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The presence of carbapenem–resistant bacteria and carbapenem resistance genes (CRGs) in livestock is increasing. To evaluate the presence of carbapenemase-producing Enterobacteriaceae (CPE) and the main CRGs along swine food chains of the Marche Region (Central Italy), samples of faeces, feed, and animal-food derived products were collected from seven small/medium, medium, and large-scale pig farms. A total of 191 samples were analysed using a culture-dependent method, with the aim of isolating CPE. Isolates were analysed for their resistance to carbapenems using a modified Hodge test and the microdilution method for the minimum inhibitory concentration (MIC) determination. Moreover, the extraction of microbial DNA from each sample was performed to directly detect selected CRGs via qPCR. Among the 164 presumptive resistant isolates, only one strain from a liver sample, identified as Aeromonas veronii, had an ertapenem MIC of 256 μg/mL and carried a carbapenemase- (cphA) and a β-lactamase- (blaOXA-12) encoding genes. A low incidence of CRGs was found; only nine and four faecal samples tested positive for blaNDM-1 and blaOXA-48, respectively. Overall, the importance of monitoring CPE and CRGs in livestock and their food chains should be stressed to control all potential non-human CPE and CRGs reservoirs and to determine safety levels for human health. [ABSTRACT FROM AUTHOR]

Objectives To investigate the optrA -carrying genetic elements and their transferability in two linezolid-resistant Streptococcus dysgalactiae subsp. equisimilis (SDSE) strains of swine origin. Methods SDSE strains (V220 and V1524) were phenotypically and genotypically characterized. Transferability of oxazolidinone resistance genes (filter mating), genetic elements and relatedness between isolates (WGS) were analysed. Excision of the genetic elements was assayed by inverse PCR. Results SDSE isolates were resistant to chloramphenicol, florfenicol and linezolid, but susceptible to tedizolid and both carried the optrA gene. In SDSE V220 optrA was located on a 72.9-kb ICE Sdy V220 inserted in the 3′ end of the chromosomal rum gene. It was 94%–96% identical (coverage, from 31% to 61%) to other optrA -carrying ICEs. In-depth ICE Sdy V220 sequence analysis revealed that optrA was carried by an IME Sdy V220 (17.9 kb), also containing the tet (O/W/32/O) gene. Inverse PCR assays excluded the ICE Sdy V220 mobility. In SDSE V1524, optrA was carried by the Φ Sdy V1524 prophage, integrated near the 5′ end of the chromosomal had gene, showing a genetic organization similar to that of other streptococcal phage. Conjugation and transduction assays failed to demonstrate the optrA transferability to streptococcal recipients. V220 and V1524 belonged to two novel sequence types (ST704 and ST634, respectively). Conclusions To the best of our knowledge, this is the first identification of the optrA gene on a prophage and an ICE in SDSE isolates from swine brain. These findings are consistent with the current belief in the key role of bacteriophages and ICEs in the streptococcal evolution and adaptation. [ABSTRACT FROM AUTHOR]

Objectives: To characterize a linezolid-resistant Enterococcus gallinarum isolate of porcine origin co-carrying cfr, optrA and poxtA genes.Methods: The genome was sequenced using the Illumina and Nanopore platforms. The presence of circular intermediates was examined by inverse PCR. Transferability of oxazolidinone resistance genes was investigated by transformation and conjugation.Results: Two plasmids, the cfr- and optrA-carrying pEgFS4-1 (35 kb) and the poxtA-harbouring pEgFS4-2 (38 kb), were identified. pEgFS4-1 disclosed a distinctive mosaic structure with two cargo regions bounded by identical IS1216 elements interpolated into a backbone related to that of Enterococcus faecium vanA-containing pVEF2. The first cargo region included the cfr and optrA contexts, whereas the second one carried a Tn554 remnant and the lnu(A) gene. Both regions were able to excise in circular form as a unique translocable unit. pEgFS4-2 plasmid was 99% identical to a not fully described E. faecium pSBC1 plasmid. The poxtA environment, flanked by IS1216, was proved to be unstable. pEgFS4-2 also exhibited another cargo region containing the tet(M)-tet(L) genes arranged in tandem and its circular form was detected. Transformation and conjugation experiments failed to demonstrate the transferability of both plasmids to enterococcal recipients. Both plasmids persisted in the absence of selective pressure.Conclusions: To the best of our knowledge, this is the first description of a linezolid-resistant E. gallinarum isolate of swine origin carrying cfr, optrA and poxtA genes. The co-presence of three linezolid resistance determinants in an intrinsically vancomycin-resistant enterococcal species is cause of concern. [ABSTRACT FROM AUTHOR]

Oxazolidinones are critically important antibiotics to treat human infections caused by multidrug-resistant bacteria, therefore the occurrence of linezolid-resistant enterococci from food-producing animals poses a serious risk to human health. In this study, Enterococcus avium 38157 and 44917 strains, isolated from the brain of two unrelated piglets, were found to carry the linezolid resistance genes cfr (D)- optrA , and cfr (D2)- poxtA , respectively. Whole genome sequencing analysis of E. avium 38157 revealed that the genes were co-located on the 36.5-kb pEa_ cfr (D)- optrA plasmid showing high identity with the pAT02-c of Enterococcus faecium AT02 from pet food. The optrA region, was 99% identical to the one of the pAv- optrA plasmid from a bovine Aerococcus viridans strain, whereas the cfr (D) genetic context was identical to that of the plasmid 2 of E. faecium 15–307.1. pEa_ cfr (D)- optrA was not transferable to enterococcal recipients. In E. avium 44917 a cfr (D)-like gene, named cfr (D2), and the poxtA gene were co-located on the transferable 42.6-kb pEa- cfr (D2)- poxtA plasmid 97% identical to the Tn 6349 transposon of the human MRSA AOUC-0915. The cfr (D2) genetic context, fully replaced the Tn 6644 that in S. aureus AOUC-0915 harbor the cfr gene. In conclusion, this is, the best of our knowledge, the first report of the new cfr (D2) gene variant. The occurrence of plasmids co-carrying two linezolid resistance genes in enterococci from food-producing animals needs close surveillance to prevent their spread to human pathogens. • Enterococcus avium strains isolated from two different swine brain. • Characterization of two plasmids containing the cfr (D)/ optrA and cfr (D2)/ poxtA genes. • Identification of a new cfr (D) variant, here named cfr (D2). • Spread of plasmids co-carrying oxazolidinones resistance genes in enterococci. [ABSTRACT FROM AUTHOR]