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.

Disease transmission among wild boars, domestic animals and humans is a public health concern, especially in areas with high wild boar densities. In this study, fecal samples of wild boars (n = 200) from different locations of the Metropolitan Area of Barcelona were analyzed by PCR to explore the frequency of β-lactamases and extended cephalosporin and carbapenem resistance genes (ESBLs) in Escherichia coli strains and the presence of toxigenic Clostridioides difficile. The prevalence of genes conferring resistance to β-lactam antimicrobials was 8.0% (16/200): blaCMY-2 (3.0%), blaTEM-1b (2.5%), blaCTX-M-14 (1.0%), blaSHV-28 (1.0%), blaCTX-M-15 (0.5%) and blaCMY-1 (0.5%). Clostridioides difficile TcdA+ was detected in two wild boars (1.0%), which is the first report of this pathogen in wild boars in Spain. Moreover, the wild boars foraging in urban and peri-urban locations were more exposed to AMRB sources than the wild boars dwelling in natural environments. In conclusion, the detection of E. coli carrying ESBL/AmpC genes and toxigenic C. difficile in wild boars foraging in urban areas reinforces the value of this game species as a sentinel of environmental AMRB sources. In addition, these wild boars can be a public and environmental health concern by disseminating AMRB and other zoonotic agents. Although this study provides the first hints of the potential anthropogenic sources of AMR, further efforts should be conducted to identify and control them.

Escherichia coli is considered one of the most common agents associated with neonatal diarrhea in piglets. The aim of this work was to characterize the pathogenic and antimicrobial resistance (AMR) profiles of 122 E. coli strains isolated from pigs suffering diarrhea (n = 94) and pigs without diarrhea (n = 28) of 24 farms in Spain. Virulence factors, toxins and AMR (ESBL and colistin) genes and AMR phenotypes of E. coli isolates were analyzed. Low prevalence of pathogenic E. coli strains (26%) was found in both groups. However, ETEC and VTEC strains were more frequently isolated from diarrheic piglets. Irrespectively of diarrhea occurrence, 97.5% of the strains showed a multidrug-resistance (MDR) profile to aminopenicillins, sulfonamides and tetracyclines. It was found that 22% of E. coli was CTX-M+, with CTX-M-14 being the principal allelic variant. Remarkably, 81.5% of CTX-M+ strains were isolated from diarrheic animals and presented an extended MDR profile to aminopenicillins, quinolones and aminoglycosides. Finally, low frequencies of colistin resistance genes mcr-1 (4/122) and mcr-4 (1/122) were found. MDR E. coli strains are circulating in pig farms of Spain, representing a serious threat to animal and public health. More appropriate diagnostic approaches (genetic and AMR phenotypic analysis) should be implemented in animal health to optimize antibiotic treatments.

The purpose of this study was to compare the immune response generated by the intramuscular and the intradermal vaccination route against the porcine reproductive and respiratory syndrome virus (PRRSV). Piglets from a seronegative and a seropositive farm were selected (n = 28 piglets per farm), and each group was divided into two groups and vaccinated after weaning with modified live vaccine Unistrain® PRRS (Laboratorios Hipra Amer, Spain) by the intramuscular (IM) or the intradermic (ID) route. For the following 6 weeks, animals were weekly bled to assess the humoral response by PRRSV-specific antibody ELISA and viral neutralisation test. At 0-, 3-, 4- and 6 weeks post-vaccination, peripheral mononuclear blood cells (PBMC) from eight animals per group were recovered to analyse cellular response by IFN-γ ELISPOT and lymphoproliferation. Serum IL-12 was also quantified by ELISA. Seroconversion was first detected 14 days post-vaccination (dpv) for both IM and ID routes, and peaked at 35 dpv (both IM groups and ID seropositive) or 42 dpv (ID seronegative). At 3 weeks after vaccination, 6/27 (22.22%) animals from negative origin had not seroconverted, and neutralising titres were significantly lower at 35 dpv compared to the seropositive origin (mean log2 titres of 1.36 and 4.25 respectively) Also, it was 10 times more probable for them to have high levels of IL-12 a week after vaccination than for animals of seropositive origin. Cellular immune response analysed by lymphoproliferation and IFN-γ ELISPOT was already present at 21 dpv and until 42 dpv, with no significant differences between groups except for a higher lymphoproliferation at 35 dpv in the IM seropositive group (Kruskal-Wallis, p < 0.05). These results indicate that the intradermal route induces an immune response equivalent to the classical intramuscular route even in presence of non-neutralising maternal immunity, which in this study has proven to facilitate seroconversion after vaccination with an heterologous strain.

Wildlife has been suggested to be a good sentinel of environmental health because of its close interaction with human populations, domestic animals, and natural ecosystems. The alarming emergence of antimicrobial resistance (AMR) in human and veterinary medicine has activated/triggered the awareness of monitoring the levels of AMR pollution in wildlife. European hedgehogs (Erinaceus europaeus) are common wild species habiting urban areas in Europe. However, there are few studies conducted in hedgehogs as reservoirs of AMR bacteria or genes. The aim of this study was to assess the occurrence and distribution of ESBL, AmpC, and carbapenem-resistant enterobacteria and AMR genes in wild European hedgehogs in Catalonia, a densely populated region of NE Spain. A total of 115 hedgehogs admitted at the Wildlife Rehabilitation Center of Torreferrussa were studied. To our knowledge, this is the first description of β-lactam resistant enterobacteria in wild hedgehogs. Interestingly, 36.8% (42/114) of the animals were detected as carriers of β-lactamase/carbapenemase resistance genes. Klebsiella spp. (59.6%), and specifically K. pneumoniae (84.6%), were the bacteria with the highest proportion of resistance genes, followed by E. coli (34.6%) and C. freundii (5.8 %). The most frequently detected genetic variants were blaCTX-M-15 (19.3%), blaSHV-28 (10.5%), blaCMY-1 (9.7%), blaCMY-2 (8.8%), and blaOXA-48 (1.7%). In addition, 52% (27/52) of the isolates presented a multidrug resistance (MDR) phenotype and 31% had an extended drug resistance (XDR) profile. No clustering of animals with AMR genes within the study region was shown in the spatial analysis, nor differences in the proportion of positive animals among regions, were detected. The results of this study suggest that wild European hedgehogs could be good sentinels of AMR environmental pollution, especially in areas with a high human population density, because they either inhabit and/or feed in an anthropogenic environment. In conclusion, it is crucial to raise awareness of the strong interconnection between habitats and compartments, and therefore this implies that AMR issues must be tackled under the One Health approach.