Prevalence and Characteristics of Phenicol-Oxazolidinone Resistance Genes in Enterococcus Faecalis and Enterococcus Faecium Isolated from Food-Producing Animals and Meat in Korea

The use of phenicol antibiotics in animals has increased. In recent years, it has been reported that the transferable gene mediates phenicol-oxazolidinone resistance. This study analyzed the prevalence and characteristics of phenicol-oxazolidinone resistance genes in Enterococcus faecalis and Enterococcus faecium isolated from food-producing animals and meat in Korea in 2018. Furthermore, for the first time, we reported the genome sequence of E. faecalis strain, which possesses the phenicol-oxazolidinone resistance gene on both the chromosome and plasmid. Among the 327 isolates, optrA, poxtA, and fexA genes were found in 15 (4.6%), 8 (2.5%), and 17 isolates (5.2%), respectively. Twenty E. faecalis strains carrying resistance genes belonged to eight sequence types (STs), and transferability was found in 17 isolates. The genome sequences revealed that resistant genes were present in the chromosome or plasmid, or both. In strains EFS17 and EFS108, optrA was located downstream of the ermA and ant(9)-1 genes. The strains EFS36 and EFS108 harboring poxtA-encoding plasmid cocarried fexA and cfr(D). These islands also contained IS1216E or the transposon Tn554, enabling the horizontal transfer of the phenicol-oxazolidinone resistance with other antimicrobial-resistant genes. Our results suggest that it is necessary to promote the prudent use of antibiotics through continuous monitoring and reevaluation.


Introduction
Enterococci are symbiotic bacteria of the gastrointestinal tract of humans and animals [1]. However, enterococci infection can cause urinary tract and soft tissue infections, and in severe cases, it causes life-threatening infections such as septicemia and meningitis [2]. Enterococci of animal origin have the potential risk of transferring their antimicrobial resistance genes to human enterococci or disseminating to humans through the food chain [3]. Enterococci are also known as nosocomial pathogens, and Enterococcus species, especially Enterococcus faecalis and E. faecium, have become a particular clinical problem [1,4,5]. The use of florfenicol, phenicol antibiotics, has been increasing in animals to treat diseases caused by E. faecalis and E. faecium infections [5].
Linezolid, which belongs to oxazolidinone, is classified as a critically important category of antibiotics by the World Health Organization, meaning that it is a major antibiotic to pigs (4/22) and fresh meat products including beef (2/22) and pork (3/22). Enterococci harboring multiple phenicol-oxazolidinone resistance genes were identified. Thirteen, four, and one E. faecalis isolates were shown to carry optrA and fexA, poxtA and fexA, and optrA, poxtA, and fexA, respectively. On the other hand, one E. faecalis had optrA only, and two of each E. faecium and E. faecalis isolates harbored poxtA only.
indicates the presence of phenicol-oxazolidinone resistance genes or transferability; −, indicates absence of resistance genes or not transferability.
The minimum inhibitory concentration (MIC) of three linezolid-resistant strains was confirmed to be 8 mg/L, and the MIC of five linezolid-intermediate strains was determined to be 4 mg/L ( Table 2). Among the florfenicol-resistant strains, a breakpoint of 16 mg/L or higher was confirmed in 21 strains, and of these, seven strains had a high MIC of 64 mg/L.

Transferability of Phenicol-Oxazolidinone Resistance Genes
The transferability of optrA, poxtA, and fexA genes was tested by the broth-mating method. It was confirmed that the resistance gene was transferred in 17 out of 22 (77%) enterococcal isolates. In two E. faecium strains, gene transfer was not identified. As shown Figure 1. Graphical presentation of multilocus sequence typing data. Each ST analyzed in this study marked in red. The lines connect single-locus variants. The clonal relatedness between sequence types (STs) was analyzed against the entire E. faecalis database. Clonal complexes (CCs) are indicated. Eight sequence types (STs) were identified among the 20 E. faecalis isolates: ST593, ST100, ST16, ST27, ST338, ST47, ST585, and ST915. The eBURST algorithm clustered the STs into one singleton and six clonal complexes: CC100, CC16, CC27, CC4, CC47, and CC915.

Transferability of Phenicol-Oxazolidinone Resistance Genes
The transferability of optrA, poxtA, and fexA genes was tested by the broth-mating method. It was confirmed that the resistance gene was transferred in 17 out of 22 (77%) enterococcal isolates. In two E. faecium strains, gene transfer was not identified. As shown in Table 1, each of the optrA, poxtA, and fexA genes possessed by each donor strain was successfully delivered to the recipient strain.

Genome Features of Three Isolates
Complete genome sequencing was performed to identify the underlying antimicrobial resistance mechanisms. Genome analysis showed that the EFS17 strain consists of one chromosome and one plasmid, and EFS36 and EFS108 strains had two and five plasmids, respectively ( Table 4). The genomic sizes of the strains EFS17, EFS36, and EFS108 consisted of 2,914,315 bp, 3,095,754 bp, and 3,093,936 bp with a GC content of 37.4%, 37.3%, and 37.3%, respectively. The genomic features of the EFS17 strain included 2803 coding genes and 73 coding regions for RNAs, of which 61 were transfer RNAs (tRNAs). Meanwhile, the EFS36 genome contained 3057 coding sequences and 78 coding regions for RNAs, including 66 tRNAs. The EFS108 genome displayed 2980 coding genes and 73 coding regions for RNAs, of which 61 were tRNAs.

In Silico Identification of Antimicrobial Resistance Genes
A blastn search for known resistance genes revealed that three strains possessed the phenicol-oxazolidinone resistance optrA gene or poxtA gene; two strains also carried the cfr(D) gene ( Figure 2). EFS17 and EFS108 strains harbored the optrA gene, whereas EFS36 and EFS108 strains had the cfr(D) and poxtA genes. The phenicol-oxazolidinone resistance genes were present on the chromosome in the EFS17 strain; for the EFS36 strain, this gene was present on the plasmid (pEFS36_2). In the EFS108 strain, phenicol-oxazolidinone resistance genes were present in both the chromosome and plasmid (pEFS108_1) (Figure 2).

In Silico Identification of Antimicrobial Resistance Genes
A blastn search for known resistance genes revealed that three strains possessed phenicol-oxazolidinone resistance optrA gene or poxtA gene; two strains also carried cfr(D) gene ( Figure 2). EFS17 and EFS108 strains harbored the optrA gene, whereas EF and EFS108 strains had the cfr(D) and poxtA genes. The phenicol-oxazolidinone resista genes were present on the chromosome in the EFS17 strain; for the EFS36 strain, this g was present on the plasmid (pEFS36_2). In the EFS108 strain, phenicol-oxazolidinone sistance genes were present in both the chromosome and plasmid (pEFS108_1) ( Figure   Figure 2. Schematic representation of phenicol-oxazolidinone resistance genes loci in three E. faecalis genomes. Genes and their orientation are shown with arrows and labeled; red, green, black, blue, and gray indicate antibiotic resistance gene, insertion sequences (ISs), transposase, known proteins, and hypothetical proteins, respectively. Csps, cold shock protein of CSP family; ecm18, class I SAM-dependent methyltransferase; radC, DNA repair protein RadC; folC, dihydrofolate synthase; yrkL, NAD(P)H oxidoreductase YRKL. The EFS17 strain harbored optrA and fexA phenicol resistance genes. In the EFS17 strain, the fexA gene was encoded 689 bp upstream of the optrA gene, whereas the EFS108 strain lacked fexA (Table S1). Transposon Tn554 was detected downstream of optrA in EFS17 and EFS108 strains ( Figure 2). Moreover, the ermA and ant(9)-I genes conferring resistance to erythromycin and spectinomycin antibiotic were located upstream of the optrA genes in both EFS17 and EFS108 isolates. Additionally, analysis of complete genome sequence revealed that the EFS17 strain included additional genes for resistance to macrolide-lincosamide-streptogramin B (ermA, ermB, lunB, lasA, and lasE), aminoglycosides (aac(6 )-aph(2"), aph(3 )-III, str, and ant(6)-Ia), and tetracyclines (tetM and tetL) ( Table 5). EFS36 and EFS108 strains harbored a poxtA-carrying plasmid named pEFS36_2 and pEFS108_1, respectively (Figure 3). Plasmid pEFS36_2 was 35,757 bp in length with a GC content of 34.3%. Plasmid pEFS108_1 was 97,455 bp in length with a GC content of 33.8%. These plasmids coharbor fexA, cfr(D), and IS1216E, which were identified in two or three places. Therefore, EFS17, EFS36, and EFS108 contained several mobile gene elements and transposase-associated genes, enabling the horizontal transfer of the phenicoloxazolidinone resistance genes. The EFS17 strain harbored optrA and fexA phenicol resistance genes. In the EFS17 strain, the fexA gene was encoded 689 bp upstream of the optrA gene, whereas the EFS108 strain lacked fexA (Table S1). Transposon Tn554 was detected downstream of optrA in EFS17 and EFS108 strains ( Figure 2). Moreover, the ermA and ant(9)-I genes conferring resistance to erythromycin and spectinomycin antibiotic were located upstream of the op-trA genes in both EFS17 and EFS108 isolates. Additionally, analysis of complete genome sequence revealed that the EFS17 strain included additional genes for resistance to macrolide-lincosamide-streptogramin B (ermA, ermB, lunB, lasA, and lasE), aminoglycosides (aac(6′)-aph(2″), aph(3′)-III, str, and ant(6)-Ia), and tetracyclines (tetM and tetL) ( Table 5). EFS36 and EFS108 strains harbored a poxtA-carrying plasmid named pEFS36_2 and pEFS108_1, respectively ( Figure 3). Plasmid pEFS36_2 was 35,757 bp in length with a GC content of 34.3%. Plasmid pEFS108_1 was 97,455 bp in length with a GC content of 33.8%. These plasmids coharbor fexA, cfr(D), and IS1216E, which were identified in two or three places. Therefore, EFS17, EFS36, and EFS108 contained several mobile gene elements and transposase-associated genes, enabling the horizontal transfer of the phenicol-oxazolidinone resistance genes.

Discussion
Knowledge of the distribution of antimicrobial-resistant strains in the food chain and food animals is important in determining the potential risk to human health [13]. Here, we investigated the prevalence and genetic characterization of phenicol-oxazolidinone resistance genes to understand better the phenicol-oxazolidinone resistance profiles of enterococci isolates obtained from food animals, animal carcass, and meat in Korea. Moreover, for the first time in Korea, this study reports the complete genome sequence of the E. faecalis EFS108 strain harboring phenicol-oxazolidinone resistant genes on both the chromosome and the plasmid.
Overall, 6.7% (22/327) of the enterococci isolates tested harbored phenicol-oxazolidinone resistance genes. Among them, optrA, poxtA, and fexA were detected in 15 isolates (4.6%), 8 isolates (2.5%), and 17 isolates (5.2%), respectively, confirming that these genes were distributed at a low level. Consistent with this study, a low phenicol-oxazolidinone resistance rate was reported in E. faecalis and E. faecium from food animals and animal carcasses in Korea [1,3,14,15], China [16], and Europe [17]. In a previous study, enterococci strains with phenicol-oxazolidinone resistance genes were isolated at slightly higher rates in E. faecalis than E. faecium, and the optrA gene was confirmed only in E. faecalis isolates [18]. Our results are congruent with reports from previous studies where optrA is predominantly present in E. faecalis, and the reason is presumed that the loss of conjugated resistance plasmids occurs in E. faecium [4,19]. Moreover, none of the phenicol-oxazolidinone resistant enterococci strains carried the cfr gene, but the cfr(D) gene was confirmed through complete genome sequencing in two E. faecalis isolates (EFS36 and EFS108). Therefore, when monitoring the phenicol-oxazolidinone resistance gene, it will be necessary to examine the cfr gene and its variants. The reason is that horizontal gene transfer is possible between cfr gene-carrying strains. In addition to the cfr gene, variants cfr(B), cfr(C), and cfr(D) have been reported in clinical isolates [9,10].
Twenty-two enterococcal isolates harboring phenicol-oxazolidinone resistance genes were resistant to one or more antimicrobial agents. Multidrug-resistant isolates were observed in both E. faecalis (95%) and E. faecium (100%) with high frequencies. A high resistance rate was observed for quinupristin/dalfopristin, florfenicol, tetracycline, erythromycin, and tylosin. Antimicrobial patterns were similar to those of other food animals. The high-level resistance to phenicols, tetracycline, and macrolides (erythromycin and tylosin) was also found in food animals and their carcass in Korea [1] and European countries [20]. These antimicrobial agents are generally administered to food animals in Korea [1]. Resistance to linezolid was observed in three E. faecalis isolates but not in E. faecium. All linezolid-resistant E. faecalis isolates were also resistant to florfenicol and chloramphenicol.
In the MLST analysis to investigate the clonal relationship of phenicol-oxazolidinone resistance strains, the STs of enterococci isolated from meat, slaughterhouses, and farms could be classified into eight different types. Consistent with this study, the diversity of STs was also reported in enterococci isolated from food animals and animal carcass in Korea [3,8] and humans and animals in China [21]. Some E. faecalis strains isolated from different samples had optrA and fexA genes commonly, and the ST was the same as 593. From this result, resistance could be acquired through antibiotics for animals such as florfenicol in the farm. It can be estimated that resistance genes optrA and fexA may be spread to animals within the farm through feed or the farm environment [1]. ST16 was identified in three optrA-positive E. faecalis; two isolates cocarried fexA, and one isolate has only optrA. This ST has been identified in food animals and humans in Korea and other countries and is known as an optrA-carrying clone with a worldwide distribution [11,19]. In addition, EFS17 isolated from pork was identified as ST585, which coharbored optrA and fexA. ST585 E. faecalis harboring optrA has also been reported in the United States, China, and Germany [11].
Complete genome sequencing was performed on three E. faecalis isolates (EFS17, EFS36, and EFS108), which have different combinations of phenicol resistance genes, to investigate the location and genetic environment of optrA and poxtA. According to a report investigating the genetic environment of optrA-carrying plasmid in E. faecalis originated from humans and animals, the IS1216E element was detected either upstream or downstream of the optrA gene [22]. In addition, fexA and erm(A)-related genes were found upstream and downstream of optrA, respectively [22]. Conversely, when optrA is located in the chromosome, the optrA gene was located immediately downstream of the transcriptional regulator gene araC or downstream of the fexA gene [22]. Alternatively, the integration of the optrA gene was observed between Tn558 and Tn554 relics located in the chromosomal radC gene [22]. The radC gene is the common integration site for transposons of the Tn554 family [22]. Our study found that the optrA gene was located on the EFS17 and EFS108 genomes but not in the EFS36 genome. As in a previous study [15], the optrA gene on the EFS108 chromosome was identified downstream of araC, whereas the optrA gene on the EFS17 chromosome was found downstream of fexA. Moreover, the chromosomal radC gene was found in both EFS17 and EFS108.
Phenicol-oxazolidinone resistance has been reported to be mediated by the transferable phenicol-oxazolidinone resistance gene in recent years [1,16]. The optrA gene in strains EFS36 and EFS108 was located adjacent to Tn554 transposon. Tn554 was also found upstream of the optrA gene in other Enterococcus strains and Staphylococcus sciuri [23]. The functionally active Tn554 can be excised from their host DNA and generate circular forms that precede the integration of the transposon into a target sequence [23,24]. A similar genetic arrangement of optrA and Tn554 was identified in enterococci and staphylococci [13,16], suggesting that optrA can be mediated for transfer between different bacterial genus and species. The optrA gene was flanked by transposon or insertion sequences, indicating that a mobile genetic element mediates the horizontal transfer of optrA between bacteria of different genera, which should be given more attention to avoid disseminating the oxazolidinone resistance gene in the environment.
The poxtA gene was located on plasmids in two enterococci strains. The poxtA gene is often surrounded by bacteria insertion sequences on the plasmid in staphylococci or enterococci strains [13,16]. Our data showed that all poxtA found on plasmids were flanked by IS1216E, similar to the previous studies [9,16,25]. IS1216E belongs to the IS6 family of the bacteria insertion sequence, which among other things mediates transmission of the macrolide-lincosamide-streptogramin B resistance genes erm(B) in E. hirae, the oxazolidinone resistance gene cfr in E. faecalis, vancomycin resistance gene vanA in E. faecium, and tetracycline resistance gene tetS in Streptococcus infantis [23,[26][27][28]. This indicates that poxtA can be transferred between different bacterial genera by IS-mediated recombination events, which plays an important role in disseminating antimicrobial resistance genes. Furthermore, two plasmids (pEFS36_2 and pEFS108_1) carrying poxtA gene also coharbored other resistance genes, such as fexA and cfr(D). This result suggests that poxtA together with fexA and cfr(D) genes may be disseminated among different strains via IS1216E.
The transferability of phenicol-oxazolidinone resistance genes was confirmed through an experiment by broth mating and a genetic context analysis using complete genome sequencing. The transferable phenicol-oxazolidinone resistant genes, optrA and poxtA, were detected by the broth mating method in 12 and four E. faecalis isolates. All of them, except for two isolates harboring poxtA, cocarried the phenicol exporter gene fexA. These results were also confirmed by whole-genome sequencing analysis. Genome analysis showed that transposons or insertion sequences mediating resistance genes were adjacent to optrA, poxtA, and fexA genes.
In conclusion, although the occurrence of a phenicol-oxazolidinone resistance gene in enterococci is still rare among food animals, a high rate of transferable phenicoloxazolidinone genes was observed in these strains. The emergence of these genes in enterococci isolates from food animals is a serious problem, as they can be transmitted to humans through the food chain. The spread of these genes can significantly limit the treatment against multidrug-resistant bacteria. Hence, active surveillance of phenicol-oxazolidinone-resistant bacteria and related resistance genes is essential to prevent the spread of resistant enterococci isolates.

Enterococcus Strains
In 2018, 282 E. faecalis strains and 45 E. faecium strains were isolated in Korea from 43 meat (19 beef and 24 pork), 24 slaughterhouses, and 16 farms. In detail, 128 E. faecalis and 9 E. faecium strains were isolated from meat, and 48 E. faecalis and 13 E. faecium strains were isolated from the slaughterhouse. The remaining 106 E. faecalis and 23 E. faecium strains were isolated from the farm. All isolates were used in the experiment after being identified as enterococci using VITEK ® MS (bioMérieux, Marcy l'Etoile, France). All enterococci strains were cultured on tryptone soya agar (TSA) medium at 37 • C for 24 h.

Screening of Phenicol-Oxazolidinone Resistance Gene by Polymerase Chain Reaction (PCR)
Genomic DNA from Enterococcus isolates was extracted using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The presence or absence of the phenicol-oxazolidinone resistance genes, such as optrA, poxtA, cfr, and fexA, in the isolates was detected by PCR according to the previous studies [9,24]. The primer sequences of each gene are shown in Table 6. The PCR mixture (25 µL) contained 1 µL of each primer (0.4 µM), 0.1 µL of Taq polymerase (5 U/µL), 2.5 µL of 10× buffer, 16.4 µL of distilled water, and 2 µL of template DNA. Amplification for the optrA, poxtA, and cfr genes was performed at 95 • C for 2 min, and 25 cycles of 95 • C for 15 s, 53 • C for 15 s, 68 • C for 1 min, and final elongation at 68 • C for 5 min. PCR conditions for the fexA gene were as follows: initial denaturation at 94 • C for 5 min, 35 cycles at 94 • C for 1 min, 57 • C for 1 min, 72 • C for 1.5 min, and final elongation at 72 • C for 5 min. The PCR-positive control DNA for optrA, poxtA, cfr, and fexA was kindly provided from the Animal and Plant Quarantine Agency in Korea.

Antimicrobial Susceptibility Testing
An antimicrobial susceptibility test was performed on the 22 E. faecalis and E. faecium isolates harboring at least one of the phenicol-oxazolidinone resistance genes optrA, poxtA, cfr, and fexA. The MIC of 16 antimicrobial agents was determined by the broth microdilution method using commercially available Sensititre1 panel KRVP2F (TREK Diagnostic Systems, West Sussex, UK), according to the manufacturer's instructions. Staphylococcus aureus ATCC 29213 and E. faecalis ATCC 29212 were used as the quality control strains. The interpretation of the results followed the Clinical and Laboratory Standards Institute guidelines [29]. Sixteen antibiotics used in this study were as follows:

MLST Analysis
MLST analysis of 20 E. faecalis isolates harboring phenicol-oxazolidinone resistance genes was performed according to a previous study [30]. Seven housekeeping genes, such as gdh, gyd, pstS, gki, aroE, xpt, and yqiL, were amplified by PCR and then sequenced. Sequence types of E. faecalis were determined using the E. faecalis MLST database in PubMLST (http://pubmlst.org/efaecalis/, accessed on 22 September 2021). The clonal relationship between STs was analyzed by the goeBURST algorithm using the PHYLOViZ software v 2.0 [31].

Conjugation
Twenty-two E. faecalis and E. faecium isolates harboring phenicol-oxazolidinone resistance genes were used as the donor cells, and with Escherichia coli strain J53 served as the recipient strain for the conjugation experiments using methods previously described by Aljahdali et al. with minor modifications [32]. Briefly, each donor and recipient cells were cultured on TSA agar and incubated at 37 • C for 24 h. Cultured donor and recipient cells were mixed in 500 µL LB broth (1:1) and incubated at 37 • C for 3 h. Transconjugants were streaked on LB agar containing 200 mg/L sodium azide and incubated at 37 • C for 24 h. Several colonies were picked and plated on MacConkey agar at 37 • C for 24 h. Transconjugants from MacConkey agar were subcultured on TSA, incubated at 37 • C for 24 h, and subjected to PCR for the optrA, poxtA, cfr, and fexA genes, respectively, according to Table 6. 4.6. Complete Genome Sequencing 4.6.1. Genome Sequencing, Assembly, and Annotation Total genomic DNAs from three strains were extracted using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Libraries were prepared using the SMRTbell Express Template Prep kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA) and subsequently sequenced using PacBio Sequel IIe (Pacific Biosciences). Raw reads were processed using demultiplex barcodes protocol, which is part of SMRT Link version 10.1, to obtain clean reads. Clean data were assembled using the microbial assembly protocol in the SMRT Link software. Genome annotations were performed using Rapid Annotation using Subsystem Technology server version 2.0 with default parameters [33]. The complete genome sequences of E. faecalis EFS17, EFS36, and EFS108 were deposited in GenBank under the accession numbers SUB10526591, SUB10526592, and SUB10526593, respectively.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/ijms222111335/s1, Table S1: Location of antimicrobial resistance genes identified in the three E. faecalis genomes.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.