Abstract

Introduction

Staphylococci are common colonizers of the skin and mucous membranes of humans and animals and opportunistic pathogens in humans [1]. Staphylococcus aureus causes endocarditis, septicemia, pneumonia, abscesses, and meningitis. The combination of toxin-mediated virulence and antibiotic resistance complicates the treatment of S. aureus infection. Additionally, methicillin-resistant S. aureus (MRSA), which has limited treatment options, has spread globally [2]. Its intrinsic resistance to several common antibiotics and ability to acquire new antibiotic resistance genes increase treatment costs and the risk of treatment failure [3]. S. aureus also inhabits food-producing animals and livestock manure. S. aureus-related antibiotic resistance genes can be transferred from food-producing animals to humans, posing a significant potential risk to public health [4].

Linezolid, an oxazolidinone antibiotic, is the antibiotic of last resort for treating clinical infections caused by multidrug-resistant (MDR) Gram-positive bacteria, including MRSA, penicillin-resistant Streptococcus pneumoniae, and vancomycin-resistant Enterococcus species [5]. Linezolid resistance is mediated by chromosomal mutations in the V domain of 23S ribosomal RNA (rRNA), mainly G2576T/G2505A, or mutations in the L3/L4 ribosomal proteins [6]. In addition, linezolid resistance occurs through the acquisition of transferable resistance determinants (optrA, poxtA, and the chloramphenicol–florfenicol resistance gene [cfr]) via mobile genetic elements [7]. cfr encodes a methyltransferase that modifies 23S rRNA, spreads through plasmids, and confers cross-resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics [8]. fexA confers resistance to chloramphenicol and florfenicol. The presence of fexA in mobile genetic elements might contribute to the persistence of linezolid resistance [9].

In addition to antibiotic resistance, S. aureus carries several virulence factors [10] granting it the ability to evade, invade, and penetrate host immune defenses. Toxins commonly secreted by S. aureus include enterotoxin, leukotoxin, hemolysin, and toxic shock syndrome toxin-1 [11], in addition to surface proteins and enzymes. The secretion of enzymes (staphylokinase, coagulase, and lipase) and surface proteins (fibronectin proteins, collagen adhesin, and protein A) allows bacteria to evade host defenses and facilitates bacterial attachment, host tissue invasion, and penetration [12]. Most of these virulence factors act by degrading host molecules or interfering with metabolic pathways in the host [13].

Recent studies isolated linezolid-resistant S. aureus from humans, livestock, and food [14, 15]. Although the prevalence of linezolid resistance is low compared to that of other antibiotics, studies on transferable linezolid resistance genes and their virulence factors are ongoing [16]. These genes are commonly embedded in mobile genetic elements, such as plasmids and phages, or present as composite transposons in the bacterial chromosome [17, 18]. Mobile genetic elements allow the rapid distribution of linezolid resistance genes and virulence factors throughout the bacterial population. The acquisition of linezolid resistance and virulence genes by S. aureus from animals can lead to difficult to treat infections in livestock workers [14]. Therefore, it is important to understand the distribution of linezolid resistance and virulence genes and monitor S. aureus strains from food and livestock.

Whole-genome sequencing (WGS) can rapidly and consistently predict multiple genes and mutations associated with antibiotic resistance and simultaneously provide surveillance data [19]. This technique can additionally reveal phylogenetic relationships among pathogenic bacterial species, and typing methods based on genome sequence data are suitable for tracking foodborne outbreaks [14]. Phenotyping complements genotyping in making treatment decisions, as WGS primarily offers predictive insights. Moreover, depending on the sequencing technology used, it can be faster to determine the minimum inhibitory concentration (MIC). However, WGS can detect various antibiotic resistance gene mutations and functional alterations, providing deeper insights into the mechanisms of antibiotic resistance [20]. The present study analyzed the genetic properties of cfr and fexA-carrying S. aureus isolated from pig carcasses by genome sequencing and evaluated the phylogenetic relationships among the strains. Furthermore, we aimed to provide basic data on the interactions between S. aureus and its hosts by analyzing the virulence factors in strains isolated from pig carcasses in Korea.

Materials and methods

Results

Phylogenetic analysis of S. aureus

To investigate the potential sources of strains, phylogenetic analysis was performed according to the presence and absence of accessory genes for 111 publicly available genomes of S. aureus isolated from pig-related sources and 15 genomes sequenced in this study (Fig. 1). Phylogenetic analysis revealed that the LNZ_R_SAU_37 and LNZ_R_SAU_46 strains were most closely related to S. aureus ISU 998 isolated in the United States (GCA_002274235.1). The LNZ_R_SAU_31, LNZ_R_SAU_58, and LNZ_R_SAU_62 strains were closely related to S. aureus NRRL B-41012 isolated in the United States (GCA_005153985.1). The LNZ_R_SAU_21, LNZ_R_SAU_26, LNZ_R_SAU_27, and LNZ_R_SAU_64 strains were closely related to S. aureus CFSAN018749 isolated in Denmark (GCA_003030065.1). The LNZ_R_SAU_25 strain was closely related to S. aureus GDB9P195A isolated in China (GCA_024916795.1). The LNZ_R_SAU_10, LNZ_R_SAU_22, LNZ_R_SAU_23, LNZ_R_SAU_24, and LNZ_R_SAU_57 strains were most closely related to S. aureus S681 isolated in Switzerland (GCA_002204735.1).

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Fig. 1

Phylogenetic tree based on presence/absence of core and accessory genes for 111 publicly available genomes of S. aureus isolated from pig-related sources and 15 genomes sequenced in this study. The colored range represents the country from which the strains were isolated. Internal tree scale and axis are displayed within the tree. Locations of genomes sequenced in this study are shown in the red circle. Strains carrying the cfr gene are indicated by blue circles.

Multilocus sequence typing (MLST) analysis

The sequence types of the 15 strains sequenced in this study were ST398, ST541, ST433, ST9, ST5, and ST8004 (Additional file 2). The most frequent type was ST541 (26.7%; n=4), followed by ST433 (20%; n=3) and ST9 (20%; n=3). The LNZ_R_SAU_64 isolate was identified with novel alleles and assigned a novel ID (ST8004) by the PubMLST team for curation and maintenance in the Bacterial Isolate Genome Sequence Database. A minimum spanning tree was generated by MLST analysis with the 15 strains sequenced in this study and other S. aureus strains of pig origin to evaluate phylogenetic relationships between strains isolated in various countries. MLST revealed the high diversity of S. aureus with distantly related strains (Fig. 2). The sequence types of 126 strains (111 publicly available genomes and 15 genomes sequenced in this study) were confirmed as ST398, ST5, ST9, ST188, ST1, ST541, ST93, ST433, ST3333, and ST8004. The most frequent type was ST398 (43.0%; n=52), followed by ST5 (20.7%; n=25) and ST9 (9.9%; n=12). ST541 and ST8004 were identified only in the strains sequenced in this study.

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Fig. 2

Minimum spanning tree by multilocus sequence type of S. aureus isolated from pigs or pig-associated environments. Each node within the tree represents a single sequence type. Node size is proportional to the number of genomes with that sequence type. Node color represents the country from which the strains were isolated. The branch length between each node indicates the number of allelic differences between the linked sequence types. Star indicates the sequence type related to the 15 isolates from this study.

Detection of virulence factors

The genome sequences of S. aureus were analyzed for genetic features associated with virulence and drug resistance. The toxin gene distribution was associated with the ST type. Both livestock- and human-associated S. aureus strains carried enterotoxins (ST433, ST9, and ST5). In addition, two ST5 S. aureus strains carried leukocidin genes (lukD and lukE). However, livestock-associated S. aureus only carried hemolysin genes (hlgA, hlgB, and hlgC). Fifteen virulence genes were identified among the 15 strains sequenced in this study (Fig. 3). A heatmap was drawn to visualize the presence or absence of virulence genes. aur, hlgA, hlgB, and hlgC were the most widespread genes, being detected in all 15 strains.

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Fig. 3

The heatmap of (A) antibiotic resistance gene and (B) virulence gene profiles across 15 S. aureus strains harboring cfr and fexA genes isolated in our previous study. Dark blocks represent the presence of genes and bright blocks represent absence. The labels on the bottom specify the tested isolates. Each row denotes one antibiotic resistance or virulence gene. The tree on the upper displays the relatedness of the isolates according to their antibiotic resistance and virulence profiles.

Genetic environment of cfr and fexA

All 15 strains sequenced in this study harbored cfr and fexA. In all strains, fexA was located 2668 or 2818 bp upstream of cfr (Fig. 4). Tn554 contains three transposition genes named tnpA, tnpB, and tnpC [26]. tnpA and tnpB of transposon Tn554 were detected downstream of cfr in the LNZ_R_SAU_10, LNZ_R_SAU_25, LNZ_R_SAU_26, LNZ_R_SAU_31, and LNZ_R_SAU_62 strains, whereas tnpC was detected upstream of cfr. In the remaining strains, ISSau9 was detected downstream of cfr, whereas tnpC was detected upstream. Analysis of the flaking region of cfr revealed that this gene was located around the plasmid replicon repUS5 in 11 strains (LNZ_R_SAU_10, LNZ_R_SAU_21, LNZ_R_SAU_26, LNZ_R_SAU_27, LNZ_R_SAU_31, LNZ_R_SAU_37, LNZ_R_SAU_46, LNZ_R_SAU_57, LNZ_R_SAU_58, LNZ_R_SAU_62, and LNZ_R_SAU_64; Fig. 5 and Additional file 4), indicating its potential for transmission. Among them, 10 strains harbored sequences highly similar to that of the S. aureus strain 004–737 X plasmid pSA737 (accession no. KC206006.1, identity 99%, query coverage 84–100%), whereas the sequence of the LNZ_R_SAU_57 strain had high identity to that of the S. aureus strain 359 plasmid unnamed2 (accession no. CP077935.1, identity 100%, query coverage 97%). The LNZ_R_SAU_22, LNZ_R_SAU_23, LNZ_R_SAU_24, and LNZ_R_SAU_25 strains did not feature a replicon flanking cfr.

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Fig. 4

Schematic representation of the genetic environment of cfr gene in 15 S. aureus strains and S. aureus strain 004-737X plasmid pSA737 (accession no. KC206006.1) harboring cfr and fexA genes. Gene orientation is shown with arrows. The cfr and fexA genes are shown in red and green colored arrows. Grey lines connect regions with>21% identity, and dark color indicates a higher percentage of identity.

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Fig. 5

Plasmid map of cfr and fexA genes-containing plasmids of (A) LNZ_R_SAU_10, (B) LNZ_R_SAU_21, (C) LNZ_R_SAU_31, (D) LNZ_R_SAU_37, and (E) LNZ_R_SAU_64. Gene and their orientation are indicated by arrows as follows: red, green, blue, purple, and gray represent antibiotic resistance genes, IS elements, plasmid replicon, other proteins, and hypothetical proteins, respectively.

Discussion

Linezolid is an antibiotic of last resort against highly resistant and complicated S. aureus infections in humans [27]. Although it is not approved for use in veterinary medicine, linezolid-resistant staphylococcal strains have been found on livestock farms and in foods [15, 21, 28–31]. The emergence of linezolid-resistant staphylococci poses a major threat to human and animal health because of the possibility of horizontal gene transfer between animals and humans and through direct contact or the food chain [27]. Consequently, linezolid-resistant staphylococci can be transmitted to humans through contact with livestock or food-producing animals, and infections caused by linezolid-resistant staphylococci could be difficult to treat [14]. The emergence of cfr-like gene-mediated linezolid resistance in staphylococci from humans and livestock has also been reported in clinical isolates in the United States, pigs and humans in Belgium, pigs in Korea, and turkeys in Egypt [15, 28, 32, 33]. Moreover, MRSA strains positive for cfr-like genes [cfr, cfr(B), cfr(C), cfr(D), and cfr(E)] have been reported sporadically in livestock in several countries, such as Portugal, Germany, and Taiwan, and in hospitalized patients in China [14, 27, 31, 33]. Interestingly, linezolid-resistant staphylococci were recently detected in pig carcasses in Korea. A previous study found that 2.3% of more than 2500 S. aureus strains were resistant to linezolid [21]. These findings highlight the urgency of monitoring linezolid resistance in gram-positive pathogenic bacteria, including staphylococci, isolated from animals in Korea.

In this study, cfr- and fexA-carrying S. aureus obtained from pig carcasses at slaughterhouses in five provinces in Korea were analyzed in detail by WGS (Additional file 1) and compared with other genomes. The 111 publicly available genomes isolated from pig-related sources were associated with eight MLST types, and the most frequent allele profiles were ST398 (55 strains), ST5 (23 strains), and ST188 (10 strains). The predominant prevalence of genomes of ST398 strains in the database might be attributable to the scope of the studies in which these strains were obtained and sequenced, such as livestock-associated MRSA studies. The 15 strains were associated with six MLST types, indicating high diversity and complexity regarding their genomic backgrounds. Five of these sequence types (ST398, ST541, ST433, ST9, and ST8004) are livestock-associated S. aureus sequence types, whereas ST5 is the only human-associated S. aureus sequence type [21, 34]. ST9 is associated with pigs and human workers on livestock-related farms. Excluding ST8004, all S. aureus lineages belong to these MLST types, suggesting the possibility of transmission between livestock and humans. Among all strains, five MRSA strains belonging to sequence types ST398 and ST541 were methicillin-resistant. In particular, three strains belonged to ST541, a livestock-associated MRSA clone in Korea, and the remaining two strains were classified as ST398 [35, 36]. ST433 is known as linezolid-resistant MRSA carrying enterotoxin genes with host specificity [37, 38]. However, although ST433 harbored enterotoxin genes, it was not identified as MRSA in this study. In previous studies, most MRSA isolates from Asia were categorized as ST9, whereas most European strains were categorized as ST398 [33, 39]. Recently, the emergence of ST398 in pigs was reported in China and Japan, illustrating the possibility of transmission of the clone from livestock to humans [31, 40, 41]. Moreover, MRSA ST398 strains were detected in pigs or humans living close to pig farms in previous studies [27, 28, 40]. The MRSA ST398 strains isolated in this study harbored genes conferring methicillin and phenol resistance flanked by mobile genetic elements. Strains belonging to ST398 can be transmitted via livestock, consistent with previous reports describing the potential of ST398 to be transmitted from animal reservoirs to humans [31].

The ability of S. aureus to infect humans and animals is attributable to its arsenal of virulence factors, such as genes encoding proteins for tissue attachment, enzyme-degrading proteins, and leukocidins [42]. The genomes of S. aureus were sequenced and analyzed to identify known virulence factors. In total, 15 virulence genes were observed among the 15 strains. aur, hlgA, hlgB, and hlgC are highly widespread virulence factors within S. aureus genomes [11, 42], and these genes were present in all strains in this study. However, two strains (LNZ_R_SAU_37 and LNZ_R_SAU_46) harbored lukD/lukE related to leukotoxins and splA/splB encoding serine protease-like proteins [43, 44]. Nine strains harbored an enterotoxin gene cluster (seg, sei, sem, sen, seo, or seu) causing foodborne outbreaks [45]. Previous studies revealed that the enterotoxin gene cluster in S. aureus is located in plasmids or genomic islands [42]. Consistent with previous studies, enterotoxin gene clusters were found on genomic islands in nine strains. These genomic islands did not contain antibiotic resistance genes. This suggests that enterotoxin gene clusters on genomic islands can be transmitted between bacteria via horizontal transfer. Another study reported that human-associated ST5 strains are more virulent than other strains [46]. In this study, ST5 strains (LNZ_R_SAU_37 and LNZ_R_SAU_46) carried the highest number of most virulence genes. These strains, which harbor leukotoxin and enterotoxin, can spread to humans through the food chain and cause illness.

Linezolid resistance is associated with mutations in the 23S rRNA gene and ribosomal proteins and/or the acquisition of cfr, optrA, and poxtA, which are carried in mobile genetic elements [14]. Accordingly, several studies detected optrA, poxtA, and cfr in linezolid-resistant S. aureus and Enterococcus faecalis strains from livestock and food [14, 15, 35]. In this study, linezolid-resistant, methicillin-sensitive S. aureus linezolid-resistant MRSA strains harbored cfr. In addition, these strains carried fexA. Both cfr and fexA were closely co-localized within the contig. Among the 111 publicly available genomes, 10 carried cfr, and all but one belonged to ST398. Conversely, optrA and poxtA, which can also mediate linezolid resistance and which are often present in mobile genetic elements, were not identified in this study. This is likely because these genes are typically observed in enterococci rather than staphylococci. All S. aureus strains carrying cfr and fexA exhibited rather strong linezolid (MIC≥8 mg/L) and chloramphenicol resistance (MIC>64 mg/L) [21]. The frequent use of phenols and pleuromutilins on Korean livestock farms could be associated with the co-selection of linezolid resistance [21].

S. aureus is considered a reservoir of antibiotic resistance genes, and insertion sequences or transposons play an important role in the propagation of genes, requiring monitoring to detect transferable antibiotic-resistant strains [32]. The repUS5 was found in all strains, excluding four ST541 strains. The repUS5 was previously found in S. aureus isolated from poultry, and it has been linked to antimicrobial resistance gene transfer in staphylococci [47]. BLAST analysis revealed that the cfr and fexA-carrying fragments had high similarities with a plasmid of the S. aureus strain 004-737X (pSA737) isolated from a clinical sample in the United States and a plasmid of S. aureus strain 359 (unnamed2) isolated from human in Germany [48]. Meanwhile, cfr was flanked by Tn554-related tnpA, tnpB, and tnpC or ISSau9 (also called IS21-558) in all strains. Among the 111 publicly available genomes, 10 harbored cfr. These strains were isolated from pig-related sources in Australia (one strain), the Netherlands (one strain), China (three strains), and Belgium (five strains). Consistent with our findings, cfr was flanked by Tn554-related tnpA, tnpB, and tnpC or ISSau9 in these strains (Additional file 5). In previous studies, Tn554-mediated optrA transfer was detected in the chromosome carrying optrA, indicating that this gene can be transmitted between bacterial species [49, 50]. ISSau9, originally detected in the pSCFS3 plasmid recovered from an S. aureus strain of pig origin in Germany, was also found in the pGMI17-006 plasmid from a human S. aureus strain from Denmark [27, 51]. The cfr in these strains is flanked by ISSau9 and Tn554. Therefore, Tn554 and ISSau9 could play important roles in the horizontal transmission of cfr and fexA in other pathogenic bacteria. These data indicate the need for continued surveillance of linezolid-resistant S. aureus carrying mobile genetic elements.

In the phylogenetic analysis, the LNZ_R_SAU_10 and LNZ_R_SAU_57 strains were grouped with S. aureus strains isolated from pigs in Belgium (Fig. 1). These strains belong to ST393, and they harbor cfr flanked by Tn554-related tnpA, tnpB, and tnpC. LNZ_R_SAU_31, LNZ_R_SAU_58, and LNZ_R_SAU_62 were grouped with S. aureus strains isolated from pig skin in the United States, and all of these strains belonged to ST9. LNZ_R_SAU_37 and LNZ_R_SAU_46 were grouped with the ISU 998 strain (sapig_32) isolated from a swine facility in the United States, and all of these strains belonged to ST5. LNZ_R_SAU_21, LNZ_R_SAU_26, and LNZ_R_SAU_27 strains were grouped with S. aureus strains isolated from a pig farm in Australia, and all of these strains belonged to ST433. Four strains (LNZ_R_SAU_22, LNZ_R_SAU_23, LNZ_R_SAU_24, and LNZ_R_SAU_25) belonging to ST541 were grouped with ST398 strains isolated from pig farm dust and pig farms in Italy and China. ST541 is a single-locus variant of ST398. Although ST541 and ST398 are closely related, they exhibit different antibiotic resistance patterns [27]. ST541 has been occasionally found in MRSA strains in Korea, but it has not yet been reported in other countries. Similarly, four linezolid-resistant S. aureus ST541 strains were detected in this study, and all but one (LNZ_R_SAU_25) harbored mecA conferring methicillin resistance. The LNZ_R_SAU_64 strain (ST8004) was grouped with the CFSAN018749 strain (ST433) isolated from tissue and/or biological fluid from swine in Denmark. This study detected the ST8004 strain (LNZ_R_SAU_64) in pig carcasses for the first time. This strain was resistant to linezolid (MIC=8 mg/L), chloramphenicol (MIC=64 mg/L), clindamycin (MIC>4 mg/L), tiamulin (MIC>4 mg/L), and quinupristin/dalfopristin (MIC>4 mg/L) [21]. ST8004 (allele profile 2-2-637-2-6-3-72) is a single-locus variant of ST433 (allele profile 2-2-2-2-6-3-72). These two sequence types featured different antibiotic resistance genes. Specifically, all ST433 strains commonly carried cfr, fexA, and fosD, whereas the ST8004 isolate harbored cfr, fexA, fosD, and vga(A). This difference in antibiotic resistance genes between extremely close types, such as ST433 and ST8004, could be attributable to the acquisition of mobile genetic elements.

This study highlighted that transferable linezolid resistance and virulence genes in S. aureus strains could persist in pig carcasses. Mobile genetic elements, such as plasmid replicons (repUS5), Tn554, and ISSau9, might mediate the horizontal transfer of cfr and fexA in S. aureus strains in pig carcasses, whereas genomic islands could play a similar role in the horizontal transfer of enterotoxin gene clusters. These results suggest that linezolid resistance and virulence genes in S. aureus strains have diverse transmission properties in livestock or food-producing animals. In addition, linezolid-resistant S. aureus can be transmitted from pigs to humans, and S. aureus in pigs used for food production could represent an important repository of transferable linezolid resistance and virulence genes. The prevalence and transmission of transferable genes in S. aureus strains from livestock or food-producing pigs should be continually monitored.

Author contributions

EK, H-SK, S-KL, and H-YK conceived and designed this study. EK and S-MY analyzed the data and carried out the experiments. S-MY and B-YM validated the experiments. H-YK supervised the study. EK wrote the draft manuscript. H-SK, S-KL, and H-YK reviewed and edited the final manuscript. All authors read and approved the final manuscript.

Availability of data and materials

The datasets are available from the corresponding author on reasonable request.

Declarations

Footnotes

Contributor Information

Suk-Kyung Lim, Email: rk.aerok@9400ksmi.

Hae-Yeong Kim, Email: rk.ca.uhk@mikyh.

References