CTX-M-14 and CTX-M-15 enzymes are the dominant type of extended-spectrum β-lactamase in clinical isolates of Escherichia coli from Korea

This study was performed to assess the prevalence and genotypes of plasmid-borne extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases in Escherichia coli in Korea. A total of 576 isolates of E. coli was collected from 12 Korean hospitals during May and July 2007. A phenotypic confirmatory test detected ESBLs in 82 (14.2 %) of the 576 E. coli isolates. The most common types of ESBLs identified were CTX-M-14 (n=32) and CTX-M-15 (n=27). The prevalence and diversity of the CTX-M mutants, including CTX-M-15, CTX-M-27 and CTX-M-57, with significant hydrolytic activity against ceftazidime were increased. PCR experiments detected genes encoding plasmid-borne AmpC β-lactamases in 15/56 cefoxitin-intermediate or cefoxitin-resistant isolates, and the most common type of AmpC β-lactamase identified was DHA-1 (n=10). These data suggest that the incidence of ESBLs in E. coli has increased as a result of the dissemination of CTX-M enzymes in Korea. In addition, CTX-M-22, CTX-M-27 and CTX-M-57 have appeared in Korea.


INTRODUCTION
The predominant mechanism for acquired resistance to blactams in Escherichia coli is the synthesis of plasmid-borne extended-spectrum b-lactamases (ESBLs) and AmpC blactamases. The clavulanic acid (CA)-inhibitory ESBLs are within molecular class A of the Ambler classification scheme and confer resistance to oxyimino-cephalosporins on their bacterial hosts (Paterson & Bonomo, 2005). CTX-M enzymes are spreading rapidly and are now the dominant type of ESBL in E. coli in many parts of the world (Rossolini et al., 2008), although classical TEM and SHV ESBLs are still dominant in the USA (Bush, 2008). In Korea, members of the CTX-M-1 and CTX-M-9 clusters have repeatedly been reported in Enterobacteriaceae since the first finding of CTX-M-14 in clinical isolates of Shigella sonnei, E. coli and Klebsiella pneumoniae in 2001 (Pai et al., 2001). A nationwide survey in 2003 reported 23/246 clinical isolates (9.3 %) of E. coli with an ESBL phenotype, and only 8 of these had CTX-M enzymes .
E. coli is intrinsically susceptible to 7-a-methoxy-cephalosporins [e.g. cefoxitin (FOX) and cefotetan] because of the low-level expression of the non-inducible species-specific ampC gene (Philippon et al., 2002). In 1989, Bauernfeind et al. (1989 described a FOX-resistant K. pneumoniae isolate, which produced a plasmid-borne AmpC blactamase, named CMY-1, from Korea. Subsequently, various types of plasmid-borne AmpC enzymes have been found worldwide (Philippon et al., 2002). In Korea, FOXresistant E. coli have increasingly been noted, and a study performed in 2003 showed that a high portion (53.4 %, 62/ 116) of FOX resistance in E. coli was due to plasmid-borne AmpC b-lactamase production . The aim of the present study was to determine the prevalence and shift of plasmid-borne AmpC b-lactamases and ESBLs, with a special focus on the CTX-M enzymes, in E. coli in Korea.

METHODS
Bacterial strains. Consecutive non-duplicate isolates of E. coli were collected during May and July 2007 from 12 hospitals in nine cities in Korea. The isolates were identified using the API-20 E system (bioMérieux). The azide-resistant E. coli J53 was used as a recipient strain for conjugative transfer. E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as MIC reference strains.
Antimicrobial susceptibility testing. Antibiotic-containing discs (BBL) were used for routine antibiograms by disc diffusion assay (CLSI, 2006a). The phenotypic confirmatory test for ESBL and/or AmpC blactamase production using boronic acid (BA) as an AmpC b-lactamase inhibitor was performed as described previously (Song et al., 2007). MICs were determined by the agar dilution method using Mueller-Hinton agar (MHA; Difco Laboratories) with an inoculum of 10 4 c.f.u. (CLSI, 2006b). The MICs of b-lactams were determined alone or in combination with a fixed concentration (4 mg ml 21 ) of CA.
Mating-out assays. Conjugation experiments were carried out between donors and the azide-resistant recipient strain E. coli J53 on MHA plates. Transconjugants were selected on MHA plates supplemented with 2 mg ceftazidime (CAZ) ml 21 or 2 mg cefotaxime (CTX) ml 21 and 100 mg sodium azide ml 21 .
Characterization of genes encoding b-lactamases. Detection of genes encoding plasmid-borne ESBLs and AmpC b-lactamases was performed by PCR amplification with the primers listed in Table 1, as described previously Song et al., 2006). Templates for PCR amplification in the clinical isolates were plasmid preparations from the clinical isolates. The PCR products were subjected to direct sequencing. Both strands of all PCR products were sequenced twice using an automatic sequencer (model 3730xl; Applied Biosystems).

E. coli isolates harbouring ESBLs
In the study period, clinical E. coli isolates (n5576) were obtained from outpatients (46.2 %), inpatients of general

E. coli isolates harbouring AmpC b-lactamases
Among 56 FOX-intermediate or FOX-resistant isolates, 26 exhibited positive results in the phenotypic confirmatory test, i.e. a ¢5 mm increase in the zone diameter of either the FOX or the cefotetan disc in the presence of BA. PCR experiments detected genes encoding plasmid-borne AmpC b-lactamases in 15 isolates. The most common type of AmpC b-lactamase identified was DHA-1 (n510), and genes encoding CMY-2 (n53), CMY-10 (n51) and CMY-11 (n51) were also detected. Nine of the fifteen isolates simultaneously harboured ESBLs.

Phenotypic characteristics
All of the isolates producing CTX-M-3 (n510), CTX-M-9 (n51), CTX-M-14 (n530) or CTX-M-22 (n51) had more than eightfold higher MICs for CTX than for CAZ (Table 3). The isolates producing CTX-M-15, CTX-M-27 or CTX-M-57 exhibited a high level of resistance to CAZ. Fourteen isolates producing plasmid-borne AmpC blactamases were highly resistant to FOX (MICs ¢128 mg l 21 ), except for two producing DHA-1. All of the isolates producing ESBLs exhibited a high level of resistance to ciprofloxacin (MICs ¢64 mg l 21 ), except for three (data not shown). A total of 15 out of 82 isolates (18.3 %) with an ESBL phenotype were resistant to amikacin (MICs ¢32 mg l 21 ).

DISCUSSION
Compared with a survey in 1997 , the prevalence of ESBL-producing E. coli in Korea has increased threefold from 4.8 to 14.2 %. We have shown previously that only 3.3 % (8/246) of clinical E. coli isolates produced CTX-M ESBLs in 2003 , but the prevalence of these enzymes increased to 13.4 % (77/576) in the present study. These results indicate that the significant increase in ESBL incidence in E. coli may be due to dissemination of CTX-M enzymes. The incidence of CTX-M-14 and CTX-M-15 increased from 0.4 % (1/246) and 1.6 % (4/246) to 5.6 % (32/576) and 4.7 % (27/576), respectively, during this period .
Dissemination of E. coli harbouring members of the CTX-M-1 and CTX-M-9 clusters has repeatedly been reported in Asian countries (Hawkey, 2008   DThe BA disc test for ESBLs was positive, but no ESBL or ampC genes were detected.