Skip to main content
NCBI home page
Springer logoLink to Springer
. 2026 Jan 31;42(2):70. doi: 10.1007/s11274-026-04786-4

Environmental occurrence of optrA-mediated linezolid resistance in Enterococcus isolates and genomic insights into Enterococcus faecium ST54 co-harboring optrA, poxtA, and cfr(D) genes

Lucas David Rodrigues Dos Santos 1, João Pedro Rueda Furlan 1,2, Rafael da Silva Rosa 1, Micaela Santana Ramos 1, Letícia Franco Gervasoni 1, Eduardo Angelino Savazzi 3, Teresa Nogueira 4,5, Eliana Guedes Stehling 1,✉
PMCID: PMC12858486  PMID: 41617929

Abstract

Linezolid-resistant Enterococcus species have emerged worldwide, with resistance mainly driven by plasmid-borne optrA, poxtA, and cfr genes. While the optrA gene has been increasingly identified in humans and animals, its presence in the environmental sector remains poorly studied, especially in South America. This study aimed to investigate and characterize linezolid resistance genes in isolates of Enterococcus faecium and Enterococcus faecalis obtained from aquatic ecosystems in 51 cities in the state of São Paulo, Brazil. Phenotypic, molecular, and genomic analyses were used for this proposal. Accordingly, 181 Enterococcus isolates were obtained, with 67 (37%) harboring the optrA gene. Most isolates exhibited multidrug resistance, and the minimum inhibitory concentration to linezolid ranged from 0.5 to > 64 mg/L. Several virulence genes and plasmid replicons were observed, with gelE and rep9 being most prevalent, respectively. Ten isolates co-harbored the optrA and poxtA genes and belonged to the known sequence type (ST) 1221 (E. faecium) and ST283, ST253, ST234, and ST1230 (E. faecalis), as well as to the new ST3018, ST3022, ST3026, and ST3027 (E. faecium), and ST2126 (E. faecalis). Moreover, one E. faecium isolate (EW1587) carried optrA, poxtA, and cfr(D) genes and, therefore, was submitted to genomic characterization. Isolate EW1587 belonged to ST54 and was closely related to an animal-derived Brazilian strain. In silico analysis predicted that optrA, poxtA, and cfr(D) genes were plasmid-borne, whereas in vitro stability tests demonstrated that these genes remained stable for 30 days. These results highlight the environmental spread of transferable oxazolidinone resistance genes, with E. faecium ST54 co-carrying optrA, poxtA, and cfr(D) genes. Therefore, continuous monitoring is essential to fully elucidate the mechanisms driving the spread and evolution of linezolid resistance across environmental reservoirs.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11274-026-04786-4.

Keywords: Brazil, Enterococci, Linezolid, Multidrug resistance, Plasmid, Water

Introduction

Enterococci are resilient bacteria found as commensal organisms in the gut and have been utilized as indicators for monitoring fecal contamination in the environment and food products (Shen et al. 2023). Among enterococci species, Enterococcus faecium and Enterococcus faecalis have been reported causing healthcare-associated infections worldwide (Monteiro et al. 2023). These pathogens pose significant health risks to immunocompromised individuals, causing severe conditions such as bacteremia, pneumonia, urinary tract infections, and meningitis (Ngbede et al. 2023; Al Rubaye et al. 2024). Notably, human diseases caused by these species have become increasingly difficult to treat due to intrinsic and acquired resistance to clinically important antimicrobial agents (Yi et al. 2022; Bender et al. 2024). Indeed, antimicrobial-resistant enterococci have emerged rapidly, with vancomycin-resistant E. faecium (VRE) classified as a global priority-resistant bacteria by the World Health Organization (Jesudason 2024).

Linezolid, a synthetic antimicrobial agent, was approved for clinical use by the United States Food and Drug Administration (FDA) as the first member of the oxazolidinone class. (Hashemian et al. 2018). It has been widely employed to treat infections caused by multidrug-resistant and vancomycin-resistant enterococci and is considered a last-resort therapeutic option (Ni et al. 2023). However, linezolid-resistant Enterococcus has also emerged, posing serious challenges to public health. Accordingly, linezolid resistance is primarily mediated by mutations in the central loop of domain V of the 23S rRNA gene, particularly the G2576U and G2505U mutations according to Escherichia coli numbering, which have been the most prevalent and well-characterized ones (Wilson et al. 2008).

Furthermore, linezolid resistance also occurs by dissemination of transferable oxazolidinone resistance genes through mobile genetic elements, including conjugative plasmids, integrative and conjugative elements (ICEs), which facilitate their rapid spread among different strains and environments (Strateva et al. 2025). Among these genes, optrA is the most frequently reported worldwide (Shen et al. 2023). This gene encodes an ATP-binding cassette (ABC-F) protein that protects the bacterial ribosome, mediating resistance to both oxazolidinones and phenicols (Almeida et al. 2020). The optrA gene also confers resistance to tedizolid, which was approved by the FDA in 2014 as a second-generation oxazolidinone. (Fu et al. 2024). To date, several optrA variants have been described, including OptrA V1, V2, V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, and V13. In Brazil, some of these variants, such as V9, V12, and V13, have already been reported in Enterococcus faecalis, highlighting the genetic diversity and geographical spread of this resistance determinant (Almeida et al. 2020). Another important gene is poxtA, which encodes an ABC-F ribosomal protection protein that reduces susceptibility to oxazolidinones and phenicols (Antonelli et al. 2018; Crowe-McAuliffe et al. 2022). Additionally, the cfr gene encodes a methyltransferase that methylates nucleotide A2503 of the 23S rRNA after transcription. This modification confers resistance not only to oxazolidinones but also to phenicols, lincosamides, pleuromutilins, and streptogramin A (denominated PhLOPSA phenotype) (Deshpande et al. 2015; Schwarz et al. 2021).

Recently, novel variants of the cfr gene, such as cfr(B) and cfr(D), have been identified at the human-animal-environmental interface (Guerin et al. 2020; Ruiz-Ripa et al. 2020). While the cfr(D) gene is not as well-characterized as other variants, it has been identified in clinically relevant Enterococcus and Staphylococcus species across various continents (Gao et al. 2022; Kim et al. 2024). Its genetic configuration is often associated with insertion sequences (IS) on conjugative plasmids (Cinthi et al. 2022a, b).

From the anthropogenic perspective, contaminated water sources have contributed to the global evolution and spread of antimicrobial resistance (AMR). In this context, surface waters represent important drivers of AMR, as they harbor a dynamic microbial community influenced by several characteristics (Reddy et al. 2022; Dos Santos et al. 2023). Despite the documented rise in linezolid-resistant enterococci (LRE) in clinical settings, their occurrence, genomic characteristics, and potential role as environmental reservoirs remain poorly understood. Therefore, this study aimed to characterize LRE isolates recovered from surface waters in Brazil using phenotypic, molecular, and genomic approaches.

Materials and methods

Environmental samples and bacterial isolation

During a surveillance study to monitor the presence of LRE in aquatic ecosystems, 136 surface water samples were collected from rivers and streams across 51 cities in São Paulo State, Brazil, between July 2021 and January 2022. The sampling points were located in areas exposed to multiple anthropogenic influences, including urban activity, recreational water use, livestock farming, and points containing discharge of domestic and hospital wastewater. For bacterial isolation, each water sample (1 L) was filtered using a sterile membrane filter with a pore size of 0.45 μm, which was then added to plates of Kanamycin Esculin Azide agar (HiMedia, India) supplemented with 4 mg/L of linezolid (Sigma-Aldrich, USA). After that, the plates were incubated for 48 h at 37 °C. Finally, dark (black or brown) colonies, morphologically typical of Enterococcus species, were selected and stored at − 80 °C using Brain Heart Infusion broth (Kasvi, Spain) supplemented with 15% glycerol.

Molecular identification

Genomic DNA of each colony was extracted according to Ramos et al. (2023), with modifications. Specifically, a pre-incubation step with 50 mg/mL of lysozyme (Sigma-Aldrich, USA) at 37 °C for 30 min was included to enhance bacterial cell wall lysis and improve DNA extraction efficiency. The identification was performed by conventional polymerase chain reactions (PCR) using a genus-specific gene for the Enterococcus genus and species-specific genes for E. faecium and E. faecalis (Supplementary Table S1). Furthermore, the Sanger sequencing of the 16S rRNA gene was also performed, and the sequences were analyzed using the blastn suite of BLAST® (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Antimicrobial susceptibility testing and high-level AMR

Antimicrobial susceptibility was initially determined using the disk diffusion method. The antimicrobials tested were linezolid, vancomycin, teicoplanin, ampicillin, imipenem, tetracycline, doxycycline, minocycline, ciprofloxacin, levofloxacin, norfloxacin, erythromycin, chloramphenicol, fosfomycin, nitrofurantoin, and rifampicin (Cecon, Brazil). The broth microdilution method was performed to determine the minimum inhibitory concentration (MIC) for linezolid and vancomycin with concentrations ranging from 1 to 256 µg/mL. The susceptibility results for all antimicrobials were interpreted according to the Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST; v.10.0, 2023), except for tetracycline, doxycycline, minocycline, chloramphenicol, rifampicin, fosfomycin, and erythromycin, for which the Clinical and Laboratory Standards Institute guidelines (CLSI, M100, 30th, 2020) were used. The enterococci isolates were classified as multidrug-resistant (MDR) using the criteria of Magiorakos et al. (2012). The high-level ciprofloxacin resistance (HLCR) and high-level aminoglycoside resistance (HLAR) were evaluated by agar dilution method using ciprofloxacin (64 µg/mL) and gentamicin (500 µg/mL)/streptomycin (2000 µg/mL), respectively (CLSI, M100, 30th, 2020; Leavis et al. 2006; Adhikari et al. 2010).

Detection of antimicrobial resistance genes (ARGs), virulence genes, and plasmid replicons

Conventional PCR was used in this step. Genes that confer resistance to linezolid [optrA, poxtA, cfr, cfr(B), and cfr(D)], aminoglycosides [aph(2’’)-Ib, ant(4’)-Ia, aac(6’)-Ie-aph(2’’)-Ia, ant(6’)-Ia, aph(3’)-IIIa, aph(2’’)-Id, and aph(2’’)-Ic], tetracyclines [tet(K), tet(L), tet(M), and tet(O)], vancomycin (vanA and vanB), and erythromycin [erm(A), erm(B), erm(C), and mefAE] were screened. In addition, virulence genes encoding adhesin to collagen (ace), gelatinase (gelE), enterococcal surface protein (esp), aggregation substances (agg), and hyaluronidase (hyl), as well as plasmid replicons (rep-1 to rep-19), were also investigated. All primers and conditions are described in the Supplementary Tables S2, S3, and S4.

Identification of mutations in the 23S rRNA

The amplified region corresponds to domain V of the 23S rRNA gene and was analyzed in all optrA-positive isolates. The 23S rRNA region was amplified by PCR using 48 °C annealing temperature and the primers 5′-GACGGAAAGACCCCATGG-3′ and 5′-ACACTTAGATGCTTT-3′, which target nucleotide positions 2049 to 2767 and result in a 718-bp fragment (Prystowsky et al. 2001). The amplicons were purified using the Illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, UK) and sequenced using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) on an ABI 3500xL Genetic Analyzer. The obtained sequences were analyzed by Geneious Prime® 2025.0.2 using 23S rRNA regions from E. faecalis V583 (GenBank: NC_004668.1) and E. faecium DO (GenBank: CP003583) as references.

Multilocus sequence typing (MLST)

The housekeeping genes used for E. faecium were adk, atpA, ddl, gyd, gdh, purK, and pstS, while for E. faecalis were gdh, pstS, gyd, gki, xpt, aroE, and yqiL. The genes were amplified and sequenced using the primers and conditions described in the Supplementary Table S5. The results were analyzed using the PubMLST databases for E. faecalis (https://pubmlst.org/organisms/enterococcus-faecalis) and E. faecium (https://pubmlst.org/organisms/enterococcus-faecium).

Whole-genome sequencing (WGS) and analysis

Genomic DNA extraction was performed using the PureLink™ Genomic DNA Mini Kit (Thermo Fisher Scientific, USA), and the WGS was conducted on the Illumina MiSeq platform (Illumina Inc., USA). Subsequently, the draft genome was de novo assembled using SPAdes v.3.15.2 (https://github.com/ablab/spades) and annotated using RAST (https://rast.nmpdr.org/rast.cgi). To identify the acquired ARGs, genes, and mutations leading to linezolid resistance, acquired virulence genes, plasmid replicons, and sequence type (ST), we used the bioinformatic tools ResFinder v.4.7.2, LRE-Finder v.1.0, VirulenceFinder v.2.0, PlasmidFinder v.2.0, and MLST v.2.0, respectively, with default parameters available from the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/). Plasmid assembly was predicted using RFPlasmid (http://klif.uu.nl/rfplasmid/) and further refined using combined strategies involving BLASTn and Geneious Prime® v.2022.2.2 (Biomatters Ltd.). The analysis of insertion sequence elements was performed using ISfinder (https://www-is.biotoul.fr/index.php).

Comparative analysis of ST54 isolates

A phylogenetic tree was constructed based on single-nucleotide polymorphism (SNP) analysis to investigate the genetic relatedness among E. faecium isolates belonging to ST54. The isolate EW1587 (this study) and public genomes (n = 25) available on the PathogenWatch database (https://pathogen.watch/) on May 1, 2025, were included in this analysis. SNP calling and phylogenetic inference were performed using the CSI Phylogeny v.1.4 (https://cge.food.dtu.dk/services/CSIPhylogeny/). Based on analyses performed using QUAST v.5.3.0 and CheckM v.1.2.3, the 11F10-MSG5009 genome (GenBank: NZ_NGMA00000000.1) was selected as reference for ST54. The phylogenetic tree was visualized by iTOL v.7 (https://itol.embl.de/). The plasmid reconstruction was predicted by PLACNETw (Vielva et al. 2017).

Plasmid stability test

Isolate E. faecium EW1587 co-harboring the optrA, poxtA, and cfr(D) genes was subjected to a plasmid stability assay for 30 days following the protocol described by Fukuda et al. (2024), with modifications. The EW1587 isolate was first inoculated into 5 mL of Brain Heart Infusion (BHI) broth (Kasvi, Spain) and incubated overnight at 37 °C. Then, 5 µL of this culture was transferred into fresh BHI, corresponding to a 1:1000 dilution. Subcultures were maintained for 30 consecutive days under three conditions: (i) room temperature, (ii) static incubation at 37 °C, and (iii) incubation at 37 °C with shaking at 124 rpm. Samples were seeded daily onto plates of BHI agar (Oxoid, UK) supplemented with linezolid (4 mg/L) and without antimicrobial, which were incubated at 37 °C for 24 h. Approximately ten colonies per plate were randomly selected for PCR-based screening of optrA, poxtA, and cfr(D) genes and MIC determination for linezolid.

Results

Enterococcus isolates harboring transferable oxazolidinone resistance genes

A total of 181 Enterococcus spp. isolates were obtained, of which 67 (37%) harbored the optrA gene. These isolates were identified as E. faecalis (n = 52, 77%) and E. faecium (n = 15, 23%) (Supplementary Table S6). Interestingly, five isolates from each species (E. faecalis: EW1636, EW1662, EW1668, EW1670, and EW1672; E. faecium: EW1637, EW1638, EW1639, EW1681, and EW1682) co-carried the optrA and poxtA genes. Furthermore, the E. faecium isolate EW1587 harbored the three transferable oxazolidinone resistance genes optrA, poxtA, and cfr(D) (Fig. 1).

Fig. 1.

Fig. 1

Open in a new tab

Map showing the 51 cities where samples were collected. The Brazilian cities are as follows: 1: Araraquara; 2: Barrinha; 3: Batatais; 4: Bebedouro; 5: Bocaina; 6: Brotas; 7: Catanduva; 8: Catiguá; 9: Dourado; 10: Dumont; 11: Franca; 12: Guaíra; 13: Guapiaçu; 14: Ibitinga; 15: Icém; 16: Ipiguá; 17: Itapuí; 18: Itirapina; 19: Ituverava; 20: Jaborandi; 21: Jaboticabal; 22: Luís Antônio; 23: Miguelópolis; 24: Monte Alto; 25: Monte Aprazível; 26: Morro Agudo; 27: Nova Granada; 28: Olímpia, 29: Ouroeste; 30: Palestina; 31: Palmares Paulista; 32: Paraíso; 33: Pedranópolis; 34: Pirassununga; 35: Pitangueiras; 36: Pontal; 37: Populina; 38: Porto Ferreira; 39: Restinga; 40: Ribeirão Bonito; 41: Ribeirão Preto; 42: Rifaina; 43: São Carlos; 44: São José da Barra; 45: São João da Bela Vista; 46: São José do Rio Preto; 47: Santa Rita do Passa Quatro; 48: Santo Antônio da Alegria; 49: Sertãozinho; 50: Tabapuã; 51: Votuporanga. The numbers highlighted in red indicate the locations where optrA-positive enterococci were obtained

Notably, different isolates carrying the optrA gene were obtained from the same river systems across different municipalities, indicating the spread of linezolid-resistant isolates through waterways. Specifically, E. faecalis isolates EW1648, EW1649, and EW1650 were recovered from the São Domingos Stream in three different cities. Similarly, E. faecium isolates EW1670 and EW1672 originated from the Mogi-Guaçu River in two distinct locations. Furthermore, E. faecium isolates EW1681 and EW1682, both carrying optrA and poxtA genes, were isolated from the Jacaré-Guaçu River (Supplementary Table S6).

AMR profiles, HLAR, and HLCR

Antimicrobial susceptibility testing was performed exclusively on the 67 optrA-positive isolates. All isolates showed resistance to imipenem, and 94% were resistant to linezolid. In E. faecalis, the MICs for linezolid ranged from 0.5 mg/L to 128 mg/L, whereas in E. faecium, the MICs for linezolid ranged from 2 mg/L to 128 mg/L. Regarding the MICs for vancomycin, E. faecalis exhibited values ranging from 1 mg/L to 8 mg/L, while in E. faecium, MICs ranged from 1 mg/L to 4 mg/L. Overall, all E. faecalis isolates were classified as MDR, presenting elevated resistance rates to tetracycline (88%), fluoroquinolones (63%), and chloramphenicol (69%). In contrast, lower resistance frequencies were identified for nitrofurantoin (19%) and ampicillin (3%). For E. faecium, 46% of isolates were classified as MDR; nonetheless, these isolates were resistant to at least four distinct antimicrobial classes. The most frequent resistance rates were observed for rifamycins (86%), fosfomycin (66%), and fluoroquinolones (40%). High-level gentamicin resistance (HLGR) was identified in 29 isolates (43%), while high-level streptomycin resistance (HLSR) was detected in 23 isolates (34%). Notably, HLGR + HLSR was found in 21 isolates (31%). Besides, HLCR was detected in six E. faecalis isolates (9%) (Supplementary Table S7).

Enterococci harboring ARGs, virulence genes, and plasmid replicons

In addition to transferable oxazolidinone resistance genes, ARGs to other antimicrobial classes were also detected. In E. faecalis isolates, the most prevalent ARGs were tet(M) (n = 52, 100%), tet(L) (n = 50, 96%), erm(B) (n = 51, 98%), and aac(6′)-Ie-aph(2″)-Ia (n = 40, 77%) (Fig. 2). Among E. faecium isolates, the most frequently detected genes were tet(L) (n = 12, 80%), tet(M) (n = 11, 73%), tet(K) (n = 9, 60%), and erm(B) (n = 12, 80%). Only E. faecium isolates harbor tet(K), and aph(2’’)-Ic genes. Furthermore, the aac(6′)-Ie-aph(2″)-Ia gene was found in 25 HLAR-positive isolates (83%), while the aph(3′)-IIIa gene was detected in four HLAR-positive isolates (12%) (Fig. 2).

Fig. 2.

Fig. 2

Open in a new tab

Overview of E. faecalis (Efc) and E. faecium (Efm) isolates obtained in this study. Colored squares indicate the presence of specific genetic determinants. 1 Sequence type (ST). 2 Minimum inhibitory concentration (MIC). 3 Antimicrobial resistance gene (ARG). 4 Virulence gene (VG). 5 High-level aminoglycoside resistance (HLAR); High-level gentamicin resistance (HLGR); and high-level streptomycin resistance (HLSR)

Virulence genotyping revealed that the gelE gene, which encodes a gelatinase, was the most detected virulence factor (n = 48, 71%), followed by the ace gene (encoding collagen adhesin) detected in 32 isolates (47%), and the esp gene (encoding enterococcal surface protein) found in 23 isolates (34%). The cytolysin gene cylA was found in 12 isolates (18%), while the aggregation substance gene asa1 was the least prevalent, as it was detected in three isolates (4%). Several plasmid replicons were observed, with 56 (83%) isolates harboring at least one rep-type. The most prevalent families were rep9 (n = 41, 61%), rep1 (n = 37, 55%), rep2 (n = 19, 28%), and rep6 (n = 12, 18%). Notably, 46 isolates (68%) carried two or more rep-types. Species-specific analysis revealed distinct plasmid profiles. E. faecalis isolates showed greater plasmid diversity, with rep9 being the most frequent (n = 39; 75%), but 11 isolates (21%) did not harbor the rep-type searched. In contrast, all E. faecium isolates harbored plasmid replicons and exhibited a more conservative profile, with rep1 detected in all isolates, followed by rep6 (n = 8, 53%) and rep2 (n = 6, 40%) (Fig. 2).

Plasmid stability

In vitro stability experiments showed that isolate EW1587 maintained optrA, poxtA, and cfr(D) genes and plasmid replicons for 30 days, allowing their long-term persistence.

Analysis of 23S rRNA mutations

The known mutations A2571G and G2595C associated with linezolid resistance were identified in two E. faecalis isolates (EW1472, EW1477) and one E. faecium isolate (EW1587). In addition to these well-established mutations, several other nucleotide changes were detected in the 23S rRNA gene. However, these additional mutations have not yet been associated with linezolid resistance in Enterococcus species. Accordingly, among the 52 E. faecalis isolates, 11 (21%) carried at least one of these uncharacterized mutations, with T2131A, G2134A, C2136T, G2153A, A2154T, T2179C, T2182C, A2189G, A2190C, C2192A, and A2210G being the most prevalent. On the other hand, E. faecium isolates (n = 7, 46%) presented the predominant A2211G mutation (Supplementary Table S8).

Enterococcal clones

MLST analysis was performed for all E. faecium and E. faecalis isolates that co-carry the optrA and poxtA genes. Among E. faecalis isolates, four were successfully assigned to known sequence types: ST283 (EW1636), ST253 (EW1668), ST234 (EW1670), and ST1230 (EW1662). In E. faecium, only one isolate (EW1637) was typed as ST1221. For the remaining isolates, E. faecalis (EW1672) and E. faecium (EW1638, EW1639, EW1681, and EW1682), the MLST scheme yielded novel allelic combination profiles that were signed as new sequence types as follows: ST2126 (E. faecalis) and ST3018, ST3022, ST3026, and ST3027 (E. faecium) (Supplementary Table S9).

Genomic insights into E. faecium ST54 co-harboring optrA, poxtA, and cfr(D) genes

The EW1587 isolate carried three transferable oxazolidinone resistance genes [optrA, poxtA, and cfr(D)] (GenBank: JBQEQP000000000). In addition, this isolate harbored other ARGs, including the acquired erm(B), fexB, fexA, tet(M), and tet(L), as well as the intrinsic aac(6’)-I, msr(C). Furthermore, the OptrA_13 variant (Tyr176Asp and Gly393Asp) was identified. No known mutations were found in GyrA and ParC, while 13 amino acid substitutions (V24A, S27G, R34Q, G66E, E100Q, K144Q, T172A, L177I, A216S, T324A, N496K, A499T, and E525D) were detected in penicillin-binding protein 5 (PBP5) and are known to be related to the PBP5-R (resistant) isoform. Virulome analysis revealed the presence of various virulence genes, including bepA, ccpA, empA, empB, empC, fms1, fms13, fms14, fms15, fms16, fms17, fms19, fms20, fms21, fnm, gls20, gls33, glsB1, sagA, scm, and sgrA. These genes have been mainly associated with biofilm formation, hydrolase enzymes, surface proteins, and adhesive matrix components. The plasmid replicons rep29, rep1, rep2, rep7a, repUS43, and repUS15 were found.

Isolate EW1587 belonged to ST54. SNP differences among globally distributed ST54 genomes ranged from 5 to 2034. EW1587 formed an external branch relative to ST54 isolates from animals and humans in Australia, Brazil, and the United States, differing by 206–289 single-nucleotide polymorphisms (SNPs) (Fig. 3). The environmental isolate EW1587 showed a difference of 206 SNPs from the animal-derived isolate 11F10-MSG5009 from Brazil, indicating a distant common ancestor and supporting long-term diversification within the ST54 lineage rather than recent transmission. Across all ST54 genomes, a broad resistome, a conservative virulome, and diverse plasmid replicons were identified. Notably, none of the ST54 genomes carried oxazolidinone resistance genes, suggesting that acquisition of such determinants likely occurred by recent horizontal gene transfer with local microbial communities.

Fig. 3.

Fig. 3

Open in a new tab

SNP-based phylogenetic tree of E. faecium ST54 genomes. Colored squares indicate the presence of specific genetic determinants as follows: red color for antimicrobial resistance; dark yellow color for virulence genes; and green color for plasmid replicons. United States (USA). Not determined (nd). The tree was rooted at the midpoint

The location of the optrA, poxtA, and cfr(D) genes was predicted in silico as plasmid-associated based on short-read data. Moreover, by using PLACNETw, plasmid-associated contigs were identified through the detection of relaxase and/or replication elements, and the optrA gene was detected on the same contig as erm(B) and rep7a. Although poxtA and cfr(D) genes were located singly on different contigs, they were connected by solid lines, suggesting they belong to the same scaffold and are likely part of the same genetic environment. Additionally, tet(M) and tet(L) genes were found on another contig grouped within the same plasmid cluster (Supplementary Fig. S1). The genetic context of optrA from this study was similar to that found in the pL14 plasmid (GenBank: CP043725) of E. faecalis ST330 from swine in Brazil. Accordingly, the araC gene from the core araC-hp1-optrA of the EW1587 isolate was truncated (ΔaraC 125 bp) when compared with that of the pL14 plasmid (araC 1,155 bp).

Discussion

To address the environmental spread of linezolid resistance in Latin America, studies conducted in Brazil have provided valuable insights. Environmental analyses of water and soil samples have identified E. faecium and E. casseliflavus isolates resistant to linezolid, although these isolates had exhibited a low prevalence of transferable resistance genes (Dos Santos et al. 2021, 2023). Similar findings have been reported in Enterococcus isolates from recreational waters, where linezolid resistance was observed despite the absence of detectable transferable resistance genes (Santiago et al. 2024). Nevertheless, metagenomic analyses of DNA extracted from treated sewage samples revealed the presence of the optrA and poxtA genes (Dias et al. 2020). Based on the available data, the widespread dissemination of linezolid resistance genes in environmental sources appears to remain limited. In contrast to these reports, our findings reveal a high frequency of transferable oxazolidinone resistance genes in surface water-derived enterococci, highlighting the environmental compartment as a relevant and underexplored reservoir.

Data on the prevalence of linezolid-resistant Enterococcus in Brazilian hospitals are also scarce. A low resistance rate (2.9%) was reported and associated with the G2576T mutation in the 23S rRNA gene in clinical isolates (Jones et al. 2013). Similarly, a study conducted in a hospital in Curitiba, Paraná (southern Brazil), found that all VRE isolates were susceptible to linezolid (Vasconcelos et al. 2024). Therefore, the data suggest that linezolid resistance in Enterococcus remains relatively low in Latin America, which is predominantly associated with chromosomal mutations rather than mediated by plasmids. Conversely, in the present study, a high frequency of optrA gene detection was observed, including isolates carrying multiple oxazolidinone resistance genes in environmental samples, suggesting that these genes may be maintained outside clinical settings.

This study identified a high frequency of the optrA gene, as well as isolates co-harboring multiple oxazolidinone resistance genes, in environmental samples. The optrA gene was described in 2015 and has been found in species other than enterococci, including Streptococcus suis, Streptococcus parasuis, Staphylococcus sp., Listeria monocytogenes, Clostridium perfringens, and Vagococcus lutrae (Shen et al. 2022). Retrospective analyses suggest that optrA-positive LRE isolates have been circulating in hospitals worldwide since 2005 (Freitas et al. 2019; Almeida et al. 2020). Accordingly, the optrA gene has been considered a major contributor to the rising incidence of LRE (Wang et al. 2015; Schwarz et al. 2021; Freitas et al. 2020). The variant and context of OptrA from the EW1587 isolate were V13 (Tyr176Asp and Gly393Asp) and P3-like, respectively. These characteristics were previously observed in a swine-derived isolate in Brazil (Almeida et al. 2020).

The dispersion of linezolid-resistant Enterococcus species has been mainly associated with humans and food-producing animals. Although not approved for farm use, linezolid resistance may be driven by florfenicol and oxytetracycline overuse in livestock and aquaculture (Gharbi et al. 2024). The selective pressure mediated by these antimicrobials may select plasmids co-harboring optrA, tet(L), tet(M), erm(A), and erm(B) genes (Yoon et al. 2020; McHugh et al. 2022; Dos Santos et al. 2023). Furthermore, insertion sequences (IS1216E) and transposons (Tn554 and Tn558) also contribute to the spread of the optrA gene. Accordingly, these mobile genetic elements enable horizontal gene transfer across diverse bacterial hosts and environmental niches (Fu et al. 2024; Liu et al. 2024; Yang et al. 2024).

A broad distribution of rep-type plasmid genes was observed, with rep9 being the most prevalent. These findings corroborate previous reports of rep9 plasmids as harboring the optrA in E. faecalis (Mikalsen et al. 2015; Zou et al. 2020). This plasmid family includes several pheromone-responsive and conjugative plasmids, including pAD1, which play a central role in the dissemination of antimicrobial resistance determinants (Jensen et al. 2010; Zou et al. 2020). Therefore, the high prevalence of rep9 identified in this study supports its species-specific association and suggests that pheromone-responsive transfer systems may contribute to the maintenance and spread of the optrA gene.

Mutational linezolid resistance was also identified in optrA-positive isolates. The well-characterized 23S rRNA mutations were detected as follows: A2571G in E. faecalis and G2595C in E. faecium. In addition, other mutations outside the commonly reported resistance-associated regions were also observed and may also be related to linezolid resistance, although such reports have so far only been described in Staphylococcus aureus and Mycobacterium smegmatis (Sander et al. 2002; Wilson et al. 2008; Long et al. 2010; Long et al. 2012). In this context, new point mutations in linezolid resistance determinants should be further explored for their roles in high-level resistance.

While optrA and poxtA have been extensively reported, emerging variants like cfr(D) remain poorly identified. The cfr(D) gene was first identified in a clinical E. faecium isolate from France in 2015 (Guerin et al. 2020). Despite its structural similarity to other cfr-like genes, functional studies have demonstrated that cfr(D) alone does not confer linezolid resistance in enterococci (Hu et al. 2022). In contrast, phenotypic resistance has been observed in Escherichia coli carrying cfr(D), suggesting that its activity may be host-dependent or influenced by specific genetic contexts (Hu et al. 2022). A previous study demonstrated that cfr(D) can frequently coexist with the IS1216, which facilitates its mobilization through the formation of transposable units with variable structures (Gao et al. 2025). Consequently, the presence of cfr(D) in diverse bacterial species further supports its potential for interspecies transfer (Zhu et al. 2021). In this study, we report the first identification of cfr(D) in South America (Supplementary Fig. S2).

The identification of Enterococcus species co-carrying transferable oxazolidinone resistance genes is noteworthy. The co-occurrence of optrA, poxtA2, and cfr(D) genes was detected in E. faecalis from duck meat in China, E. gallinarum of swine origin in Italy, and E. faecium from swine in Nigeria. Additionally, clinical isolates of E. faecalis and E. faecium also harbored these genes (Shen et al. 2023; Ngbede et al. 2023; Coccitto et al. 2022; Torabi et al. 2023). Notably, in the EW1587 isolate, cfr(D) is located near poxtA, potentially within the same resistance region that can be transmitted at a high rate through horizontal transfer (Cinthi et al. 2022a, b; Nüesch-Inderbinen et al. 2022; Shen et al. 2023). Furthermore, acquired ARGs that confer resistance to other antimicrobials, including macrolides, tetracyclines, and aminoglycosides, as well as substitutions that reduce the binding affinity of PBP5 for ampicillin, have also been identified, highlighting the multidrug resistance phenotype that limits therapeutic options for infections caused by enterococci (Pietta et al. 2014; Novais et al. 2016; Said et al. 2019; Andrea et al. 2025).

The detection of new sequence types among enterococci studied highlights the genetic diversity of the environmental linezolid-resistant isolates. E. faecalis ST1230 was singly identified in South Korea from a non-hospitalized patient (PubMLST ID: 2314), whereas ST234 and ST253 have been previously associated with linezolid resistance mediated by the optrA gene in human and animal medicine (Mortelé et al. 2024; Gagetti et al. 2023; Nüesch-Inderbinen et al. 2023). By using the PubMLST database, E. faecalis ST283 was identified in animals and surface water in Europe and humans from Mexico, the latter harboring the optrA gene (Martínez-Ayala et al. 2025).

Among the E. faecium isolates, only ST1221 was assigned using the conventional MLST technique. This clone was previously reported in an environmental sample from the United Kingdom (E. faecium ID PubMLST: 2962). Genomic analysis identified ST54, with its first genome-associated report in 2014. In Brazil, there is only one genomic report in a green turtle (GenBank: NZ_NGMA00000000.1). In this context, the scarcity of published studies on these Enterococcus clones limits our understanding of their diversity and dissemination. Taken together, these findings indicate that environmental enterococci comprise diverse and poorly characterized lineages carrying transferable oxazolidinone resistance genes, supporting the role of surface waters as reservoirs that may contribute to the long-term persistence and evolution of linezolid resistance.

Conclusion

This study reports the occurrence of transferable oxazolidinone resistance genes in Enterococcus species isolated from surface waters in Brazil. The high prevalence of optrA in the environment likely results from a multifaceted interplay of factors, including indirect selection pressure from non-linezolid antimicrobials, plasmid-mediated co-resistance, and efficient horizontal gene transfer mechanisms. These findings highlight the environment as an underappreciated reservoir and disseminator of transferable oxazolidinone resistance genes. Therefore, continuous monitoring is essential to comprehensively understand the processes involved in the spread and evolution of linezolid resistance within the environmental sector.

Supplementary information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (DOCX 1.19 MB) (1.2MB, docx)

Acknowledgements

The authors thank the FAPESP (grant number 2023/12947-4, 2023/04555-9), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (grant numbers 8887.824722/2023-00 and Finance code 001), and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant numbers 304905/2022-4, 130086/2021-5, and 141016/2021-3) for fellowships.

Author contributions

Lucas David Rodrigues dos Santos: Conceptualization, Methodology, Software, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing. João Pedro Rueda Furlan: Conceptualization, Methodology, Software, Formal analysis, Investigation, Data curation, Writing - review & editing. Rafael da Silva Rosa: Methodology, Formal analysis, Data curation. Micaela Santana Ramos: Methodology, Formal analysis, Data curation. Letícia Franco Gervasoni: Methodology, Formal analysis, Data curation Eduardo Angelino Savazzi: Conceptualization, Investigation. Teresa Nogueira: Methodology, Formal analysis, Data curation, Writing - review editing. Eliana Guedes Stehling: Conceptualization, Investigation, Data curation, Supervision, Project administration, Funding acquisition, Writing - review editing,

Funding

The Article Processing Charge (APC) for the publication of this research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (ROR identifier: 00x0ma614). This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grant number 2023/12947-4).

Data availability

Data availabilityThe genome assembly of strain EW1587 was submitted to GenBank under accession number: JBQEQP000000000.

Declarations

Ethical approval

Not required.

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.

References

  1. Adhikari L (2010) High-level aminoglycoside resistance and reduced susceptibility to vancomycin in nosocomial enterococci. J Global Infect Dis 2:231. 10.4103/0974-777X.68534 [Google Scholar]
  2. Almeida LM, Gaca A, Bispo PM, Lebreton F, Saavedra JT, Silva RA, Basílio-Júnior ID, Zorzi FM, Filsner PH, Moreno AM, Gilmore MS (2020) Coexistence of the oxazolidinone resistance–associated genes cfr and optrA in Enterococcus faecalis from a healthy piglet in Brazil. Front Public Health 8:518. 10.3389/fpubh.2020.00518
  3. Al Rubaye M, Janice J, Bjørnholt JV, Löhr IH, Sundsfjord A, Hegstad K (2024) The first vanE-type vancomycin-resistant Enterococcus faecalis isolates in Norway – phenotypic and molecular characteristics. J Glob Antimicrob Resist 36:193–199. 10.1016/j.jgar.2023.12.021 [DOI] [PubMed] [Google Scholar]
  4. Andrea A, Mourão J, Hendriksen R (2025) DTU Genomic Proficiency Test 2024 Report: Interpretation of Submitted Data and Scoring System for Escherichia coli, Staphylococcus aureus and Enterococcus spp. Available online: https://www.food.dtu.dk/english/-/media/institutter/foedevareinstituttet/temaer/antibiotikaresistens/dtu-genomic-proficiency-test-2024-report.pdf. (accessed on 25 December 2025)
  5. Antonelli A, D’Andrea MM, Brenciani A, Galeotti CL, Morroni G, Pollini S, Varaldo PE, Rossolini GM (2018) Characterization of poxtA, a novel phenicol–oxazolidinone–tetracycline resistance gene from an MRSA of clinical origin. J Antimicrob Chemother 73:1763–1769. 10.1093/jac/dky088 [DOI] [PubMed] [Google Scholar]
  6. Bender JK, Fleige C, Funk F, Moretó-Castellsagué C, Fischer MA, Werner G (2024) Linezolid resistance genes and mutations among linezolid-susceptible Enterococcus spp.—a loose cannon? Antibiotics 13:101. 10.3390/antibiotics13010101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cinthi M, Coccitto SN, D’Achille G, Morroni G, Simoni S, Fioriti S, Magistrali CF, Brenciani A, Giovanetti E (2022) Characterization of a novel cfr(D)/poxtA-carrying plasmid in an oxazolidinone-resistant Enterococcus casseliflavus isolate from swine manure, Italy. J Glob Antimicrob Resist 30:308–310. 10.1016/j.jgar.2022.07.007 [DOI] [PubMed] [Google Scholar]
  8. Cinthi M, Coccitto SN, Fioriti S, Morroni G, Simoni S, Vignaroli C, Magistrali CF, Albini E, Brenciani A, Giovanetti E (2022) Occurrence of a plasmid co-carrying cfr (D) and poxtA2 linezolid resistance genes in Enterococcus faecalis and Enterococcus casseliflavus from porcine manure, Italy. J Antimicrob Chemother 77:598–603. 10.1093/jac/dkab456
  9. Coccitto SN, Cinthi M, Fioriti S, Morroni G, Simoni S, Vignaroli C, Garofalo C, Mingoia M, Brenciani A, Giovanetti E (2022) Linezolid-resistant Enterococcus gallinarum isolate of swine origin carrying cfr, OptrA and PoxtA genes. J Antimicrob Chemother 77:331–337. 10.1093/jac/dkab408 [DOI] [PubMed] [Google Scholar]
  10. Crowe-McAuliffeC, Murina V, Turnbull KJ, Huch S, Kasari M, Takada H, Nersisyan L, Sundsfjord A, Hegstad K, Atkinson GC, Pelechano V, Wilson DN, Hauryliuk V (2022) Structural basis for PoxtA-mediated resistance to phenicol and oxazolidinone antibiotics. Nat Commun 13(1):1860. 10.1038/s41467-022-29274-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Deshpande LM, Ashcraft DS, Kahn HP, Pankey G, Jones RN, Farrell DJ, Mendes RE (2015) Detection of a new cfr -like gene, cfr (B), in Enterococcus faecium isolates recovered from human specimens in the United States as part of the SENTRY antimicrobial surveillance program. Antimicrob Agents Chemother 59:6256–6261. 10.1128/AAC.01473-15 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dias MF, Da Rocha Fernandes G, Cristina De Paiva M, Christina De Matos Salim A, Santos AB, Amaral Nascimento AM (2020) Exploring the resistome, virulome and microbiome of drinking water in environmental and clinical settings. Water Res 174:115630. 10.1016/j.watres.2020.115630 [DOI] [PubMed] [Google Scholar]
  13. Dos Santos LDR, Furlan JPR, Gallo IFL, Ramos MS, Savazzi EA, Stehling EG (2021) Occurrence of multidrug-resistant Enterococcus faecium isolated from environmental samples. Lett Appl Microbiol 73:237–246. 10.1111/lam.13508 [DOI] [PubMed] [Google Scholar]
  14. Dos Santos LDR, Furlan JPR, Rosa RDS, Ramos MS, Savazzi EA, Stehling EG (2023) Linezolid-resistant Enterococcus casseliflavus strains of surface water and coexistence of cfr and optrA genes on an Inc18-type plasmid. J Glob Antimicrob Resist 33:18–20. 10.1016/j.jgar.2023.02.008 [DOI] [PubMed] [Google Scholar]
  15. Freitas AR, Tedim AP, Novais C, Lanza VF, Peixe L (2020) Comparative genomics of global OptrA-carrying Enterococcus faecalis uncovers a common chromosomal hotspot for OptrA acquisition within a diversity of core and accessory genomes. Microb Genomics 6. 10.1099/mgen.0.000350
  16. Freitas AR, Tedim AP, Novais C, Peixe L (2019) Comparative genomics of global optrA -carrying Enterococcus faecalis uncovers common genetic features and a chromosomal hotspot for optrA acquisition. 10.1101/785295
  17. Fukuda A, Nakajima C, Suzuki Y, Usui M (2024) Transferable linezolid resistance genes (optrA and poxtA) in enterococci derived from livestock compost at Japanese farms. J Glob Antimicrob Resist 36:336–344. 10.1016/j.jgar.2024.01.022 [DOI] [PubMed] [Google Scholar]
  18. Fu Y, Deng Z, Shen Y, Wei W, Xiang Q, Liu Z, Hanf K, Huang S, Lv Zexun, Cao T, Peng C, Zhang R, Zou X, Shen J, Schwarz S, Wang Y, Liu D, Lv Ziquan, Ke Y (2024) High prevalence and plasmidome diversity of optrA-positive enterococci in a Shenzhen community, China. Front. Microbiol. 15:1505107. 10.3389/fmicb.2024.1505107 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gagetti P, Faccone D, Ceriana P, Lucero C, Menocal A, Argentina GL, Corso A (2023) Emergence of optrA-mediated linezolid resistance in clinical isolates of Enterococcus faecalis from Argentina. J Glob Antimicrob Resist 35:335–341. 10.1016/j.jgar.2023.10.014 [DOI] [PubMed] [Google Scholar]
  20. Gao X, Luo X, Qian R, Gao G, Liu J, Hong J, Yue C, Liu JH, Liu YY (2025) Emergence of linezolid resistance genes OptrA and cfr(D) in an Enterococcus saccharolyticus from chicken. Antibiotics 14:337. 10.3390/antibiotics14040337 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gao Y, Wang Z, Fu J, Cai J, Ma T, Xie N, Fan R, Zhai W, Feßler AT, Sun C, Wu C, Schwarz S, Zhang R, Wang Y (2022) Spreading of cfr-carrying plasmids among staphylococci from humans and animals. Microbiol Spectr 10:e0246122. 10.1128/spectrum.02461-22 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gharbi M, Tiss R, Hamdi C, Hamrouni S, Maaroufi A (2024) Occurrence of florfenicol and linezolid resistance and emergence of <Emphasis Type="Italic">optrA</Emphasis> gene in <Emphasis Type="Italic">Campylobacter coli</Emphasis> isolates from Tunisian avian farms. Int J Microbiol 1694745. 10.1155/2024/1694745 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Guerin F, Sassi M, Dejoies L, Zouari A, Schutz S, Potrel S, Auzou M, Collet A, Lecointe D, Auger G, Cattoir V (2020) Molecular and functional analysis of the novel cfr(D) linezolid resistance gene identified in Enterococcus faecium. J Antimicrob Chemother 75:1699–1703. 10.1093/jac/dkaa125 [DOI] [PubMed] [Google Scholar]
  24. Hashemian SMR, Farhadi T, Ganjparvar M (2018) Linezolid: a review of its properties, function, and use in critical care. Drug Des Devel Ther 12:1759–1767. 10.2147/DDDT.S164515 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hu Y, Won D, Nguyen LP, Osei KM, Seo Y, Kim J, Lee Y, Lee H, Yong D, Choi JR, Lee K (2022) Prevalence and genetic analysis of resistance mechanisms of linezolid-non susceptible enterococci in a tertiary care hospital examined via whole-genome sequencing. Antibiotics (Basel) 11:1624. 10.3390/antibiotics11111624 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jensen LB, Garcia-Migura L, Valenzuela AJS, Løhr M, Hasman H, Aarestrup FM (2010) A classification system for plasmids from enterococci and other Gram-positive bacteria. J Microbiol Methods 80:25–43. 10.1016/j.mimet.2009.10.012 [DOI] [PubMed] [Google Scholar]
  27. Jesudason T (2024) WHO publishes updated list of bacterial priority pathogens. Lancet Microbe 5(9):100940 [DOI] [PubMed] [Google Scholar]
  28. Jones RN, Guzman-Blanco M, Gales AC, Gallegos B, Castro ALL, Martino MDV, Vega S, Zurita J, Cepparulo M, Castanheira M (2013) Susceptibility rates in Latin American nations: report from a regional resistance surveillance program (2011). Braz J Infect Dis 17:672–681. 10.1016/j.bjid.2013.07.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kim E, Yang SM, Kwak HS, Moon BY, Lim SK, Kim HY (2024) Genomic characteristics of cfr and fexA carrying Staphylococcus aureus isolated from pig carcasses in Korea. Vet Res 55:21. 10.1186/s13567-024-01278-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Leavis HL, Willems RJL, Top J, Bonten MJM (2006) High-level ciprofloxacin resistance from point mutations in gyrA and parC confined to global hospital-adapted clonal lineage CC17 of Enterococcus faecium. J Clin Microbiol 44:1059–1064. 10.1128/JCM.44.3.1059-1064.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu S, Yang X, Li R, Wang S, Han Z, Yang M, Zhang Y (2024) IS6 family insertion sequences promote optrA dissemination between plasmids varying in transfer abilities. Appl Microbiol Biotechnol 108:132. 10.1007/s00253-023-12858-w [DOI] [PubMed] [Google Scholar]
  32. Long KS, Munck C, Andersen TMB, Schaub MA, Hobbie SN, Böttger EC, Vester B (2010) Mutations in 23S rRNA at the peptidyl transferase center and their relationship to linezolid binding and cross-resistance. Antimicrob Agents Chemother 54:4705–4713. 10.1128/AAC.00644-10 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Long KS, Vester B (2012) Resistance to linezolid caused by modifications at its binding site on the ribosome. Antimicrob Agents Chemother 56:603–612. 10.1128/AAC.05702-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL (2012) Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 18:268–281. 10.1111/j.1469-0691.2011.03570.x [DOI] [PubMed] [Google Scholar]
  35. Martínez-Ayala P, Perales-Guerrero L, Gómez-Quiroz A, Avila-Cardenas BB, Gómez-Portilla K, Rea-Márquez EA, Vera-Cuevas VC, Gómez-Quiroz CA, Briseno-Ramírez J, De Arcos-Jiménez JC (2025) Whole-genome sequencing of Linezolid-Resistant and Linezolid-Intermediate-Susceptibility Enterococcus faecalis clinical isolates in a Mexican Tertiary Care University Hospital. Microorganisms 13:684. 10.3390/microorganisms13030684 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McHugh MP, Parcell BJ, Pettigrew KA, Toner G, Khatamzas E, El Sakka N, Karcher AM, Walker J, Weir R, Meunier D, Hopkins KL, Woodford N, Templeton KE, Gillespie SH, Holden MTG (2022) Presence of optrA-mediated linezolid resistance in multiple lineages and plasmids of Enterococcus faecalis revealed by long read sequencing. Microbiology 168. 10.1099/mic.0.001137
  37. Mikalsen T, Pedersen T, Willems R, Coque TM, Werner G, Sadowy E, Van Schaik W, Jensen LB, Sundsfjord A, Hegstad K (2015) Investigating the mobilome in clinically important lineages of Enterococcus faecium and Enterococcus faecalis. BMC Genomics 16:282. 10.1186/s12864-015-1407-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Monteiro Marques J, Coelho M, Santana AR, Pinto D, Semedo-Lemsaddek T (2023) Dissemination of enterococcal genetic lineages: a one health perspective. Antibiotics 12:1140. 10.3390/antibiotics12071140 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mortelé O, van Van Kleef– Koeveringe S, Vandamme S, Jansens H, Goossens H, Matheeussen V (2024) Epidemiology and genetic diversity of linezolid-resistant Enterococcus clinical isolates in Belgium from 2013 to 2021. J Glob Antimicrob Resist 38:21–26. 10.1016/j.jgar.2024.04.010 [DOI] [PubMed] [Google Scholar]
  40. Nüesch-Inderbinen M, Biggel M, Haussmann A, Treier A, Heyvaert L, Cernela N, Stephan R (2023) Oxazolidinone resistance genes in florfenicol-resistant enterococci from beef cattle and veal calves at slaughter. Front Microbiol 14:1150070. 10.3389/fmicb.2023.1150070 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nüesch-Inderbinen M, Haussmann A, Treier A, Zurfluh K, Biggel M, Stephan R (2022) Fattening pigs are a reservoir of Florfenicol-Resistant enterococci harboring Oxazolidinone resistance genes. J Food Prot 85:740–746. 10.4315/JFP-21-431 [DOI] [PubMed] [Google Scholar]
  42. Ngbede EO, Sy I, Akwuobu CA, Nanven MA, Adikwu AA, Abba PO, Adah MI, Becker SL (2023) Carriage of linezolid-resistant enterococci (LRE) among humans and animals in Nigeria: coexistence of the cfr, optrA, and poxtA genes in Enterococcus faecium of animal origin. J Glob Antimicrob Resist 34:234–239. 10.1016/j.jgar.2023.07.016 [DOI] [PubMed] [Google Scholar]
  43. Ni J, Long X, Wang M, Ma J, Sun Y, Wang W, Yue M, Yang H, Pan D, Tang B (2023) Transmission of linezolid-resistant Enterococcus isolates carrying optrA and poxtA genes in slaughterhouses. Front. Sustain. Food Syst. 7:1179078. 10.3389/fsufs.2023.1179078 [Google Scholar]
  44. Novais C, Tedim AP, Lanza VF, Freitas AR, Silveira E, Escada R, Roberts AP, Al-Haroni M, Baquero F, Peixe L, Coque TM (2016) Co-diversification of Enterococcus faecium core genomes and PBP5: evidences of pbp5 horizontal transfer. Front Microbiol 7:1581. 10.3389/fmicb.2016.01581 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pietta E, Montealegre MC, Roh JH, Cocconcelli PS, Murray BE (2014) Enterococcus faecium PBP5-S/R, the missing link between PBP5-S and PBP5-R. Antimicrob Agents Chemother 58(11):6978–6981. 10.1128/AAC.03648-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Prystowsky J, Siddiqui F, Chosay J, Shinabarger DL, Millichap J, Peterson LR, Noskin GA (2001) Resistance to Linezolid: characterization of mutations in rRNA and comparison of their occurrences in Vancomycin-Resistant Enterococci. Antimicrob Agents Chemother 45:2154–2156. 10.1128/AAC.45.7.2154-2156.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ramos MS, Furlan JPR, Dos Santos LDR, Rosa RDS, Savazzi EA, Stehling EG (2023) Patterns of antimicrobial resistance and metal tolerance in environmental Pseudomonas aeruginosa isolates and the genomic characterization of the rare O6/ST900 clone. Environ Monit Assess 195:713. 10.1007/s10661-023-11344-0 [DOI] [PubMed] [Google Scholar]
  48. Reddy S, Kaur K, Barathe P, Shriram V, Govarthanan M, Kumar V (2022) Antimicrobial resistance in urban river ecosystems. Microbiol Res 263:127135. 10.1016/j.micres.2022.127135 [DOI] [PubMed] [Google Scholar]
  49. Ruiz-Ripa L, Feßler AT, Hanke D, Eichhorn I, Azcona-Gutiérrez JM, Pérez-Moreno MO, Seral C, Aspiroz C, Alonso CA, Torres L, Alós JI, Schwarz S, Torres C (2020) Mechanisms of linezolid resistance among enterococci of clinical origin in Spain—detection of optrA- and cfr(D)-carrying E. faecalis. Microorganisms 8:1155. 10.3390/microorganisms8081155 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Said HS, Abdelmegeed ES (2019) Emergence of multidrug resistance and extensive drug resistance among enterococcal clinical isolates in Egypt. Infect Drug Resist 12:1113–1125. 10.2147/IDR.S189341 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sander P, Belova L, Kidan YG, Pfister P, Mankin AS, Böttger EC (2002) Ribosomal and non-ribosomal resistance to oxazolidinones: species‐specific idiosyncrasy of ribosomal alterations. Mol Microbiol 46:1295–1304. 10.1046/j.1365-2958.2002.03242.x [DOI] [PubMed] [Google Scholar]
  52. Santiago GS, Dropa M, Martone-Rocha S, Dos Santos TP, De Moura Gomes VT, Barbosa MRF, Razzolini MTP (2024) Antimicrobial resistance characterization of Enterococcus faecium, Enterococcus faecalis and Enterococcus hirae isolated from marine coastal recreational waters in the State of São Paulo, Brazil. J Water Health 22:1628–1640. 10.2166/wh.2024.098 [DOI] [PubMed] [Google Scholar]
  53. Schwarz S, Zhang W, Du XD, Krüger H, Feßler AT, Ma S, Zhu Y, Wu C, Shen J, Wang Y (2021) Mobile oxazolidinone resistance genes in gram-positive and gram-negative bacteria. Clin Microbiol Rev 34:e00188-20. 10.1128/CMR.00188-20 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Shen W, Huang Y, Cai J (2022) An optimized screening approach for the oxazolidinone resistance gene optrA yielded a higher fecal carriage rate among healthy individuals in Hangzhou, China. Microbiol Spectr 10:e02974-22. 10.1128/spectrum.02974-22 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Shen W, Zhang R, Cai J (2023) Co-occurrence of multiple plasmid-borne linezolid resistance genes optrA, Cfr, poxtA2 and Cfr (D) in an Enterococcus faecalis isolate from retail meat. J Antimicrob Chemother 78:1637–1643. 10.1093/jac/dkad142 [DOI] [PubMed] [Google Scholar]
  56. Strateva TV, Hristova P, Stoeva TJ, Hitkova H, Peykov S (2025) First detection and genomic characterization of linezolid-resistant Enterococcus faecalis clinical isolates in Bulgaria. Microorganisms 13:195. 10.3390/microorganisms13010195 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Torabi M, Faghri J, Poursina F (2023) Detection of genes related to linezolid resistance (poxtA, cfr, and optrA) in clinical isolates of Enterococcus spp. From humans: A first report From Iran. Adv Biomed Res 12. 10.4103/abr.abr_74_23
  58. Vasconcelos TM, Mesa D, Rodrigues LS, Medeiros Dos Santos É, Krul D, Siqueira AC, De Abreu RBV, Motta FDA, Conte D, Dalla-Costa LM (2024) Outbreak of vancomycin-resistant Enterococcus faecium ST1133 in paediatric patients with acute lymphoblastic leukaemia from southern Brazil. J Glob Antimicrob Resist 36:41–44. 10.1016/j.jgar.2023.11.005 [DOI] [PubMed] [Google Scholar]
  59. Vielva L, De Toro M, Lanza VF, De La Cruz F (2017) PLACNETw: a web-based tool for plasmid reconstruction from bacterial genomes. Bioinformatics 33:3796–3798. 10.1093/bioinformatics/btx462 [DOI] [PubMed] [Google Scholar]
  60. Wang Y, Lv Y, Cai J, Schwarz S, Cui L, Hu Z, Zhang R, Li J, Zhao Q, He T, Wang D, Wang Z, Shen Y, Li Y, Feßler AT, Wu C, Yu H, Deng X, Xia X, Shen J (2015) A novel gene, optrA, that confers transferable resistance to Oxazolidinones and phenicols and its presence in Enterococcus faecalis and Enterococcus faecium of human and animal origin. J Antimicrob Chemother 70:2182–2190. 10.1093/jac/dkv116 [DOI] [PubMed] [Google Scholar]
  61. Wilson DN, Schluenzen F, Harms JM, Starosta AL, Connell SR, Fucini P (2008) The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning. Proc Natl Acad Sci U S A 105:13339–13344. 10.1073/pnas.0804276105 [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Yang P, Li J, Lv M, He P, Song G, Shan B, Yang X (2024) Molecular epidemiology and horizontal transfer mechanism of optrA -carrying Linezolid-resistant Enterococcus faecalis. Pol J Microbiol 73:349–362. 10.33073/pjm-2024-031 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Yi M, Zou J, Zhao J, Tang Y, Yuan Y, Yang B, Huang J, Xia P, Xia Y (2022) Emergence of optrA-mediated Linezolid resistance in Enterococcus faecium: a molecular investigation in a tertiary hospital of Southwest China from 2014–2018. Infect Drug Resist 15:13–20. 10.2147/IDR.S339761 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yoon S, Son SH, Kim YB, Seo KW, Lee YJ (2020) Molecular characteristics of optrA-carrying Enterococcus faecalis from chicken meat in South Korea. Poult Sci 99:6990–6996. 10.1016/j.psj.2020.08.062 [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zhu Y, Yang Q, Schwarz S, Yang W, Xu Q, Wang L, Liu S, Zhang W (2021) Identification of a Streptococcus parasuis isolate co-harbouring the oxazolidinone resistance genes cfr (D) and optrA. J Antimicrob Chemother 76:3059–3061. 10.1093/jac/dkab297 [DOI] [PubMed] [Google Scholar]
  66. Zou J, Tang Z, Yan J, Liu H, Chen Y, Zhang D, Zhao J, Tang Y, Zhang J, Xia Y (2020) Dissemination of linezolid resistance through sex pheromone plasmid transfer in Enterococcus faecalis. Front Microbiol 11:1185. 10.3389/fmicb.2020.01185 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

Supplementary Material 1 (DOCX 1.19 MB) (1.2MB, docx)

Data Availability Statement

Data availabilityThe genome assembly of strain EW1587 was submitted to GenBank under accession number: JBQEQP000000000.

Articles from World Journal of Microbiology & Biotechnology are provided here courtesy of Springer

RESOURCES