CTX-M enzymes are the most common extended-spectrum ß-lactamases among Escherichia coli in a tertiary Greek hospital

Spyros Pournaras1, Alexandros Ikonomidis1, Ioulia Kristo1, Athanassios Tsakris2,* and Antonios N. Maniatis1

1 Department of Medical Microbiology, University of Thessalia, Mezourlo, Larissa; 2 Department of Microbiology, Faculty of Nursing, School of Health Sciences, University of Athens, 123 Papadiamantopoulou Street, 11527 Athens, Greece

Keywords: ESBLs , hospital-acquired infections , CTX-M ß-lactamases , Etest , PFGE , PCR , Greece

Sir,

Extended-spectrum ß-lactamases (ESBLs) have emerged among Gram-negative bacteria; predominantly Klebsiella pneumoniae and, to a lesser extent, Escherichia coli and other species. Most ESBLs are mutants of the classical TEM and SHV enzymes, but since 1995 a rapid increase in the number of CTX-M variants has been reported among enterobacterial isolates from hospitalized patients.1 CTX-M enzymes hydrolyse and confer resistance to cefotaxime preferentially over ceftazidime and exhibit a higher susceptibility to tazobactam than to clavulanic acid. Currently, the CTX-M family includes almost 40 variants, divided between five major amino acid sequence subtypes (see www.lahey.org/studies/other.asp#table1).1 They are most prevalent in South America and the Far East, but have also been disseminated in several European countries.13 Recently, CTX-M-type ß-lactamases were reported among E. coli isolates from Greece.4

During February 2003–February 2004, 426 E. coli isolates were recovered consecutively from hospital-acquired infections of separate patients hospitalized at the University Hospital of Larissa, Thessalia, Greece. Twenty-three (5.4%) of the isolates were confirmed as ESBL producers by the Etest ESBL screening method (AB Biodisk, Solna, Sweden) using cefotaxime and ceftazidime plus clavulanate. MICs of ß-lactams against these isolates were determined using Etest, whereas susceptibility testing against other antimicrobials was performed by the disc diffusion method on Mueller–Hinton agar.5 Against all but four of these isolates, the cefotaxime MIC was at least eight-fold higher than that of ceftazidime. Third-generation cephalosporins and aztreonam exhibited a notably variable efficacy against the ESBL producers (Table 1).


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Table 1. Susceptibility and transferability data of the 20 CTX-M-positive E. coli isolates

 
To type the ß-lactamase genes carried by the ESBL-producing isolates, PCR was performed using primers specific for blaTEM, blaSHV, blaCTX-M and blaIBC.6 Genes encoding CTX-M-type determinants were detected in 20 of the ESBL-positive isolates with two others being positive for both blaSHV and blaTEM and one was negative for all bla genes tested. Fourteen of the CTX-M-positive isolates produced an additional ß-lactamase of the TEM-type. Sequencing of the 873 bp blaCTX-M amplicons on both strands revealed that six encode CTX-M-1 and 14 encode CTX-M-3. Genotyping using pulsed-field gel electrophoresis (PFGE) of XbaI-digested genomic DNA7 showed that the CTX-M-producers belonged to 13 distinct groups. CTX-M-producing isolates were mated in nutrient agar with E. coli 26R793 (lac rifR) and transconjugants were selected on Muller–Hinton agar containing rifampicin 100 mg/L and cefotaxime 2 mg/L. Transfer of cefotaxime resistance was obtained in all but four cases along with other antibiotic-resistance determinants, including chloramphenicol, gentamicin, tetracycline and trimethoprim/sulfamethoxazole (Table 1). Transfer frequencies varied from 3.8 x 10–5–4.1 x 10–3 per donor cell. Following plasmid isolation by an alkaline lysis method,8 transconjugants contained one to three plasmids that varied in size from 8–130 kb. The four isolates with non-transferable resistance contained plasmids that ranged from 30–150 kb and were different in size from those with transferable resistance.

The CTX-M-positive isolates were recovered from seven children and 13 adults who were hospitalized in seven different units of the hospital. Four of them had been treated with third-generation cephalosporins prior to the isolation. Sixteen of the patients had a severe urinary tract infection whereas the remaining four exhibited purulent infections.

This report documents the predominance of blaCTX-M genes among ESBL-positive E. coli recovered from clinical infections in a tertiary Greek hospital. The isolates exhibited plasmid-mediated resistance that affected the antimicrobial activity of penicillins and cephalosporins but also to several alternative antibiotics used to treat E. coli infections. The observation that different CTX-M subtypes, encoded by plasmids of varying size, are being carried by distinct strains of E. coli implies that the genes, and not simply the organisms carrying the genes, are spreading within our hospital. Our findings support the hypothesis that CTX-M enzymes will become the dominant ESBLs among E. coli worldwide. Since ESBL detection procedures are not always sensitive, the predominance of CTX-M enzymes suggests that it is important for our laboratories to perform synergy tests with cefpodoxime, which is degraded by TEM, SHV and CTX-M ESBLs.2

Footnotes

* Corresponding author. Tel: +30-210-7461483; Fax: +30-210-7461489; Email: atsakris{at}med.uoa.gr

References

1 . Bonnet, R. (2003). Growing group of extended-spectrum ß-lactamases: the CTX-M enzymes. Antimicrobial Agents and Chemotherapy 48, 1–14.[ISI]

2 . Mushtaq, S., Woodford, N., Potz, N. et al. (2003). Detection of CTX-M-15 extended-spectrum ß-lactamase in the United Kingdom. Journal of Antimicrobial Chemotherapy 52, 528–9.[Free Full Text]

3 . Baraniak, A., Fiett, J., Hryniewicz, W. et al. (2002). Ceftazidime-hydrolysing CTX-M-15 extended-spectrum ß-lactamase (ESBL) in Poland. Journal of Antimicrobial Chemotherapy 50, 393–6.[Abstract/Free Full Text]

4 . Mavroidi, A., Tzelepi, E., Miriagou, V. et al. (2002). CTX-M-3 ß-lactamase-producing Escherichia coli from Greece. Microbial Drug Resistance 8, 35–7.[CrossRef][ISI][Medline]

5 . National Committee for Clinical Laboratory Standards (2001). Performance Standards for Antimicrobial Disk Susceptibility Tests: Approved Standard M2-A7. NCCLS, Wayne, PA, USA.

6 . Tzelepi, E., Magana, C., Platsouka, E. et al. (2003). Extended-spectrum ß-lactamase types in Klebsiella pneumoniae and Escherichia coli in two Greek hospitals. International Journal of Antimicrobial Agents 21, 285–8.[CrossRef][ISI][Medline]

7 . Jalaluddin, S., Devaster, J. M., Scheen, R. et al. (1998). Molecular epidemiological study of nosocomial Enterobacter aerogenes isolates in a Belgian hospital. Journal of Clinical Microbiology 36, 1846–52.[Abstract/Free Full Text]

8 . Olsen, J. E. (1990). An improved method for rapid isolation of plasmid DNA from wild-type gram-negative bacteria for plasmid restriction profile analysis. Letters in Applied Microbiology 10, 209–12.[ISI][Medline]