Identification of the Multiresistance Gene poxtA in Oxazolidinone-Susceptible Staphylococcus haemolyticus and Staphylococcus saprophyticus of Pig and Feed Origins

Previous studies on the prevalence and transmission mechanism of oxazolidinone resistance gene poxtA in CoNS are lacking, which this study addresses. By screening 763 CoNS isolates from different sources of several livestock farms in Guangdong, China, 2018–2020, we identified that the poxtA was present in seven CoNS isolates of pig and feed origins. Species identification and multilocus sequence typing (MLST) confirmed that seven poxtA-positive CoNS isolates were composed of five ST64-Staphylococcus haemolyticus and two Staphylococcus saprophyticus isolates. All poxtA-positive Staphylococcus haemolyticus isolates shared similar pulsed-field gel electrophoresis (PFGE) patterns. Transformation assays demonstrated all poxtA-positive isolates were able to transfer poxtA gene to Staphylococcus aureus RN4220. S1-PFGE and whole-genome sequencing (WGS) revealed the presence of poxtA-carrying plasmids in size around 54.7 kb. The plasmid pY80 was 55,758 bp in size and harbored the heavy metal resistance gene czcD and antimicrobial resistance genes, poxtA, aadD, fexB and tet(L). The regions (IS1216E-poxtA-IS1216E) in plasmid pY80 were identified in Staphylococcus spp. and Enterococcus spp. with different genetic and source backgrounds. In conclusion, this was the first report about the poxtA gene in Staphylococcus haemolyticus and Staphylococcus saprophyticus, and IS1216 may play an important role in the dissemination of poxtA among different Gram-positive bacteria.


Introduction
Coagulase negative staphylococci (CoNS) are one of the most common opportunistic pathogens found on human skin and mucous membranes as a component of normal flora [1,2]. Besides their role in keeping homeostasis, CoNS have been involved in a series of infectious processes, ranging from nosocomial infections to livestock bacterial sepsis and mastitis [3,4]. In addition to their virulence, the emergence of antibiotic resistance in CoNS and then horizontal dissemination among staphylococci should be alarming. The increasing drug resistance of CoNS significantly limited the treatment options [5,6]. Among CoNS, Staphylococcus haemolyticus is the second most frequently isolated from human blood culture and Staphylococcus saprophyticus is one of the most common pathogens responsible for community urinary tract infections [5,7]. Oxazolidinones such as linezolid and tedizolid are antibacterially active against Grampositive pathogens including methicillin-resistant Staphylococcus aureus (MRSA), methicillinresistant CoNS and vancomycin-resistant enterococci (VRE) [8]. However, the discovery of transferable oxazolidinone resistance genes such as cfr, cfr(B), cfr(C), optrA and poxtA as well as the mutations in 23S rRNA and ribosomal proteins L3 and L4 challenged the clinical use of oxazolidinones [9]. Worriedly, linezolid-resistant staphylococci have been detected worldwide [10]. The plasmid-mediated oxazolidinone resistance genes including cfr and optrA spread among a number of bacterial species of different origins around the world shortly after they were reported [11]. The fact that cfr and optrA genes can be selected by phenicols and other ribosomal-targeted drugs that are widely used in livestock and veterinary hospitals is closely associated with global spread of the resistance genes [12][13][14]. The recently described plasmid-mediated oxazolidinone resistance gene poxtA could decrease susceptibility to phenicols and tetracyclines, so the poxtA gene posed a threat to disseminate in bacteria from animal setting [15,16]. The poxtA gene has been identified in MRSA and Enterococcus strains of human and animal origins [15][16][17][18]. Livestock is widely recognized as a reservoir of antimicrobial resistance genes [19].
In this study, we described for the first time the identification and characterization of poxtA gene in S. haemolyticus and S. saprophyticus isolates from pig and chicken farms in Guangdong province, China.

Identification of poxtA Gene in CoNS Isolates
The poxtA gene was detected in seven CoNS isolates in 2018 including five S. haemolyticus isolates (GDY8P33P, GDY8P50P, GDY8P58P, GDY8P60P and GDY8P80P all of pig origin) and two S. saprophyticus isolates (GDY8P136P of pig origin, GDH8C97P of feed origin) ( Table 1).

Distribution of ARGs in poxtA-Positive CoNS Isolates and the Electrotransformants
In total, 16 additional ARGs were detected among the poxtA-positive CoNS isolates ( Figure 1). ARGs were widespread in the poxtA-positive isolates of both feed and pig origins. Except for the widely distributed resistance genes aadD, ant(6)-Ia, blaZ, mecA, lsa(E), lnu(B), erm(C), fexB, tet(L) and dfrG in the poxtA-positive CoNS isolates, the distribution of individual ARG varied among the poxtA-positive CoNS isolates. For example, the cfr, tet(M) and aac(6 )-aph(2") genes were also identified in the poxtA-positive S. haemolyticus isolates ( Figure 1). All poxtA-positive CoNS isolates were able to transfer the fexB, poxtA and tet(L) genes to S. aureus strain RN4220 (Table 2). In addition, the five poxtA-positive S. haemolyticus isolates were able to transfer the aadD gene to S. aureus strain RN4220 (Table 2).

Phylogenetic Relatedness of poxtA-Positive CoNS Isolates
All poxtA-positive S. haemolyticus isolates derived from swine nasal swabs in pig farm D represented ST64 by MLST analysis and were closely related by phylogenetic analysis (Table 1 and Figure 1). In addition, 121 SNPs were identified in all poxtA-positive S. haemolyticus isolates ( Figure 1). The S. saprophyticus isolates GDH8C97P recovered from feed in chicken farm A and GDY8P136P recovered from swine nasal swab in pig farm D shared 1957 SNPs difference ( Figure 1).

Plasmids Analysis
S1-PFGE and WGS analysis confirmed that the poxtA gene in the seven CoNS isolates and corresponding electrotransformants was located on plasmids ranging in size around 54.7 kb (Figure 2 and Figure S1). Plasmid pY80 carrying the poxtA gene was 55,758 bp in size and exhibited <38% coverage with other plasmids in NCBI database, with an average GC content of 34.0%. In total, 51 ORFs coding for proteins of >50 amino acids were identified ( Figure 2). Except for the 14 ORFs encoding hypothetical proteins with no defined function, the products of the remaining 37 ORFs exhibited identities ranging from 76.3% to 100% to proteins with known functions, including antimicrobial resistance, heavy metal resistance, conjugative transfer or transposition, plasmid replication and other function ( Figure 2).

Genetic Environment of poxtA Gene
The poxtA-carrying segments (IS1216-poxtA-IS1216-fexB-IS431mec-tet(L)-aadD-IS431mec) of 17287 bp in plasmid pY80 of pig origin) were selected to conduct comparative analysis with other poxtA-carrying segments. The IS1216-poxtA-IS1216 segment of 4130 bp showed >98% identity to corresponding sequences in two Enterococcus hirae plasmids (pHDC14-2.27K and pfas4-1 both of pig origins), two Enterococcus faecalis plasmids (pM18/0011 of human origin and pC10 of pig origin), 10 Enterococcus faecium plasmids (pSDGJQ5 of chicken origin, pM160954 of human origin, pE1077-23 of pig origin, pSCBC1 of pig origin, pSDGJP3 of pig origin, pYN2-1 of pig origin, pHN11 of pig origin, pGZ8 of pig origin, pSC3-1 of chicken origin and pC25-1 of pig origin) and the genome of S. aureus AOUC-0915 of human origin (Figure 3). In addition, the IS1216-poxtA segment of 2363 bp showed >98% identity to corresponding sequences in the genomes of Enterococcus faecium P36 of pig origin and Pediococcus acidilactici BCC1 of chicken origin (Figure 3). The 11951 bp region (fexB-IS431mec-tet(L)-aadD-IS431mec) downstream of IS1216-poxtA-IS1216 in plasmid pY80 was identified. Within this region, the 4200 bp segment (tet(L)-aadD- (iv) positions and directions of predicted coding sequences are indicated by colored arrows according to their predicted functions. Red arrows represent resistance genes, teal arrows represent heavy meatal resistance genes, orange arrows represent genes involved in transfer or transposition, gray arrows represent plasmid replication genes, blue arrows represent genes of unknown functions or other functions.
IS431mec) showed >98% identity to the S. aureus plasmid (pERGB of human origin) ( Figure  3). The poxtA-carrying fragments that often harbor additional resistance genes such as fexB, tet(L) and tet(M) were identified in different bacterial species (Figure 3).

Discussion
CoNS are recognized as significant opportunistic pathogens that cause infections in humans and animals [4,5], and CoNS carrying important antimicrobial resistance genes such as oxazolidinone resistance genes could pose a huge burden on the healthcare system and breeding industry [10]. The transferable oxazolidinone resistance gene poxtA in different enterococci was the most recently reported [17]. Attention should be paid to the fact that the poxtA gene was originally detected in a linezolid-resistant MRSA strain [16]. Therefore, there was a risk that the poxtA could spread to other bacterial strains. The observation that the poxtA gene was mainly detected in S. haemolyticus and S. saprophyticus isolates might suggest an S. haemolyticus and S. saprophyticus reservoir. In this study, the results indicated that poxtA-positive ST64-S. haemolyticus isolates from swine nasal swabs in a pig farm shared low SNPs difference and were closely related, and that poxtA-positive S. saprophyticus isolates from a pig farm and a chicken farm shared a high SNPs difference.

Discussion
CoNS are recognized as significant opportunistic pathogens that cause infections in humans and animals [4,5], and CoNS carrying important antimicrobial resistance genes such as oxazolidinone resistance genes could pose a huge burden on the healthcare system and breeding industry [10]. The transferable oxazolidinone resistance gene poxtA in different enterococci was the most recently reported [17]. Attention should be paid to the fact that the poxtA gene was originally detected in a linezolid-resistant MRSA strain [16]. Therefore, there was a risk that the poxtA could spread to other bacterial strains. The observation that the poxtA gene was mainly detected in S. haemolyticus and S. saprophyticus isolates might suggest an S. haemolyticus and S. saprophyticus reservoir. In this study, the results indicated that poxtA-positive ST64-S. haemolyticus isolates from swine nasal swabs in a pig farm shared low SNPs difference and were closely related, and that poxtA-positive S. saprophyticus isolates from a pig farm and a chicken farm shared a high SNPs difference. Therefore, ST64-S. haemolyticus isolates carrying poxtA can spread among pigs in the pig farm, and the potential spread of S. saprophyticus isolates carrying poxtA between the pig farm and chicken farm should arouse people's attention.
It was reported that IS1216 played a major role in the processes of aiding the dissemination and persistence of poxtA among enterococci [20]. The presence of these homologous gene regions (IS1216-poxtA-IS1216 or IS1216-poxtA) in Staphylococcus spp., Enterococcus spp. and Pediococcus spp. confirmed that IS1216 was closely related to the spread of poxtA Pathogens 2021, 10, 601 7 of 11 among these Gram-positive bacteria with different genetic and source backgrounds. This was a further reminder that the poxtA gene might have spread widely in these bacteria. It has been found from reported studies that most of these homologous gene regions are located on transferable plasmids which often harbor additional resistance genes such as the tetracycline resistance genes tet(M) and tet(L), and the phenicol exporter gene fexB [15][16][17][18]. In this study, plasmid pY80 carried heavy metal resistance gene czcD and aminoglycosidemodifying enzyme gene aadD in addition to tet(L), fexB and poxtA genes. The co-occurrence of poxtA with other antimicrobial and heavy metal resistance genes on the transferable plasmids may lead to the co-selection of poxtA, contributing to its persistence and accelerating its dissemination [15]. The poxtA gene was identified in the new plasmid. Once the poxtA gene is inserted into a plasmid with strong transmission ability, it will bring great difficulties to control the further transmission of the poxtA gene. In China, florfenicol has been widely used in food-producing animals [21]. It was reported that the emergence of oxazolidinone resistance genes such as poxtA is closely related to the use of florfenicol in breeding farms [22]. Antimicrobial susceptibility testing showed that all donors and electrotransformants were resistant to florfenicol, indicating poxtA-positive plasmids could be directly selected by florfenicol. The phenomenon that the strains and electrotransformants investigated in this study were phenotypically oxazolidinone-susceptible despite the fact that they carry up to two oxazolidinone resistance genes (cfr and poxtA) is very interesting. This may be due to the possibility that cfr was not transcribed [23] and poxtA played a relatively low role on oxazolidinone susceptibility [16]. The fact that electrotransformants with aadD and tet(L) genes did not show resistance to kanamycin, neomycin, doxycycline and tetracycline might be related to the silencing of these genes. It is easy for people to ignore the resistance genes without corresponding drug-resistant phenotypes, resulting in the widespread spread of them [24]. That antimicrobial agents used in livestock could exert selective pressures on bacteria [25] and all the poxtA-positive CoNS isolates exhibited multidrug resistance and carried additional resistance genes should account for the spread of poxtA gene in the CoNS isolates [15]. All isolates were screened for the presence of poxtA by PCR using previously described primers [15]. Species identification was performed using MALDI-TOF MS (Bruker Daltonik GmbH, Bremen, Germany) and further confirmed by 16S rDNA sequence analysis.

Molecular Epidemiology Analysis and Transformation Assays
Multilocus sequence typing (MLST) was conducted for identification of clonal correlation of the poxtA-positive S. haemolyticus (http://www.shaemolyticus.mlst.net Accessed on: 29 January 2021) [26]. Plasmid DNA from all poxtA-positive CoNS isolates was extracted using a Qiagen Prep Plasmid Midi Kit (Qiagen, Hilden, Germany) and transferred into a recipient S. aureus strain RN4220 by electroporation using Gene Pulser apparatus (Bio-Rad, Hercules, CA, United States) [27]. Electrotransformants were selected on brain heart infusion (BHI) agar containing 10 µg/mL of florfenicol. Electrotransformants were further confirmed for the presence of poxtA gene by PCR analysis. The successful electrotransformants were further screened for the presence of aadD, fexB, tet(L) and tet(M) genes by PCR.

S1-PFGE and WGS Analysis
Genomic DNA of all poxtA-positive CoNS isolates and corresponding electrotransformants were digested with S1 endonuclease and separated by PFGE as previously described [30]. Whole-cell DNA of all poxtA-positive CoNS isolates were prepared using the HiPure Bacterial DNA Kit (Magen, Guangzhou, China), following the manufacturer's instructions, and then preceded by library construction on Novaseq 6,000 sequencing platform, which produced 150 bp paired-end reads (Novogene Company, Beijing, China). Novaseq sequences were assembled using CLC Genomics Workbench 10 (CLC Bio, Aarhus, Denmark). The GDY8P80P isolate carrying poxtA and cfr genes was further used for whole-genome sequencing on PacBio RS II sequencing platform (Biochip Company, Tianjin, China). Pacbio sequences were assembled using hierarchical genome-assembly process [31]. The assembled Pacbio sequences were corrected through Burrows-Wheeler Aligner's Smith-Waterman Alignment (BWA-SW) software to ensure their integrity according to Novaseq sequences [32]. The plasmids carrying poxtA were annotated using the Rapid Annotation of microbial genomes using Subsystems Technology annotation server (http://rast.nmpdr.org/ Accessed on: 29 January 2021) [33]. Acquired resistance genes (ARGs) were identified in the genomes using ResFinder 4.0 [34]. The genetic comparison of the poxtA gene from different species was generated using Easyfig 2.1 [35]. Based on the draft genome sequences, a phylogenic tree was constructed for all sequenced poxtA-positive CoNS isolates by CSI Phylogeny 1.4 (https:// cge.cbs.dtu.dk/services/CSIPhylogeny/ Accessed on: 29 January 2021), with the genome of GDY8P50P used as a reference. The tree was visualized using software Fig Tree 1.4.2. Single nucleotide polymorphism (SNP) divergence among various isolates carrying poxtA was calculated using snippy (https:// www.github.com/heilaaks/snippy/ Accessed on: 29 January 2021).

Ethical Considerations
The study was approved by the South China Agriculture University (SCAU) Animal Ethics Committee. The research was conducted in strict accordance with Section 20 of the Animal Diseases Act of 1984 (Act No 35 of 1984) and the Declaration of Helsinki, and was approved by the SCAU Institutional Animal Care and Use Committee.

Conclusions
In conclusion, this is the first study to report on the presence of the poxtA gene in livestock-derived S. haemolyticus and S. saprophyticus. The presence of IS1216-poxtA-IS1216 in Staphylococcus spp., Enterococcus spp. and Pediococcus spp. with different genetic and source backgrounds indicated an important role of IS1216 in the dissemination of poxtA. Moreover, the co-occurrence of poxtA with other antimicrobial and heavy metal resistance genes on the transferable plasmids may lead to the co-selection of poxtA, contributing to its persistence and accelerating its dissemination even in the absence of direct selective pressure by the use of phenicols, tetracyclines and oxazolidinones. Attention should be paid to the potential risks of the transfer of the plasmid-borne poxtA from enterococci and staphylococci to other Gram-positive bacteria. Therefore, routine surveillance for the spread of poxtA in different Gram-positive bacteria and the prudent use of antimicrobial agents in food-producing animals are urgently warranted.