One hundred forty-five florfenicol-resistant enterococci, isolated from swine fecal samples collected from 76 pig farms, were investigated for the presence of optrA, cfr, and poxtA genes by PCR. Thirty florfenicol-resistant Enterococcus isolates had at least one linezolid resistance gene. optrA was found to be the most widespread linezolid resistance gene (23/30), while cfr and poxtA were detected in 6/30 and 7/30 enterococcal isolates, respectively. WGS analysis also showed the presence of the cfr(D) gene in Enterococcus faecalis (n = 2 isolates) and in Enterococcus avium (n = 1 isolate). The linezolid resistance genes hybridized both on chromosome and plasmids ranging from ~25 to ~240 kb. Twelve isolates were able to transfer linezolid resistance genes to enterococci recipient. WGS analysis displayed a great variability of optrA genetic contexts identical or related to transposons (Tn6628 and Tn6674), plasmids (pE035 and pWo27-9), and chromosomal regions. cfr environments showed identities with Tn6644-like transposon and a region from p12-2300 plasmid; cfr(D) genetic contexts were related to the corresponding region of the plasmid 4 of Enterococcus faecium E8014; poxtA was always found on Tn6657. Circular forms were obtained only for optrA- and poxtA-carrying genetic contexts. Clonality analysis revealed the presence of E. faecalis (ST16, ST27, ST476, and ST585) and E. faecium (ST21) clones previously isolated from humans. These results demonstrate a dissemination of linezolid resistance genes in enterococci of swine origin in Central Italy and confirm the spread of linezolid resistance in animal settings.

Objectives To investigate the genetic elements and the transferability of linezolid resistance genes in three enterococci co-carrying cfr(D) and poxtA2 isolates from manure of a swine farm in central Italy. Methods Two Enterococcus faecalis isolates and one Enterococcus casseliflavus isolate carrying both cfr(D) and poxtA genes were tested for their susceptibility to florfenicol, chloramphenicol, linezolid, tedizolid, tetracycline and vancomycin. Linezolid resistance genes transfer (filter mating), localization (S1-PFGE/hybridization), genetic elements and relatedness between isolates (WGS) were analysed. Results Two E. faecalis isolates and one E. casseliflavus isolate carried the cfr(D) gene and the recently described poxtA2 variant. In the three enterococci, cfr(D) and poxtA2 were co-located on a 33 480 bp plasmid, pV386, 95%–100% identical (coverage 84%) to the Tn6349 transposon of Staphylococcus aureus AOUC-0915. In all isolates, both genes also showed a chromosomal location. Same sequence identities were found from the comparison with currently known poxtA2 genetic elements. In the plasmid pV386, poxtA2 gene was not bounded by two IS1216, as described in pIB-BOL, but closely associated to the cfr(D) and fexA genes. pV386 was always transferred by filter mating to Enterococcus faecium 64/3 recipient. Conclusions The occurrence of the pV386 plasmid in E. faecalis and E. casseliflavus from swine manure is of great concern and highlights the need for control measures to contain its spread to other enterococcal species.

Linezolid is a last resort antibiotic for the treatment of severe infections caused by multi-resistant Gram-positives; although linezolid resistance remains uncommon, the number of linezolid-resistant enterococci has increased during recent years due to worldwide spread of acquired resistance genes (cfr, optrA and poxtA) in clinical, animal and environmental setting. In this study we investigated the occurrence of linezolid-resistant enterococci in marine samples from two coastal areas in Italy. Isolates grown on florfenicol-supplemented Slanetz-Bartley were investigated for their carriage of optrA, poxtA and cfr genes: optrA was found in one E. faecalis, poxtA in three E. faecium and two E. hirae and cfr was not found. Two of the three poxtA-carrying E. faecium and the two E. hirae showed related PFGE profiles. Two E. faecium belonged to the new ST1710, which clustered in the clonal complex CC94, encompassing nosocomial strains. S1-PFGE/hybridization assays showed a double (chromosome and plasmid) location of poxtA and plasmid location of optrA. WGS revealed that poxtA was contained in a Tn6657-like element carried by two plasmids (pEfm-EF3 and pEh-GE2) of similar size, found in different species, and that poxtA were flanked by two copies of IS1216 in both plasmids. In mating experiments all but one (E. faecalis EN3) strains were able to transfer the poxtA gene to E. faecium 64/3. The occurrence of linezolid resistance genes in enterococci from marine samples is of great concern and highlights the need to improve practices aimed at limiting the transmission of linezolid resistant strains to humans from the environmental reservoirs. Importance Linezolid is one of the few antimicrobials available to treat severe infections due to drug-resistant Gram-positive bacteria, thus the emergence of linezolid-resistant enterococci carrying transferable resistance determinants is of great concern for public health. Linezolid resistance genes (cfr, optrA and poxtA), often plasmid located, can be transmitted via horizontal gene transfer and have the potential to spread globally. This study highlights the first detection of enterococci carrying linezolid resistance genes from sediment and zooplankton samples in two coastal urban areas in Italy. The presence of clinically relevant resistant bacteria, such as linezolid-resistant enterococci, in marine environment could reflect their spillover from human and/or animal reservoirs and could indicate that also coastal seawaters could represent a source of these resistance genes.

A retrospective study of the epidemiology of vancomycin-resistant enterococci (VRE) in a regional hospital of central Italy in 2001-2018 demonstrated an increased VRE prevalence since 2016. A total of 113 VRE isolates, 89 E. faecium (VREfm) and 24 E. faecalis (VREfs), were collected in the study period. All strains showed highlevel resistance to vancomycin; 107 also showed teicoplanin resistance. Altogether, 84 VREfm and 20 VREfs carried vanA, whereas 5 VREfm and 1 VREfs carried vanB. MLST analysis documented that 89 VREfm isolates mainly belonged to ST78, ST80, and ST117. Most strains were isolated from 2001 to 2007, ST78 being the predominant clone. VREfm re-emerged in 2016 with a prevalence of the ST80 lineage. Most VREfs were isolated from 2001 to 2006; although they belonged to 7 different STs, there was a prevalence of ST88 and ST6. Notably, ST88 was sporadically recovered throughout the study period. The increasing rate of VREfm isolation from 2016 to 2018 may be related to the influx of new successful clones and to the renewed and widespread use of vancomycin. Improved infection control measures in hospital wards should be adopted to limit the spread of new epidemic VRE strains.

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