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Detection of extended-spectrum beta-lactamases in E. coli and Klebsiella spp. by isoelectric focusing and PCR

Abstract number: p1414

Oksuz  L., Gurler  N.

Objectives: 

The aim of this study was the detection of beta lactamases types in extended spectrum beta lactamases producing E. coli and Klebsiella spp strains. The study strains consisted of 12 E. coli and 32 Klebsiella spp (28 K. pneumoniae and 4 K. oxytoca) isolated from various specimens of patients hospitalized in different units (Intensive Care, Hematology, Oncology, Neonatology, Transplantation and Pediatric Surgery Units) of the Istanbul Medical Faculty between July 2002–March 2004.

Methods: 

Antimicrobial susceptibility tests were performed by disc diffusion according to the CLSI guidelines. MICs of cefotaxime and ceftazidime were determined by the agar dilution method and by the E-test ESBL. Isoelectring focusing was performed by the method of Matthew et al (J Gen Microbiol, 1976). Polimerase chain reaction was performed using primers specific for the bla TEM, blaSHV and blaCTX-M genes. Conjugation was carried out by broth mating. A rifampicin-resistant E.coli strain (J62-2) was used as the recipient. Plasmid extraction was performed by the alkaline lysis method.

Results: 

Resistance to imipenem or meropenem was not detected. Amikacin was the second most effective antibiotic against all strains, after carbapenems. All isolates demonstrated an ESBL phenotype using the double-disc synergy test. MIC50 and MIC90 for cefotaxime were found 16 mg/ml and 64 mg/ml in Klebsiella spp and E.coli strains, respectively. IEF results indicated that all isolates produced 1–4 beta lactamases. Isoelectric points of the isolates were between 5.4–9.0. Among K. pneumoniae and E.coli strains, rates of TEM, SHV, CTX-M beta lactamases were found 64.3%, 92.9%, 67.9%; 66.7%, 25%, 83.3%, respectively. The profiles generated with ERIC-2 PCR primer contained several bands, ranging in size from 170 to 1500 bp. Ten, three and six groups were separated for K. pneumoniae, K. oxytoca and E. coli, respectively. Thirty-one of the isolates (70.45%) were able to transfer their resistance to recipient E. coli strains by conjugation. Twenty of the transkonjugants (64.5%) were found to carry only one plasmid (>48a). Eleven strains (35.5%) harbored more than one plasmid, with sizes ranging from 10 to 100 kb.

Conclusions: 

In conclusion, we found that CTX-M was more common than TEM and SHV beta lactamases in E. coli strains isolated from patients hospitalized in Istanbul Medical Faculty, however SHV was the most common beta lactamase in K. pneumoniae. The present work was supported by the Research Fund of Istanbul University. Project no T223/06032003.

Session Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Subject:
Location: Oxford, UK
Presentation type:
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