A ten-year-surveillance program of zoonotic pathogens in feral pigeons in the City of Madrid (2005-2014): The importance of a systematic pest control.

Feral pigeons have increased in urban settings worldwide becoming a potential health risk for humans and other animals. Control and surveillance programs are essential to prevent the possible transmission of zoonotic pathogens carried by pigeons. A surveillance program was carried out in Madrid City (Spain) during 2005-2014 to determine the role of urban pigeons as carriers of zoonotic agents comparing these results with studies performed elsewhere in the last fifteen years. A total of 1372 pigeons were randomly captured and tested for detection of Antimicrobial susceptibility and genetic heterogeneity of Campylobacter and Salmonella isolates were determined. During the first phase (August 2005-July 2010), 428 animals were analyzed individually, while in the second period (August 2010-December 2014), 944 pigeons were analyzed in pools (n = 2-3 in 2010 and n = 5-6 in 2013 and 2014). The most prevalent pathogen during the first phase was Campylobacter spp., (6.57%, 95% confidence interval 3.05-12.10%) followed by Salmonella spp. (4.41%, 95% CI: 2.30-7.58%) and C. psittaci (2.56%, 95% CI: 0.70-6.53%)]. The PCR techniques, used during the 2010-2014 phase of the study, confirmed the presence of Campylobacter spp. (prevalence of 0-14.83%) and C. psittaci (0-12,94%) among pigeons of Madrid. Antimicrobial susceptibility testing suggested low levels of resistance. Presence of zoonotic agents in feral pigeons highlights the importance of surveillance programs on this species, although the relative low prevalence found suggests a limited risk to Public and Animal Health in Madrid.

Gene pool transmission of multidrug resistance among Campylobacter from livestock, sewage and human disease.

The use of antimicrobials in human and veterinary medicine has coincided with a rise in antimicrobial resistance (AMR) in the food-borne pathogens Campylobacter jejuni and Campylobacter coli. Faecal contamination from the main reservoir hosts (livestock, especially poultry) is the principal route of human infection but little is known about the spread of AMR among source and sink populations. In particular, questions remain about how Campylobacter resistomes interact between species and hosts, and the potential role of sewage as a conduit for the spread of AMR. Here, we investigate the genomic variation associated with AMR in 168 C. jejuni and 92 C. coli strains isolated from humans, livestock and urban effluents in Spain. AMR was tested in vitro and isolate genomes were sequenced and screened for putative AMR genes and alleles. Genes associated with resistance to multiple drug classes were observed in both species and were commonly present in multidrug-resistant genomic islands (GIs), often located on plasmids or mobile elements. In many cases, these loci had alleles that were shared among C. jejuni and C. coli consistent with horizontal transfer. Our results suggest that specific antibiotic resistance genes have spread among Campylobacter isolated from humans, animals and the environment.

Food-borne zoonotic pathogens and antimicrobial resistance of indicator bacteria in urban wild boars in Barcelona, Spain.

Wildlife is increasingly abundant in urban environments, but little is known about the zoonotic pathogens carried by these populations. Urban wild boars are of particular concern because this species is well-known as a pathogen reservoir, and thus, we studied selected zoonotic pathogens in urban wild boars in Barcelona, Spain (n=41). Salmonella enterica was found in 5.00% (95% CI 0.61-16.91) and Campylobacter coli in 4.88% (95% CI 0.6-16.53) of the animals. E. coli O157:H7 and C. jejuni were not found. Other thermophilic Campylobacter were moderately prevalent (19.51%, 95% CI 8.82-34.87). Additionally, we screened for antimicrobial resistance in indicator bacteria: resistance was most frequent in Enterococcus faecium (95% of the isolates were resistant to at least one antimicrobial agent), followed by Enterococcus faecalis (50%) and Escherichia coli (10%). For the first time resistance to linezolid in bacteria carried by wildlife is reported. These findings pose a concern for public health, and thus, further research is needed on wildlife in urban environments.

Effect of cattle on Salmonella carriage, diversity and antimicrobial resistance in free-ranging wild boar (Sus scrofa) in northeastern Spain.

Salmonella is distributed worldwide and is a pathogen of economic and public health importance. As a multi-host pathogen with a long environmental persistence, it is a suitable model for the study of wildlife-livestock interactions. In this work, we aim to explore the spill-over of Salmonella between free-ranging wild boar and livestock in a protected natural area in NE Spain and the presence of antimicrobial resistance. Salmonella prevalence, serotypes and diversity were compared between wild boars, sympatric cattle and wild boars from cattle-free areas. The effect of age, sex, cattle presence and cattle herd size on Salmonella probability of infection in wild boars was explored by means of Generalized Linear Models and a model selection based on the Akaike's Information Criterion. Prevalence was higher in wild boars co-habiting with cattle (35.67%, CI 95% 28.19-43.70) than in wild boar from cattle-free areas (17.54%, CI 95% 8.74-29.91). Probability of a wild boar being a Salmonella carrier increased with cattle herd size but decreased with the host age. Serotypes Meleagridis, Anatum and Othmarschen were isolated concurrently from cattle and sympatric wild boars. Apart from serotypes shared with cattle, wild boars appear to have their own serotypes, which are also found in wild boars from cattle-free areas (Enteritidis, Mikawasima, 4:b:- and 35:r:z35). Serotype richness (diversity) was higher in wild boars co-habiting with cattle, but evenness was not altered by the introduction of serotypes from cattle. The finding of a S. Mbandaka strain resistant to sulfamethoxazole, streptomycin and chloramphenicol and a S. Enteritidis strain resistant to ciprofloxacin and nalidixic acid in wild boars is cause for public health concern.

Genetic basis for dissemination of armA.

OBJECTIVES AND METHODS: armA is a novel plasmid-borne 16S rRNA methyltransferase that confers high-level resistance to 4,6-disubstituted deoxystreptamines. Recently, we have isolated from a high-level broad-spectrum aminoglycoside-resistant Escherichia coli animal isolate a plasmid, pMUR050, that bore the armA gene. In order to elucidate the genetic basis for the spread of armA, we have determined the complete nucleotide sequence of pMUR050. RESULTS: armA was borne by a complex transposon composite flanked by two direct repeats of IS26. The transposon composite included a class one integron with sul1 for resistance to sulphonamides and ant3''9 conferring resistance to spectinomycin-streptomycin, and a macrolide efflux pump and mefE/mel conferring high-level resistance to erythromycin. We identified in GenBank that another plasmid, pCTX-M3, from a Polish Citrobacter freundii human isolate, bore the same genetic structure, including armA. CONCLUSIONS: armA is present in human and animal isolates within a novel transposon composite. Further spread of armA between bacteria of diverse origin is to be expected.

Beta-lactamase characterization in Escherichia coli isolates with diminished susceptibility or resistance to extended-spectrum cephalosporins recovered from sick animals in Spain.

A total of 1439 Escherichia coli isolates from sick animals were received from the Spanish Network of Veterinary Antimicrobial Resistance Surveillance (VAV) from 1997 to 2001. Antimicrobial susceptibility tests were performed and diminished susceptibility to cefotaxime and ceftazidime was identified in 2.5% and 2.8% of the isolates, respectively. Beta-lactamase characterization was carried out in the group of 20 E. coli isolates with both characteristics. The MIC ranges of different beta-lactams showed by these 20 isolates were as follows (in microg/ml): ampicillin (64-->256), amoxicillin-clavulanic acid (4-64), ticarcillin (8-->128), cefazolin (32-->256), cefoxitin (4-->128), cefotaxime (1-64), ceftazidime (2-->64), ceftriaxone (0.5-64), imipenem (< or = 0.06-0.25), and aztreonam (2-->32). TEM, SHV, CMY, and FOX beta-lactamase genes were analyzed by PCR and sequencing. The beta-lactamase genes detected were the following ones (number of isolates): bla(TEM-1b) (3), bla(TEM-1a) (1), bla(TEM-30f) (2), bla(TEM-1b) + bla(CMY-2) (2), and bla(SHV-12) (1). Sequences of the promoter and/or attenuator region of the chromosomal ampC gene were studied in all the 20 isolates. Mutations at position -42 or -32 were detected in 16 isolates and these mutations were associated with the presence of a TEM type beta-lactamase in 6 isolates. Besides, a high variety of plasmidic beta-lactamases was detected including TEM-30 and CMY-2. To our knowledge, this is the first time that TEM-30 beta-lactamase has been detected in E. coli isolates of animal origin.

Detection of CMY-2, CTX-M-14, and SHV-12 beta-lactamases in Escherichia coli fecal-sample isolates from healthy chickens.

Genes encoding the CMY-2, CTX-M-14, and SHV-12 beta-lactamases were detected in three of five Escherichia coli isolates from fecal samples from healthy chickens which showed resistance or diminished susceptibility to extended-spectrum cephalosporins. A -42 mutation at the promoter region of the ampC gene was detected in the other two isolates.