Carbapenem-Resistant KPC-2-Producing Escherichia coli in a Tel Aviv Medical Center, 2005 to 2008

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2010, p Vol. 54, No /10/$12.00 doi: /aac Copyright 2010, American Society for Microbiology. All Rights Reserved. Carbapenem-Resistant KPC-2-Producing Escherichia coli in a Tel Aviv Medical Center, 2005 to 2008 Moran G. Goren, Shiri Navon-Venezia, Inna Chmelnitsky, and Yehuda Carmeli* The Molecular Epidemiology and Antibiotic Resistance Laboratory, Division of Epidemiology, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Received 27 September 2009/Returned for modification 22 November 2009/Accepted 7 March 2010 All of the carbapenem-resistant Escherichia coli (CREC) isolates identified in our hospital from 2005 to 2008 (n 10) were studied. CREC isolates were multidrug resistant, all carried bla KPC-2, and six of them were also extended-spectrum beta-lactamase producers. Pulsed-field gel electrophoresis indicated six genetic clones; within the same clone, similar transferable bla KPC-2 -containing plasmids were found whereas plasmids differed between clones. Tn4401 elements were identified in all of these plasmids. Carbapenem resistance in Escherichia coli is usually attributed to the acquisition of -lactamases such as AmpC (14, 23, 24, 27, 31), metallo -lactamases (4, 17, 25, 33), or KPC-type carbapenemases (2, 3, 26, 32). In 2005, KPC-2-mediated carbapenem-resistant E. coli (CREC) clinical strains were first identified in our hospital (21). The increasing prevalence of carbapenem-resistant Enterobacteriaceae in Israel (30), along with concerns regarding the emergence of highly epidemic clones, led to the study of carbapenem resistance in E. coli in our hospital. We determined CREC prevalence, elucidated the molecular mechanisms contributing to carbapenem resistance, and explored the molecular epidemiology and plasmids associated with this resistance. All of the CREC isolates identified in our hospital from February 2005 to October 2008 were included in this study. Strains were identified as resistant to at least one carbapenem using the Vitek-2 and agar dilution (MIC of imipenem or meropenem, 4 g/ml; MIC of ertapenem, 2 g/ml). Antibiotic susceptibilities were determined by Vitek-2 (biomérieux Inc., Marcy l Etoile, France), and MICs of carbapenems were determined by agar dilution according to the Clinical and Laboratory Standards Institute (CLSI) protocols (8). MICs of tigecycline and colistin and MICs of imipenem, meropenem, and ertapenem lower than 0.5 g/ml were determined by Etest (AB Biodisk, Solna, Sweden). The criteria used for the interpretation of carbapenem MICs were based on the CLSI 2010 guidelines (9). The interpretive criterion used for tigecycline was based on FDA breakpoint values for Enterobacteriaceae that define a MIC of 2 as susceptible. -Lactamases were analyzed by analytical isoelectric focusing (IEF) (16) on crude enzyme preparations from sonicated cell cultures as described elsewhere (21). The following -lactamases were used as controls: TEM-1, pi 5.4; TEM- 26, pi 5.6; K1, pi 6.5; SHV-1, pi 7.6; P99, pi 7.8; ACT-1, pi 9. * Corresponding author. Mailing address: Division of Epidemiology, Tel Aviv Sourasky Medical Center, 6 Weizmann St., Tel Aviv 64239, Israel. Phone: Fax: Published ahead of print on 15 March The genetic relatedness between isolates was determined using pulsed-field gel electrophoresis (PFGE) as previously described (7). DNA macrorestriction patterns were compared according to the Dice similarity index (1.5% tolerance interval) (9a) using GelCompar II version 2.5 (Applied Maths, Kortrijk, Belgium). A PFGE clone was defined as a group of strains showing 85% banding pattern similarity (19). Multilocus sequence typing (MLST) was performed on two representative E. coli clones according to the protocol at the E. coli MLST website ( /PF8/mlst/EColi.html). Epidemiological links and potential contact between patients were analyzed using data on room location, consulting physicians, and other procedures performed during their hospitalization. PCR molecular screening of -lactamase genes and Tn4401 elements (18) was performed using the primers listed in Table 1. PCR products were sized on an agarose gel and sequenced using an ABI PRISM 3100 genetic analyzer (PE Biosystems). Nucleotide and deduced protein sequences were identified using the BLAST algorithm ( Plasmids were purified as described previously (12) and transformed into E. coli DH10B by electroporation (Electroporator 2510; Eppendorf, Hamburg, Germany). Transformants were selected on LB agar plates containing 100 g/ml ampicillin, and selected colonies were screened by PCR for the presence of bla KPC. Plasmid size estimation was performed by digestion of plasmid DNA prepared as described previously (7, 22), followed by S1 nuclease (190 U; Promega, Madison, WI) (1) and PFGE. Electrophoresis was carried out as described previously (7), using the Lambda ladder marker (New England Biolabs, Boston, MA). Comparison of KPC-encoding plasmids was performed using restriction length polymorphism (RFLP) following digestion with the BglII, EcoRV, SmaI, and KpnI endonucleases (New England Biolabs, Boston, MA). Southern analysis was performed as described previously (21), using a radioactively labeled bla KPC-2 probe (892 bp) obtained with bla KPC primers (5). Ten CREC isolates were studied. They originated from various isolation sites of 10 patients with no apparent epidemiological connection. The overall prevalence of carbapenem re- 2687

2 2688 GOREN ET AL. ANTIMICROB. AGENTS CHEMOTHER. Screened gene and primer type bla KPC TABLE 1. Primers used in this study Sequence a Forward ATGTCACTGTATCGCCGTCT 5 Reverse TTTTCAGAGCCTTACTGCCC bla SHV group Forward TTTATCGGCCYTCACTCAAGG 5 Reverse GCTGCGGGCCGGATAACG bla TEM group Forward KACAATAACCCTGRTAAATGC 5 Reverse AGTATATATGAGTAAACTTGG bla CTX-M-2 group Forward ATGATGACTCAGAGCATTCG 5 Reverse TTATTGCATCAGAAACCGTG bla CTX-M-3 group Forward GTTGTTGTTATTTCGTATCTTCC 5 Reverse CGATAAACAAAAACGGAATG bla CTX-M-9 group Forward GTGACAAAGAGAGTGCAACGG 5 Reverse ATGATTCTCGCCGCTGAAGCC bla CTX-M-25 group Forward CACACGAATTGAATGTTCAG 5 Reverse TCACTCCACATGGTGAGT Reference bla CMY-1 group Forward CAACAACGACAATCCATCCTGTG This paper Reverse CAACCGGCCAACTGCGCCAGGA bla CMY-2 group Forward ATGAAAAAATCGTTATGCTGCGCTCTG This paper Reverse ATTGCAGCTTTTCAAGAATGCGCC bla OXA-9 Forward GCGGACTCGCGCGGCTTTAT This paper Reverse GCGAGATCACCAAGGTAGTCGGC bla OXA-40 Forward GCAAATAMAGAATATGTSCC 10 Reverse CTCMACCCARCCRGTCAACC bla OXA-58 Forward CGATCAGAATGTTCAAGCGC 28 Reverse ACGATTCTCCCCTCTGCGC ISKpn6 Forward GAAGATGCCAAGGTCAATGC 19 Reverse GGCACGGCAAATGACTA ISKpn7 Forward GCAGGATGATTTCGTGGTCT 13 Reverse AGGAAGTCGGTGAAGCTGAA tnpa Forward CACCTACACCACGACGAACC 19 Reverse GCGACCGGTCAGTTCCTTCT tnpr Forward ACTGTGACGCATCCAATGAG 13 Reverse ACCGAGGGAGAATGGCTACT a KisGorT,MisAorC,RisAorG,SisGorC,andYisCorT. sistance in E. coli during this study period was 0.063% (10 cases out of 15,918 E. coli isolates). All 10 isolates were multidrug resistant (Table 2). The MIC 50 s and MIC 90 s of imipenem and meropenem were 4 and 8 g/ml, those of ertapenem were 16 and 32 g/ml, and those of doripenem were 1 and 4 g/ml, respectively. All of the isolates were susceptible to tigecycline (MIC 50 and MIC 90 of 0.19 and 0.75 g/ml) and to colistin (MIC 50 and MIC 90 of and 0.19 g/ml) (Table 2). TABLE 2. Antibiotic susceptibility testing results of clinical CREC strains isolated at the Tel Aviv Sourasky Medical Center from 2005 to 2008 and their transformants MIC ( g/ml) a -Lactamase gene(s) PFGE cluster Isolation site Date of isolation E. coli isolate CRO CAZ FEP ATM TZP AMK GEN CIP LVX IPM b MEM b ETP b DP b TGC c CST c 157 2/2005 Urine III KPC-2 CTX-M-15 TEM T d KPC /2005 Blood II KPC-2 CTX-M-2 TEM T KPC /2005 Peritoneal fluid V KPC-2 TEM T KPC /2005 Urine I KPC-2 CTX-M-15 OXA-9 TEM T KPC-2 OXA /2005 Wound I KPC-2 CTX-M-15 OXA-9 TEM T KPC-2 OXA /2006 Synovial fluid IV KPC-2 TEM /2006 Synovial fluid IV KPC-2 CTX-M-15 TEM T KPC /2006 Abscess IV KPC-2 TEM /2006 Blood IV KPC-2 TEM T KPC /2007 Peritoneal fluid VI KPC-2 SHV-12 TEM T KPC-2 TEM DH10B recipient a CRO, ceftriaxone; CAZ, ceftazidime; FEP, cefepime; ATM, aztreonam; TZP, piperacillin-tazobactam; AMK, amikacin; GEN, gentamicin; CIP, ciprofloxacin; LVX, levofloxacin; IPM, imipenem; MEM, meropenem; ETP, ertapenem; DP, doripenem; TGC, tigecycline; CST, colistin. Unless otherwise noted, MICs were determined by Vitek-2. b MIC determined by agar dilution; carbapenem MICs lower than 0.5 (except for DP) were determined by Etest. c MIC determined by Etest. d T, transformant.

3 VOL. 54, 2010 CARBAPENEM-RESISTANT KPC-PRODUCING ESCHERICHIA COLI 2689 FIG. 1. PFGE of clinical CREC isolates. Shown are DNA restriction patterns and a dendrogram showing the level of similarity between SpeI-restricted patterns of CREC isolates. Isolates with asterisks were described previously (21). The scale indicates the degree of genetic relatedness between the strains. Isolates were placed into six different clusters based on GelCompar Dice algorithm coefficients, which range from 0 to 100%, as illustrated by the scale to the left of each dendrogram. The year of isolation of each isolate is shown at the right. PFGE of the 10 CREC isolates revealed six distinct genetic clones (Fig. 1): four clones from 2005 (21), a new clone consisting of four isolates in 2006, and a different clone in Isolates belonging to the 2006 clone, although genetically identical, originated from four patients hospitalized in different wards during a 1-month period with no apparent epidemiological relatedness. MLST of CREC isolate 386 from 2005 identified this strain as being of sequence type 471 (ST471) reported before in France (11). CREC isolate 547, which belonged to the 2006 clone (Fig. 1), possessed a novel sequence type, ST39. IEF demonstrated the production of more than one -lactamase by each isolate (data not shown). A -lactamase with an apparent pi of 6.7 was observed in 9 out of 10 isolates, consistent with the pi of KPC-type carbapenemases. PCR screening for -lactamase genes, followed by sequencing, revealed the presence of bla KPC-2 in all of the strains. Six of the 10 isolates were also extended-spectrum beta-lactamase (ESBL) producers (Table 2).The strains carrying CTX-M enzymes showed higher MICs of ceftazidime and cefepime than the non-esbl producers (Table 2). Transformation experiments were performed with eight CREC isolates (Table 2). Plasmid DNA derived from an E. coli 1679 transformant showed a plasmid size different from that of the donor, suggesting rearrangements of plasmid DNA within this strain; therefore, it was excluded from further analysis. Plasmid DNA analysis of transformants indicated that each has acquired a single plasmid (Fig. 2). PCR screening results of plasmid DNA confirmed the presence of bla KPC in all of them, while not all -lactamases were transferred (Table 2). Acquisition of the bla KPC-2 -containing plasmids usually elevated the MICs of cephalosporins, aztreonam, aminoglycosides, and carbapenems, yet none of the transformants presented the same level of carbapenem resistance as the respective donor strain (Table 2). CREC isolates possessed two or three plasmids which differed in size (Fig. 2A, lanes D). Isolates from the same year and belonging to the same clone carried highly similar-sized plasmids. Southern blot analysis of plasmid DNA from clinical isolates and their transformants showed that each clinical isolate carried a single plasmid encoding bla KPC-2 and that these plasmids varied in size, ranging from 45 kb (Fig. 2A, lanes 4 and 6) to 100 kb (carried by E. coli strain 157 isolated in 2005) (lane 2). The plasmid DNA RFLP patterns of seven transformants, obtained by using several endonucleases, revealed different restriction patterns but a shared common region, especially between strains isolated in the same year. Southern analysis of the resulting fragments with a labeled-bla KPC-2 probe revealed the same hybridization signal, suggesting that these plasmids share a large fragment harboring bla KPC-2 (Fig. 3), similar to what we found previously in the 2005 isolates (21). Southern blot analysis following restriction with the SmaI endonuclease, which digests bla KPC-2 at nucleotide 790, led to two hybridization signals, suggesting the presence of a single copy of bla KPC-2 in all of the transferred plasmids (Fig. 3B). PCR screening and sequencing of all Tn4401 elements (18) revealed the presence of tnpa transposase, tnpr, and the insertion sequences ISKpn6 and ISKpn7 in all of the isolates and transformants. Based on the sequence recognition of SmaI, bla KPC-2 should be digested, as well as the two genes surrounding it IstB (part of ISKpn7) and ISKpn6 in a single site, resulting in two DNA fragments of 1 and 1.7 kb. These two fragments were visualized by Southern hybridization (Fig. 3B), which may indicate that the structure of Tn4401 in the close vicinity surrounding bla KPC-2 in our strains is conserved. While carbapenem resistance in Enterobacteriaceae is increasing worldwide, CREC isolates are still rare. However, carbapenem resistance in E. coli is considered to be a great public health threat due to its potential to spread in hospital and community settings (29). This is the first study focusing on the molecular epidemiology and nature of carbapenem resistance in a collection of E. coli isolates within a hospital setting. This paper presents an extension of our previous study in which we first described CREC isolates residing outside the United States (21). Isolates showed a multidrug resistance phenotype, like all

4 2690 GOREN ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 2. PFGE after S1 restriction of donor clinical strains and transformants (A) and Southern blotting using a bla KPC-2 probe (B). Plasmid profiles of six CREC isolates representing genetic clusters I to V and their transformants as determined by S1 nuclease treatment, followed by PFGE (A) and Southern blot analysis using bla KPC-2 probe (B). Lane M, Lambda Ladder PFG Marker (New England Biolabs, Boston, MA); lanes 1 to 12, E. coli clinical isolates (D) and their respective transformants (T). KPC-producing Enterobacteriaceae; however, they possessed lower carbapenem MICs (4- to 8-fold lower) compared to the MICs of carbapenem-resistant Klebsiella pneumoniae ST258 (12). Genotyping of the isolates revealed that resistance to carbapenems in E. coli from 2005 to 2008 was not clonally related, except for four cases in 2006 that were genetically identical, but epidemiological data did not prove an apparent linkage among them. Sixty percent of the KPC-2-producing strains were also ESBL producers but apparently belonged to clones different from those described before (7). Carbapenem resistance in E. coli during the years studied was rendered by a KPC-2 carbapenemase encoded on varioussized plasmids, which differed between clones but had regions in common. This is the first report describing the presence of Tn4401 elements in the vicinity of bla KPC-2 in E. coli previously described (18). The exact source of the bla KPC-2 gene from E. FIG. 3. Restriction analysis of bla KPC-2 -harboring plasmids (A) and Southern blotting using a bla KPC-2 probe (B). Restriction analysis of bla KPC-2 -containing plasmids derived from six CREC isolates after EcoRV (A) or SmaI (B) digestion, followed by Southern blotting using a bla KPC-2 probe. The two isolates belonging to cluster I (isolates 360 and 386) display the same restriction pattern; therefore, isolate 386 was chosen to depict the restriction pattern of both isolates. SmaI endonuclease cleaves bla KPC-2 at position 790 and the IstB and ISKpn6 open reading frames at positions 203 and 676, respectively, creating fragments of 1 and 1.5 kb. Lanes M, 1-kb DNA ladder (New England Biolabs, Boston, MA).

5 VOL. 54, 2010 CARBAPENEM-RESISTANT KPC-PRODUCING ESCHERICHIA COLI 2691 coli identified in our hospital is still uncertain. Originally, KPC-2 was detected from Enterobacter cloacae in our hospital in 2004 (6, 15), suggesting that they may have acted as a reservoir for the bla KPC-2 gene. In contrast to epidemic K. pneumoniae clone ST258 (20), carbapenem-resistant E. coli clones did not spread significantly during the last 4 years since their emergence in our hospital or worldwide. However, the potential transfer of bla KPC-2 genes into highly fit, rapidly spreading E. coli strains is disturbing. Strict infection control policies, together with joint efforts, will aid in limiting the further dissemination of bla KPC into E. coli, the most common clinical pathogen. This work was supported in part by European Commission FP7: SATURN Impact of Specific Antibiotic Therapies on the Prevalence of Human Host Resistant Bacteria research grant We thank George Jacoby, Lahey Clinic, Burlington, MA, for providing the strains used as positive controls in the IEF and PCR experiments. Doripenem was kindly provided by Karen Bush and Anne Marie Queenan, R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ. We thank Tali Kotlovsky for her helpful computerized analysis of epidemiological links and potential contact between patients. This work was performed in partial fulfillment of the requirements for an M.S. degree of Moran G. Goren, Department of Microbiology and Clinical Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. REFERENCES 1. Barton, B. M., G. P. Harding, and A. J. Zuccarelli A general method for detecting and sizing large plasmids. Anal. Biochem. 226: Bratu, S., S. Brooks, S. Burney, S. Kochar, J. Gupta, D. 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