To investigate the gradual genetic changes occurred in heteroresistant vancomycin-variable (VVE) Enterococcus faecium ST80 isolate - belonging to the vanA ST80 nosocomial clone although recovered from marine environment - contributing to its reversion to a resistant phenotype after exposure to increasing concentrations of vancomycin. WGS of the parental strain and three revertants obtained by exposure to vancomycin was performed. vanA copy number and difference in vanA expression between VVE and revertants were evaluated by RT-qPCR. Heteroresistance was demonstrated by vancomycin E-test and population analysis profiling. The E. faecium JSEG15 isolate, susceptible to vancomycin although carrying a Tn1546-like transposon on 36-kb plasmid, gave rise to three different resistant mutants following gradual passages on increasing vancomycin concentrations. Revertants showed increased vancomycin MICs, with the highest resistance in JSEG15-rev3 (MIC = 128 mg/L). All revertants presented a deleted Tn1546-like on a 34-kb plasmid and JSEG15-rev2 and JSEG15-rev3 also showed an additional chromosomal copy of the vanRS and vanHAX clusters, and higher vanA gene copies and expression. A mutation in the chromosomal D-Ala-D-Ala ligase gene was identified in JSEG15-rev3 which also transferred the vanA carrying plasmid by in vitro conjugation. The parental strain and all revertants exhibited heteroresistance, with higher frequency of resistant subpopulations in the revertants. These findings emphasize the dynamic genetical changes enabling VVE to regain full resistance, underscoring the need for vigilant treatment strategies to address enterococcal infections and for monitoring the spread of clinically relevant strains in the environment for public health protection.

The swine production chain represents a critical point for the spread of antimicrobial resistance, with slaughterhouses acting as potential hotspots for cross-contamination. The veterinary use of phenicols may promote resistance to oxazolidinones, a last-resort class of antibiotics. This study investigated the presence of oxazolidinone-resistant enterococci in a pig slaughterhouse in central Italy, analyzing 376 samples, 25.8% of which tested positive for Enterococcus spp. resistant to florfenicol. Environmental samples collected at the end of the day of slaughter showed higher contamination levels compared to those collected beforehand. PCR analysis of 42 isolates of Enterococcus faecalis and Enterococcus faecium revealed a high prevalence of the optrA gene, followed by poxtA and cfr(D). The data indicated high resistance rates to tetracycline and erythromycin, consistent with existing literature. Linezolid resistance was observed in 73.8% of isolates, while no vancomycin resistance was detected. Whole Genome Sequencing (WGS) analysis revealed diverse sequence types, complex resistomes, multiple virulence factors, and considerable variability in the genetic elements carrying cfr/cfr(D), optrA, and poxtA. These determinants were often plasmid-borne and co-localized with other antibiotic and heavy metal resistance genes, which could contribute to their persistence and dissemination through co-selection mechanisms. Overall, the findings highlight the widespread occurrence of oxazolidinone-resistant enterococci in the pig production chain and their potential circulation across different production stages. An integrated approach encompassing prudent antibiotic use in livestock, good hygiene practices at the slaughterhouses, and increased awareness of the problem will be essential to mitigate the spread of antimicrobial resistance along the food production chain.

Environmental dissemination of antimicrobial resistance is a growing global concern. This study investigated the occurrence, genetic context, and mobility of the oxazolidinone resistance genes in enterococci isolated from high-altitude river sediments in the Gilgit-Baltistan region of Pakistan. A total of 17 florfenicol-resistant enterococci were recovered from 3 of the 12 sampling sites analyzed. Four multidrug-resistant strains (two Enterococcus faecium, one E. faecalis, and one E. hirae) harboring the optrA gene were selected for whole-genome sequencing using Illumina and Nanopore platforms. Genomic analyses showed that optrA was plasmid-borne in all isolates and co-localized with additional resistance genes, including macrolide, phenicol, and tetracycline resistance determinants. The optrA genetic context was frequently associated with insertion sequences, suggesting their role in the mobilization of multidrug-resistance elements. Mini-translocatable units of the optrA genetic context were detected as circular forms in two of four strains, further supporting their mobility potential. Conjugation assays demonstrated successful horizontal transfer of the optrA gene mediating linezolid resistance, with transfer frequencies ranging from 2.03 × 10⁻⁶ to 6.36 × 10⁻⁴ transconjugants per recipient cell. optrA-carrying plasmids remained stable after serial passages without antibiotic pressure. Phylogenetic analysis showed that isolates belonged to distinct sequence types and clustered with strains of diverse geographic and ecological origins, suggesting dissemination of optrA across different genetic backgrounds rather than clonal expansion. The occurrence of linezolid-resistant enterococci in high-altitude river sediments suggests their widespread distribution and emphasizes the need for broader environmental surveillance programs to monitor clinically relevant resistances even in remote ecosystems. IMPORTANCE:This study provides important insights into the environmental dissemination of antimicrobial resistance by demonstrating the occurrence and genetic diversity of clinically relevant resistance determinants in enterococci isolated from remote high-altitude river sediments. The findings emphasize that natural environments could serve as reservoirs and exchange hubs for antimicrobial resistance, even in ecosystems with limited direct anthropogenic impact. Therefore, integrated One Health surveillance strategies that include environmental compartments alongside human and animal populations are required to better monitor and mitigate the emergence of clinically significant resistant bacteria.

The spread of the transferable optrA gene poses an increasing threat to the clinical efficacy of oxazolidinones. Here, we characterized a novel optrA-carrying plasmid, pEfa-optrA-Arg, from a linezolid-resistant Enterococcus faecalis clinical isolate from Argentina. The 68,653-bp conjugative plasmid harbored optrA together with multiple antimicrobial resistance genes and showed high similarity to a plasmid previously identified in a bovine isolate from Switzerland. pEfa-optrA-Arg, or a closely related variant, was also detected in E. faecalis isolates from several Argentinian hospitals, highlighting the role of horizontal gene transfer in the spread of antimicrobial resistance across human and animal reservoirs within the One Health continuum.

Oxazolidinones (linezolid and tedizolid) are last-resort antibiotics used to treat severe infections caused by multidrug-resistant Gram-positive bacteria. Although linezolid is not approved for veterinary use, resistant bacteria—particularly enterococci—have increasingly been reported in food-producing animals worldwide. Slaughterhouses represent potential hotspots for the transmission of antimicrobial-resistant pathogens along the food chain. This study investigated the presence of oxazolidinone resistance genes and relevant genetic elements in florfenicol-resistant enterococci and staphylococci isolated from wastewater collected from an Italian swine slaughterhouse. In total, five enterococci, six staphylococci, and one Mammaliicoccus sciuri (previously known as Staphylococcus sciuri) isolate were recovered. PCR screening revealed that all staphylococci and M. sciuri carried the cfr gene, while enterococci were positive for either optrA or poxtA. Nine isolates were selected for whole genome sequencing. Enterococcus hirae EM2 carried poxtA on the novel pEhEM2-poxtA plasmid. In E. faecium ED1 and EF1, optrA was located on the novel pEfmED1-optrA plasmid. E. durans EF2 carried poxtA within a Tn6657 transposon integrated into a pEgFS4-2-like plasmid also containing tet(M), tet(L), and genes related to transposition and plasmid replication. In Staphylococcus simulans SN1 and SF3, cfr was found on the novel pSsSN1-cfr plasmid and on p12-02300 plasmid, respectively. In Staphylococcus cohnii SD1, cfr was located on the previously described plasmid unnamed1, while in S. cohnii SF2 it was inserted into a chromosomal Tn558-like transposon along with fexA. M. sciuri SN4 harboured cfr on a small plasmid showing high similarity to the pK8D55P-cfr plasmid previously found in an M. sciuri isolate from ducks. These results highlight the role of slaughterhouse wastewater as a potential reservoir of oxazolidinone resistance genes and emphasize the need for continued monitoring to limit the environmental spread of antimicrobial resistance from animal production systems.

Biofilm plays a crucial role in the pathogenesis and chronicity of urinary tract infections (UTIs). The present work aimed to evaluate the anti-biofilm effects of Formulation (DIF17BRO® plus NAC) in combination with ciprofloxacin (CPX) on Escherichia coli strains. The antimicrobial activity of ciprofloxacin was evaluated by minimum inhibitory concentration (MIC) determination, and the antibiofilm effects of ciprofloxacin alone and combined with Formulation were evaluated on E. coli ATCC700926, E. coli ATCC10536, E. coli PNT, and E. coli PCA mature biofilms in terms of CFU/mL and biomass quantifications. Moreover, the potential protective effects of Formulation plus ciprofloxacin was tested in a Galleria mellonella in vivo infection assay. Our results underlined the increased microbial reduction in the mature biofilm in the presence of the combination Formulation and CPX, even at a lower concentration of CPX. Formulation increased the percentage of biofilm biomass reduction, inducing a disruption of the biofilm structure itself. Our present findings confirm that MIC CPX combined with Formulation also induced an antimicrobial effect in the G. mellonella assay. Formulation facilitated the perturbation of the biofilm polymeric matrix, enhancing the antibiotic penetration and its antimicrobial action on bacteria, underlining Formulation’s role as an enhancer of ciprofloxacin antibacterial action.

OBJECTIVES:To characterize the optrA-, cfr(D)- and vanA-carrying linear plasmids detected in three MDR enterococcal clinical isolates. METHODS:Enterococcus faecium (868), E. faecium (1001) and Enterococcus faecalis (2048), which were linezolid- and vancomycin-resistant due to the presence of optrA, cfr(D) and vanA genes, were tested for their susceptibility to several antibiotics. Characterization of the genetic elements carrying antibiotic resistance genes and ST determination were achieved using WGS data. The plasmid topology was evaluated by S1-PFGE. Resistance gene transferability was assessed by filter-mating experiments. RESULTS:The linezolid- and vancomycin-resistant enterococci also showed resistance to tedizolid, chloramphenicol, tetracycline, erythromycin, ampicillin and levofloxacin. Both E. faecium 868 and E. faecium 1001 belonged to ST80 (included in clade A1), whereas E. faecalis 2048 was associated with ST6. WGS analysis revealed a plasmid co-localization of the optrA, cfr(D) and vanA genes. optrA was carried by Tn6674-like or Tn7695-like transposons; cfr(D) was associated with a truncated guaA gene, both flanked by IS1216 with opposite polarity; vanA was found on a Tn1546-like transposon containing IS1542 and IS1251 transposases. PFGE of S1 nuclease-treated and untreated DNAs displayed the linear topology of optrA-, cfr(D)- and vanA-harbouring plasmids. Only E. faecium 868 was able to transfer linezolid and vancomycin genes to an enterococcal recipient. CONCLUSIONS:To the best of our knowledge this is the first report on the occurrence of a linear plasmid in E. faecalis. Linear plasmids can play a key role in the spread of oxazolidinone and glycopeptide resistance with serious consequences for public health.

Objectives To clarify the genetic basis of high-level linezolid resistance in three Clostridioides difficile strains from calves. Methods A WGS approach was used to comprehensively characterize C. difficile A501, A505 and A516 strains exhibiting high linezolid MICs, and to clarify their phylogenetic relationships. Linezolid resistance gene transferability was assessed by filter mating experiments. Results WGS analysis revealed the presence of cfr(B) in C. difficile A501 and A516, both exhibiting the ST11, and cfr(C) in C. difficile A505 belonging to a non-toxigenic ST15 clone. The cfr(B) gene was on a novel 25 791 bp integrative conjugative element (ICE), named ICECd-cfr(B), similar to an uncharacterized region of Clostridium sp. C1, but significantly different from the Tn6218 transposon typically associated with this gene. The cfr(C) gene was found on a novel 32 770 bp ICE, named ICECd-cfr(C), which was identical to an uncharacterized region of the C. difficile DSM 104450 chromosome. ICECd-cfr(C) exhibited high nucleotide identity, but low coverage, with a cfr(C)-carrying region previously detected in C. difficile 020482; whereas in C. difficile A505 this region was interrupted by a 16.6 kb DNA insertion. Conjugation assays failed to demonstrate the transferability of cfr(B) and cfr(C) genes. Conclusions To the best of our knowledge, this is the first report of C. difficile isolates from calves with high linezolid MICs due to novel cfr(B)- and cfr(C)-carrying ICEs. C. difficile animal isolates, belonging to ST11 and ST15 clones with zoonotic potential, could act as reservoirs for the spread of linezolid resistance genes to human intestinal pathogens, with serious consequences for public health.

Actinobacillus pleuropneumoniae is responsible for porcine pleuropneumonia, a highly contagious lung infection. The control of this respiratory disease remains heavily reliant on antibiotics, with phenicols being one of the primary classes of antibiotics used in pig farming. In the present study, we describe three isolates (B2278, B2176 and B2177) of A. pleuropneumoniae resistant to florfenicol attributed to the presence of the floR gene, which were obtained from two pig farms in Italy. Florfenicol susceptibility tests indicated that B2176 exhibited an intermediate susceptibility profile, while B2177 and B2278 were resistant. All three isolates belonged to serovar 6 and tested positive for the presence of the floR gene. Whole genome sequencing analysis revealed that isolates B2176, B2177 and B2278 harbored genes encoding the toxins ApxII and ApxIII, characteristic of strains with moderate virulence. Moreover, phylogenetic analysis demonstrated that these isolates were closely related, with single nucleotide polymorphisms (SNPs) ranging from 8 to 19. The floR gene was located on a novel 5588 bp plasmid, designated as pAp-floR. BLASTN analysis showed that the pAp-floR plasmid had high nucleotide identity (99 %) and coverage (60 %) with the pMVSCS1 plasmid (5621 bp) from Mannheimia varigena MVSCS1 of porcine origin. Additionally, at least under laboratory conditions, pAp-floR was stably maintained even in the absence of direct selective pressure, suggesting that it does not impose a fitness cost. Our study underscores the necessity of monitoring the spread of florfenicol-resistant A. pleuropneumoniae isolates in the coming years.

Purpose To investigate the occurrence of vancomycin-variable enterococci (VVE) in a hospital in central Italy. Methods vanA positive but vancomycin-susceptible Enterococcus faecium isolates (VVE-S) were characterized by antibiotic susceptibility tests, molecular typing (PFGE and MLST), and WGS approach. The reversion of VVE-S to a resistant phenotype was assessed by exposure to increasing vancomycin concentrations, and the revertant isolates were used in filter mating experiments. qPCR was used to analyze the plasmid copy number. Results Eleven putative VVE-S were selected. WGS revealed two categories of vanA cluster plasmid located: the first type showed the lack of vanR , the deletion of vanS , and an intact vanH / vanA / vanX cluster; the second type was devoid of both vanR and vanS and showed a deletion of 544-bp at the 5′-end of the vanH . Strains ( n = 7) carrying the first type of vanA cluster were considered VVE-S and were able to regain a resistance phenotype (VVE-R) in the presence of vancomycin, due to a 44-bp deletion in the promoter region of vanH/vanA/vanX , causing its constitutive expression. VVE-R strains were not able to transfer resistance by conjugation, and the resistance phenotype was unstable: after 11 days of growth without selective pressure, the revertants were still resistant but showed a lower vancomycin MIC. A higher plasmid copy number in the revertant strains was probably related to the resistance phenotype. Conclusion We highlight the importance of VVE transition to VRE under vancomycin therapy resulting in a potential failure treatment. We also report the first-time identification of VVE-S isolates pstS -null belonging to ST1478.

OBJECTIVES:To investigate the global distribution of an optrA-harbouring linezolid-resistant Enterococcus faecalis ST476 clonal lineage. METHODS:Comprehensive searches of the NCBI database were performed to identify published peer-reviewed articles and genomes of E. faecalis ST476. Each genome was analysed for resistome, virulome, OptrA variant and optrA genetic contexts. A phylogenetic comparison of ST476 genomes with publicly available genomes of other STs was also performed. RESULTS:Sixty-six E. faecalis ST476 isolates from 15 countries (China, Japan, South Korea, Austria, Denmark, Spain, Czech Republic, Colombia, Tunisia, Italy, Malaysia, Belgium, Germany, United Arab Emirates and Switzerland) mainly of human and animal origin were identified. Thirty available ST476 genomes compared with genomes of 591 STs indicated a progressive radiation of E. faecalis STs starting from ST21. The closest ancestral node for ST476 was ST1238. Thirty E. faecalis ST476 genomes exhibited 3-916 SNP differences. Several antimicrobial resistance and virulence genes were conserved among the ST476 genomes. The optrA genetic context exhibited a high degree of or complete identity to the chromosomal transposon Tn6674. Only three isolates displayed an optrA-carrying plasmid with complete or partial Tn6674. The WT OptrA protein was most widespread in the ST476 lineage. CONCLUSIONS:Linezolid-resistant optrA-carrying E. faecalis of the clonal lineage ST476 is globally distributed in human, animal and environmental settings. The presence of such an emerging clone can be of great concern for public health. Thus, a One Health approach is needed to counteract the spread and the evolution of this enterococcal clonal lineage.

The presence of erm(T) gene conferring resistance to macrolides, lincosamides and streptogramin B (MLSB), was screened in 296 enterococci collected from clinical samples in a central Italy hospital and seven Enterococcus faecium isolates resulted positive to erm(T) by PCR. All isolates were resistant to erythromycin, tetracycline, ciprofloxacin and ampicillin but susceptible to vancomycin and chloramphenicol. Whole Genome Sequencing analysis revealed that in five E. faecium isolates, all belonging to the sequence type ST80 included in the clonal complex CC17 responsible of nosocomial infections, erm (T) gene was chromosome-located, in different genetic contexts. In E. faecium 735,236, erm (T) was on a 4,159-bp region flanked by two IS1216 and inserted at the 3′ end of the mp gene. In E. faecium 711,448 and 739,437, erm (T) was found in a 4,463-bp region identical to that detected in E. faecium 735,236 except for 319 bp. In E. faecium 713,729 and 757,415, erm (T) was on a 7,038-bp region flanked by IS1251 and ISEfm2 transposases and encompassed between the genes encoding a recombinase and three hypothetical proteins. erm(T)-carrying minicircles were detected in all isolates by inverse PCR assays demonstrating that erm(T) was included in mobile elements. However, in conjugation assays by filter mating, the erm(T) transferability was unsuccessful. Although macrolides are not used to treat enterococcal infections, the resistance is nonetheless widespread. These antibiotics are critically important in human medicine, but only few studies focused on erm (T)-harbouring clinical enterococci. The emergence of erm (T)-mediated erythromycin resistance among enterococci, potentially transferable to other nosocomial pathogens, should be constantly monitored.

Aims To investigate enterococci carrying linezolid and vancomycin resistance genes from fecal samples recovered from wild boarsMethods and results Florfenicol- and vancomycin-resistant enterococci, isolated on selective agar plates, were screened by PCR for the presence of linezolid and vancomycin resistance genes. Five isolates carried optrA or poxtA linezolid resistance genes; one strain was resistant to vancomycin for the presence of vanA gene. All isolates were tested for their antibiotic susceptibility and subjected to Whole Genome Sequencing (WGS) analysis. In Enterococcus faecalis (E. faecalis) V1344 and V1676, the optrA was located on the new pV1344-optrA and pV1676-optrA plasmids, respectively, whereas in Enterococcus faecium (E. faecium) V1339 this gene was on a 22 354-bp chromosomal genetic context identical to the one detected in a human E. faecium isolate. In both E. faecium V1682 and E. durans V1343, poxtA was on the p1818-c plasmid previously found in a human E. faecium isolate. In E. faecium V1328, the vanA gene was on the Tn1546 transposon in turn located on a new pV1328-vanA plasmid. Only E. faecium V1682 successfully transferred the poxtA gene to an enterococcal recipient in filter mating assays.Conclusions The occurrence of genetic elements carrying linezolid and vancomycin resistance genes in enterococci from wild boars is a matter of concern, moreover, the sharing of plasmids and transposons between isolates from wild animals, human, and environment indicates an exchange of genetic material between these settings.

Seafood is a crucial global food source, but its role as a reservoir for antibiotic-resistant pathogens is concerning. This study focuses on the prevalence of antibiotic resistance in retail seafood, using Enterococcus spp. as an indicator. Seven categories of raw seafood were collected from retail markets in Central Italy. Out of 422 samples, 288 tested positive for Enterococcus spp., resulting in a prevalence rate of 68.25% (CI95%: 63.66%-72.51%). The most common species were E. faecalis (48%) and E. faecium (17.4%). Enterococcus spp. was most frequently found in cephalopods, salmon, bivalves, and crustaceans than in sea bass and bream. The odds of Enterococcus spp. recovery were higher in frozen than in fresh samples, while there was no difference between farm-raised and wild-caught seafood. A subset of 102 isolates was selected for antibiotic resistance testing, showing the highest resistance rates to quinupristin/dalfopristin (52.94%) and tetracycline (27.45%). Over 70% of isolates were multidrug-resistant. Additionally, strains resistant to vancomycin and oxazolidinones, two last-resort antimicrobials, were detected. Vancomycin resistance was observed in E. casseliflavus and E. gallinarum, which are naturally resistant due to the vanC chromosomal cluster. The optrA gene, responsible for resistance to oxazolidinones and phenicols, was found in E. thailandicus and E. faecium, where it was located on to a transferable plasmid. In conclusion, this study confirms the widespread presence of Enterococcus spp. in retail seafood. The detection of multi-resistant isolates and resistance to last-resort antimicrobials highlights significant human health risks, stressing the importance of including Enterococcus spp. in seafood antibiotic resistance surveillance.

OBJECTIVES:Staphylococcus epidermidis is a member of the human skin microbiome. However, in recent decades, multidrug-resistant and hospital-adapted S. epidermidis clones are increasingly involved in severe human infections associated with medical devices and in immunocompromised patients. In 2016, we reported that a linezolid- and methicillin-resistant S. epidermidis ST2 clone, bearing the G2576T mutation, was endemic in an Italian hospital since 2004. This study aimed to retrospectively analyse 34 linezolid- and methicillin-resistant S. epidermidis (LR-MRSE) strains collected from 2018 to 2021 from the same hospital. METHODS:LR-MRSE were typed by Pulsed-Field Gel Electrophoresis and multilocus sequence typing and screened for transferable linezolid resistance genes. Representative LR-MRSE were subjected to whole-genome sequencing (WGS) and their resistomes, including the presence of ribosomal mechanisms of linezolid resistance and of rpoB gene mutations conferring rifampin resistance, were investigated. RESULTS:ST2 lineage was still prevalent (19/34; 55.9%), but, over time, ST5 clone has been widespread too (15/34; 44.1%). Thirteen of the 34 isolates (38.2%) were positive for the cfr gene. Whole-genome sequencing analysis of relevant LR-MRSE displayed complex resistomes for the presence of several acquired antibiotic resistance genes, including the SCCmec type III (3A) and SCCmec type IV (2B) in ST2 and ST5 isolates, respectively. Bioinformatics and polymerase chain reaction (PCR) mapping also showed a plasmid-location of the cfr gene and the occurrence of previously undetected mutations in L3 (ST2 lineage) and L4 (ST3 lineage) ribosomal proteins and substitutions in the rpoB gene. CONCLUSION:The occurrence of LR-MRSE should be carefully monitored in order to prevent the spread of this difficult-to-treat pathogen and to preserve the efficacy of linezolid.