OBJECTIVES: The global occurrence of antibiotic resistance genes in bacteria in water environments is an increasing concern. Treated wastewater was sampled daily over a 45 day period from the outflow of a municipal wastewater treatment plant in Brno, Czech Republic, and examined for extended-spectrum β-lactamase (ESBL)-producing bacteria. METHODS: Water samples were cultivated on MacConkey agar with cefotaxime (2 mg/L) and individual colonies were examined for ESBL production. Phenotypic ESBL-positive bacteria identified as Escherichia coli or Klebsiella spp. were tested for the presence of antibiotic resistance genes, the virulence gene afa/dra and the bla(CTX-M) upstream region. Genetic relatedness was analysed by PFGE, multilocus sequence typing and plasmid analysis. RESULTS: A total of 68 ESBL-producing Enterobacteriaceae isolates were detected in 34 out of 45 wastewater samples. ESBL-producing isolates included 26 E. coli isolates, 4 Klebsiella pneumoniae isolates and 1 Klebsiella oxytoca isolate. The pandemic and multiresistant B2-O25b-ST131 clone was predominant, being detected among 19 E. coli isolates, and 17 of the B2-O25b-ST131 isolates were positive for the FIA replicon and the afa/dra operon and had an IS26 element flanking bla(CTX-M-15). Seventeen of the B2-O25b-ST131 isolates showed closely related PFGE profiles (defined by 84% band similarity) and belonged to identical clonal groups. CONCLUSIONS: The results highlight the inadequacy of the treatment process in removing multiresistant bacteria from municipal wastewater and point to a risk of transmission of clinically important multiresistant strains, such as the pandemic ST131 clone, to the environment. This is the first study demonstrating the pandemic ST131 clone in wastewater.

CEITEC VFU University of Veterinary and Pharmaceutical Sciences Brno Brno Czech Republic

CEITEC VFU University of Veterinary and Pharmaceutical Sciences Brno Palackeho 1 3 612 42 Brno

Department of Biology and Wildlife Diseases Faculty of Veterinary Hygiene and Ecology University of Veterinary and Pharmaceutical Sciences Brno Brno

Department of Biology and Wildlife Diseases Faculty of Veterinary Hygiene and Ecology University of Veterinary and Pharmaceutical Sciences Brno Brno Czech Republic

Department of Microbiology Faculty of Veterinary Medicine Wroclaw University of Environmental and Life Sciences C K Norwida 30 50 375 Wroclaw Poland

Institute of Microbiology and Infectious Diseases Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences Brno Brno Czech Republic

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$a Dolejská, Monika, $d 1982- $7 mub2011634219 $u Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic. m.dolejska@centrum.cz

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$a CTX-M-15-producing Escherichia coli clone B2-O25b-ST131 and Klebsiella spp isolates in municipal wastewater treatment plant effluents / $c M. Dolejska, P. Frolkova, M. Florek, I. Jamborova, M. Purgertova, I. Kutilova, A. Cizek, S. Guenther, I. Literak

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$a OBJECTIVES: The global occurrence of antibiotic resistance genes in bacteria in water environments is an increasing concern. Treated wastewater was sampled daily over a 45 day period from the outflow of a municipal wastewater treatment plant in Brno, Czech Republic, and examined for extended-spectrum β-lactamase (ESBL)-producing bacteria. METHODS: Water samples were cultivated on MacConkey agar with cefotaxime (2 mg/L) and individual colonies were examined for ESBL production. Phenotypic ESBL-positive bacteria identified as Escherichia coli or Klebsiella spp. were tested for the presence of antibiotic resistance genes, the virulence gene afa/dra and the bla(CTX-M) upstream region. Genetic relatedness was analysed by PFGE, multilocus sequence typing and plasmid analysis. RESULTS: A total of 68 ESBL-producing Enterobacteriaceae isolates were detected in 34 out of 45 wastewater samples. ESBL-producing isolates included 26 E. coli isolates, 4 Klebsiella pneumoniae isolates and 1 Klebsiella oxytoca isolate. The pandemic and multiresistant B2-O25b-ST131 clone was predominant, being detected among 19 E. coli isolates, and 17 of the B2-O25b-ST131 isolates were positive for the FIA replicon and the afa/dra operon and had an IS26 element flanking bla(CTX-M-15). Seventeen of the B2-O25b-ST131 isolates showed closely related PFGE profiles (defined by 84% band similarity) and belonged to identical clonal groups. CONCLUSIONS: The results highlight the inadequacy of the treatment process in removing multiresistant bacteria from municipal wastewater and point to a risk of transmission of clinically important multiresistant strains, such as the pandemic ST131 clone, to the environment. This is the first study demonstrating the pandemic ST131 clone in wastewater.

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$a Florek, Magdalena $u Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic; Department of Microbiology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, C.K. Norwida 30, 50-375 Wroclaw, Poland

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