Most of the studies focused on antimicrobial resistance (AMR) performed in wildlife describe Escherichia coli as the principal indicator of the selective pressure. In the present study, several species of Enterobacterales with a large panel of cephalosporin resistant (CR) genes have been isolated from wildlife in Catalonia. A total of 307 wild animals were examined to determine the prevalence of CR enterobacteria, AMR phenotypes and the presence of common carbapenem and CR genes. The overall prevalence of CR-phenotype was 13% (40/ 307): 17.3% in wild mammals (18/104) and 11.5% in wild birds (22/191) (p<0.01). Hedgehogs showed the highest prevalence (13.5% of 104) of the mammal specimens, and raptors the highest in bird specimen (7.3% of 191). Although CR E. coli was the most frequently isolated (45%), other CR-Enterobacterales like Klebsiella pneumoniae (20%), Citrobacter freundii (15%), Enterobacter cloacae (5%), Proteus mirabilis (5%), Providencia spp (5%) and Serratia marcescens (2.5%) were also isolated. A high diversity of CR genes was identified among the isolates, with 50% yielding blaCMY-2, 23% blaSHV-12, 20% blaCMY-1 and 18% blaCTX-M-15. Additionally, resistance to carbapenems associated to OXA-48 gene was found. Most of the CR isolates, principally K. pneumoniae and C. freundii, were multiresistant with co-resistance to fluoroquinolones, tetracycline, sulphonamides and aminoglycosides. This study reports high prevalence of Enterobacterales harbouring a variety of CR genes and OXA-48 mediated-carbapenem resistance, all of them frequently associated to nosocomial human infections, for the first time in wild mammals and wild birds. Implementation of control measures to reduce the impact of anthropogenic pressure in the environment is urgently needed.

Two hundred and fifteen diarrheic samples from 1 to 7 days old piglets were tested for a panel of enteric pathogens. In 19 of the studied farms additional fecal samples from apparently healthy pen-mates were collected and tested for the same panel of infectious agents. Samples were bacteriologically cultured and tested by PCR for E. coli virulence factors genes, C. perfringens types A and C toxins (Cpα, Cpβ, Cpβ2) and C. difficile toxins (TcdA, TcdB). Moreover, Rotavirus A (RVA), Rotavirus B (RVB), Rotavirus C (RVC), porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) were also determined by RT-qPCR. More than one pathogen could be detected in all of the outbreaks. Nevertheless, RVA was the only agent that could be statistically correlated with the outcome of diarrhea. For the other viruses and bacteria analyzed significant differences between the diseased pigs and the controls were not found. In spite of this, the individual analysis of each of the studied farms indicated that other agents such as RVB, RVC, toxigenic C. difficile or pathogenic E. coli could play a relevant role in the outbreak of diarrhea. In conclusion, the large diversity of agent combinations and disease situations detected in neonatal diarrhea outbreaks of this study stand for a more personalized diagnosis and management advice at a farm level.

A 42-d experiment was conducted to evaluate the effect of Cu and Zn source and Cu level on pig performance, mineral status, bacterial modulation, and the presence of antimicrobial-resistant genes in isolates of Enterococcus spp. At weaning, 528 pigs (5.9 ± 0.50 kg) were allotted to 48 pens of a randomized complete block design in a 2 × 2 factorial arrangement with two Cu and Zn sources (SF: sulfate and HCl: hydroxychloride) and two Cu levels (15 and 160 mg/kg). As a challenge, the pigs were reared in dirty pens used by a previous commercial batch. Two-phase diets were offered: the pre-starter (PS) phase from day 1 to 14 and the starter phase (ST) from day 14 to 42. At days 14 and 42, pigs were individually weighed and blood samples from one pig per pen were taken. At the end of the experiment, one pig per pen was euthanized to collect the samples. Feeding high levels of Cu increased body weight (BW) from 16.6 to 17.7 kg (P &lt; 0.001). Furthermore, average daily gain, gain to feed (G:F) ratio, average daily feed intake (ADFI), and mineral status were enhanced with Cu at 160 mg/kg (P &lt; 0.05) compared with Cu at 15 mg/kg. There was no effect of the interaction between source × level on any of the growth performance responses except for ADFI (P = 0.004) and G:F (P = 0.029) at the end of the ST period and for G:F (P = 0.006) for entire nursery period (day 0 to 42). At the end of the ST period, pigs fed Cu at 160 mg/kg as HCl had not only higher ADFI but also lower G:F than those fed Cu as SF at 160 mg/kg. Meanwhile, for the entire nursery period, G:F did not differ between pigs fed Cu at 160 mg/kg as HCl or SF. In colonic digesta, the relative abundance of Streptococcus, Enterobacter, Escherichia, among others, decreased (P-adjust &lt; 0.05), while Lachnospira and Roseburia tended (P-adjust &lt; 0.10) to increase in pigs fed Cu at 160 mg/kg as HCl compared with those fed Cu SF at 160 mg/kg. An increase (P-adjust &lt; 0.05) in Methanosphaera and Roseburia was observed in pigs fed Cu at 160 mg/kg. From colon digesta, Enterococcus spp. was isolated in 40 samples, being E. faecalis the most dominating (65%) regardless of the experimental diet. Genes of ermB (7.5%) and tetM (5%) were identified. No genes for Cu (tcrB) or vancomycin (vanA, vanB, vanC1, and vanC2) were detected. In conclusion, European Union permissible levels of Cu (160 mg/kg), of both sources, were able to increase performance, mineral status, and bacterial modulation compared with nutritional level. Different effects on growth performance, mineral tissue content, and microbial modulation were observed between Cu and Zn sources.

Free-living raptors (birds of prey) can act as reservoirs of potentially zoonotic agents, but they also can be affected by microorganisms as target hosts. In this retrospective study, microbiological results (n = 663) and antibiotic sensitivity profiles (n = 108) of bacterial isolates were analysed from diseased free-living raptors. Sixty-nine percent of cases (n = 457) yielded bacteria: 58% were in pure culture and 42% were of different species. Remarkably, samples from necropsies (47%) had higher percentage of pure isolations than those obtained from clinical (31%) samples (P < 0.001). Among bacterial isolates, Escherichia coli was the most common agent (35%), principally recovered from necropsied birds with clinical signs of septicaemia or respiratory disorders. Pseudomonas aeruginosa (7%) was isolated from birds with systemic infection and from oral lesions, especially in nocturnal raptors (P < 0.001). Staphylococcus spp. (5%), mainly Staphylococcus aureus, was found to be the most prevalent cause of pododermatitis (35%) and Staphylococcus hyicus was isolated from conjunctivitis (18.2%). Interestingly, 8% of samples with lesions compatible with avian tuberculosis were positive to the Mycobacterium avium complex. The most frequent fungi associated with pneumonic lesions and ingluvitis were Aspergillus spp. and Candida spp., respectively. More than 50% of the 108 isolates (34 different bacterial spp.) demonstrated resistance to clindamycin, ampicillin, tetracycline, cefuroxime, enrofloxacin and trimethoprim/sulphamethoxazole. Among the E. coli strains, 71% (27/38) presented a multidrug-resistance pattern to >3 antimicrobials. Detection in wildlife of antimicrobial-resistant pathogens that might be significant at the animal-human-ecosystem interface is of great relevance under the 'One Health' approach.

The frequent usage of antibiotics in livestock has led to the spread of resistant bacteria within animals and their products, with a global warning in public health and veterinarians to monitor such resistances. This study aimed to determine antibiotic resistance patterns and genes in pig farms from Spain during the last twenty years. Susceptibility to six antibiotics commonly used in pig production was tested by qualitative (disk diffusion) and quantitative (minimum inhibitory concentration, MIC) methods in 200 strains of Escherichia coli which had been isolated between 1999 and 2018 from clinical cases of diarrhoea in neonatal and post-weaned piglets. Results showed resistance around 100% for amoxicillin and tetracycline since 1999, and a progressive increase in ceftiofur resistance throughout the studied period. For colistin, it was detected a resistance peak (17.5% of the strains) in the 2011-2014 period. Concerning gentamicin, 11 of 30 strains with intermediate susceptibility by the disk diffusion method were resistant by MIC. Besides, the most frequent antimicrobial resistance genes were the extendedspectrum beta-lactamase (ESBL) bla CTX-M (13.5% of strains, being CTX-M-14, CTX-M-1 and CTX-M-32 the most prevalent genomes, followed by CTX-M-27, CTX-M-9 and CTX-M-3), AmpC-type beta-lactamase (AmpC) bla CMY-2 (3%) and colistin resistance genes mcr-4 (13%), mcr-1 (7%) and in less proportion mcr-5 (3%). Interestingly, these mcr genes were already detected in strains isolated in 2000, more than a decade before their first description. However, poor concordance between the genotypic mcr profile and the phenotypical testing by MIC was found in this study. These results indicate that although being a current concern, resistance genes and therefore antimicrobial resistant phenotypes were already present in pig farms at the beginning of the century.