Emergence of Clonally Related SHV-5 ESBL-producing Escherichia coli Strains in the Community

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Background: Extended-spectrum β -lactamase (ESBL)-producing Escherichia coli is an emergent cause of urinary tract infections in nonhospitalized patients in different countries. The aim of this study was the molecular characterization of ESBL-producing E. coli strains isolated from urine of outpatients in Zagreb region. Materials and Methods: During the five-month study period a total of 2, 451 E. coli strains were isolated from urine of nonhospitalized patients with significant bacteriuria. ESBL production was detected by double-disk diffusion technique and by >3-dilution reduction in the minimal inhibitory concentration of ceftazidime in the presence of clavulanate. A total of 39 ESBL-producing E. coli strains (1.59%) were collected and characterised. Molecular relatedness between the strains was analyzed by pulsed-field gel electrophoresis (PFGE) and isolates were considered to be genetically related if the Dice coefficient correlation was >80%.  -lactamases were characterized by isoelectric focusing, substrate profile determination, polymerase chain reaction and sequencing of blaSHV genes. PCR products were subjected to PCR Nhe test to distinguish between SHV-1 and SHV-ESBL. Plasmids were extracted by alkaline lysis method and digested with EcoR1 enzyme. Results: Chromosomal DNA analysis by PFGE exhibited a great genomic similarity among ESBL strains. The Dice coefficient of similarity for 25 strains was ≥ 83.49%. All strains produced a  -lactamase with the isoelectric point of 8.2. Enzymes produced by the strains antagonized the activity of the disks containing ceftazidime, cefotaxime, ceftriaxone and aztreonam but not cefoxitin and imipenem. All strains yielded an amplicon with primers specific for SHV β -lactamases. PCR Nhe test demonstrated SHV-ESBL in all tested strains. Fifteen strains possessed an additional TEM β -lactamase. No CTX-M β -lactamases was found. Based on sequencing of blaSHV genes enzymes of five strains were identified as SHV-5  -lactamase which conferred on the producing isolates high level of ceftazidime and aztreonam resistance. Mutations at the Ambler positions 238 and 240 were found in all gene sequences. The blaSHV genes were encoded on related plasmids which also carried resistance genes for aminoglycosides. Plasmids were assigned to nine fingerprinting patterns (A-F). Conclusion: The study demonstrated community-associated emergence of clonally related SHV-5 β -lactamase– producing E. coli strains. Other studies on ESBLs in the outpatient populations reported CTX-M ESBLs to be most prevalent in the community setting. The fact that the strains were clonally related, that produced the same type of ESBL and that the high proportion of strains were resistant to aminoglycosides which are not prescribed outside of hospitals, could indicate the possible food origin of ESBL E. coli strains.