Monika Dolejska

High prevalence of antimicrobial-resistant genes and integrons in Escherichia coli isolates from black-headed gulls in the Czech Republic

Aims:  To carry out an assessment of the occurrence of resistance to antimicrobials in Escherichia coli that has been isolated from young Black‐headed Gulls in three nesting colonies.

Antibiotic-Resistant Escherichia coli Bacteria, Including Strains with Genes Encoding the Extended-Spectrum Beta-Lactamase and QnrS, in Waterbirds on the Baltic Sea Coast of Poland

ABSTRACT Individual cloacal swabs of mallards (Anas platyrhynchos) and of herring gulls (Larus argentatus), as well as samples of waterbird feces obtained in 2008 and 2009, were cultivated for Escherichia coli. Isolates of E. coli were tested for susceptibilities to 12 antimicrobial agents by the disk diffusion method. Moreover, the samples were subcultivated on MacConkey agar (MCA) containing cefotaxime (2 mg liter−1) to detect E. coli with extended-spectrum beta-lactamase (ESBL) and subsequently on MCA supplemented with ciprofloxacin (0.05 mg liter−1) and MCA with nalidixic acid (20 mg liter−1) to isolate fluoroquinolone-resistant E. coli. PCR was used to detect specific antibiotic resistance genes. We found 9 E. coli isolates producing ESBL with bla genes: blaCTX-M-1 (6 isolates), blaCTX-M-9 plus blaTEM-1b (1 isolate), blaCTX-M-15 plus blaOXA-1 (1 isolate), and blaSHV-12 (1 isolate). In the isolate with blaCTX-M-15, the gene aac(6)-Ib-cr was also detected. The bla genes were harbored by transferable plasmids of the IncN and IncI1 groups. Nine quinolone-resistant E. coli isolates with qnrS genes were found and characterized. The gene qnrS was associated with a Tn3-like transposon on the IncX1 plasmid together with blaTEM-1 in two isolates. The gene qnrS was also harbored by conjugative plasmids of the IncN and IncX2 groups. Even if populations of wild birds are not directly influenced by antibiotic practice, we have demonstrated that antibiotic-resistant E. coli strains, including strains with various ESBL and qnrS genes, are found in the feces of wild birds on the coast of the Baltic Sea in Poland.

Antimicrobial-resistant faecal Escherichia coli in wild mammals in central Europe: multiresistant Escherichia coli producing extended-spectrum beta-lactamases in wild boars

Aims:  To determine the presence of antibiotic‐resistant faecal Escherichia coli in populations of wild mammals in the Czech Republic and Slovakia.

Antibiotic-resistant Salmonella and Escherichia coli isolates with integrons and extended-spectrum beta-lactamases in surface water and sympatric black-headed gulls

Aim:  To examine surface water from a pond in the northeastern part of the Czech Republic and young black‐headed gulls (Larus ridibundus) nesting on the same pond for the presence of antibiotic‐resistant Salmonella and Escherichia coli.

CTX-M-15-producing Escherichia coli clone B2-O25b-ST131 and Klebsiella spp. isolates in municipal wastewater treatment plant effluents

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.

Complete sequencing of an IncHI1 plasmid encoding the carbapenemase NDM-1, the ArmA 16S RNA methylase and a resistance–nodulation–cell division/multidrug efflux pump

OBJECTIVES To characterize the pNDM-CIT plasmid identified in Citrobacter freundii carrying genes encoding the metallo-β-lactamase NDM-1 and the 16S RNA methylase ArmA. METHODS The complete DNA sequence of pNDM-CIT was obtained by using the 454-Genome Sequencer FLX procedure on a library obtained using plasmid DNA purified from the pNDM-CIT Escherichia coli J53 transconjugant. Contig assembly and predicted gaps were confirmed and filled by PCR-based gap closure. Comparative analysis with IncHI1 incompatibility group plasmids was performed using BLASTN and BLASTP algorithms. RESULTS Plasmid pNDM-CIT was 288::920 bp and revealed an IncHI1 plasmid scaffold, showing novel resistance and potential virulence determinants. The bla(NDM-1) gene was identified within a novel genetic context, flanked by a duplication of the class 1 integron on both sides. The replicase gene repAciN, originating from Acinetobacter spp. plasmids, was identified in a close association with the Tn1548::armA transposon and the macrolide resistance mel-mph2 cluster. The same structure was identified in silico from a series of enterobacterial plasmids carrying the armA gene. The repAciN gene probably represents a remnant sign of the original occurrence of the armA gene in Acinetobacter plasmids. A CP4-like prophage sequence was identified in pNDM-CIT, containing a resistance-nodulation-cell division/multidrug resistance (RND/MDR) efflux pump cluster surrounded by two IS1-like elements. This resistance determinant, associated with such a prophage sequence, has never been reported on plasmids. CONCLUSIONS Plasmid pNDM-CIT differed significantly from all known bla(NDM-1)-carrying plasmids identified in Enterobacteriaceae, since it combines the metallo-β-lactamase NDM-1, the 16S RNA methylase ArmA and a cryptic prophage carrying the RND/MDR efflux pump.

Antibiotic resistant Escherichia coli and Salmonella in Russian rooks (Corvus frugilegus) wintering in the Czech Republic.

Aims:  To characterize antibiotic resistant Escherichia coli and Salmonella isolates in rooks wintering in the Czech Republic.

Antibiotic resistance in faecal bacteria (Escherichia coli, Enterococcus spp.) in feral pigeons.

Aims:  To determine the presence of antibiotic‐resistant faecal Escherichia coli and Enterococcus spp. in feral pigeons (Columba livia forma domestica) in the Czech Republic.

Plasmids carrying blaCTX-M-1 and qnr genes in Escherichia coli isolates from an equine clinic and a horseback riding centre

OBJECTIVES The aim of this study was to determine the occurrence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli at an equine clinic and a horseback riding centre, and to discuss the impact of antimicrobial treatment on resistance selection. METHODS Faeces from horses, environmental smears and flies were sampled at both the clinic and riding centre. Staff at the equine clinic were also examined. The samples were cultivated on MacConkey agar with cefotaxime (2 mg/L) to isolate ESBL-producing E. coli. The presence of bla and qnr genes was tested by PCR, and transferability was determined by conjugation. Replicon typing and restriction analysis of plasmids harbouring ESBL and qnr genes were performed. RESULTS E. coli with the blaCTX-M-1 gene were isolated from horses, staff, environmental smears and flies at the two sites. E. coli isolates from the equine clinic harboured an IncHI1 conjugative 235-285 kb plasmid containing blaCTX-M-1, catA1, strA, sul2 and tet(B) genes. Some of these were positive for qnrS1 and/or qnrB19, and were located on 40 or 45 kb IncN or IncX1 conjugative plasmids. The gene blaCTX-M-1 in isolates from the riding centre was carried by IncN (30 kb) and IncI1 (85 kb) conjugative plasmids. Horizontal gene transfer seems to be involved in disseminating E. coli with ESBL and qnr genes at the clinic and riding centre. CONCLUSIONS The study illustrates that ESBL-producing E. coli, as well as plasmids carrying ESBL genes of clinical interest, can be easily transferred among horses, humans and flies living in close contact.

Characterization of IncN plasmids carrying blaCTX-M-1 and qnr genes in Escherichia coli and Salmonella from animals, the environment and humans

OBJECTIVES The aim of the study was to characterize a collection of Escherichia coli and Salmonella harbouring qnr and bla(CTX-M-1) genes on IncN plasmids isolated from humans, food-producing, companion and wild animals, and the environment from six European countries. METHODS Nineteen IncN plasmids were compared using restriction fragment length polymorphism (RFLP), plasmid multilocus sequence typing (pMLST) and hybridization with repN, qnrS1, qnrB19 or bla(CTX-M-1) probes. Plasmids pKT58A and pHHA45 were sequenced using the 454-Genome Sequencer FLX platform on a library constructed from plasmid DNA purified from the respective E. coli transformants. RESULTS Three types of IncN plasmids carrying bla(CTX-M-1), qnrS1 and qnrB19 genes were identified in strains isolated from the Czech Republic, Poland, Slovakia, Denmark, Italy and the Netherlands, corresponding to pMLST sequence type (ST) 1, ST3 and ST8, respectively. Related plasmids circulating in human and animal isolates were identified. Complete nucleotide sequences of the ST1 pHHA45 plasmid carrying bla(CTX-M-1), isolated from E. coli from pigs in Denmark, and the ST3 pKT58A plasmid harbouring qnrS1, identified in E. coli from a water bird, were obtained. CONCLUSIONS Our results demonstrated wide distribution of specific IncN plasmids disseminating bla(CTX-M-1) and qnr genes among animals and humans in Europe.