Genomic Information on Linezolid-Resistant Sequence-Type 398 Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolated from a Pig

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important pathogens causing health care and community-associated (CA) infections with high mortality and morbidity rates (David et al., 2010). The recent emergence of a livestock-associated (LA) clone of MRSA with multilocus sequence-type (ST) 398 featuring a staphylococcal cassette chromosome mec type V (SCCmec V) has been a serious public health problem (He et al., 2018; Pirolo et al., 2019; Back et al., 2020). Although ST398 LA-MRSA has mainly been detected in pigs and pig farm environments, zoonotic transmission of ST398 LA-MRSA to humans through direct or indirect contact is increasing (Cuny et al., 2009; He et al., 2018; Pirolo et al., 2019). Furthermore, fatal cases of infections with human-adapted clones of ST398 CA-MRSA have been reported in China (He et al., 2018). Previous studies about virulence determinants of ST398 and closely related STs within clonal complex (CC) 398 revealed that CC398 LA-MRSA lacked several major staphylococcal virulence genes encoding immune evasion cluster (IEC), toxic shock syndrome toxin 1, exfoliative toxins, and Panton–Valentine leukocidin (PVL) (Price et al., 2012; Sahibzada et al., 2017). However, several uncommon antimicrobial resistance genes for S. aureus, such as phenicol exporter gene fexA, linezolid resistance gene cfr (chloramphenicol–florfenicol resistant), tetracycline resistance gene tet(L), and macrolide–lincosamide–streptogramin B (MLS_B) resistance genes erm(T), have been identified in CC398 LA-MRSA strains (Kehrenberg et al., 2006; Kadlec et al., 2009; Gomez-Sanz et al., 2013).

The cfr gene encodes methyltransferase, which facilitates methylation of the adenosine residue at position 2503 in 23S rRNA. This modification results in a multiresistance phenotype, with resistance to phenicols, lincosamides, oxazolidinones, pleuromutilin, and streptogramin A (PhLOPS_A) antibiotics (Long et al., 2006). The cfr gene was first reported in a S. sciuri isolate from a calf and was thereafter detected in a number of staphylococcal isolates worldwide (Schwarz et al., 2000). Although linezolid-resistant S. aureus is still relatively rare in Korea, Moon et al. (2015) reported the detection of cfr-positive CC398 LA-MRSA strains isolated from pig carcass samples. Given the clinical significance of linezolid as one of the last resort for the treatment of human infections caused by multidrug-resistant Gram-positive bacteria (Choo et al., 2016; Wessels et al., 2018), it is important to investigate the genetic mechanism of linezolid resistance and epidemiological relatedness of the linezolid-resistant LA-MRSA strains. Thus, in this study, whole-genome sequencing was used to evaluate the epidemiological characteristics and genomic features of antimicrobial resistance in a pig-associated linezolid-resistant CC398 LA-MRSA strain.

Materials and Methods

Results

Genomic features of the ST398 LA-MRSA strain PJFA-521M

The draft genome of ST398 MRSA strain PJFA-521M contains a circular chromosome with a length of 2,895,108 bp, coverage of 150.9 × , and GC content of 33%. A total of 2760 annotated genes possessed 2695 open-reading frames (ORFs), 57 tRNAs, and 7 rRNAs. Among 2695 ORFs, 2011 genes (74.6%) were predicted to encode functional proteins, whereas 684 genes (25.4%) were uncharacterized or hypothetical.

The functional categorizations of COG and SEED are given in Figure 1. In the COG categorization, the five major COG categories were as follows: 211 ORFs of amino acid transport and metabolism (7.8%, category E); 161 of inorganic ion transport and metabolism (6%, category P); 150 of translation, ribosomal structure, and biogenesis (5.6%, category J); 143 of replication, recombination, and repair (5.3%, category L); and 140 of carbohydrate transport and metabolism (5.2%, category G). Likewise, ORFs belonging to amino acids and derivatives (273), carbohydrates (178), protein metabolism (160), cofactors, vitamins, prosthetic groups, pigments (117), and DNA metabolism (91) were mainly distributed within the SEED subsystem categorization. In addition, 68 ORFs and 25 ORFs belonged to the virulence, disease-defense category and MGEs, respectively. The functional characterization of genes in PJFA-521M revealed that metabolic functions comprise 32% of the S. aureus genome.

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FIG. 1.

Analysis of functional categories in the genome of strain PJFA-521M based on the (A) COG and (B) SEED databases. COG, Clusters of Orthologous Groups. Color images are available online.

Phylogenetic lineage and antibiogram of CC398 isolates

A phylogenetic tree of 90 different CC398 S. aureus strains including PJFA-521M is given in Figure 2. PJFA-521M was most closely related to four of the ST398 LA-MRSA strains (UB08116, 12152-5, 44523-1, and 30116 strains) isolated in European countries. All the CC398 MRSA and methicillin-susceptible Staphylococcus aureus (MSSA) strains were resistant to tetracycline and carried one or more tetracycline resistance genes. Of note, all the CC398 S. aureus strains had the tet(38) gene except one pig-originated MRSA strain (AV6) (Fig. 2). However, only 18% (16/90) of the CC398 MRSA strains carried the dfrG gene, which was identified in PJFA521M for trimethoprim resistance. The frequencies of two phenicol resistance genes, cat and fexA, among the 90 CC398 MRSA strains were 4% and 6%, respectively (data not shown). None of the strains carried genes associated with mupirocin and fusidic acid resistance. Of importance, only 2/90 CC398 S. aureus strains (PJFA-521M and 09/01691/3 strains) were positive for the cfr gene. The genetic environments of the cfr gene in PJFA-521M and 09/01691/3 were identical (Supplementary Fig. S2B).

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FIG. 2.

Phylogenetic relationship and antibiogram of CC398 strains. Color images are available online.

Discussion

Although CC398 LA-MRSA has mainly colonized pigs, human infections with CC398 LA-MRSA through direct or indirect contact with various livestock animals have been increasingly reported in recent years (Cuny et al., 2009; He et al., 2018; Pirolo et al., 2019). Genomic analyses of the CC398 lineage of LA-MRSA revealed that gain or loss of certain genetic components was frequent during its transmission between animal and human hosts (Price et al., 2012).

The absence of pvl genes and ϕSa3 prophage containing IEC in porcine-derived CC398 strains supports the notion that these genes are associated with immunomodulation in humans but not in nonhuman hosts (Price et al., 2012). Similarly, the PJFA-521M strain was also negative for lukS/F genes encoding PVL. However, novel leukocidins designated lukG and lukH (also known as lukAB) were detected in the genome of PJFA-521M. LukGH displaying cytotoxic potency in human phagocytes was initially described in the USA300 CA-MRSA TCH1516 strain and subsequently identified in a ST398 LA-MRSA isolate (Schijffelen et al., 2010; Trstenjak et al., 2019). The sequence analyses of LukGH identified in the PJFA-521M strain resulted in 99% and 89% amino acid identity to those of the ST398 LA-MRSA S0385 and USA300 CA-MRSA TCH1516 (CP000730) strains, respectively. Based on previous studies, LukGH displays a marked host-species specificity, and variations in LukH may greatly impact the pathogenesis of MRSA during its transmission between pig and human hosts (Ventura et al., 2010; Badarau et al., 2015; Trstenjak et al., 2019).

A homolog of staphylococcal pathogenicity island (SaPI-S0385) harboring scn and vwb was also identified during the PJFA-521M genome analysis (Schijffelen et al., 2010). Staphylococcal complement inhibitor (SCIN), a product of the scn gene, interferes with complement activation by blocking C3 convertases. The vwb homolog in the PJFA-521M genome encodes von Willebrand factor-binding protein (vWbp), which binds to the vWf protein. It has been shown that both human and porcine serums displayed similar levels of coagulation rates after exposure to vWbp (Bjerketorp et al., 2004). Although a complete understanding of the host range for scn and vwb homologs on SaPI-S0385 is not available, the presence of these genes may broaden host specificity during transmission between human and animal hosts.

In addition to immunomodulators, various genes encoding adhesion factors were identified in the genome of the PJFA-521M strain. A number of cell wall-anchored proteins and secreted proteins appear to be involved in adherence to host cells during staphylococcal colonization (Tulinski et al., 2014). In addition to their binding to host cell molecules, such as fibrin and fibrinogen, ClfA, ClfB, FnBPA, FnBPB, SdrC, SdrD, and SdrE, the role of enhanced staphylococcal immune escape or host cell invasion was reported (Tulinski et al., 2014). In a previous report (Schijffelen et al., 2010), it was demonstrated that the ST398 LA-MRSA strain S0385 carried several unique MGEs containing genes involved in host-cell adhesion. The presence of MGEs containing scn, vwb, and tet(M) was identified only in animal-derived ST398 MRSA strains (Schijffelen et al., 2010). Similarly, the PJFA-521M strain harbored various unique MGEs, especially a homolog of vwb-containing MGEs, in ST398 LA-MRSA S0385 strain. SCIN encoded by scn identified in SaPI-S0385 and ϕ3, which inhibits the complement activation by interfering with C3b deposition, resulted in a reduction in pathogen killing of human neutrophils (Rooijakkers et al., 2005). Proteins encoded by vwb (vWbp), which binds to vWf, play multifunctional roles in the coagulation activity with host prothrombin in plasma (Viana et al., 2010; Thomer et al., 2013). Moreover, surface-bound protein A of S. aureus also increases the adherence ability and damage of host cells by interacting with vWf (Viela et al., 2019). These results suggest that the carriage of scn and vwb genes may help evade host innate immunity and facilitate adaptation of ST398 LA-MRSA in human host.

The carriage of multiple antimicrobial resistance genes responsible for multidrug-resistant phenotypes has recently been reported in ST398 LA-MRSA strains (Sarrou et al., 2016; Fessler et al., 2018). The tetracycline resistance phenotype along with the resistance genes have frequently been found in CC398 LA-MRSA strains (Price et al., 2012; Eom et al., 2019). The presence of the chromosomal tet(M) gene has been considered a robust genetic marker for CC398 LA-MRSA strains (Price et al., 2012). In addition, dual carriage of tet(M) and plasmid-borne tet(K) or tet(L) genes was observed in many LA-MRSA strains, which displayed higher MIC values for tetracycline than the strains carrying a single gene (Kadlec et al., 2010; Back et al., 2020). In this study, the presence of both tet(M) and tet(L) in the PJFA-521M strain has also been confirmed, which may explain the high MIC (64 μg/mL) and minimum bactericidal concentration values (128 μg/mL) to tetracycline.

Recently, the detection of rather uncommon resistance genes has been reported in CC398 LA-MRSA isolates (Kadlec et al., 2010; Gomez-Sanz et al., 2013). The MLS_B resistance gene, erm(T), the trimethoprim resistance gene dfrG, and the tetracycline resistance gene tet(L) were identified in the genome of the PJFA-521M strain (Supplementary Fig. S1). The novel gene cluster of erm(T), dfrK and tet(L) was initially found on the plasmid pKKS25 of a ST398 LA-MRSA isolate from a sow (Kadlec et al., 2010). Instead of drfK, the PJFA-521M strain carried dfrG in contig 33, which showed 100% nucleotide sequence similarity to that of the CM.S2 MRSA strain (GenBank Accession No. IMCJ1454) (Sekiguchi et al., 2005). Although occurrence of dfrG and dfrK genes has been rare in animals, the presence of dfrK and tet(L) gene cluster may affect emergence of trimethoprim resistance genes under the selective pressure of tetracycline in pig farms (Back et al., 2020). As presented in Supplementary Figure S1, the genetic composition of contig 31 containing aadE, spw, lsa(E), and lnu(B) genes in the PJFA-521M strain showed 99% nucleotide sequence identity to the chromosomal integration region of MSSA C2828 strain (GenBank Accession No. JQ861959) (Lozano et al., 2012). The novel aminoglycoside and lincosamide resistance gene clusters in a multiresistance plasmid have been previously described in animal isolates (Wendlandt et al., 2013; Sarrou et al., 2016). Collectively, these data indicate that colocalization of multiple resistance genes conferring resistance to various classes of antimicrobial agents may enable their co-selection and persistence even in the absence of a direct selective pressure in pig farms.

Linezolid has been exclusively used for clinical practice as a last-resort antimicrobial for the treatment of MRSA and Gram-positive bacterial infections (Choo et al., 2016; Wessels et al., 2018). Although it is not approved for use in veterinary medicine, the occurrence of linezolid-resistant S. aureus and staphylococci in food-producing animals has caused significant public health concerns, especially owing to the cfr-mediated multiresistance PhLOPS_A phenotype (Cuny et al., 2017; Li et al., 2018). In the PJFA-521M strain, the cfr gene was identified within 3216-bp contig 44, which exhibited 100% nucleotide sequence identity with the plasmid p14-01514 of S. epidermidis (NZ_KX520649) (Wessels et al., 2018). Moreover, identification of the florfenicol–chloramphenicol exporter gene fexA in contig 42 suggests that fexA and cfr are colocalized on a plasmid of the PJFA-521M strain (Supplementary Fig. S1). Co-selection of cfr and fexA in MRSA and other staphylococci has been linked to the widespread use of chloramphenicol and florfenicol in swine farms (Kehrenberg et al., 2006; Shen et al., 2013; Zhao et al., 2016; Sun et al., 2018) and coexistence of cfr and fexA with additional resistance gene such as aacA-aphD, tet(L), dfrK, erm(A), erm(B), or lsa(B) may allow the cfr and fexA genes to be co-selected under nonphenicol use in farms (Kehrenberg et al., 2007; Shen et al., 2013). Besides the cfr gene, a novel oxazolidinone resistance gene, optrA, has been reported in staphylococci from pigs (Fan et al., 2016, 2017). In this study, whole-genome sequence analysis confirmed the absence of the optrA gene in the PJFA-521M strain. However, large-scale investigation of the surveillance of the cfr and optrA genes in staphylococci and enterococci of livestock origin is needed to assess the prevalence of these genes in the farm environment. To the best of our knowledge, this is the first report of whole-genome sequence analysis of an ST398 LA-MRSA strain isolated from a pig in Korea.

Conclusions

Our results demonstrate that: (i) the linezolid-resistant ST398 LA-MRSA strain isolated from a pig carries the cfr gene for the PhLOPS_A resistance phenotype; (ii) in addition to cfr for linezolid resistance, the tet(L), erm(T), dfrG, aadE, spw, lsa(E), lnu(B), and fexA genes were identified in PJFA-521M, which may help co-select the strain under exposure to various antimicrobial agents in the pig farm environment; and 3) the PJFA-521M strain harbored various unique MGEs, some of which contain host-specific virulence factors for human and animal host infections.