Occurrence of cfr-Positive Linezolid-Susceptible Staphylococcus aureus and Non-aureus Staphylococcal Isolates from Pig Farms

The emergence and spread of cfr-mediated resistance to linezolid in staphylococci have become a serious global concern. The acquisition of cfr confers multidrug resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A (PhLOPSA phenotype). However, occurrence of cfr-positive and linezolid-susceptible staphylococci has been identified. To investigate the mechanism underlying linezolid susceptibility in cfr-positive Staphylococcus aureus and non-aureus staphylococci (NAS) isolates from pig farms in Korea. Eleven cfr-positive and linezolid-susceptible staphylococci were analyzed for mutations in domain V of 23S rRNA, ribosomal proteins (L3, L4, and L22), cfr open reading frames (ORFs), and cfr promoter regions. The effect of the cfr mutation (Q148K) on the PhLOPSA phenotype was determined using plasmid constructs expressing either the mutated (cfrQ148K) or nonmutated cfr genes. All 11 (six S. aureus and five NAS) cfr-positive and linezolid-susceptible isolates had a point mutation at position 442 in cfr ORFs (C to A) that resulted in the Q148K mutation. No mutations were detected in 23S rRNA, L3, L4, or L22. The Q148K mutation in Cfr is responsible for phenotypes susceptible to PhLOPSA antimicrobial agents. To our knowledge, this is the first study to report the causal role of a single nucleotide mutation (Q148K) in cfr of S. aureus and NAS isolates in PhLOPSA resistance. Continued nationwide surveillance is necessary to monitor the occurrence and dissemination of mutations in cfr that affect resistance phenotypes in staphylococci of human and animal origin.


Introduction
Linezolid, the first member of oxazolidinones approved solely for use in humans, has been considered a last resort antimicrobial agent in treatment of serious infections caused by antimicrobial-resistant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) [1]. Because of its unique mode of action, which inhibits prokaryotic protein synthesis from a very early stage, and the synthetic nature of the drug, it was proposed that the development of resistance to linezolid is rare [2]. However, since the first report of linezolid resistance in a clinical isolate of MRSA in 2001 [1], the occurrence of linezolid-resistant S. aureus and non-aureus staphylococci (NAS) has been increased [3]. Moreover, although linezolid is strictly prohibited for use in livestock, the worldwide emergence of linezolid-resistant staphylococcal isolates has also been reported in various food-producing animals [4][5][6][7].
In staphylococci, linezolid resistance is mostly mediated by point mutations in the central loop of domain V region of the 23S rRNA [8,9]. As the primary target site of oxazolidinones, point mutations in these regions have previously been shown to be associated with the linezolid resistance phenotype [2]. In addition to the mutations in 23S rRNA, muta-tions in the genes encoding the 50S ribosomal proteins L3 (rplC), L4 (rplD), and L22 (rplV) at peptidyl transferase center (PTC) have been identified in linezolid-resistant bacteria [2,10].
Besides the point mutations in bacterial chromosomes, the transferable multidrug resistance gene cfr, encoding a 23S rRNA methyltransferase, has recently been reported [11,12]. The Cfr protein mediates methylation of the C8 carbon in the adenine residue at position 2503 (m 8 A2503, Escherichia coli numbering) in the 23S rRNA, resulting linezolid resistance [13]. Moreover, due to the proximal location of A2503 to the ribosomal binding sites of other antimicrobial agents targeting the bacterial 50S ribosome, the Cfr-mediated methylation in A2503 confers distinctive multidrug resistance (MDR) phenotypes to at least five classes of antimicrobial agents, including, phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A (PhLOPS A ) [14]. Since the first detection of cfr gene in a bovine isolate of Staphylococcus sciuri [11], carriage of cfr gene has been reported in various staphylococcal species in farm animals and farm environments [5,15,16]. In Korea, cfr-mediated linezolid resistance has also been identified in S. aureus and NAS isolates collected from pig farms and slaughterhouses [4,17,18].
Recently, it has been reported that staphylococcal isolates carrying the cfr gene failed to show resistance phenotype to linezolid [16,[19][20][21][22][23]. In this study, S. aureus and NAS isolates that possess a cfr gene but are phenotypically susceptible to linezolid were identified in pigs and farm environments. Although the cfr-positive and linezolid-susceptible phenotypes have also been reported in C. difficile and E. faecalis [24][25][26], molecular mechanisms involved in the phenomenon are still unknown. Thus, to address the genotypic-phenotypic discrepancy in linezolid resistance in staphylococci, cfr-positive linezolid-susceptible S. aureus and NAS strains were analyzed for point mutations within the cfr open reading frame (ORF) and promoter sequences. Moreover, the impact of a point mutation in the cfr ORF on linezolid resistance was evaluated in a previously described plasmid system [27] expressing a point-mutated form of cfr gene derived from the cfr-positive linezolid-susceptible isolates.

Identification of cfr-Positive Linezolid-Susceptible Staphylococci in Pig Farms
As shown in Table 1, a total of 11 staphylococcal isolates that were susceptible to linezolid but positive for cfr gene were obtained from the pig farms. Of the 11 cfr-positive linezolid-susceptible isolates, six were methicillin-susceptible S. aureus (MSSA) strains, all belonging to sequence type (ST) 398, and isolated from healthy pigs (SA16, SA17, SA18, and SA19) and farm environment (SA20 and SA21). The other five isolates were four different species of coagulase-negative staphylococci (CoNS), consisting of two S. epidermidis (SE9 and SE10) strains from pigs, two S. sciuri (SSC1 and SSC2) strains from farm environments, and one S. simulans (SSM1) from a pig. Except for the two methicillin-resistant S. sciuri strains, which had non-typeable SCCmec, all the other four CoNS were methicillinsusceptible isolates. MLST analyses revealed that the two S. epidermidis (SE9 and SE10) and one S. sciuri (SSC2) strains were ST570 and ST85, respectively. An MLST scheme has not yet been developed for S. simulans. The six cfr-positive linezolid-susceptible ST398 MSSA strains displayed identical linezolid MIC of 2 mg/L, while cfr-positive linezolid-susceptible CoNS strains showed linezolid MIC range of 0.75 to 4 mg/L ( Table 1). The SSM1 strain showed the lowest level of resistance to linezolid (MICs of 0.75 µg/mL) even with the carriage of cfr gene. All 11 cfr-positive linezolid-susceptible staphylococci were also carrying the fexA gene, which was correlated with the chloramphenicol resistance phenotype.

Genetic Assessment of cfr ORF and Its Promoter in LZD-Susceptible Staphylococci
Sequencing analyses of the cfr ORF revealed that all 11 cfr-positive linezolid-susceptible staphylococci had a single point mutation (C to A) at position 442 in the cfr gene versus the cfr sequences of linezolid-resistant staphylococci (SA2 and SE7 strains) (Supplementary Figure S1). This point mutation results in a glutamine-to-lysine substitution at position 148 (cfr Q148K ). In contrast, analyses of cfr promoter sequences (523-bp regions upstream of the ATG start codon) revealed no differences between the linezolid-resistant and -susceptible staphylococci. Moreover, no mutations in domain V of 23S rRNA or ribosomal proteins (L3, L4, and L22) were identified in the 11 cfr-positive linezolid-sensitive staphylococci (Supplementary Table S2).

Impact of cfr Q148K on PhLOPS A Resistance Phenotype
As shown in Table 2, pRB474 constructs expressing either the point-mutated forms of cfr (cfr Q148K ) or non-mutated cfr (wild-type cfr) were generated and then electroporated into the two linezolid-susceptible strains of RN4220 and ST398 MRSA SA100, which were negative for both cfr and fexA genes.
As expected, the cfr-positive SA12 strain showed resistant phenotype to linezolid (MIC of 16 mg/L) and all the other four classes of antimicrobial agents tested ( Table 3). Complementation of the linezolid-susceptible S. aureus strains, RN4220 and ST398 MRSA SA100, with plasmids expressing the wild-type cfr resulted in 4-fold increase in linezolid MICs compared to those of the control strains carrying empty pRB474 plasmid (Table 3). In addition to the increase in linezolid MICs, expression of the wild-type cfr caused significant increases in phenicols, lincosamides, pleuromutilins, and streptogramins, leading to the PhLOPS A resistance phenotypes. However, similar to the RN4220 and ST398 MRSA strains bearing empty pRB474 plasmids, the two strains complemented with plasmids expressing cfr Q148K displayed susceptible or low-level resistance phenotypes to the five classes of antimicrobial agents. Table 2. Stains and plasmids used in this study.

Linezolid Population Analysis Profiles
For the two S. aureus strains (RN4220 and SA100) expressing the wild-type cfr, the linezolid population curves were significantly shifted to the right versus those for strains complemented with the empty plasmid or pRB4747::cfr Q148K gene ( Figure 1A,B). Areaunder-the curve (AUC) values for linezolid population analyses were~7-fold less for the two strains expressing cfr Q148K gene than for the strains expressing the nonmutated cfr gene or carrying empty plasmids.  AUC values of RN4220 and SA100 strains expressing the wild-type cfr were 44.12 ± 1.48 and 45.69 ± 0.48, respectively. AUC values of RN4220 and SA100 strains expressing cfr Q148K were 5.95 ± 0.03 and 5.90 ± 0.04, respectively.

Genetic Environment of cfr Q148K
Four cfr-positive linezolid-susceptible staphylococcal strains (SA16, SA19, SE10, and SSC2) were selected for comparative analysis of the genetic regions harboring the cfr Q148K genes. Genome sequence data indicated that the all cfr-positive strains possessed 38-kb plasmids (pSA16, pSA19, pSE10-1, and pSSC2-1) carrying cfr Q148K genes. As shown in Figure 2, the identical structures of the cfr Q148K containing regions flanked by the Tn558 transposon elements were found on the pSA16, pSA19, pSE10-1, and pSSC2-1 plasmids. BLASTn analysis showed that the 10-kb cfr-carrying segments of S. aureus (SA16 and SA19), S. epidermidis (SE10), and S. sciuri (SSC2) strains shared 99.9% nucleotides sequence identities. The cfr Q148K harboring regions showed 99% nucleotide sequence identities with the previously reported wild-type cfr-carrying plasmid pSA12 (CP049977) of a linezolidresistant ST398 MRSA strain SA12, except for sequence variations in transposon elements (tnpA and tnpB). The cfrQ148K genes are located on 38-kb plasmids (pSA16, pSA19, pSE10-1, and pSSC2-1) of staphylococci strains. Different colored arrows indicate that different genes with the direction of transcription; red arrows represent cfr genes; yellow arrows represent fexA genes; green arrows represent IS elements; blue arrows represent orf138 genes; gray arrows represent transposon elements. Shaded regions indicate nucleotide sequence identities ranging from 95% to 100%.

Discussion
Although linezolid resistance in staphylococci still remains rare, recent studies demonstrated a worldwide increase in the occurrence of linezolid-resistant S. aureus and NAS [4,7,17]. Unlike the chromosomal mutations in genes encoding 23S rRNA or ribosomal L3 and L4 proteins of linezolid-resistant isolates [9,34], the transmissible nature of cfrmediated linezolid resistance in staphylococci has raised a significant concern in terms of horizontal transfer of resistance within and between different species of staphylococci [4,17]. In particular, carriage of the cfr on transferable plasmids and/or colocalization with insertion sequence (IS) elements has been reported in staphylococcal isolates of various animal and human origins [3,34]. Recently, the occurrence of cfr-mediated linezolid resistance in livestock-associated MRSA (LA-MRSA) and CoNS isolates obtained from pig farms and slaughterhouses was identified in Korea [4,17,18]. Whole genome sequence analyses of the linezolid-resistant LA-MRSA and CoNS isolates revealed that the cfr genes were located on plasmids and were usually associated with mobile genetic elements, such as transposons and conjugative elements [4,15,17,35].
In the current study, 11 cfr-positive but linezolid-susceptible staphylococci were identified in pig farms in Korea. As shown in Table 1, except for two methicillin-resistant S. sciuri isolates, 9 of the 11 cfr-positive linezolid-susceptible isolates were methicillin-sus- Figure 2. The schematic presentation of cfr Q148K harboring regions in staphylococci strains. The cfr Q148K genes are located on 38-kb plasmids (pSA16, pSA19, pSE10-1, and pSSC2-1) of staphylococci strains. Different colored arrows indicate that different genes with the direction of transcription; red arrows represent cfr genes; yellow arrows represent fexA genes; green arrows represent IS elements; blue arrows represent orf138 genes; gray arrows represent transposon elements. Shaded regions indicate nucleotide sequence identities ranging from 95% to 100%.
In addition, fexA genes flanked by the Tn558 transposon elements were co-located in the downstream of cfr Q148K . Genetic analysis revealed that the fexA genes identified in this study were 100% identical in nucleotide sequence identity to those in the pSA12.

Discussion
Although linezolid resistance in staphylococci still remains rare, recent studies demonstrated a worldwide increase in the occurrence of linezolid-resistant S. aureus and NAS [4,7,17]. Unlike the chromosomal mutations in genes encoding 23S rRNA or ribosomal L3 and L4 proteins of linezolid-resistant isolates [9,34], the transmissible nature of cfr-mediated linezolid resistance in staphylococci has raised a significant concern in terms of horizontal transfer of resistance within and between different species of staphylococci [4,17]. In particular, carriage of the cfr on transferable plasmids and/or colocalization with insertion sequence (IS) elements has been reported in staphylococcal isolates of various animal and human origins [3,34]. Recently, the occurrence of cfr-mediated linezolid resistance in livestock-associated MRSA (LA-MRSA) and CoNS isolates obtained from pig farms and slaughterhouses was identified in Korea [4,17,18]. Whole genome sequence analyses of the linezolid-resistant LA-MRSA and CoNS isolates revealed that the cfr genes were located on plasmids and were usually associated with mobile genetic elements, such as transposons and conjugative elements [4,15,17,35].
In the current study, 11 cfr-positive but linezolid-susceptible staphylococci were identified in pig farms in Korea. As shown in Table 1, except for two methicillin-resistant S. sciuri isolates, 9 of the 11 cfr-positive linezolid-susceptible isolates were methicillin-susceptible staphylococci. Sequencing analyses of the cfr ORFs in these isolates revealed a specific point mutation of cfr Q148K . Although recently published studies have also described cfrpositive but linezolid-susceptible S. aureus [19][20][21][22]36] or NAS strains [16,23], the molecular genetic mechanism of the linezolid-susceptible phenotype in these strains has not yet been elucidated [2]. A recent study from Italy described a frameshift mutation within the chromosomal cfr gene (lack of adenine residue at position 379) in a cfr-positive linezolid-susceptible ST398 LA-MRSA of porcine origin [36]. Although we identified the cfr Q148K mutation in previously published sequences of the MRSA strain SR153 [21] and S. haemolyticus strains VB5326 and VB19548 [34], the present study is the first to report the Q148K mutation in cfr ORF in association with a linezolid-susceptible phenotype in cfr-positive S. aureus and NAS isolates. Analyses of the cfr promoter regions in the 11 cfr-positive linezolid-susceptible staphylococci showed no variation in sequences compared to those of SA2, SA3, and SE7 isolates, suggesting that the linezolid-susceptible phenotype was not attributed to changes in transcription of cfr by cis-acting elements.
Next, as shown in Table 3, the effect of the Q148K mutation in cfr ORF on susceptibility to PhLOPS A antimicrobial agents was determined. The incorporation of wild-type cfr into the complementation plasmid, pRB474, resulted in increased MICs of all the five classes of PhLOPS A antimicrobials in RN4220 and SA100 strains. In contrast, expression of cfr Q148K from the same plasmid was unable to increase MICs to any of the PhLOPS A antibiotics, indicating that the Q148K mutation in the cfr ORF is responsible for the linezolid-susceptible phenotype identified in the 11 cfr-positive linezolid-susceptible staphylococci. In addition, there were significant leftward shifts in linezolid population analysis AUCs in the S. aureus strains expressing Cfr Q148K compared to the strains expressing wild-type Cfr ( Figure 1A,B). Unlike the PhLOPS A antibiotics, susceptibility to tetracycline and vancomycin was unaffected by the expression of wild-type cfr or cfr Q148K, confirming the specific effect of cfr on PhLOPS A agents. The impact of the Q148K mutation in cfr on PhLOPS A phenotype is likely to be similar in NAS strains. However, attempts to electroporate the pRB474 constructs expressing wild-type cfr and cfr Q148K into S. epidermidis or S. sciuri isolates were unsuccessful because of the much lower frequency of transformation in CoNS than in S. aureus strains [37].
WGS analyses of cfr-positive linezolid-susceptible S. aureus (SA16 and SA19) and NAS strains (S. epidermidis SE10, and S. sciuri SSC2) revealed the location of cfr Q148K genes on 38-kb plasmids (pSA16, pSA19, pSE10-1, and pSSC2-1) in these strains (Figure 2), which showed >99% nucleotide sequence similarity. These results indicate that the plasmids possessing cfr Q148K gene can be transmitted among staphylococci in the pig farm environments via horizontal transfer. Moreover, these plasmids showed >99% nucleotide sequence homology to the previously reported 38-kb plasmid, pSA12 [4], suggesting that the wild-type cfr and cfr Q148K genes are located on the same plasmid backbone.
In line with the report by LaMarre et al., which reported low fitness cost of the cfr [38], there was no difference in growth rates between the S. aureus strains carrying the wild-type cfr and cfr Q148K (Supplementary Figure S2).
Moreover, all 11 linezolid-susceptible staphylococci were positive for fexA. Previous studies from our laboratory and others reported that the cfr and fexA genes are frequently colocalized on a transferable plasmid [3,4,7,17,35]. In this study, WGS analyses also confirmed co-localization of fexA and cfr Q148K on a transferable plasmid, indicating that fexA may contribute to the maintenance of the cfr Q148K -carrying plasmid under antibiotic selective pressure in pig farms. Although there was no significant difference in fitness costs between the wild-type cfr and cfr Q148K without antibiotic selective pressure, future research is warranted to investigate effect of the mutation on long-term stability and transmissibility of the resistant plasmids, especially under selective pressure. Recently, the Q148K mutation within cfr ORF was detected in a linezolid-susceptible clinical MRSA isolate [21], suggesting that continuous monitoring on occurrence of cfr Q148K and other cfr variants in staphylococci originated from human and animal is necessary. It should be recognized that our data in this study were generated from a limited number of staphylococcal isolates. Moreover, molecular mechanisms involved in resistance to other antibiotics were not included in this study. Nonetheless, this is the first to report the mechanisms underlying linezolid susceptibility in cfr-positive livestock-associated staphylococci isolated from pig farms in Korea.

Bacterial Strains
Previous studies from our laboratory revealed the emergence of cfr-mediated linezolid resistance in livestock-associated MRSA (LA-MRSA) and NAS strains isolated from pig farms in Korea [4,17]. In a retrospective investigation of the prevalence of cfr among staphylococci on pig farms from 2017 to 2021, cfr-positive linezolid-susceptible S. aureus (n = 6) and NAS isolates (two S. epidermidis, two S. sciuri, and one S. simulans) were identified in a same pig farm. The staphylococcal isolates used in this study are listed in Table 1. Three linezolid-resistant S. aureus (SA2, SA3, and SA12) and one S. epidermidis (SE7) isolates were selected from the recently described staphylococcal strains isolated from pig farms [4,17].
All S. aureus and NAS isolates were cultured in Mueller-Hinton broth (Difco Laboratories, Detroit, MI, USA) or tryptic soy broth (Difco Laboratories) for each assay. All isolates were stored at −75 • C until used for each experiment.

DNA Isolation and cfr Sequencing
PCR amplification of the cfr ORF and the cfr promoter region (523 bp upstream of ATG start codon) was carried out using the specific primer pair cfr-F (5 -GCGAAATGGCTCAAT-TTTCA-3 ) and cfr-R (5 -TTCCACCCAGTAGTCCATTCA-3 ) based on the sequence information of a cfr-harboring plasmid pSA12 (GenBank accession no. CP049977). DNA sequencing of the PCR product was performed at Bionics (Seoul, Korea). The presence of fexA gene, which encodes the florfenicol-chloramphenicol exporter, was determined in all isolates as described before [46].

Genetic Manipulations and cfr Cloning
Genomic DNA samples from staphylococci were prepared as previously before [48]. Plasmid DNA was extracted from Escherichia coli and S. aureus using PureYield TM plasmid miniprep kit (Promega, Madison, WI, USA). The preparation of competent cells and transformation of E. coli DH5α were performed as described before [49]. Electroporation of plasmid DNA into staphylococcal isolates was performed as described previously [50,51]. Briefly, 100 µL of electro-competent S. aureus cells were mixed with 1 µg of the plasmids, transferred into 1 mm electroporation cuvettes (Bio-Rad, Hercules, CA, USA), and kept on ice for 15 min. After application of electropulse at 2.3-2.5 kV, resistance 100 Ω, and capacity 25 µF, 1 mL of fresh TSB was added, and the cells were cultured at 37 • C for 1 h. The cells were then spread on tryptic soy agar (Difco Laboratories) plates containing chloramphenicol (10 µg/mL) and putative transformants were selected.
For the in trans complementation constructs, nonmutated and point mutated cfr ORFs were PCR-amplified with primers cfr-HindIII (5 -CCCAAGCTTGCAAATTGTGAAAGGA-TGAAA-3 ) and cfr-XbaI (5 -CCCTCTAGATCCACCCAGTAGTCCATTCA-3 ) using purified DNA samples from SA12 and SA16 strains, respectively ( Table 2). The PCR products were then cloned into the HindIII and XbaI sites of the pRB474 expression vector [30], which places expression of the cloned crf gene under the control of vegII promoter (Table 2). DNA sequences of the cfr ORFs ligated into pRB474 were confirmed by sequencing analyses and enzyme digestions.

Population Analysis
Population analyses were carried out using linezolid as previously described with minor modifications [52]. Briefly, staphylococcal inoculum of~10 8 CFU/mL was plated onto Mueller-Hinton Agar (MHA) supplemented with different concentrations of linezolid (0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 µg/mL). The range of linezolid concentrations tested was selected to encompass sublethal to lethal linezolid levels based on the linezolid MIC data. After incubation at 37 • C for 24 h, visible colonies on the plates were counted. At least three independent assays were performed for each strain.

Whole Genome Sequencing Analysis
Whole genome sequence (WGS) data of cfr-positive linezolid-susceptible staphylococci were generated using a combination of Oxford Nanopore MinION (Oxford Nanopore Technologies, Oxford, UK) and Illumina iSeq (Illumina Inc., San Diego, CA, USA). Sequencing data were assembled de novo using Unicycler v.0.5.0. Functional annotation of assembled genome was carried out using the Prokka (v1.14.6) and Rapid Annotation in Subsystem Technology server tool. Integrative data from ResFinder (https://cge.cbs.dtu.dk/services/ ResFinder/, accessed on 10 November 2022) of the Center for Genomic Epidemiology and the Comprehensive Antibiotic Resistance Database were used to confirm the presence and location of cfr genes. To analyze the cfr-containing regions, comparative sequence analyses were conducted on the strains sequenced in this study and the previously reported cfr-carrying plasmid pSA12 in a linezolid-resistant ST398 LA-MRSA strain SA12 (GenBank accession no. CP049977) [4].

Conclusions
In the current study, we identified the occurrence of cfr-positive linezolid-susceptible S. aureus and NAS on pig farms in Korea. Our results suggest that (i) the Q148K mutation within the cfr ORF can recapitulate the linezolid-susceptible phenotype observed in the six ST398 MSSA and five CoNS strains (two S. epidermidis, two S. sciuri, and one S. simulans) collected from pigs and pig farm environments, (ii) the Q148K mutation in cfr confers susceptibilities to all the five classes of PhLOPS A antimicrobial agents; and (iii) in addition to the cfr-mediated linezolid resistance, ST398 MSSA and NAS isolates likely acquire cfr Q148K -containing plasmids through intra-or inter-species interactions.