU per spot confirmed high level MICs of ceftazidime (> µg/ml) and cefotaxime (> µg/ml) and low level MICs of imipenem (0. µg/ml) and meropenem (0. µg/ml) for the isolate BD0 (). Clavulanic acid (Sigma, St. Louis, MO) at a fixed concentration of µg/ml lowered MICs of ceftazidime and cefotaxime to 1 µg/ml and µg/ml, respectively (Table 1). Isolate BD0 showed a positive result in Hodge test, which was performed on a MacConkey agar plate with an ertapenem disk, indicating the production of a carbapenemase (). However, the isolate showed a negative result in EDTA-based double disk synergy test, which was performed on a Mueller-Hinton agar plate with an imipenem disk and a disk containing EDTA (0g) plus sodium mercaptoacetic acid ( mg), indicating that the isolate does not produce metallo-β-lactamases (). In the isoelectric focusing analysis, three β-lactamase activities with isoelectric points of.,.1, and., corresponding to GES-, OXA-1, and CTX-M-1 respectively, were detected. Downloaded from on July, 01 by guest

4 PCR and sequencing experiments were performed to detect genes encoding CTX-M-, GES-, PER-, SHV-, TEM-, and VEB-type ESBLs as previously described (). Isolate BD0 carried two β-lactamase genes, the bla CTX-M-1 and the bla GES-. The isolate transferred cefotaxime resistance to the E. coli J azide R recipient in mating experiments (), in which transconjugants were selected on MacConkey agar (Difco Laboratories, Detroit, MI) plates supplemented with cefotaxime ( µg/ml) and sodium azide (0 µg/ml). The location of β-lactamase genes was identified by hybridization of I-CeuIdigested genomic DNA or S1 nuclease-treated linearized plasmids with probes specific for the β-lactamase genes, various replicons of plasmids, and 1S rdna as described previously (,). The probe specific for the bla CTX-M-1 gene hybridized with a ~0-kbp plasmid in both isolate BD0 and its transconjugant, but that for the bla GES- gene did not. The ~0-kbp plasmid carried two types of replicon, FIA and FIC. Replicon sequence typing was performed on the plasmid as previously described (1), and STs of the replicons were identified to be FIA (1) and FIC (1). This result is in agreement with the previous study which has confirmed that all the variants of ST producing CTX-M-1 ESBL harboured the corresponding gene on IncF-type plasmid (). The probe specific for the bla GES- gene hybridized with an I-CeuI-macrorestriction fragment of ~0-kbp in isolate BD0. The probe specific for 1S rdna also hybridized with the I-CeuImacrorestriction fragment, indicating chromosomal location of the bla GES- gene in the isolate. To investigate genetic environments surrounding the bla GES- gene, several overlapping PCR fragments obtained from whole DNA of the E. coli isolate were sequenced with primers corresponding to internal region of class 1 integron as previously described (1). Downloaded from on July, 01 by guest Identical to the bla GES- gene located on a plasmid in K. pneumoniae CHAK, the bla GES-

5 1 1 1 gene was located on a class 1 integron as a gene cassette downstream of the atti1 recombination site. The integron had three unique gene cassettes (bla GES- -aac( )-IIabla OXA-1 /orf) between the two conserved elements, -CS and -CS, but the -base element of the third gene cassette was interrupted by a putative transposase gene, orf. In summary, E. coli isolate BD0 of ST harbored two β-lactamase genes, the bla CTX-M-1 gene on a ~0-kbp plasmid carrying FIA and FIC replicons and the bla GES- gene on a class 1 integron in a chromosome. Recently, the intercontinental dissemination of E. coli clone of ST producing CTX-M-1, which was mostly carried on an IncF plamids, has been reported (). Emergence of E. coli clone of ST simultaneously producing CTX-M-1 and GES- could be a serious threat to global health. This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (00-00). (word no. = ) Downloaded from on July, 01 by guest

6 REFERENCES Bae, I. K., et al. 00. Genetic and biochemical characterization of GES-, an extended-spectrum class A β-lactamase from Klebsiella pneumoniae. Diagn. Microbiol. Infect. Dis. :-.. Barton, B.M., G. P. Harding, and A. J. Zuccarelli. 1. A general method for detecting and sizing large plasmids. Anal. Biochem. :-0.. Bermudes, H., et al. 1. Molecular characterization of TEM- (IRT-1), a novel inhibitor-resistant TEM-derived β-lactamase in a clinical isolate of Klebsiella oxytoca. Antimicrob. Agents Chemother. :1-.. Bogaerts, P., et al. 0. GES extended-spectrum β-lactamases in Acinetobacter baumannii isolates in Belgium. Antimicrob. Agents Chemother. :-.. Clinical and Laboratory Standards Institute. 00. Performance standards for antimicrobial susceptibility testing; nineteenth informational supplement. M0-S1, Wayne, PA.. Dhanji H., et al. 0. Molecular epidemiology of fluoroquinolone-resistant ST Escherichia coli producing CTX-M extended-spectrum β-lactamases in nursing homes in Belfast, UK. J. Antimicrob. Chemother. :-0.. Lee, K., et al. 0. Improved performance of the modified Hodge test with MacConkey agar for screening carbapenemase-producing Gram-negative bacilli. J. Microbiol. Methods :1-1.. Lee, K., Y. S. Lim, D. Yong, J. H. Yum, and Y. Chong. 00. Evaluation of the Hodge test and the imipenem-edta double-disk synergy test for differentiating metallo-β-lactamase-producing isolates of Pseudomonas spp. and Acinetobacter spp. Downloaded from on July, 01 by guest

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8 Table 1 MICs of E. coli wild strain (BD0), its transconjugant, and the recipient E. coli J MIC (µg/ml) Antimicrobial agent a Wild strain Transconjugant Recipient E. coli BD0 E. coli trcbd0 E. coli J (bla GES- + bla CTX-M-1 ) (bla CTX-M-1 ) Amoxicillin-clavulanic acid Piperacillin-tazobactam > > 1. Cefoxitin 1 1 Ceftazidime > 1 0. Ceftazidime-clavulanic acid Cefotaxime > 0.0 Cefotaxime-clavulanic acid Cefepime > Aztreonam 0. Imipenem Imipenem-clavulanic acid Meropenem Meropenem-clavulanic acid Ertapenem Ertapenem-clavulanic acid a Clavulanic acid were added at a fixed concentration of µg/ml. Downloaded from on July, 01 by guest