Plasmid Fusion and Recombination Events That Occurred during Conjugation of poxtA-Carrying Plasmids in Enterococci

ABSTRACT Linezolid plays a crucial role in the treatment of infections caused by multiresistant Gram-positive bacteria. The poxtA gene not only confers oxazolidinone and phenicol resistance but also decreases susceptibility to tetracycline. In this study, we investigated structural changes in mobilizable poxtA-carrying plasmids in enterococci which occurred during conjugation experiments using S1-PFGE (pulsed-field gel electrophoresis), Southern blot hybridization, and whole-genome sequencing (WGS) analysis. Two poxtA-carrying strains were identified in Enterococcus faecalis E006 and Enterococcus lactis E843, respectively. E. faecalis E006 contains the 121,520-bp conjugative plasmid pE006-121 and the 19,832-bp mobilizable poxtA-carrying plasmid pE006-19, while E. lactis E843 contains the 171,930-bp conjugative plasmid pE843-171 and the 27,847-bp mobilizable poxtA-carrying plasmid pE843-27. Moreover, both poxtA-carrying plasmids were mobilized by their respective conjugative plasmid in enterococci by plasmid fusion; one was generated by homologous recombination in E. faecalis through an identical 864-bp homologous region in the plasmids of the parental strain, while another was generated by an IS1216E-mediated plasmid integration in E. lactis, involving a replicative transposition. IMPORTANCE Until now, all the poxtA genes described in enterococci, including E. faecalis, E. faecium, and E. hirae, are plasmid-borne, suggesting that plasmids play an important role in the dissemination of the poxtA gene among enterococci. This study showed that the mobilizable poxtA-carrying plasmid could transfer with the help of conjugative plasmid in enterococci via plasmid fusion, with one generated by homologous recombination in E. faecalis, and another by replicative transposition in E. lactis. During both the fusion events, the poxtA-carrying plasmids changed from nonconjugative to conjugative, leading to the generation and enhanced dissemination of the larger phenicol-oxazolidinone-tetracycline resistance-encoding plasmids in enterococci.

IMPORTANCE Until now, all the poxtA genes described in enterococci, including E. faecalis, E. faecium, and E. hirae, are plasmid-borne, suggesting that plasmids play an important role in the dissemination of the poxtA gene among enterococci. This study showed that the mobilizable poxtA-carrying plasmid could transfer with the help of conjugative plasmid in enterococci via plasmid fusion, with one generated by homologous recombination in E. faecalis, and another by replicative transposition in E. lactis. During both the fusion events, the poxtA-carrying plasmids changed from nonconjugative to conjugative, leading to the generation and enhanced dissemination of the larger phenicol-oxazolidinone-tetracycline resistance-encoding plasmids in enterococci.
KEYWORDS enterococci, linezolid resistance, poxtA, plasmid integration L inezolid is a one of the last therapeutic options for the treatment of clinical infections caused by MDR Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP). Florfenicol is a broad-spectrum phenicol drug exclusively used in animals to control respiratory tract infections (1,2). However, the emergence of cross-resistance to linezolid and florfenicol poses a serious challenge to both human and veterinary medicine. To date, at least seven acquired linezolid/florfenicol resistance genes, belonging to three different groups, have been reported, including cfr, cfr(B), cfr(C), cfr(D), cfr(E), optrA, and poxtA (3)(4)(5)(6)(7)(8)(9)(10). The cfr gene and its variants code for 23S rRNA methylases, while the optrA and poxtA genes encode the ribosome-protective proteins of the ABC-F family. The cfr gene confers resistance to phenicols, lincosamides, oxazolidinones (linezolid but not tedizolid), pleuromutilins, and streptogramin A (the socalled PhLOPSA phenotype). It was first described in Staphylococcus sciuri (recently reclassified as Mammaliicoccus sciuri) and thereafter detected in various Gram-positive and Gram-negative bacteria. Unlike cfr, the optrA gene confers resistance to tedizolid, a novel oxazolidinone, in addition to linezolid and phenicol resistance. It was first identified in Enterococcus faecalis and E. faecium (4) but has also been detected in various other Gram-positive bacteria (11). The poxtA gene, which confers decreased susceptibility to tetracycline in addition to phenicol and oxazolidinone resistance, has recently been identified in MRSA and enterococci (9)(10)(11)(12)(13)(14). More recently, poxtA2 was detected in Enterococcus gallinarum (15).
The prevalence of the poxtA gene has been investigated in enterococci from foodproducing animal farms in China, Italy, and Korea, as well as in linezolid-resistant Enterococcus spp. (LRE) from patients in Ireland, Spain, and France (13,(16)(17)(18)(19)(20)(21)(22). In China, Lei et al. reported that the poxtA gene was present in 5.5% (19/345) of enterococci isolated from 36 food-producing animal farms across 12 provinces in 2017 (16). However, in our previous study, the poxtA gene was present in 57.9% (66/114) of florfenicol-resistant enterococci isolated from two swine farms in Henan Province in 2018, possibly the result of widespread use of florfenicol and doxycycline in these swine farms (13). In Italy, Fioriti et al. reported the poxtA gene with the prevalence of 20.7% (30/145) in florfenicol-resistant enterococci isolated from swine fecal samples (17). In Korea, 156 linezolid-resistant isolates were detected in 5,482 enterococci isolated from food-producing animals during 2008 to 2018, and 23.1% of these (36/156) had the poxtA gene (18). In addition, Kim et al. reported the poxtA gene with a prevalence of 2.5% (8/ 327) in enterococci isolated from food-producing animals and meat in Korea in 2018 (19). In Ireland, 15 poxtA-carrying enterococci were identified in 154 LRE which were recovered from patients in 14 hospitals between June 2016 and August 2019 (20). In Spain, clinical LRE isolates from different hospitals were submitted to the Spanish Reference Laboratory from 2015 to 2018, and 6.2% of them (6/97) were poxtA-positive (21). In France, out of 466 LRE received by the National Reference Centre for Enterococci from French hospitals between 2016 and 2020, 47 (10.1%) were poxtA-positive (22).
Previously, all of the poxtA genes described in enterococci have been plasmidborne, suggesting that plasmids play an important role in the dissemination of the poxtA gene among enterococci (22,23). In this study, two mobilizable poxtA-carrying plasmids were identified in E. faecalis and E. lactis, respectively. In addition, two fusion events involving homologous recombination and replicative transposition were investigated, which transferred the poxtA gene from mobilizable plasmids into conjugative plasmids. Consequently, continuous monitoring of poxtA-mediated linezolid resistance in enterococci is urgently needed.

RESULTS
Linezolid resistance mediated by plasmids in enterococci was transferable. The florfenicoland linezolid-resistant E. faecalis E006 (ST692) and E. lactis E843 were poxtApositive. Conjugation assays showed that the poxtA gene in E. faecalis E006 and E. lactis E843 could be transferred to the recipient strains E. faecalis JH2-2 (ST8) and E. faecium GE-1 (ST515), respectively. The corresponding transconjugants were designated E. faecalis E006ÂJH2-2-TC1 (ST8) and E. faecium E843ÂGE-1-TC1 (ST515), respectively. Antimicrobial susceptibility testing (AST) results for the donor strains, their transconjugants, and the recipient strains are shown in Table 1. Both transconjugants displayed elevated MICs of the respective antimicrobial agents compared with those of the recipient strain. S1-PFGE (pulsed-field gel electrophoresis) showed that there was a single plasmid in both the transconjugant E. faecalis E006ÂJH2-2-TC1 and E. faecium E843ÂGE-1-TC1, but two plasmids in their parental strains (Fig. 1). Moreover, the size of the plasmids in the transconjugants was almost equivalent to that of one of the plasmids in their parental strains. Southern blot hybridization confirmed that the poxtA gene was located on the smaller plasmid in the parental strains and on the single plasmid in the transconjugants (Fig. 1). To investigate how the poxtA gene could be transferred from the small plasmid in the parental strain to the larger plasmid in the transconjugants, whole-genome DNA sequencing was performed for all four stains (E. faecalis E006, E. lactis E843, transconjugants E. faecalis E006ÂJH2-2-TC1, and E. faecium E843ÂGE-1-TC1). In addition, the larger plasmids in E. faecalis E006ÂJH2-2-TC1 and E. faecium E843ÂGE-1-TC1 could further be transferred to the recipient strains E. faecalis E533 (ST69) and E. faecalis JH2-2 at frequencies of 1.7 Â 10 29 and 1.4 Â 10 26 , respectively, indicating that the poxtA-carrying plasmids can change from mobilizable to conjugative and become self-transferable.
Genetic basis of transmission of linezolid resistance in E. faecalis. For the two plasmids in E. faecalis E006, the larger plasmid was designated pE006-101 and the smaller one was designated pE006-19. The conjugative plasmid pE006-101 is 101,692 bp in size and harbors three copies of the macrolide-lincosamide-streptogramin B resistance gene erm(B) and one copy of erm(A). In addition, it also carries single copies of the florfenicol/ oxazolidinone resistance gene optrA, the lincosamide resistance gene lnu(B), the trimethoprim resistance gene dfrG, the gentamicin-tobramycin-kanamycin resistance gene aac(A)aph(D), the streptomycin resistance gene aadE, and the bacitracin resistance operon bcrABDR. Moreover, like the previously described optrA-carrying pheromone responsive  plasmid pEF10748, pE006-101 is a rep9-type plasmid (pCF10 prototype, the best-studied pheromone responsive plasmid) by plasmid typing, and harbors the essential sex pheromone response genes, such as prgA, prgB, and prgC ( Fig. 2A; 24). The mobilizable plasmid pE006-19 is 19,832 bp in size and carries the florfenicol resistance gene fexB, the gene poxtA, and bcrR, which is a part of the bacitracin resistance operon bcrABDR ( Fig. 2A). Only one conjugative plasmid, 121,537 bp in size and designated pE006-TC-121, was detected in the transconjugant E006ÂJH2-2-TC1. Sequence analysis revealed that pE006-TC-121 was obtained through fusion between pE006-101 and pE006-19 (Fig. 2). Detailed analysis of these three plasmids suggested that the mechanism of plasmid fusion was due to homologous recombination involving an 864-bp homologous region (HR) which included bcrR. One copy each of this HR was detected in pE006-101 and pE006-19, but two copies were detected in pE006-TC-121. The HRs in all three plasmids were identical. According to these results, we propose that pE006-101 and pE006-19 underwent homologous recombination at the HR, resulting in the formation of the fusion plasmid pE006-TC-121 (Fig. 3).
Genetic basis of transmission of linezolid resistance in E. lactis. A plasmid fusion event was also observed in E. lactis E843. There are two plasmids in the parental strain, designated pE843-171 and pE843-27, as well as one plasmid in the transconjugant E843ÂGE-1-TC1, designated pE843-200. Plasmid pE843-171 is 171,930 bp in size and harbors two copies of the streptomycin resistance gene aadE, as well as single copies of the macrolide-lincosamide-streptogramin B resistance gene erm(B), the neomycin-kanamycin resistance gene aphA3, the lincosamide resistance gene lnu(B), the pleuromutilin-lincosamide-streptogramin A (PLS A ) resistance gene lsa(E), and the spectinomycin resistance gene spw. All resistance genes were clustered in an ;14-kb region. pE843-171 was found to be a pLG1-like conjugative plasmid with essential type IV secretion system (T4SS) genes (25), such as virB1, virB4, and virD4 homologues (Fig. 4A). The mobilizable plasmid pE843-27 is 27,847 bp in size and carries the florfenicol resistance genes poxtA and fexB and the tetracycline resistance genes tet(M) and tet(L). In pE843-27, there are a total of six copies of IS1216E, with five of them located in the same orientation (Fig. 4A).
As in E006ÂJH2-2-TC1, there was only one conjugative plasmid in the transconjugant E843ÂGE-1-TC1. This plasmid, designated pE843-TC-200, is 200,566 bp in size (Fig. 4A). Based on the detailed sequence analysis, the formation of this fusion plasmid was due to a series of genetic events generated by IS1216E. We propose that IS1216E in the mobilizable plasmid pE843-27 attached to the target site (AAATCTCT) in the pLG1-like plasmid pE843-171; then, the replicative transposition occurred, and pE843-27 was inserted into pE843-171, forming a characteristic 8-bp target site duplication (TSD, 59-AAATCTCT-39) and generating an additional copy of IS1216E in the fusion plasmid pE843-200 ( Fig. 4 and 5).

DISCUSSION
Enterococci are opportunistic pathogens which have become one of the main causes of nosocomial and community-acquired human infections, including septicemia, endocarditis, and urinary tract infections (26). Their intrinsic resistances and ability to acquire resistances through mobile genetic elements (MGEs) have compromised the choice of therapeutic options to treat enterococcal infections. Among MGEs, conjugative plasmids play a key role in the dissemination of resistance genes, as conjugative plasmids are self-transmissible and can mobilize nonconjugative plasmids present in the same enterococcal cell (27).
In Gram-negative bacteria, IS26-mediated replicative transposition and translocation play an important role in mobilizing antimicrobial resistance genes (28,29). IS1216 in Gram-positive bacteria and IS26 in Gram-negative bacteria belong to the IS6/IS26 family of bacterial insertion sequences and are assumed to exert similar functions. To our knowledge, the genetic environment of the poxtA gene, as described previously, contains IS1216E (23), suggesting its potential role in the dissemination of the poxtA gene. However, how IS1216E promotes the transmission of the poxtA gene is not yet fully explored.
In our previous study, the role of IS1216E-mediated transposition and translocation was shown in the formation of poxtA fusion plasmids (30). By IS1216E-mediated replicative transposition, a fusion poxtA-carrying plasmid was generated through a poxtA-carrying transposon fused into another small plasmid. Moreover, a new, larger fusion plasmid was formed via IS1216E-mediated translocation, generated by the integration of an IS1216E-based transmissible unit into the mobilizable poxtA-carrying plasmid (30). In both cases, the fused poxtA plasmids could be mobilized with the aid of conjugative plasmids in the same strains.
Transmissible plasmids can be classified as conjugative or mobilizable plasmids. In order to realize the transfer of the mobilizable plasmids, at least two processes must be taken into account: (i) the mobilizable plasmids can be mobilized by functions encoded in trans provided by other auxiliary conjugative elements, where a distinct relaxase gene expressing a Mob protein on the majority of mobilizable plasmids targets an oriT and the resulting relaxosome is recruited to the T4SS encoded by a coresident conjugative element (27), as observed in our previous study (30); or (ii) the mobilizable plasmids could be directly fused to conjugative plasmids by IS-mediated replicative transposition or homologous recombination through an identical HR, which caused the mobilizable plasmids to become conjugative, as observed in this study.
Several studies have shown that replicative transposition or homologous recombination played an important role in the evolution of plasmid backbones and the acquisition of antimicrobial resistance genes in Gram-negative bacteria (28,29,(31)(32)(33). To the best of our knowledge, this is the first time that the roles of replicative transposition and homologous recombination were shown in the shaping of poxtA-carrying plasmids from mobilizable to conjugative in enterococci, which are important health care-associated Gram-positive opportunistic pathogens. Because the mobilizable plasmids are not self-transmissible, their transmission usually requires the assistance of conjugative plasmids or other conjugative elements. In this study, replicative transposition and homologous recombination have been shown to participate in changing two mobilizable poxtA-carrying plasmids from nonconjugative to conjugative; as a result, these fused poxtA-carrying plasmids become self-transmissible, which accelerates their transmission.
Pheromone responsive plasmid transfer systems are highly efficient for genetic exchanges that allow antimicrobial resistance genes to be spread within bacterial populations (34). In our previous study, two pheromone-responsive conjugative optrA-carrying plasmids which showed high transfer frequencies of ;10 22 were identified in porcine E. faecalis isolates (35). In a recent study, pheromone-responsive plasmids were proven to be the predominant plasmids in optrA-carrying E. faecalis clinical isolates, and most of them were conjugative (24). In this study, the conjugative optrA-carrying pheromone-responsive plasmid pE006-101 fused with the mobilizable poxtA-carrying plasmid, suggesting that linezolid resistance co-conferred by both optrA and poxtA genes in the same plasmid could be widely disseminated through sex pheromone plasmids.
In summary, this study showed that mobilizable poxtA-carrying plasmids can transfer through fusion into conjugative plasmids in enterococci. Two fusion events were identified: one through homologous recombination via an identical HR, the other through replicative transposition mediated by IS1216E. In both fusion events, the poxtA-carrying plasmids changed from mobilizable to conjugative, leading to the generation of new plasmids and the potentially enhanced dissemination of the poxtA gene in enterococci.

MATERIALS AND METHODS
Bacterial strains and antimicrobial susceptibility testing. E. faecalis E006 and E. lactis E843 were isolated from fecal samples during a routine survey of antimicrobial resistance in bacteria from pigs in Henan Province, China, in 2018. Strain species of E843 and E006 were identified by PCR with 16s rRNA primers, followed by sequencing. E. faecalis JH2-2 (RIF r ), E. faecium GE-1 (RIF r ), and E. faecalis E533 (a strain without plasmids, identified and stored in our lab, TET r ) served as recipient strains in transfer experiments. AST was performed using broth microdilution according to the recommendations given in the M100 (31st ed.) of the Clinical and Laboratory Standards Institute (CLSI; 36). S. aureus ATCC 29213 served as the quality control strain.
PCR analysis and transfer experiments. The presence of the resistance gene poxtA in E. faecalis E006 and E. lactis E843 was determined by PCR using the primers described previously (13). All PCR products were subjected to Sanger sequencing. Conjugation experiments were performed as previously described (37). In brief, the overnight cultures of donors and recipients were mixed at a ratio of 1:4 (200:800 mL), centrifuged, and suspended with 100 mL fresh brain heart infusion (BHI) broth; the mixed bacteria were then plated on BHI agar at 37°C for 18 h. The bacteria grown on the BHI agar were sluiced off with 4 mL BHI broth, and 100 mL of the culture was plated on selective BHI agar. When E. faecalis E006 was used as the donor, E. faecalis JH2-2 was used as the recipient; when E. lactis E843 was used as the donor, E. faecium GE-1 served as the recipient. Transconjugants were selected on BHI agar containing rifampicin (50 mg/L) and florfenicol (10 mg/L) and further confirmed by AST and multilocus sequence typing (MLST) following harmonized protocols (https://pubmlst.org/). The positive transconjugants were E. faecalis E006ÂJH2-2-TC1 and E. faecium E843ÂGE-1-TC1, respectively. To assess the selftransferability of the fusion plasmids, further conjugation experiments were performed. When E. faecalis E006ÂJH2-2-TC1 was used as the donor, E. faecalis E533 (TET r ) was used as the recipient, and the transconjugants were selected on BHI agar containing tetracycline (10 mg/L) and florfenicol (10 mg/L). When E. faecium E843ÂGE-1-TC1 was used as the donor, E. faecalis JH2-2 [VAL r , natural resistance due to the presence of lsa(A)] served as the recipient, and the transconjugants were selected on BHI agar containing valnemulin (64 mg/L) and florfenicol (4 mg/L). All transconjugants were further confirmed using the methods described above. S1-PFGE and Southern blot hybridization. The genomic DNA was digested with S1 endonuclease (New England Biolabs, Beverly, MA, USA) separated by PFGE as previously described (38). After transfer to Amersham Hybond-N 1 membranes (GE Healthcare), the genomic DNA was hybridized with a poxtA probe as described in other studies (30).