engleski

Predominace of SHV-5 beta-lactamase in enteric bacteria causing community-acquired urinary tract infections in Bosnia and Herzegovina

Plasmid-encoded resistance to broad-spectrum cephalosporins and aztreonam is becoming a widespread phenomenon in clinical medicine. An increase in the prevalence of ESBLs causing community urinary tract infections (CAUTI) was observed recently among children (0-6 years of age) in Zenica-Doboj canton, Bosnia and Herzegovina Thus, the aim of the present work was to characterize ESBLs in K. pneumoniae and E. coli from urinary tract infections in this region. From the May 2004 to April 2005 2059 enterobacteria were isolated consecutively from single outpatient urine samples submitted to the Laboratory. Antibiotic susceptibilities to 15 antimicrobials were tested by disk diffusion method according to CLSI . After initial screening, the production of ESBLs was confirmed by double-disk synergy test. Nineteen ESBL-producing strains were available for further testing. Minimum inhibitory concentrations (MICs) of wide range of antibiotics were determined by a twofold microdilution test according to CLSI. Transferability of resistance to oxymino cephalosporins was tested by conjugation (broth mating method) employing E. coli A15R- stain resistant to rifampicin as recipient. Crude bacterial sonicates were subjected to isoelectric focusing (IEF) in the polyacrilamide gel with the pH range of 3.5 to 10.  -Lactamases were detected by staining of the gel with nitrocefin. The enzymes of the referral strains producing TEM-1, TEM-2, SHV-1, SHV-2, SHV-3, SHV-4, SHV-5 and CTX-M-15 were used as a pH standard. BlaESBL genes were detected by PCR with primers specific for TEM, SHV and CTX-M β -lactamases BlaSHV genes of the three K. pneumoniae transconjugants (14213, 35117, 35123) were subjected to DNA sequencing. Sequencing was performed using an ABI PRISM 377 Genetic Analyser (Applied Biosystems). Transconjugant plasmid DNA was extracted by alkaline lysis procedure, digested with EcoRI and subjected to electrophoresis in 0.8% agarose gel in TBE buffer. PFGE of Xba-digested genomic DNA was performed with a CHEF-DRIII system (Bio-rad) as described previously. From the May 2004 to April 2005 2, 059 enterobacteria were isolated from consecutive single urine samples (437 of males, 1622 of females) obtained from outpatients. E. coli was isolated in 68.1%, Klebsiaellae spp. in 23.7%, Proteus spp. in 6.8%, Citrobacter spp. and Enterobacter spp. in 0.7% each, urine samples. ESBLs were detected by double-disk synergy test and 8 fold reduction in ceftazidime MIC in the presence of clavulanic acid in 2.7% of coliform isolates, of which 9.0% were among Klebsiella spp., 0.6% among E. coli isolates, and 5.5% belonged to the other enterobacteria. ESBL producers were detected in 42.6% of children up to six years of age. The prevalence of ESBLs in males was twice higher than in females, 69.1% and 30.9% respectively. The addition of clavulanic acid to ceftazidime lowered the MIC below 2 mg/L. Imipenem and meropenem were consistently active with MICs below 0.5 and 0.25 mg/L, respectively. Successful transfer of antibiotic resistance was achieved for 10 K. pneumoniae and 3 E. coli isolates. Resistance to non β -lactam antibiotics was cotransferred alongside with cephalosporins resistance in most cases. E. coli strains and their respective transconjugants showed a band of enzyme activity at the pI of 5.4 corresponding to the TEM-1  -lactamase whereas K. pneumoniae isolates and their E. coli transconjugants displayed a band at pI of 8.2 consistent with SHV-5 enzyme. PCR detected blaSHV genes in all K. pneumoniae strains and their transconjugants and blaTEM genes in all E. coli strains. No CTX-M  -lactamase producers were found. Based on sequencing of blaSHV genes, enzymes from all three tranconjugants were identified as SHV-5  -lactamase. Plasmid analysis after digestion with EcoR1 showed that transconjugants harbour distinct fingerprinting patterns (results not shown). K. pneumoniae and E. coli strains showed distinct PFGE patterns . The prevalence of 2.6% ESBL-producing strains in the community found in our study corresponded to the prevalence found in hospital settings and private health care centers in some reports. This study has shown much higher prevalence of ESBL-producing isolates among K. pneumoniae than E. coli isolates This observations is not in agreement with other studies which found E. coli to be the most prevalent ESBL-producing species in outpatient samples. Misuse or overuse of antimicrobial drugs in the Bosnia and Herzegovina and migratory flux of regional populations could result in emergence and selection of these ESBL-phenotypes in the community. According to our results SHV-ESBLs are dominant among K. pneumoniae strains and TEM-ESBLs are the most prevalent in E. coli isolates. This is in contrast with the reports from other authors who found CTX-M  -lactamases to be dominant in E. coli community specimens Both, TEM- and SHV- -lactamases are usually limited to nosocomial infections caused by Klebsiella spp. However this is the first description of SHV-5 β -lactamase predomination in the community specimens. Since the strains showed distinct PFGE patterns, we concluded that there was no clonal spread of blaTEM and blaSHV genes. It is assumed that dissemination of blaTEM and blaSHV genes was rather due to the genetic exchange between different clones and different plasmids.

extended-spectrum beta-lactamases; ceftazidime; urinary tract infections

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