Oxazolidinone resistance-associated genes cfr and optrA in MDR CoNS from healthy pigs in Italy

BACKGROUND: Oxazolidinones are relatively novel antibiotics used exclusively in human medicine as last resort drugs for resistant pathogens like MRSA, vancomycin-resistant enterococci and penicillin-resistant Streptococcus pneumoniae. However, in the last two decades, oxazolidinone resistance genes cfr and optrA have been sporadically reported worldwide in Staphylococcus spp. of livestock origin.(1) These genes can be chromosomal, but they are often transferable through mobile genetic elements, especially plasmids.(2) In Italy, they have been recently detected in enterococci of swine origin.(3) OBJECTIVES: To uncover the presence of cfr and optrA genes in methicillin-resistant CoNS (MRCoNS) originating from swine nasal swabs sampled in a high farm-density area of northwestern Italy. Healthy pigs were sampled from three productive stages (finishing, weaners and sows). After isolating pure cultures, selected staphylococci (n = 27), resulted methicillin-resistant from previous mecA identification, were phenotypically tested through Kirby–Bauer disc diffusion method for the antibiotics clindamycin, doxycycline, erythromycin, enrofloxacin, florfenicol, gentamicin, linezolid, tetracycline, tiamulin and trimethoprim/sulfamethoxazole (EUCAST v.11.0 guidelines for linezolid disc, CLSI VET08 for the other antibiotics). MIC through Etest (Liofilchem®, Roseto degli Abbruzzi, Teramo, Italy) was used for the antibiotic ceftaroline. RESULTS: All the chosen MRCoNS were MDR (MDR CoNS), as they were phenotypically resistant to more than three antibiotic classes. No strain was positive for ceftaroline resistance. Since linezolid resistance was recovered in six samples, we decided to perform PCR for the cfr gene (746 bp), which was detected in Staphylococcus sciuri from a piglet (GenBank accession number OL412394), and optrA (1395 bp), which was recovered in Staphylococcus pasteuri from a finisher, S. sciuri from a sow and Staphylococcus cohnii from a weaner (GenBank accession numbers OM165030, OM165031 and OM165032). Sanger sequencing confirmed PCR result for cfr, with 100% identity with the cfr gene detected from a clinical Italian isolate of MRSA (MH746818), and for optrA gene, which had 100% identity with the optrA previously found in a swine Italian Enterococcus faecium strain (MT723958). As far as we know, this is the first time that a cfr gene has been detected in S. sciuri from a nasal sample of animal origin in Italy. Furthermore, optrA was never detected in S. pasteuri and S. cohnii strains. CONCLUSIONS: These results are relevant from a One Health perspective, as they underline the need for oxazolidinone resistance monitoring, not only in human medicine, but also at farm level. In this way, it will be easier to prevent the dissemination of this resistance to human community and hospitals, where oxazolidinones are considered last-resort antibiotics. Furthermore, they remind the importance of surveillance of antibiotic usage in pigs, as cfr and optrA resistance in staphylococci can be elicited using certain antibiotics, like phenicols, due to cross-resistance to this antibiotic class

Whole Genome Sequencing (WGS) Analysis of Virulence and AMR Genes in Extended-Spectrum β-Lactamase (ESBL)-Producing <i>Escherichia coli</i> from Animal and Environmental Samples in Four Italian Swine Farms

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

Occurrence of Methicillin-Resistant Coagulase-Negative Staphylococci (MRCoNS) and Methicillin-Resistant Staphylococcus aureus (MRSA) from Pigs and Farm Environment in Northwestern Italy

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