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. 2026 Jan 8;26:125. doi: 10.1186/s12866-025-04666-1

Genomic analysis of Enterococcus faecium co-carrying optrA and poxtA from a swine farm: dissemination across the human–animal–environment interface

Panpan Xia 1, Huimin Wu 1, Wanzhao Chen 1, Rui Tian 1, Mengqi Yang 1, Shuqin Xu 1, Chenhui Zhang 1, Tianyuan Zeng 1, Lining Xia 1,2,✉
PMCID: PMC12908341  PMID: 41501627

Abstract

Background

The transferable resistance genes optrA and poxtA mediate cross-resistance to florfenicol and linezolid, posing serious challenges to both veterinary and human healthcare. Swine farms serve as critical ecological niches for the development and dissemination of multidrug-resistant (MDR) Enterococcus faecium (E. faecium) strains. However, the mechanisms by which E. faecium harboring optrA and poxtA disseminates and persists across the human-animal-environment interface remain unclear.

Results

In this study, 61 multidrug-resistant E. faecium isolates carrying optrA and/or poxtA were recovered from swine, farm workers, and surrounding environments. Antimicrobial susceptibility testing, conjugation assays, whole-genome sequencing, and phylogenomic analysis were performed. The predominant resistance genes were optrA (78.7%), poxtA (28.5%), and fexA (74.9%). Phylogenetic analysis of 18 representative isolates identified six distinct clades, including a novel sequence type (ST2514) shared across all three sources, suggesting potential inter-host transmission. One representative strain (RX23) harbored optrA and poxtA on two distinct multi-replicon plasmids. Experimental exposure to florfenicol increased plasmid stability (> 90% retention) and resistance levels (2–4-fold MIC elevation), indicating adaptive persistence under antibiotic pressure. Although co-transfer imposed an initial fitness cost, this burden was mitigated over serial passages, enabling long-term plasmid retention.

Conclusions

Our findings provide evidence that both plasmid-mediated transfer and ecological selection contribute to the dissemination and persistence of optrA/poxtA-positive E. faecium in swine farms. The presence of shared lineages across humans, animals, and environmental niches highlights a potential public health threat. Integrated surveillance and antimicrobial stewardship under the One Health framework are essential to prevent further dissemination along the food production chain.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-025-04666-1.

Keywords: Enterococcus faecium, OptrA, PoxtA, Cross-species transmission, Evolutionary adaptation, Swine farm, Antimicrobial resistance

Introduction

Antimicrobial resistance (AMR) has become a pressing concern in both human and veterinary medicine. Food-producing animals are well recognized as reservoirs of resistant bacteria and transferable resistance genes that may disseminate across hosts and environmental compartments. Enterococcus faecium (E. faecium), a Gram-positive intestinal commensal and opportunistic pathogen, is of particular concern due to its ability to persist in diverse environments, including water, soil, and vegetation, and its high intrinsic resistance. Importantly, E. faecium exhibits remarkable genomic plasticity, characterized by the frequent acquisition, maintenance, and exchange of antimicrobial resistance genes via mobile genetic elements (MGEs). This genomic flexibility enables the bacterium to adapt rapidly to selective pressures and facilitates the dissemination of resistance genes such as optrA and poxtA across human, animal, and environmental reservoirs, reinforcing its role in the One Health framework [1, 2]. Within intensive farming systems, multidrug-resistant (MDR) Enterococcus spp. may demonstrate bidirectional transmission between human and animal populations through pathways such as the food chain, occupational exposures, and environmental contamination [3, 4].

Florfenicol, a veterinary antimicrobial widely applied in livestock production, is commonly used to treat bacterial infections but also imposes strong selective pressure for resistance. The emergence of transferable resistance genes such as optrA and poxtA-both capable of mediating cross-resistance to florfenicol and linezolid-has raised considerable concern in recent years. The dissemination of linezolid-resistant enterococci (LRE) has been increasingly reported in animal, food, and human samples [5–8], threatening the efficacy of last-line antimicrobials in clinical settings.

The optrA and poxtA genes encode ATP-binding cassette F (ABC-F) proteins that confer reduced bacterial susceptibility to oxazolidinones and phenicols through a ribosomal protection mechanism [9]. The optrA gene, typically located on chromosomes or plasmids, protects the bacterial ribosome from antibiotic inhibition and can disseminate via horizontal gene transfer (HGT). This gene can be transferred to different genomic locations through various MGEs, including transposons, integrative and conjugative elements (ICEs), and insertion sequences (ISs), facilitating its wide spread across diverse bacterial populations [10–12]. In 2018, a related ABC-F protein, poxtA, sharing 32% amino acid identity with optrA, was first identified in a clinical methicillin-resistance Staphylococcus aureus (MRSA) strain isolated from a respiratory sample in Italy [13]. Unlike optrA, the presence of poxtA reduces bacterial susceptibility not only to oxazolidinones and phenicols but also to tetracyclines.

In recent years, the co-carriage of optrA and poxtA in the same enterococci has further exacerbated the issue of drug resistance. Plasmids carrying poxtA, optrA, and other resistance genes have been detected in E. faecium of porcine origin [14]. A 2022 study reported the simultaneous presence of optrA, cfr, and poxtA genes in a Enterococcus gallinarum (E. gallinarum) strain of porcine origin, with these resistance determinants maintained on two distinct plasmids. Remarkably, these genetic elements persisted under non-selective pressure conditions, despite the absence of detectable conjugative activity [15]. Furthermore, enterococci isolates co-carrying optrA and poxtA have been found fur animals in China [16]. Although these studies highlight the emergence of strains harboring multiple resistance determinants, knowledge about their occurrence across different hosts, their genomic contexts, and their evolutionary stability remains limited.

To address these gaps, we investigated E. faecium isolates co-carrying optrA and poxtA from swine, humans, and the farm environment in Xinjiang, China. Through phenotypic testing, conjugation assays, and whole-genome sequencing (WGS), we characterized their antimicrobial resistance profiles and genomic contexts, and examined the transferability of these resistance determinants. In addition, evolutionary and stability experiments were carried out on transconjugants in which both genes had been introduced into E. faecium, enabling us to assess the fitness costs and persistence associated with their carriage. These findings provide new insights into the ecology, dissemination, and long-term maintenance of oxazolidinone resistance determinants in farm ecosystems.

Materials and methods

Sample collection, isolation and identification of bacterial strains

In June 2021, a total of 736 samples were collected from a self-reproducing swine farm in Xinjiang, China, including swine anal swabs, environmental samples, and fecal and shoe sole swabs from farm staff. The sample composition was as follows: 423 swine anal swabs (75 from swine later identified as diarrheic based on clinical inspection and farm health records, and 348 from swine without apparent clinical symptoms; no prior sampling preference regarding health status was applied), 275 environmental samples (36 shoe sole swabs from staff, 41 environmental water samples, and 198 swabs from pen surfaces), and 38 fecal samples from farm personnel (Table S1). Bacterial isolation followed the principle of “one sample, one colony.”

Samples were enriched in brain-heart infusion (BHI) broth supplemented with 6.5% NaCl for 20 h, then streaked onto Enterococcus-selective agar plates containing 4 µg/mL florfenicol. Colonies were purified until morphological homogeneity was achieved, and single colonies were picked for further analysis.Putative enterococcal isolates were confirmed by polymerase chain reaction (PCR) amplification of species-specific genes (including ddlE. faecalis and ddlE. faecium) (Table S2). To validate the accuracy of PCR-based identification, a subset of isolates was further subjected to 16 S rRNA sequencing.

Antimicrobial susceptibility testing and resistance gene detection

The acquired oxazolidinone resistance genes (optrA, poxtA, and cfr) in the isolated strains were detected by PCR. Specific primers were designed using Primer 6.0 software (Table S2) and synthesized by Sangon Biotech (Shanghai) Co., Ltd. To confirm the specificity of PCR amplification, a subset of randomly selected PCR products was subjected to Sanger sequencing (Sangon Biotech (Shanghai) Co., Ltd.). Antimicrobial susceptibility testing (AST) against 11 antimicrobial agents was performed using the agar dilution method. Enterococcus faecalis (E. faecalis) ATCC 29212 was used as the quality control strain to ensure assay accuracy and reproducibility. MIC results were interpreted according to guidelines from the Clinical and Laboratory Standards Institute [17] and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org/clinical_breakpoints/). For antimicrobial agents without established CLSI/EUCAST breakpoints in Enterococcus spp. (e.g., florfenicol and enrofloxacin), MIC values were compared and interpreted based on reference ranges reported in previous studies [18].

Conjugative transfer experiments

To assess the transferability of antimicrobial resistance genes, donor strains were selected based on resistance gene profiles for conjugation experiments with E. faecalis JH2-2 (resistant to fusidic acid and rifampicin) as the recipient. Transconjugants were initially selected on brain heart infusion agar (BHIA) supplemented with 100 µg/mL rifampicin, 50 µg/mL fusidic acid, and 8 µg/mL florfenicol. Putative transconjugants were confirmed through PCR detection of optrA and poxtA genes, antimicrobial susceptibility testing. The conjugation transfer frequency was calculated as the number of transconjugant colonies divided by the number of recipient colonies (transconjugants/recipients).

Genomic extraction and WGS

Eighteen representative isolates were selected for WGS based on phenotypic diversity and source type. Genomic DNA was extracted using the FastPure® Bacterial DNA Isolation Mini Kit (Vazyme Biotech, China), and assessed by 1.0% agarose gel electrophoresis and NanoDrop spectrophotometry. PCR was used to confirm species identity and resistance gene presence prior to sequencing.

Short-read sequencing (2 × 150 bp) was performed on the Illumina HiSeq 2,500 platform. Quality filtering and de novo assembly were conducted using SPAdes v3.14.0. One representative isolate underwent long-read sequencing using Oxford Nanopore’s MinION platform. Reads were processed with Trimmomatic [19]. Long-read-only assemblies were generated using Flye v2.4.2, and hybrid assemblies were performed using Unicycler v0.4.8.

Bioinformatics analysis

Genome annotation was performed using the RAST server (http://rast.nmpdr.org/) with manual curation. Antibiotic resistance genes (ARGs), insertion sequence (IS) elements, and plasmid replicon types were identified using ABRicate tool (https://github.com/tseemann/abricate) with the NCBI AMRFinderPlus [20], MEGARes [21], and PlasmidFinder [22] databases. MLST was performed using the PubMLST database (https://pubmlst.org/), with novel sequence types (STs) assigned through the platform’s allele submission system. Plasmid comparative analysis was conducted using the BRIG [23], while genetic contexts of resistance gene homologs were visualized using EasyFig (http://mjsull.github.io/Easyfig/) [24]. Phylogenetic analysis was performed based on core genome single nucleotide polymorphisms (SNPs) using Roary v3.13.0 [25] and FastTree v2.1.11 [26], with final visualization and annotation using ChiPlot v4.0 [27].

Evolutionary stability assays of transconjugants

In vitro stability of optrA/poxtA-carrying plasmids was assessed using transconjugants harboring both genes (strain JH-RX23), optrA alone (strain JH-BY3), or poxtA alone (strain JH-BY51). Each was passaged for 50 generations in BHI broth with or without florfenicol (4 µg/mL). At intervals (generations 10, 20, 30, 40, and 50), bacterial colonies were plated onto non-selective BHI agar, and 50 isolates per sample were screened by PCR for optrA, poxtA, and RX23-specific plasmid markers. MICs of five representative strains per group (50th generation) were measured by broth microdilution (CLSI standard).

Evaluation of fitness cost

To evaluate fitness burden from co-carrying optrA and poxtA, growth curves were generated for the recipient JH2-2, its derivative JH-RX23 transconjugant, and passaged clones under both selective and non-selective conditions (10th to 50th generation). Each strain was grown in BHI broth (1:1,000 dilution from overnight MH broth cultures), and OD600 was measured every hour for 12 h using a 96-well plate reader. Each assay was performed in triplicate. Results were expressed as mean ± standard deviation and analyzed using IBM SPSS Statistics version 26.0, with figures prepared using GraphPad Prism 8.1.2.

Nucleotide sequence accession numbers

The DNA sequences of 18 isolates obtained have been deposited in the NCBI GenBank database under BioProject accession numbers PRJNA1207208 and PRJNA1086269.

Results

Prevalence and co-carriage of resistance genes in florfenicol resistant E. faecium

Among the 506 florfenicol-resistant Enterococcus isolates, optrA was the most prevalent resistance gene, detected in 398 isolates (78.7%), followed by fexA (379, 74.9%) and poxtA (144, 28.5%). Co-carriage of optrA and poxtA was observed in 84 isolates (16.6%), and 42 isolates (8.3%) carried all three genes simultaneously (Table S2). Isolates from swine and the environment showed the highest frequencies of multi-gene co-carriage. Focusing on E. faecium, 148 of the 506 resistant isolates (29.3%) were assigned to this species. Among them, 61 strains (41.2%) carried both optrA and poxtA, with origins including swine (n = 24), environmental samples (n = 35), and humans (n = 2). Furthermore, 29 of these 61 double-positive isolates also harbored fexA and exhibited high-level florfenicol resistance (MICs ≥ 64 µg/mL). These findings indicate that E. faecium constitutes an important reservoir of co-transmissible resistance genes, particularly within animal and environmental niches.

Analysis of antimicrobial resistance phenotypes and genotypes in E. faecium co-carrying optrA and poxtA

Antimicrobial susceptibility testing revealed that all 61 E. faecium strains co-carrying optrA and poxtA genes exhibited resistance to florfenicol, erythromycin, and tetracycline, with only one strain demonstrating susceptibility to doxycycline, confirming their MDR nature. The prevalence of linezolid-nonsusceptible E. faecium (LNSE) reached 60.7% (n = 37), predominantly isolated from porcine and environmental sources. linezolid-intermediate E. faecium (LIEfs) accounted for 47.5% (n = 29), while 8 strains (13.1%) displayed complete linezolid resistance, exclusively isolated from environmental samples. Enrofloxacin-resistant E. faecium was solely detected in porcine and environmental isolates. Notably, all 61 strains remained susceptible to vancomycin, ciprofloxacin, and levofloxacin. Multidrug resistance analysis identified all isolates as MDR strains (resistant to ≥ 3 antimicrobial classes), with the ERY-GEN-FFC-DOX-TET resistance pattern being the most prevalent (Table S5).

ARGs profiling demonstrated the presence of 8 out of 9 targeted resistance genes (excluding cfr) among the 61 E. faecium isolates. The most prevalent resistance genes were erm(B) (n = 61), tet(M) (n = 60), and tet(L) (n = 60), followed by erm(A) (n = 43), aac(6’)-aph2’’ (n = 43), and fexA (n = 29). The fexA gene was exclusively detected in 8 porcine and 21 environmental isolates, while the remaining 7 resistance genes were present across human, porcine, and environmental sources. Tetracycline and doxycycline resistance correlated with the presence of tet(M) and tet(L) genes in all strains except CF53 (isolated from a farrowing environment). Erythromycin resistance consistently associated with erm(B) carriage. These findings demonstrate concordance between phenotypic resistance patterns and genotypic determinants in all 61 E. faecium isolates (Table S5). The distribution of these resistance genes across human, porcine, and environmental isolates emphasizes the potential for the spread of these MDR strains across different ecological niches.

Transferability of optrA- or/and poxtA-harboring genetic elements

Conjugation experiments were performed using E. faecalis JH2-2 as the recipient for all 61 E. faecium isolates co-carrying optrA and poxtA. Putative transconjugants were selected on antibiotic-containing media, and their identity as E. faecalis was confirmed by species-specific PCR targeting the ddlE. faecalis gene, thereby excluding donor contamination. Transfer of optrA and/or poxtA to E. faecalis JH2-2 was detected in 17 donor strains (27.8%), including one strain showing simultaneous co-transfer of both resistance genes (Table S6). These results demonstrate the interspecies dissemination potential of optrA and poxtA between E. faecium and E. faecalis. For the remaining 44 strains, repeated conjugation attempts under varied conditions failed to yield transconjugants.

The conjugative transfer frequencies varied substantially among the different donor isolates, spanning over five orders of magnitude, from 4.0 × 10⁻⁴ (BY40) to 3.7 × 10⁻⁹ (BY33). Most frequencies clustered between 10⁻⁴ and 10⁻⁵ (e.g., BY25, BY40, BY43, BY44) and between 10⁻⁸ and 10⁻⁹ (e.g., BY7, BY33, BY51, JC175), indicating significant strain-to-strain differences in the efficiency of horizontal gene transfer.

Phylogenetic analysis and genomic characterization

To elucidate the population structure, resistome profiles, and genetic backgrounds of E. faecium co-carrying optrA and poxtA, eighteen representative isolates from swine, human, and environmental sources were subjected to WGS using the Illumina platform. These isolates were selected to capture ecological and genetic diversity across the farm ecosystem. Analysis of acquired AMR genes revealed 20 distinct resistance determinants across the 18 genomes. Aminoglycoside resistance genes were the most prevalent category, accounting for 30.0% (6/20) of the total. Core-genome phylogenetic reconstruction segregated the isolates into six distinct clades, with strains sharing the same sequence types (STs) clustering closely within phylogenetic groups (Fig. 1).

Fig. 1.

Fig. 1

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Phylogenetic analysis and basic information of the 18 E. faecium isolates in this study. The phylogenetic tree was constructed using Roary and FastTree based on core genome SNPs

Notably, isolates from humans, swine, and environmental sources were found to share a novel sequence type, ST2514. These ST2514 strains exhibited highly similar AMR gene profiles and differed by no more than 15 SNPs across the core genome. Such low SNP divergence supports the possibility of recent clonal dissemination across ecological compartments (Table S7). Additionally, ST2514 differed from ST22 of the CC17 clonal complex by only a single nucleotide in the gdh allele, suggesting evolutionary proximity to a lineage associated with hospital-adapted E. faecium. Environmental isolates exhibited greater genotypic diversity, spanning six different STs distributed across multiple clades. The nursery environment isolate BY51 harbored the highest plasmid replicon diversity, carrying five distinct replicon types. Overall, 94.1% (17/18) of all sequenced isolates co-carried the repUS15 and rep2 plasmid replicons, but environmental strains showed increased plasmid complexity and structural variation compared to swine and human isolates. Taken together, these findings suggest a potential clonal expansion of certain E. faecium lineages, particularly ST2514, across human, animal and environmental reservoirs, possibly accompanied by an enrichment of MGEs within environmental niches.

Four distinct genetic architectures flanking the optrA locus were identified across sequenced isolates (Fig. 2). Among 18 optrA-positive E. faecium isolates, 82.4% (14/18) harbored Type I configurations. The key genes tnpA and tnpB, which encode functional proteins related to encoding and transposition, were present upstream of optrA in the type I genetic background. The Type II variant (exclusively observed in strain BY51) contained an araC-optrA-hp cassette preceded by a canonical Tn554 transposon structure. Type III configurations displayed high structural homology to Type II, differing primarily through SNPs. Type IV genetic environment exhibits the phenomenon of multiple copies of erm(A), with both upstream and downstream of araC-optrA-hp-hp-hp carrying the macrolide resistance gene erm(A). The upstream of araC-optrA-hp-hp-hp is the Tn554 transposon structure. Eighteen E. faecium strains exhibited clonal relatedness and carried poxtA, although Illumina short-read sequencing failed to fully resolve the gene’s flanking genomic context due to insufficient read continuity in the poxtA-containing region. This limitation precluded a comprehensive reconstruction of the mobile genetic environment in which poxtA is embedded. In contrast, the optrA locus was found within diverse mobile genetic contexts, suggesting multiple mechanisms of acquisition and dissemination.

Fig. 2.

Fig. 2

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Schematic diagram of the comparison of the flanking regions of optrA among different genomic backbone types. The regions with ≥ 77% homology between these structures are marked by grey shading

Plasmid genomic features underlying transfer of optrA and poxtA in strain RX23

To elucidate the genetic basis underlying the observed co-transfer of optrA and poxtA, WGS was performed on the donor strain RX23, which was the only isolate in this study capable of transferring both genes simultaneously. RX23, isolated from the shoe sole of a swine farm worker, harbored two plasmids: a 151,155 bp plasmid (pAFL-R23-optrA) carrying the optrA gene and a 67,195 bp plasmid (pAFL-R23-2) carrying poxtA. The pAFL-R23-optrA plasmid also encoded the macrolide resistance gene erm(A) and multiple MGEs, including IS1216E, and shared > 75% coverage and 99% nucleotide identity with several geographically diverse plasmids, such as pW47 (China, porcine), pK70-1a-A (Switzerland, bovine), pF88_1 (Switzerland, river water), p17-318_1 (France, clinical), and K6D_p1 (Japan, clinical) (Fig. 3a). Within this plasmid, a ~ 10 kb region harboring optrA was flanked by erm(A) and the transposon Tn554, exhibiting high structural conservation with the corresponding regions of the above plasmids (Fig. 3b). All these plasmids were identified in E. faecium.

Fig. 3.

Fig. 3

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Structures of the pAFL-R23-optrA and other similar plasmids. (a) Circular comparison between optrA-bearing plasmids pAFL-R23-optrA with other similar plasmids available in the NCBI database. The outmost circle indicates the reference plasmid investigated in this study. (b) Linear alignment of optrA structures with homologous sequences. The regions with ≥ 82% homology between these structures are marked by grey shading

The poxtA-carrying plasmid pAFL-R23-2 co-harbored multiple resistance determinants, including tet(M), tet(L), aac(6’)-aph2’’, ant(6)-Ia, and aph(3’)-IIIa, and MGEs (IS1216E). Comparative genomic analysis revealed that pAFL-R23-2 shared > 80.0% coverage and 99% sequence identity with pHDC14-2 (China, porcine, E. hirae) and pR39-1-B (Switzerland, bovine, E. faecium) (Fig. 4a).

Fig. 4.

Fig. 4

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Structures of the pAFL-R23-2 and other similar plasmids. (a) Circular comparison between poxtA-bearing plasmids pAFL-R23-2 with other similar plasmids available in the NCBI database. The outmost circle indicates the reference plasmid investigated in this study. (b) Linear alignment of poxtA structures with homologous sequences. The regions with ≥ 82% homology between these structures are marked by grey shading

A conserved ~ 10 kb region containing poxtA was flanked by two direct repeats of IS1216E in the same orientation, forming a composite transposon structure, and displayed high structural similarity to regions in plasmids pT17-1-poxtA−53k, pTF51-2-41k, pC25-1, pEfM1, and pC27-2, all of which were exclusively identified in E. faecium isolates (Fig. 4b).These results suggest that both optrA and poxtA are located within mobile genetic regions exhibiting high sequence similarity and structural conservation across multiple isolates, implying their capacity for horizontal transfer among diverse plasmids and ecological environments.

Plasmid stability under florfenicol selective pressure following co-transfer of optrA and poxtA

To assess the stability of transferred resistance determinants, we selected three representative E. faecalis JH2-2 transconjugants: one carrying optrA (JH-BY3), one carrying poxtA (JH-BY51), and one simultaneously harboring both genes (JH-RX23). E. faecalis JH2-2 was used as a standard recipient strain to provide a clear and consistent model for evaluating plasmid stability.

Distinct gene maintenance patterns were observed among the transconjugants through stability assays. In the optrA-carrying transconjugant JH-BY3, gene retention decreased to 4.2% by generation 20 under non-selective conditions, with complete gene elimination (100.0% loss) achieved by generation 50. Conversely, florfenicol selection pressure maintained gene stability with minimal gene loss (< 5.0%) throughout the experimental period. The poxtA-harboring transconjugant JH-BY51 exhibited sustained gene stability, maintaining 100.0% retention through 40 generations and 95.0% retention at generation 50 under both selective and non-selective conditions (Fig. 5).

Fig. 5.

Fig. 5

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Stability dynamics of optrA and poxtA in E. faecalis transconjugants under florfenicol-free versus florfenicol selective pressure. Gene retention was monitored for 50 serial passages under conditions with florfenicol (+ FFC) or without florfenicol (-FFC). JH-BY3 (carrying optrA only) rapidly lost the gene in the absence of FFC but maintained it under selection. JH-BY51 (carrying poxtA only) retained the gene stably in both conditions. JH-RX23 (carrying both optrA and poxtA) showed stable maintenance of optrA, while poxtA gradually declined without FFC but was preserved under selection

The dual-resistant transconjugant JH-RX23 displayed differential gene retention patterns: complete maintenance(>95.0%) of both resistance determinants under florfenicol selection versus progressive loss of poxtA (retention declining from 100.0% at generation10 to < 10.0% by generation 50) in antibiotic-free conditions, while optrA retention remained > 90.0% (Fig. 5). Notably, JH-RX23 all gene loss events correlated directly with plasmid loss, as confirmed by plasmid profiling.

Post-evolution MIC analysis demonstrated 2–4 fold increases in florfenicol and linezolid resistance in both JH-RX23 and JH-BY51 lineages maintained under florfenicol selection (Figure S1). The optrA-only transconjugant JH-BY3 showed limited resistance enhancement with linezolid MIC increasing from 8 to 16 µg/mL. Notably, MIC values persisted at elevated levels even after plasmid loss.

Associated adaptability costs of optrA and poxtA co-transfer in E. faecalis

To assess the fitness impact of resistance gene acquisition in a standardized background, growth curves (OD600) were measured for E. faecalis JH2-2 and its transconjugants JH-BY3 (optrA), JH-BY51 (poxtA), and JH-RX23 (optrA-poxtA) (Fig. 6). Transfer of either optrA or poxtA alone did not affect growth compared with the recipient. In contrast, co-transfer of both genes (JH-RX23) resulted in a clear growth delay, indicating a fitness cost. Although fitness costs were apparent at the beginning of the experiment, after approximately 30th generations the growth rates of co-carrying transconjugants improved, approaching those of the recipient strain. These findings indicate that the initial fitness cost of optrA and poxtA co-transfer can be mitigated during serial passage.

Fig. 6.

Fig. 6

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Growth dynamics of optrA and poxtA transfer in E. faecalis JH2-2. (a, b) optrA-trans conjugant JH-BY3; (c, d) poxtA-transconjugant JH-BY51; (e, f) optrA-poxtA-cotransconjugant JH -BY51. Growth curves during passages 10–50 under non-selective conditions (left panels) and florfenicol-supplemented conditions (right panels)

Discussion

The persistent accumulation of antimicrobial residues at the human–animal–environment interface drives the emergence and dissemination of AMR through selective pressure and HGT. In this study, we revealed a high prevalence of E. faecium isolates carrying optrA and/or poxtA from swine, farm workers, and environmental samples collected from a single swine farm in Xinjiang. Notably, 61 (41.2%) isolates co-carried both genes, indicating that this farm ecosystem serves as a reservoir and potential amplification site for oxazolidinone resistance. WGS and conjugation-based analyses revealed that clonal expansion and plasmid-mediated transfer jointly contribute to the persistence and dissemination of these resistance genes across ecological compartments. Phylogenetic evidence further confirmed clonal transmission of the genes among swine, farm workers, and the surrounding environment. Plasmid localization analysis showed that in strain RX23, optrA and poxtA were located on distinct conjugative plasmids, while conjugation assays demonstrated that both genes could be co-transferred into an E. faecalis recipient. Although acquisition of these plasmids imposed a transient fitness cost, long-term passage experiments indicated the emergence of partial compensatory adaptation, potentially due to genetic adjustments or partial plasmid loss.

Our findings are consistent with extensive epidemiological evidence showing that the overuse of veterinary antimicrobials is a major driver of AMR in enteric bacteria such as Salmonella, Campylobacter, Escherichia coli, and Enterococcus [28–32]. Among these, the widespread application of florfenicol in livestock production has driven the selection of transferable resistance genes optrA and poxtA, which confer cross-resistance to oxazolidinones, including linezolid [9]. While the individual presence of optrA or poxtA has been reported in previous studies, their co-occurrence within the same bacterial strain, particularly in Western China, has been rarely documented. The observed co-carriage rate of 41.2% is notably higher than most previous studies. For example, surveillance in two Chinese slaughterhouses detected only two co-harboring strains out of 291 enterococcal isolates, accounting for less than 1.0% [4]. Similarly, a study from a swine farm in China reported that only 3.4% of isolates co-carried both optrA and poxtA [33]. Even in florfenicol-resistant populations, the co-carrying rate remains low; one study found that only 1.7% of enterococcal isolates co-carried both genes [34]. Environmental and food chain sources have also shown occasional detections. For instance, a Swiss water sample yielded a single E. faecium strain co-carrying both genes [35], and co-carrying E. faecium was identified in raw milk, raising concerns about zoonotic and foodborne transmission risks [36]. In clinical human samples, co-carrying of optrA and poxtA is even rarer: Spain reported only five such isolates (5.2%) between 2015 and 2018 [37], and Belgium reported just one E. faecium strain with both genes [38]. The unusually high co-occurrence rate of optrA and poxtA genes observed in this study differs significantly from most reports from domestic and international farms, environmental settings, and clinical surveillance. This finding suggests that specific epidemiological pressures, farming practices, or the background of plasmid transmission in the region may collectively contribute to the enrichment and dissemination of these multidrug resistance genes.

Despite restrictions on veterinary use of linezolid, the detection of LRE in animal and environmental reservoirs underscores the importance of cross-resistance selection driven by florfenicol and related antimicrobials [39–41]. As linezolid remains one of the last-resort agents against vancomycin-resistant enterococci (VRE), these findings raise significant public health concerns [42]. Encouragingly, China implemented a comprehensive ban on all non-traditional feed additives in 2020 and has strengthened drug residue monitoring, which may help reduce the emergence of antimicrobial resistance.

The co-localization of optrA with fexA, observed in nearly half of the co-carrying isolates, is consistent with previous reports of tandem arrangements on MGEs [43, 44]. The detection of virtually identical isolates (≤ 15 SNPs) across human, porcine and environmental samples supports clonal dissemination of this lineage within the farm ecosystem. WGS further demonstrated clonal dissemination of ST2514 across human, animal, and environmental compartments, aligning with evidence of bidirectional transmission of MDR E. faecium between livestock, wastewater, and clinical settings [7, 45]. The identification of ST2514, which is closely related to CC17, a lineage associated with nosocomial infections [46, 47], raises additional concerns about the convergence of hospital- and farm-associated lineages. The widespread detection of repUS15-type plasmids suggests ongoing plasmid recombination, which may further accelerate the acquisition of resistance determinants.

The multidrug resistance determinants optrA and poxtA exploit plasmid-mediated MGEs to facilitate horizontal gene transfer across human–animal–environment ecosystems. Genomic mapping revealed optrA and poxtA reside on distinct conjugative plasmids (pAFL-R23-optrA and pAFL-R23-2) in strain RX23, with conjugation assays confirming their transferability to E. faecalis JH2-2 under laboratory conditions, validating their interspecies dissemination potential in natural settings. The poxtA-bearing plasmid pAFL-R23-2 contains a canonical IS1216E-poxtA-IS1216E composite transposon structure in direct orientation, providing mechanistic basis for its epidemiological success across ecological niches. Comparative genomics revealed > 75% sequence homology between these plasmids and those from geographically diverse Enterococcus isolates (porcine, bovine, and environmental sources), suggesting plasmid recombination as a key mechanism for cross-regional dissemination. Previous studies have demonstrated that the formation of circular intermediates mediated by IS1216E facilitates the mobilization of resistance cassettes [48]. IS1062/ISEnfa5-driven recombination enables critical resistance transfers (e.g., vanA operon) [49, 50]. The co-occurrence of IS1216E, IS1062, and ISEnfa5 in these plasmids underscores their evolutionary capacity to propagate resistance genes through dynamic MGEs interactions. Interestingly, plasmid stability assays showed differential maintenance: optrA plasmids exhibited greater persistence, whereas poxtA plasmids were more frequently lost, suggesting size and replication efficiency may shape evolutionary trajectories.

From a fitness perspective, the acquisition of single resistance plasmids (optrA or poxtA) imposed negligible growth penalties, but dual plasmid carriage resulted in transient growth impairment that was progressively alleviated during long-term passage. This pattern supports prior observations that plasmid burdens are initially costly but may be mitigated through compensatory mutations or regulatory adaptation [51–53]. However, our current data cannot definitively distinguish whether the observed fitness recovery was driven by plasmid stabilization, genetic mutations within the host genome, or transcriptional adaptation of resistance genes. Further experiments, such as whole-genome sequencing of evolved transconjugants or transcriptomic profiling, will be needed to clarify the underlying compensatory mechanisms.

We acknowledge that our study has certain limitations. The findings, derived from a single swine farm, may not fully capture regional variability in the distribution of resistance genes, and broader, longitudinal investigations across diverse farming systems are needed to verify the generality of these observations. In addition, laboratory-based conjugation and stability assays cannot fully reproduce the complex ecological conditions of farm environments, where fluctuating antimicrobial exposure and microbial community interactions may influence plasmid behavior. Nevertheless, despite these constraints, our results provide important insights into the potential risks associated with transferable oxazolidinone resistance genes in livestock settings and their broader implications for public health.

In conclusion, our study provides genomic and experimental evidence that E. faecium strains co-carrying optrA and poxtA can disseminate within farm ecosystems and adapt to maintain resistance despite associated fitness costs. These findings underscore the need for continuous monitoring and integrated strategies to limit the spread of novel oxazolidinone resistance determinants across the human–animal–environment interface.

Conclusion

This study reveals that E. faecium co-carrying optrA and poxtA is widely distributed across swine, human, and environmental compartments within a farm ecosystem. Genomic analysis indicates that both clonal spread and horizontal gene transfer contribute to the dissemination of these resistance genes. The genetic similarity across sources suggests ongoing cross-host and environmental transmission. These findings emphasize the need for integrated One Health strategies that address both bacterial hosts and MGEs to curb the dissemination of transferable resistance in food-producing environments.

Supplementary Information

Supplementary Material 1. (256.9KB, docx)

Author’ contributions

**Panpan Xia: ** Methodology, Validation, Investigation, Formal analysis, Writing – original draft, Writing – review & editing. **Huimin Wu** : Data curation, Formal analysis, Writing – review & editing. **Wanzhao Chen** : Investigation. **Rui Tian** : Investigation. **Mengqi Yang** : Investigation. **Shuqin XU** : Investigation. **Chenhui Zhang** : Investigation. **Tianyuan Zeng** : Investigation. **Lining Xia** : Validation, Supervision, Project administration, Funding acquisition.

Funding

This work was sponsored by the National Natural Science Foundation of China (No. 32360910), and the Xinjiang Uygur Autonomous Region “Tianshan Talents” Cultivation Program—“Three Rural” Key Talent Development Project (Project No.: 2023SNGGGCC008).

Data availability

The gene sequences of the 18 *E. faecium* isolates obtained in this study have been deposited in the NCBI GenBank database under the BioProject accession numbers PRJNA1207208 and PRJNA1086269. The corresponding assembly accession numbers are provided in Table S8. All other relevant data are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study involved the collection of fecal samples from swine farm workers, swine rectal swabs, and environmental samples. All human participants were adults and voluntarily provided written informed consent prior to sample collection. Because the study involved only non-invasive sampling without the collection of identifiable personal or clinical data, the requirement for formal ethical review was waived by the Institutional Review Board of Xinjiang Agricultural University, in accordance with national and institutional regulations. All procedures involving human participants were conducted in compliance with the Declaration of Helsinki. Informed consent was also obtained from all farm owners prior to the collection of animal and environmental samples. All sampling activities were conducted in accordance with institutional, national, and international guidelines and local legislation.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (256.9KB, docx)

Data Availability Statement

The gene sequences of the 18 *E. faecium* isolates obtained in this study have been deposited in the NCBI GenBank database under the BioProject accession numbers PRJNA1207208 and PRJNA1086269. The corresponding assembly accession numbers are provided in Table S8. All other relevant data are available from the corresponding author upon reasonable request.

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