JAC Advance Access published online on May 2, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn181
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Original research |
Turkey: a further country concerned by community-acquired Escherichia coli clone O25-ST131 producing CTX-M-15
1 Department of Clinical Microbiology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey 2 Clinical Microbiology Laboratory, Hifzissihha Institute, Izmir, Turkey 3 Institut National de la Santé et de la Recherche Médicale, U-773, Faculté de Médecine D. Diderot, Université Paris 7, Paris, France 4 Institut National de la Santé et de la Recherche Médicale, ESPRI 26, Université Montpellier 1, UFR de Médecine, Nîmes, France 5 Laboratoire de Bactériologie, Virologie, Parasitologie, CHU Carémeau, Nîmes, France
* Corresponding author. Tel: +33-4-66-68-32-31; Fax: +33-4-66-68-38-24; E-mail: albert.sotto{at}chu-nimes.fr
Received 7 January 2008; returned 8 February 2008; revised 29 March 2008; accepted 31 March 2008
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Abstract |
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Objectives: The aim of this study was to assess the frequency and diversity of extended-spectrum β-lactamases (ESBLs) produced by exclusively community-acquired Escherichia coli isolates in Izmir (Turkey) and to search for isolates producing CTX-M-15 and belonging to the pandemic clone E. coli O25-ST131.
Methods: The patients with E. coli urinary tract infections (UTIs) and no hospitalization in the last 12 months, and no transfer from hospital, no stay in nursing home and no antimicrobial treatment in the previous 3 months were prospectively included over a 1 year period. Those E. coli detected positive for ESBL were characterized and compared with a representative of E. coli clone O25-ST131 with regard to bla genes, antibiotic resistance, phylogenetic groups, PFGE profiles and virulence factor genes (n = 17). O serotyping, multilocus sequence typing (MLST) and AmpC typing were performed to confirm that the Turkish isolate belonged to the clone O25-ST131.
Results: Among the 3108 UTIs diagnosed, 82 (2.6%) were due to community E. coli isolates and followed the strict inclusion criteria. Seventeen of them (21%) produced an ESBL, of which CTX-M-15 was predominant (53%). These ESBL-positive isolates, distributed equally into three phylogenetic groups, displayed 13 PFGE profiles and three clusters. A Turkish CTX-M-15-producing isolate as a member of the clone ST131 was suggested by a high similarity of its PFGE profile to that of the clone representative and was confirmed by O serotyping, AmpC typing and MLST.
Conclusions: This study describes the community emergence of CTX-M-15-producing E. coli isolates, including an isolate of clone O25-ST131, in Turkey.
Key Words: enterobacteria , epidemiology , extended-spectrum β-lactamases
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Introduction |
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Recently, CTX-M β-lactamases produced by Gram-negative bacteria have been increasingly reported worldwide, notably in the community.1 CTX-M enzymes are now widespread in isolates of Escherichia coli, the major cause of urinary tract infections (UTIs), leading to serious problems for the antimicrobial management of these infections.1 As CTX-M producers are frequently multidrug-resistant, this problem is exacerbated. Recently, an emerged, broadly disseminated, CTX-M-15-positive E. coli clonal group (E. coli O25-ST131) was described on at least three continents.2,3 In Asian countries, few data have been published about the epidemiology of the E. coli producing CTX-M enzymes isolated from inpatients and outpatients.4 A very recent publication has reported that the CTX-M-15 enzyme was highly common among the extended-spectrum β-lactamase (ESBL)-positive E. coli isolates obtained from inpatients and outpatients at a University Hospital in Istanbul (Turkey).5
The present study also reports on ESBL-positive E. coli from Turkey but on those exclusively acquired in the community by essentially young people living in Izmir, a city in the western part of Turkey. This study demonstrated that Turkey is a country also concerned by E. coli clone O25-ST131 producing CTX-M-15.
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Materials and methods |
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Data collection and bacterial isolates
The Hifzissihha Institute (also known as the Institute of Hygiene that belongs to the Turkish Health Ministry) is a laboratory related to different primary healthcare units and is in charge of the poorest outpatients living in the city of Izmir. The study was initiated on 1 September 2004 and carried out until 31 August 2005. The enrolled patients were those consulting for symptoms corresponding to a UTI. However, they were definitively included in the study when they presented a UTI (bacteriuria 
105 cfu/mL) due to an E. coli isolate and no hospitalization in the last 12 months, no transfer from hospitals, no stay in nursing home, no antimicrobial treatment in the previous 3 months and no urinary devices.
Bacterial genus and species were identified by conventional methods, and all of the E. coli isolates were stored at –80°C.
Susceptibility to antimicrobial agents was measured by using the disc diffusion assay on Mueller–Hinton. ESBL production was screened by the double-disc synergy test and by phenotypic confirmation with ceftazidime/ceftazidime clavulanate (C/CD) discs.
Characterization of β-lactamase-encoding and qnr genes
The genes blaTEM, blaSHV, blaOXA and blaCTX-M were detected by PCR using specific primers and identified by sequencing the PCR products.2 To determine the ampC variants, DNA was amplified and sequenced as described previously.2
The qnrA, qnrB and qnrS genes were screened by PCR in all of the ESBL-producing isolates.6 A search for mutations in gyrA, gyrB, parC and parE genes was performed by PCR and gene sequencing.7
The intercontinental clone of E. coli O25-ST131 corresponds to a group of strains with 68% to 100% similarity by PFGE.2,3 MTPA1, a French community strain belonging to the intercontinental clone, was included in the PFGE experiments in order to detect an eventual relationship between Turkish isolates and the clonal group. Macrorestriction analysis of XbaI-digested chromosomal DNA was performed by PFGE with the CHEF DRII system (Bio-Rad SA, Ivry-sur-Seine, France).7 The PFGE patterns were analysed with Gel compar computer software for Windows version 3.5 (Applied Math, Kortrijk, Belgium) and compared using the algorithmic clustering method known as the unweighted-pair group method using arithmetic averages with the Dice coefficient of similarity. Isolates were considered to be within a cluster if the coefficient of similarity was >80%.
Phylogenetic grouping and the O group of the ESBL-producing E. coli isolates were determined by PCR-based methods developed by Clermont et al.2,8
Sequence type (ST) determination
Multilocus sequence typing (MLST) was carried out as described previously.2 Allelic profile and ST determinations were performed as per the E. coli MLST web site scheme.
To assess the strain virulence potentiality, 17 extraintestinal virulence-associated genes were evaluated by PCR: the papG alleles, papC, sfaS, focG, afa/draBC, fimH, hlyA, cnf1, iutA, kpsMTK1 and kpsMTII, traT, iroN, malX, irp2 and ompT.2
For each antibiotic resistance, comparisons between the ESBL-producing and susceptible E. coli strains, and CTX-M- and other ESBL (TEM and SHV)-producing E. coli were performed by using Fisher’s exact test. For the number of virulence factors (VFs), comparisons between the different phylotyping groups were evaluated using the Wilcoxon test. A P value 
0.05 given by SAS®/ETS software release 8.1 (SAS Institute Inc, Cary, NC, USA) was considered as statistically significant.
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Main characteristics of patients and isolates
Among the 3108 UTIs diagnosed in the Hifzissihha Institute, E. coli isolates were identified in 2110 cases (67.9%) and, among these, 578 (18.6%) came from patients who had had no previous antimicrobial treatment in the last 3 months. However, only 82 of these 578 patients had had no contact with healthcare centres or hospitals in the previous 12 months (Table 1). Overall, 2.6% of the patients diagnosed with a UTI at the Hifzissihha Institute could be included in our study with an infection acquired in the community. Among these patients, 21% (17/82) had a UTI due to an ESBL-producing E. coli isolate and 76.5% of these isolates produced a CTX-M enzyme. CTX-M-15 was the most prevalent CTX-M enzyme (Table 2). Such a predominance of the CTX-M-15 enzyme had also been found by Gonullu et al.5 in the ESBL-producing E. coli isolates identified in Istanbul. However, the prevalence of ESBL-positive E. coli isolates was lower in the population studied by Gonullu et al. (6%) than in ours (21%). This difference might be due to only the involvement, in our study, of E. coli clinical isolates exclusively acquired in the community. Beside the CTX-M enzymes, there were TEM and SHV derivatives (Table 2). Interestingly, the different ESBL types observed in the general population in Izmir corresponded to those which were commonly identified in the ESBL-positive hospital Enterobacteriaceae before 2000 (TEM-3, SHV-12 and TEM-24) and to those which are currently dominant in the hospital (CTX-M enzyme).1 This finding suggests that the ESBLs identified in the community correspond to the most widespread ESBLs in the hospital.
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Interestingly, the ESBL-producing isolates were obtained from patients having a lower median age (38 years) than those without ESBL-positive isolates (Table 1). Moreover, 47% of the patients with ESBL-positive E. coli isolates were <9 years old. This trend has recently been described in Latin America.9 However, in this study, the CTX-M-producing E. coli isolates were obtained from the faeces of healthy children. Thus, the fact that young people had faecal carriage or community-acquired UTIs due to E. coli isolates producing CTX-M strongly suggests the large dissemination of these β-lactamases in the community.
Although the 82 patients included in our study had had no antimicrobial treatment in the previous 3 months, their community-acquired E. coli isolates were commonly resistant to the antibiotics classically used to treat UTIs (Table 1). However, those producing ESBLs were significantly more often resistant to these antibiotics than those that were ESBL-negative. Moreover, the isolates producing CTX-M enzymes were more often resistant than those producing TEM and SHV derivatives: for example, 69% of the CTX-M-producing isolates were resistant to ciprofloxacin versus 50% of the TEM- and SHV-producing isolates (P < 0.001). No qnr gene was detected in the quinolone-resistant, ESBL-producing E. coli isolates, whereas all presented substitutions in both GyrA (Ser-83
Tyr) and ParC (Ser-80Ile).
Phylogenetic groups and molecular epidemiology of ESBL-producing E. coli
The 17 ESBL-producing isolates were almost distributed equally into three of the four phylogenetic groups described in E. coli: 30% belonged to B2, 35% to D and 35% to A. Table 2 shows the distribution of VF-encoding genes within the 17 ESBL-producing isolates. Independent of the phylogenetic group, the ESBL-positive isolates appeared to lack genes encoding adhesins (except for PapG group found in 65% of the isolates) since afa/draBC, focG and papC genes were absent and fimH and sfaS were only present in 18% and 6% of the isolates, respectively.
PFGE revealed a great genomic diversity of the 17 ESBL-producing E. coli isolates as they were classified into 13 PFGE profiles (data not shown). However, PFGE also revealed three clusters (>80% similarity) of CTX-M-producing isolates (Table 2), two (CI and CIII) concerning CTX-M-15 and one (CII) concerning CTX-M-3 associated with SHV-12 (Table 2). Cluster CI comprised two isolates belonging to group D and having the same antibiotic resistance and seven VF genes. Cluster CII also comprised two isolates with identical antibiotic resistance and nine VF genes but that belonged to the phylogenotype B2. The third cluster comprised three isolates of phylogenotype A. These three isolates were identical regarding antibiotic resistance phenotype and VF gene pattern including five VF genes. The last information provided by the analysis of the PFGE profiles was a high PFGE similarity (77%) between strain KEC6691 and strain MTPA1, the strain corresponding to the representative of the intercontinental E. coli clone O25-ST131. The two strains belonged to phylogenetic group B2, had the same antibiotic resistance and had the same 10 VF determinants (Table 2).
French and Turkish E. coli isolates of clone O25-ST131
Because of a level of similarity between strain KEC6691 and strain MTPA1 (a representative of clone ST131) for both PFGE (77%) and virulence gene (94%) profiles similar to that previously described for the isolates belonging to the intercontinental clone O25-ST131, we conducted an extended comparison of these strains.2 Strain KEC6691 as strain MTPA1 displayed serogroup O25, and sequence type ST131 and had an AmpC-EC6 variant. All these data confirmed that KEC6691 belonged to the intercontinental E. coli O25-ST131 clonal group.
This report documents the presence of CTX-M enzymes in E. coli isolates, which are exclusively community-acquired and mainly coming from young Turkish people. One of the particularities of the CTX-M-producing E. coli isolates identified in the community is their high virulent potential.2,10 Our study confirms this feature and also that CTX-M-producing isolates are commonly multidrug-resistant.1–3 This study allowed us to demonstrate the presence of the clone E. coli O25-ST131 in Turkey. Although only one E. coli O25-ST131 isolate was found in this study, this finding emphasizes the worldwide propagation of the clone ST131 that harbours, as recently demonstrated, multidrug resistance-mediating plasmids of the IncFII family, a family largely diffused in E. coli.
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INSERM Espri 26 is supported by INSERM, Université de Montpellier 1, La Ville de Nîmes and La Région Languedoc Roussillon.
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None to declare.
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Nicolas-Chanoine MH, Blanco J, Leflon-Guibout V, et al. Intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15. J Antimicrob Chemother (2008) 61:273–81.
3 . Coque TM, Novais A, Carattoli A, et al. Dissemination of clonally related Escherichia coli strains expressing extended-spectrum β-lactamase CTX-M-15. Emerg Infect Dis (2008) 14:195–200.[Web of Science][Medline]
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Gonullu N, Aktas Z, Kayacan CB, et al. Dissemination of CTX-M-15 β-lactamase genes carried on Inc FI and II plasmids among clinical isolates of Escherichia coli in a University Hospital in Istanbul, Turkey. J Clin Microbiol (2008) 46:1110–2.
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Pallecchi L, Bartoloni A, Fiorelli C, et al. Rapid dissemination and diversity of CTX-M extended-spectrum β-lactamase genes in commensal Escherichia coli isolates from healthy children from low-resource settings in Latin America. Antimicrob Agents Chemother (2007) 51:2720–5.
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