CTX-M-producing Escherichia coli in pigs from a Czech farm during production cycle.

We evaluated the prevalence and epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates in pigs during production cycle on a Czech farm with the history of previous use of ceftiofur. ESBL-producing E. coli isolates were obtained from rectal swabs from pigs of different age groups (suckling piglets, weaned piglets, growers and sows). Collected samples were directly cultivated on MacConkey agar with cefotaxime (2 mg l-1 ), whereas intestinal swabs of slaughtered pigs and surface swabs from pig carcasses were also pre-enriched in buffered peptone water without antimicrobials before the cultivation. Clonal relationship of selected isolates was determined by XbaI pulse-field gel electrophoresis and multi-locus sequence typing. The transferability of plasmids carrying blaCTX-M genes was tested by conjugation experiments. From all examined samples, 141 (43·7%, n = 323) were positive for ESBL-producing E. coli. All ESBL-producing isolates showed resistance to multiple antimicrobials and were positive for blaCTX-M genes. The blaCTX-M-1 was carried by conjugative IncN/ST1 plasmids (c. 40-45 kb) while the blaCTX-M-15 was located on conjugative F plasmids with F:18:A5:B1 formula (c. 165 kb). This study demonstrated the persistence of CTX-M-positive E. coli isolates 2 months after banner of ceftiofur usage and indicated possible risk of transmission of these isolates to humans via the food chain.

Antibiotic resistance and virulence factors in mesophilic Aeromonas spp. from Czech carp fisheries.

AIMS: The aim of this study was to determine phenotypic and genotypic resistance, virulence and clonal relationship of aeromonads and related species isolated from Czech carp fisheries. METHODS AND RESULTS: Forty-nine isolates obtained from a total of 154 fish from three breeding facilities were species identified using matrix-assisted laser desorption/ionization time of flight and the sequencing of the rpoB housekeeping gene. Most Aeromonas isolates were identified as Aeromonas veronii (94%, n = 34). Susceptibility to six antibiotics (oxytetracycline, flumequine, florfenicol, sulphamethoxazole/trimethoprim, enrofloxacin and oxolinic acid) was tested using the disc diffusion method. The presence of resistance genes and virulence factors was verified by PCR and sequencing, and the clonal relationship was analysed using pulsed-field gel electrophoresis (PFGE). Phenotypic resistance to one or more antimicrobials was found in 32 isolates (65%, n = 49). Resistance to oxytetracycline was the most common (41%) and associated mainly with the presence of tet(E) gene, while the percentage of isolates resistant to florfenicol was low (2%). Isolates carried one to five of the tested virulence factors and showed high diversity of PFGE profiles. CONCLUSIONS: Since the highest percentage of antimicrobial resistance in aeromonads was found for oxytetracycline and the lowest percentage for florfenicol, it is suggested that florfenicol could be an adequate treatment alternative in carp fisheries. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing resistance of aeromonads to commonly used antimicrobials has become an emerging problem in fisheries. This study was conducted in relation to the practical needs to identify a suitable antibiotic as an alternative to oxytetracycline.

Occurrence of plasmid-mediated quinolone resistance genes in Escherichia coli and Klebsiella spp. recovered from Corvus brachyrhynchos and Corvus corax roosting in Canada.

The spread of antimicrobial resistance from human activity derived sources to natural habitats implicates wildlife as potential vectors of antimicrobial resistance transfer. Wild birds, including corvid species can disseminate mobile genetic resistance determinants through faeces. This study aimed to determine the occurrence of plasmid-mediated quinolone resistance (PMQR) genes in Escherichia coli and Klebsiella spp. isolates obtained from winter roosting sites of American crows (Corvus brachyrhynchos) and common ravens (Corvus corax) in Canada. Faecal swabs were collected at five roosting sites across Canada. Selective media isolation and multiplex PCR screening was utilized to identify PMQR genes followed by gene sequencing, pulse-field gel electrophoresis and multilocus sequence typing to characterize isolates. Despite the low prevalence of E. coli containing PMQR (1·3%, 6/449), qnrS1, qnrB19, qnrC, oqxAB and aac(6')-Ib-cr genes were found in five sequence types (ST), including E. coli ST 131. Conversely, one isolate of Klebsiella pneumoniae contained the plasmid-mediated resistance gene qnrB19. Five different K. pneumoniae STs were identified, including two novel types. The occurrence of PMQR genes and STs of public health significance in E. coli and Klebsiella pneumoniae recovered from corvids gives further evidence of the anthropogenic derived dissemination of antimicrobial resistance determinants at the human activity-wildlife-environment interface. SIGNIFICANCE AND IMPACT OF THE STUDY: This study examined large corvids as possible vector species for the dissemination of antimicrobial resistance in indicator and pathogenic bacteria as a means to assess the anthropogenic dissemination of plasmid-mediated quinolone resistance (PMQR) genes. Although rare, PMQR genes were found among corvid populations across Canada. The clinically important Escherichia coli strain ST131 containing aac(6')-Ib-cr gene along with a four-class phenotypic antimicrobial resistance (AMR) pattern as well as one Klebsiella pneumoniae strain containing a qnrB19 gene were identified in one geographical location. Corvids are a viable vector for the circulation of PMQR genes and clinically important clones in wide-ranging environments.

Complete nucleotide sequences of two NDM-1-encoding plasmids from the same sequence type 11 Klebsiella pneumoniae strain.

The sequence type 11 Klebsiella pneumoniae strain Kpn-3002cz was confirmed to harbor two NDM-1-encoding plasmids, pB-3002cz and pS-3002cz. pB-3002cz (97,649 bp) displayed extensive sequence similarity with the blaNDM-1-carrying plasmid pKPX-1. pS-3002cz (73,581 bp) was found to consist of an IncR-related sequence (13,535 bp) and a mosaic region (60,046 bp). A 40,233-bp sequence of pS-3002cz was identical to the mosaic region of pB-3002cz, indicating the en bloc acquisition of the NDM-1-encoding region from one plasmid by the other.

IncI1 plasmids associated with the spread of CMY-2, CTX-M-1 and SHV-12 in Escherichia coli of animal and human origin.

Fourteen plasmids carrying blaCTX -M-1, blaSHV -12 or blaCMY -2 genes from Escherichia coli of both avian and human origin were analysed. IncI1 plasmids were largely predominant. Plasmid mutilocus sequence typing and comparative analysis revealed that the blaCMY -2 -ST12-IncI1 plasmids from avian E. coli were identical to those previously found in Salmonella from humans, but different to those associated with human E. coli. The IncI1-ST3 plasmids carrying blaCTX -M-1 or blaSHV -12 were related to those previously identified in avian E. coli, but different to those identified in human E. coli. Overall, no plasmids shared by E. coli of both origin (human/avian) were identified; however, further investigations are needed.

Extended-spectrum beta-lactamase-producing Escherichia coli in turkey meat production farms in the Czech Republic: national survey reveals widespread isolates with bla(SHV-12) genes on IncFII plasmids.

AIM: The occurrence and epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in the environment of turkey farms in the Czech Republic were studied. METHODS AND RESULTS: Extended-spectrum beta-lactamase-producing E. coli isolates were found on 8 (20%) of 40 turkey farms surveyed. A total of 200 environmental smears were examined, and a total of 25 ESBL-producing E. coli were isolated. These isolates were analysed using XbaI pulsed-field gel electrophoresis and divided into nine pulsotypes. Most of the isolates harboured the gene bla(SHV-12) on a 40-kb plasmid of the IncFII group with an identical EcoRV restriction profile. Indistinguishable or clonally related SHV-12-producing isolates belonging to the same pulsotypes were found at some unrelated farms. CONCLUSIONS: Widespread occurrence of ESBL-producing E. coli isolates with bla(SHV-12) carried on IncFII plasmids in meat production flocks in the Czech Republic was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate vertical transmission of ESBL-producing E. coli within the turkey production pyramid. The study shows the risk of multiresistant ESBL-producing bacteria and antibiotic-resistance genes being transmitted to humans via the food chain.

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. METHODS AND RESULTS: Cloacal swabs of feral pigeons collected in the city of Brno in 2006 were cultivated for antibiotic-resistant E. coli. Resistance genes, class 1 and 2 integrons, and gene cassettes were detected in resistant isolates by polymerase chain reaction (PCR). The samples were also cultivated for enterococci. Species status of enterococci isolates was determined using repetitive extragenic palindromic-PCR. Resistance genes were detected in resistant enterococci by PCR. E. coli isolates were found in 203 of 247 pigeon samples. Antibiotic resistance was recorded in three (1·5%, n(E. coli) =203) isolates. Using agar containing ciprofloxacin, 12 (5%, n(samples) =247) E. coli strains resistant to ciprofloxacin were isolated. No ESBL-producing E. coli isolates were detected. A total of 143 enterococci were isolated: Ent. faecalis (36 isolates), Ent. faecium (27), Ent. durans (19), Ent. hirae (17), Ent. mundtii (17), Ent. gallinarum (12), Ent. casseliflavus (12) and Ent. columbae (3). Resistance to one to four antibiotics was detected in 45 (31%) isolates. Resistances were determined by tetK, tetL, tetM, tetO, aac(6')aph(2''), ant(4')-Ia, aph(3')-IIIa, ermB, pbp5, vanA and vanC1 genes. CONCLUSIONS: Antibiotic-resistant E. coli and Enterococcus spp. occurred in feral pigeons in various prevalences. SIGNIFICANCE AND IMPACT OF THE STUDY: Feral pigeon should be considered a risk species for spreading in the environment antimicrobial resistant E. coli and enterococci.

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. METHODS AND RESULTS: Rectal swabs or faeces collected during 2006-2008 from wild mammals were spread on MacConkey agar and MacConkey agar containing 2 mg l(-1) of cefotaxime. From plates with positive growth, one isolate was recovered and identified as E. coli. Susceptibility to 12 antibiotics was tested using the disk diffusion method. Resistance genes, class 1 and 2 integrons and gene cassettes were detected in resistant isolates by polymerase chain reaction (PCR). Extended-spectrum beta-lactamases (ESBL) were further characterized by DNA sequencing, macrorestriction profiling and determination of plasmid sizes. Plasmid DNA was subjected to EcoRV digestion, transferability by conjugation and incompatibility grouping by multiplex PCR. The prevalence of resistant isolates was 2% in small terrestrial mammals (rodents and insectivores, n(E. coli) = 242), 12% in wild ruminants and foxes (n(E. coli) = 42), while no resistant isolates were detected in brown bears (n(E. coli) = 16). In wild boars (Sus scrofa) (n(E. coli) = 290), the prevalence of resistant isolates was 6%. Class 1 and 2 integrons with various gene cassettes were recorded in resistant isolates. From wild boars, five (2%, n(rectal smears) = 293) multiresistant isolates producing ESBL were recovered: one isolate with bla(CTX-M-1) + bla(TEM-1), three with bla(CTX-M-1) and one with bla(TEM-52b). The bla(CTX-M-1) genes were carried on approx. 90 kb IncI1 conjugative plasmids. CONCLUSIONS: Antibiotic-resistant E. coli occurred in populations of wild mammals in various prevalences. SIGNIFICANCE AND IMPACT OF THE STUDY: Wild mammals are reservoirs of antibiotic-resistant E. coli including ESBL-producing strains which were found in wild boars.

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. METHODS AND RESULTS: A total of 16% (n = 87) of water and 24% (n = 216) of gull samples yielded Salmonella. Salmonella Enteritidis PT8 and PT4 were the most prevalent. Antibiotic resistance was found in 12% (n = 14) of water and 28% (n = 51) of gull salmonellae. Escherichia coli were found in 83 (95%) and 213 (99%) of pond water and gull samples, respectively. Totals of 18% (n = 83) of water and 28% (n = 213) of gull E. coli isolates were resistant to antimicrobial agents tested. Class 1 integrons were found in 21% (n = 14) of water and 15% (n = 60) of gull antibiotic-resistant E. coli isolates. Class 2 integrons and extended-spectrum beta-lactamase-producing E. coli isolates (with bla(CTX-M-1), bla(CTX-M-15)-like, bla(SHV-2) and bla(SHV-12)) were found in 13% (eight positive, n = 60 gull-resistant E. coli isolates) and 3% (seven positive, n = 216 gull E. coli isolates) of gull isolates, respectively. Antibiotic-resistant E. coli isolates with identical pulsed field gel electrophoresis (PFGE) patterns were found in either gulls or water, but not both. Salmonellae of the same serotype and PFGE profile were found in both gulls and water. CONCLUSION: A high prevalence of antibiotic-resistant salmonellae and E. coli were found in both pond water and in sympatric black-headed gulls. SIGNIFICANCE AND IMPACT OF THE STUDY: Intensive contamination of pond surface water by antibiotic-resistant E. coli and salmonellae was documented. Black-headed gulls were identified as important reservoirs of antibiotic-resistant salmonellae and E. coli, including extended-spectrum beta-lactamase-producing isolates.

Antimicrobial resistant Escherichia coli isolates in cattle and house sparrows on two Czech dairy farms.

Rectal smears of calves, cows and young bulls, as well as cloacal smears of house sparrows (Passer domesticus), from farms at the villages of Sumice and Troskotovice, Czech Republic, were examined for E. coli resistant to 12 antimicrobials. The resistant isolates were tested for antimicrobial-resistance genes and integrons. Totals of 40% (n=183), 3% (n=95), 0% (n=33), and 9% (n=54) of Escherichia coli isolates from calves, cows, young bulls and house sparrows, respectively, were antimicrobial resistant. The following genes were identified in cattle E. coli isolates: tetA, tetB (isolates resistant to tetracycline), bla(TEM) (beta-lactams), strA, aadA (streptomycin), sul1, sul2 (sulphonamides), and cat, floR (chloramphenicol). Seven of 16 antimicrobial-resistant calf isolates from the Sumice farm possessed class 1 integrons with the aadA1 gene cassette integrated, 1 kb in size. On the Troskotovice farm, eight of 57 antimicrobial-resistant calf isolates possessed class 1 integrons. Integrons of 1.5kb with the dhfr1- aadA1 gene cassette were found in four isolates, followed by a 1kb integron with the aadA1 gene found in three isolates, and a 1.7kb integron with the dhfr17-aadA5 gene cassette and the phenotype ASSuTSxtNaCipCCfG. The prevalence of resistant E. coli in calves compared to adult cattle was much higher and probably was influenced by oral antimicrobial usage in calves, feeding with milk and colostrum from treated cows, as well as mechanisms unrelated to antimicrobial drug selection. Although house sparrows lived together with the cattle and came into contact with cattle waste on the farm, they were not infected by resistant E. coli isolates with the same characteristics as those found in cattle.

Survey of Shiga toxigenic Escherichia coli O157 and drug-resistant coliform bacteria from in-line milk filters on dairy farms in the Czech Republic.

AIMS: To determine the occurrence of Shiga toxin-producing Escherichia coli (STEC) O157 and coliform bacteria isolates resistant to antimicrobial agents in dairy herds by examining milk filters and to analyse the influence of management factors and antibiotic use on antimicrobial resistance. METHODS AND RESULTS: A total of 192 in-line milk filters were sampled on 192 dairy farms in the Czech Republic. Information on feeding, husbandry, production, and antibiotic therapy were obtained by questionnaire. The milk filters were cultured for STEC O157 and coliform bacteria. All recovered isolates were examined for antimicrobial susceptibility and presence of antimicrobial-resistance genes. STEC O157 was detected in four (2%) of the filters. Resistant nonpathogenic E. coli and coliform bacteria isolates with specific genes were detected in 44 (23%) of the filters. CONCLUSIONS: The study demonstrated a high prevalence of resistant coliform bacteria in milk filters obtained on Czech dairy farms. SIGNIFICANCE AND IMPACT OF THE STUDY: The occurrence of resistant coliform bacteria in milk filters was significantly higher among isolates from farms where antibiotic therapy against mastitis was employed during the dry period (P < 0.05).

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. METHODS AND RESULTS: Three hundred and sixty-three faeces samples from rooks were examined for antibiotic resistant Escherichia coli and Salmonella. Altogether 13.7%E. coli isolates were resistant to antimicrobial agents tested. The dominant type of resistance was to tetracycline. Resistant E. coli isolates were examined for antibiotic resistance genes and class 1 integrons. Five of 29 antibiotic resistant isolates possessed the int1 gene. Nine Salmonella isolates (2.5%) were found in rook faeces. All the isolates belonged to serotype Salmonella enterica serovar Enteritidis phage type PT8 and PT23. CONCLUSIONS: The study suggests that rooks can be infected by antibiotic resistant E. coli and Salmonella isolates, probably reflecting the presence of such isolates in their sources of food and/or water in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Rooks can serve as reservoirs and vectors of antibiotic resistant E. coli and Salmonella isolates and potentially transmit these isolates over long distances.

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. METHODS AND RESULTS: A total of 257 isolates were tested for sensitivity to eight antibacterial substances by disk diffusion method. The polymerase chain reaction was used for detecting specific genes of antibacterial resistance and class 1 integrons in resistant E. coli isolates. A total 75 (29.9%) of 257 isolates were resistant to one or more antimicrobial agents. The dominant type of resistance was to tetracycline, detected in 49 (19.1%) isolates. Resistance to ampicillin was detected in 30 (11.7%), cephalothin in 11 (4.3%), streptomycin in 24 (9.3%), sulphonamides in 20 (7.8%) and chloramphenicol in 5 (1.9%) isolates. Nine isolates carrying integrons were detected. CONCLUSIONS: The study suggests that young Black-headed Gulls are an important host reservoir of resistant E. coli strains, probably reflecting the presence of such strains in their sources of food and/or water. SIGNIFICANCE AND IMPACT OF THE STUDY: Although Black-headed Gulls do not naturally come into contact with antibiotics, these birds can be infected with resistant E. coli and potentially serve as their reservoirs, vectors and bioindicators in the environment.