RESUMO
Whole genome sequencing (WGS) is a powerful tool to analyze bacterial genomes rapidly, and can be useful to study and detect AMR genes. We carried out WGS on a group of Escherichia coli (n = 30), sampled from healthy animals and farm environment in four pigsties in northern Italy. Two × 250bp paired end sequencing strategy on Illumina MiSeq™ was used. We performed in silico characterization of E. coli isolates through the web tools provided by the Center for Genomic Epidemiology (cge.cbs.dtu.dk/services/) to study AMR and virulence genes. Bacterial strains were further analyzed to detect phenotypic antimicrobial susceptibility against several antimicrobials. Data obtained from WGS were compared to phenotypic results. All 30 strains were MDR, and they were positive for the genes blaCTX-M and blaTEM as verified by PCR. We observed a good concordance between phenotypic and genomic results. Different AMR determinants were identified (e.g., qnrS, sul, tet). Potential pathogenicity of these strains was also assessed, and virulence genes were detected (e.g., etsC, gad, hlyF, iroN, iss), mostly related to extraintestinal E. coli pathotypes (UPEC/APEC). However, enterotoxin genes, such as astA, ltcA and stb were also identified, indicating a possible hybrid pathogenic nature. Various replicons associated to plasmids, previously recovered in pathogenic bacteria, were identified (e.g., IncN and IncR plasmid), supporting the hypothesis that our strains were pathogenic. Eventually, through WGS it was possible to confirm the phenotypic antibiotic resistance results and to appreciate the virulence side of our ESBL-producing E. coli. These findings highlight the need to monitor commensal E. coli sampled from healthy pigs considering a One Health perspective.
RESUMO
Swine farming as a source of methicillin-resistant Staphylococcus aureus (MRSA) has been well documented. Methicillin-resistant coagulase-negative staphylococci (MRCoNS) have been less studied, but their importance as pathogens is increasing. MRCoNS are indeed considered relevant nosocomial pathogens; identifying putative sources of MRCoNS is thus gaining importance to prevent human health hazards. In the present study, we investigated MRSA and MRCoNS in animals and environment in five pigsties in a high farm-density area of northwestern Italy. Farms were three intensive, one intensive with antibiotic-free finishing, and one organic. We tested nasal swabs from 195 animals and 26 environmental samples from three production phases: post-weaning, finishing and female breeders. Phenotypic tests, including MALDI-TOF MS, were used for the identification of Staphylococcus species; PCR and nucleotide sequencing confirmed resistance and bacterial species. MRCoNS were recovered in 64.5% of nasal swabs, in all farms and animal categories, while MRSA was detected only in one post-weaning sample in one farm. The lowest prevalence of MRCoNS was detected in pigs from the organic farm and in the finishing of the antibiotic-free farm. MRCoNS were mainly Staphylococcus sciuri, but we also recovered S. pasteuri, S. haemolyticus, S. cohnii, S. equorum and S. xylosus. Fifteen environmental samples were positive for MRCoNS, which were mainly S. sciuri; no MRSA was found in the farms' environment. The analyses of the mecA gene and the PBP2-a protein highlighted the same mecA fragment in strains of S. aureus, S. sciuri and S. haemolyticus. Our results show the emergence of MRCoNS carrying the mecA gene in swine farms. Moreover, they suggest that this gene might be horizontally transferred from MRCoNS to bacterial species more relevant for human health, such as S. aureus.