Alois Cizek

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.

Shiga toxin‐producing Escherichia coli O157 in feedlot cattle and Norwegian rats from a large‐scale farm

A total of 365 faecal samples from different categories of cattle, 12 samples of untreated slurry, 50 samples of fresh droppings of feral domestic pigeons, 20 samples of fresh droppings of domestic sparrows and stool samples of 19 synanthropic rodents were examined for the presence of Escherichia coli by broth enrichment culture and a subsequent immunomagnetic separation. Escherichia coli O157 was found in 72 (20%) bovine samples, six (50%) samples of untreated slurry and four (40%) of 10 rats (Rattus norvegicus). Significant differences were found in the E. coli O157 shedding frequency between different age categories of bulls. Genes stx2 and eaeA were detected in all isolates, and the stx1 gene in all but 10 isolates.

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.

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.

Antimycobacterial and herbicidal activity of ring-substituted 1-hydroxynaphthalene-2-carboxanilides.

In this study, a series of 22 ring-substituted 1-hydroxynaphthalene-2-carboxanilides were prepared and characterized. Primary in vitro screening of the synthesized compounds was performed against Mycobacterium marinum, Mycobacterium kansasii and Mycobacterium smegmatis. The compounds were also tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. Most of tested compounds showed the antimycobacterial activity against the three strains comparable or higher than the standard isoniazid. N-(3-Fluorophenyl)-1-hydroxynaphthalene-2-carboxamide showed the highest biological activity (MIC=28.4μmol/L) against M. marinum, N-(4-fluorophenyl)-1-hydroxynaphthalene-2-carboxamide showed the highest biological activity (MIC=14.2μmol/L) against M. kansasii, and N-(4-bromophenyl)-1-hydroxynaphthalene-2-carboxamide expressed the highest biological activity (MIC=46.7μmol/L) against M. smegmatis. This compound and 1-hydroxy-N-(3-methylphenyl)naphthalene-2-carboxamide were the most active compounds against all three tested strains. The PET inhibition expressed by IC50 value of the most active compound 1-hydroxy-N-(3-trifluoromethylphenyl)naphthalene-2-carboxamide was 5.3μmol/L. The most effective compounds demonstrated insignificant toxicity against the human monocytic leukemia THP-1 cell line. For all compounds, structure-activity relationships are discussed.

IncN plasmids carrying blaCTX-M-1 in Escherichia coli isolates on a dairy farm

The aim of the study was to compare the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli bovine isolates on a conventional dairy cattle farm with high consumption of parenteral and intramammary cephalosporins (farm A) and on an organic dairy farm with no cephalosporin use (farm B). ESBL-producing E. coli were isolated from rectal swabs and milk filters by selective cultivation on MacConkey agar with cefotaxime (2mg/l). ESBL genes were identified by polymerase chain reaction (PCR) and sequencing, and the genetic diversity of the isolates was determined by XbaI pulsed field gel electrophoresis (PFGE). Conjugative transfer, incompatibility group, and restriction fragment length polymorphism (RFLP) profiles of the ESBL-carrying plasmids were studied. Higher prevalence (39%, n(rectal samples in cows)=309) of CTX-M-1-producing E. coli isolates was found on farm A compared to farm B (<1%, n(rectal samples in cows)=154; 0%, n(rectal samples in calves)=46). Using PFGE, the isolates from farm A were divided into nine pulsotypes. In all ESBL-positive isolates, the bla(CTX-M-1) gene was carried on 40 kb IncN conjugative plasmids of three related HincII restriction profiles. Horizontal gene transfer through transmission of IncN plasmids harboring bla(CTX-M-1) as well as clonal dissemination of a particular clone seems to be involved in dissemination of CTX-M-1-producing E. coli isolates in cows on the farm using cephalosporins in treating bacterial infections. The study demonstrates a possible role of cephalosporin use in the widespread occurrence of CTX-M-1-producing E. coli on the conventional dairy cattle farm compared to the organic farm.

High prevalence of Salmonella and IMP-4-producing Enterobacteriaceae in the silver gull on Five Islands, Australia

Objectives The objective of this study was to investigate the silver gull as an indicator of environmental contamination by salmonellae and carbapenemase-producing Enterobacteriaceae (CPE) in south-east Australia. Methods A total of 504 cloacal samples were collected from gull chicks at three nesting colonies in New South Wales, Australia [White Bay (n = 144), Five Islands (n = 200) and Montague Island (n = 160)] and were examined for salmonellae and CPE. Isolates were tested for carbapenemase genes and susceptibility to 14 antibiotics. Clonality was determined by PFGE and MLST. Genetic context and conjugative transfer of the carbapenemase gene were determined. Results A total of 120 CPE of 10 species, mainly Escherichia coli (n = 85), carrying the gene blaIMP-4, blaIMP-38 or blaIMP-26 were obtained from 80 (40%) gulls from Five Islands. Thirty percent of birds from this colony were colonized by salmonellae. Most isolates contained the gene within a class 1 integron showing a blaIMP-4-qacG-aacA4-catB3 array. The blaIMP gene was carried by conjugative plasmids of variable sizes (80–400 kb) and diverse replicons, including HI2-N (n = 30), HI2 (11), A/C (17), A/C-Y (2), L/M (5), I1 (1) and non-typeable (6). Despite the overall high genetic variability, common clones and plasmid types were shared by different birds and bacterial isolates, respectively. Conclusions Our data demonstrate a large-scale transmission of carbapenemase-producing bacteria into wildlife, likely as a result of the feeding habits of the birds at a local waste depot. The isolates from gulls showed significant similarities with clinical isolates from Australia, suggesting the human origin of the isolates. The sources of CPE for gulls on Five Islands should be explored and proper measures applied to stop the transmission into the environment.