Genomic Insights of First ermB-Positive ST338-SCCmecVT/CC59 Taiwan Clone of Community-Associated Methicillin-Resistant Staphylococcus aureus in Poland

We report the first Polish representative of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), lukS/F-PV-positive, encoding the ermB gene, as a genetic determinant of constitutive resistance to macrolides, lincosamides, and streptogramin B antibiotics, cMLS-B. This is the first detection of the CA-MRSA strain responsible for nosocomial infection in the Warsaw Clinical Hospital. Resistance to β-lactams associates with a composite genetic element, SCCmec cassette type VT (5C2&5). We assigned the strain to sequence type ST338 (single-locus variant of ST59), clonal complex CC59, spa-type t437, and agr-type I. Genomic-based comparison was designated SO574/12 as an international Taiwan clone, which has been so far described mainly in the Asia-Pacific region. The ermB gene locates on the chromosome within the 14,690 bp mobile element structure, i.e., the MESPM1-like structure, which also encodes aminoglycoside- and streptothricin-resistance genes. The MESPM1-like structure is a composite transposon containing Tn551, flanked by direct repeats of IS1216V insertion sequences, which probably originates from Enterococcus. The ermB is preceded by the 273 bp regulatory region that contains the regulatory 84 bp ermBL ORF, encoding the 27 amino acid leader peptides. The latest research suggests that a new leader peptide, ermBL2, also exists in the ermB regulatory region. Therefore, the detailed function of ermBL2 requires further investigations.


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is described as one of the most dangerous human pathogens. For many years, community-associated MRSA, CA-MRSA, has been limited to populations outside healthcare settings. It was just in the last two decades that it were considered clinically significant and a potentially highly virulent pathogen associated with serious, highly invasive and progressive skin and soft tissue infections, necrotizing pneumonia, sepsis, and fasciitis, particularly in young healthy individuals [1][2][3]. In recent years, due to the evolving epidemiology of CA-MRSA, these strains have also emerged as the cause of hospital outbreaks. Nosocomial outbreaks of CA-MRSA have been reported in various parts of the world, often affecting particular areas of hospitals, such as neonatology, pediatric, obstetric, or ophthalmic units, where the prevalence of healthcare-acquired MRSA, HA-MRSA, has been low [1,[3][4][5][6].
CA-MRSA strains differ from HA-MRSA. They have unique epidemiology, phylogenetic origin, and genetic profile that is linked with carrying a smaller version of SCCmec (mainly type IV and V or V T ); an ability to produce toxins, e.g., Panton-Valentine leukocidin (PVL), which confer higher toxigenic potential; and expression of a unique antibiotic resistance pattern (resistance to fewer non-β-lactam antibiotics than HA-MRSA) [2,7]. tance (cMLS-B) in D-test zone method (all disc zones equal 6 mm, MIC value >256 mg/L). When using end-point PCR to detect ermA/B/C genes, only ermB gene was positively confirmed (amplicon was 359 bp in size). The main features of SO574/12 clinical isolate and details of antimicrobial resistance profile to 21 antimicrobials are listed in Tables 1 and 2, parts A and B.

Genome-Assembly Features
The MRSA ermB SO574/12 was assembled with the use of SPAdes. The coarse consistency of the genome was 100%, fine consistency was 99.8%, and completeness was 100%. There were 67 contigs (the largest 494,917 bp), an estimated genome length of 2,792,694 bp, and an average G+C content of 32.74%. The N50 length was 127,953 bp. The L50 count was 7. Key assembling data for MRSA SO574/12 isolate are presented in Table 3.

Genome Annotation and Taxonomy Confirmation
The genome of the SO574/12 strain was annotated using the RAST tool kit (RASTtk) and assigned a unique genome identifier: 1280.32586. This genome has 2643 protein coding sequences (CDS) and 60 transfer RNA (tRNA) genes (see Table S1). The annotation included 512 hypothetical proteins and 2131 proteins with functional assignments. The proteins with functional assignments included 740 proteins with Enzyme Commission (EC) numbers, 613 with Gene Ontology (GO) assignments, and 534 proteins that were mapped to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.
A circular graphical display of the distribution of the genome annotations is provided ( Figure 1). Each color informs about a different CDS type. It includes rings, from outer to inner rings: the contigs, CDS on the forward strand, CDS on the reverse strand, RNA genes, CDS with homology to known antimicrobial resistance genes (AMR), CDS with homology to know virulence factors (VF), GC content, GC skew, and others.

Comparative Genomics of CA-MRSA SO574/12
The global whole-genome and local small-scale alignment with the use of the progressive Mauve algorithm was performed for two genomes: CA-MRSA ZY05 Taiwan clone, as reference, and CA-MRSA SO574/12. The resulting alignment was visualized using the PATRIC BBRC website, providing insight into homologous regions and changes due to DNA recombination. The details of the global large-scale genome alignment are presented in Figure 2.

Genomes (KEGG) pathways.
A circular graphical display of the distribution of the genome annotations is provided ( Figure 1). Each color informs about a different CDS type. It includes rings, from outer to inner rings: the contigs, CDS on the forward strand, CDS on the reverse strand, RNA genes, CDS with homology to known antimicrobial resistance genes (AMR), CDS with homology to know virulence factors (VF), GC content, GC skew, and others.  Each type of locally collinear block (LCB) was marked with a different color. LCBs containing highly conserved homology regions were assigned with the same stain. Homology regions that were defined within both aligned genomes were connected with a line in the same color. Thirty-six common LCBs were detected. The maximum and minimum lengths of the LCBs were 372,796 bp and 641 bp, respectively, in the ZY05 genome, and 372,878 bp and 643 bp, respectively, in the SO574/12 genome. The summary length of the LCBs was 2,761,725 bp in the ZY05 genome and 2,750,602 bp in the SO574/12 genome, which were 97.85% and 98.50% of the total genome sizes, respectively. The ermB gene was detected in contig 33 (size 14,772 bp) of the CA-MRSA SO574/12 genome. The size of the homology region in the genome of CA-MRSA ZY05 was 14,767 bp.
The global whole-genome and local small-scale alignment with the use of the progressive Mauve algorithm was performed for two genomes: CA-MRSA ZY05 Taiwan clone, as reference, and CA-MRSA SO574/12. The resulting alignment was visualized using the PATRIC BBRC website, providing insight into homologous regions and changes due to DNA recombination. The details of the global large-scale genome alignment are presented in Figure 2. Each type of locally collinear block (LCB) was marked with a different color. LCBs containing highly conserved homology regions were assigned with the same stain. Homology regions that were defined within both aligned genomes were connected with a line in the same color. Thirty-six common LCBs were detected. The maximum and minimum lengths of the LCBs were 372,796 bp and 641 bp, respectively, in the ZY05 genome, and 372,878 bp and 643 bp, respectively, in the SO574/12 genome. The summary length of the LCBs was 2,761,725 bp in the ZY05 genome and 2,750,602 bp in the SO574/12 genome, which were 97.85% and 98.50% of the total genome sizes, respectively. The ermB gene was detected in contig 33 (size 14,772 bp) of the CA-MRSA SO574/12 genome. The size of the homology region in the genome of CA-MRSA ZY05 was 14,767 bp.
The local small-scale sequence alignment allows to compare homologous regions insight of defined pair of locally collinear blocks (LCBs), which encode the ermB gene for both CA-MRSA strains. Additionally, plasmid sequence pEflis48 from E. faecalis N48 strain, which was also ermB-positive, was included in the analysis. The details of the alignment are presented in Figure 3. The local small-scale sequence alignment allows to compare homologous regions insight of defined pair of locally collinear blocks (LCBs), which encode the ermB gene for both CA-MRSA strains. Additionally, plasmid sequence pEflis48 from E. faecalis N48 strain, which was also ermB-positive, was included in the analysis. The details of the alignment are presented in Figure 3.  The aligned sequences of CA-MRSA ZY05 and CA-MRSA SO574/12 strains and a part of the pEflis48 plasmid, according to the progressive Mauve algorithm, were classified into one common LCB and recognized as homologues with a high level of similarity, coverage, and structure organization.
In the genome of strains ZY05 and SO574/12, the ermB gene was located in a chromosome on the mobile element structure (MES), as opposed to E. faecalis N48, where it was found on an independent extra chromosomal mobile genetic element (MGE), plasmid pEflis48. Chromosomally encoded ermB MESs demonstrated a high similarity to The aligned sequences of CA-MRSA ZY05 and CA-MRSA SO574/12 strains and a part of the pEflis48 plasmid, according to the progressive Mauve algorithm, were classified into one common LCB and recognized as homologues with a high level of similarity, coverage, and structure organization.
In the genome of strains ZY05 and SO574/12, the ermB gene was located in a chromosome on the mobile element structure (MES), as opposed to E. faecalis N48, where it was found on an independent extra chromosomal mobile genetic element (MGE), plasmid pEflis48. Chromosomally encoded ermB MESs demonstrated a high similarity to MES PM1 in CA-MRSA PM1 CC59/ST59/SCCmecVb from Taiwan (since the genome sequence of S. aureus PM1 strain is deposited in GenBank database in the form of many parts, rather than a single molecule, it was impossible to include the PM1 genome in the whole-genome comparison analysis). Locally, small-scale multiple sequence alignment was performed, which is presented later in this publication. The ermB MES region in the SO574/12 clinical isolate was named an MES PM1-like structure. It encodes 19 coding sequences and two insertion sequences. The appropriate MESs in ZY05 MRSA and pEflis48 MGE additionally contain disrupted sat-4 gene and gentamicin resistance predicted region but, according to the methodology used, do not encode the hypothetical protein that just follows down the ORF of ermB gene. A higher content of the mobile element protein gene was also noticed.

Genomic-Based Antimicrobial Resistance Analysis
Fifty-four genes with well-predicted function related to the mechanisms of antibiotic resistance were detected. The genes are listed and characterized by the mechanism of resistance and the function of gene product in Tables 5 and S2. Determinants were divided into two essential groups: the first group containing genes directly correlated with the phenotype of resistance and the second group with genes that are either intrinsic or species-specific, or they encode the target of the drug in the tested genome, so their lack, derepression, or overexpression or other mutational changes demonstrate a resistance phenotype. The correlation of the detected genes with an antimicrobial resistance profile is presented in Table 2.

Genomic-Based Antimicrobial Resistance Analysis
Fifty-four genes with well-predicted function related to the mechanisms of antibiotic resistance were detected. The genes are listed and characterized by the mechanism of resistance and the function of gene product in Tables 5 and S2. Determinants were divided into two essential groups: the first group containing genes directly correlated with the phenotype of resistance and the second group with genes that are either intrinsic or species-specific, or they encode the target of the drug in the tested genome, so their lack, derepression, or overexpression or other mutational changes demonstrate a resistance phenotype. The correlation of the detected genes with an antimicrobial resistance profile is presented in Table 2.
The strain was positively confirmed as a Panton-Valentine leukocidin, PVL gene carrier (lukS/F-PV), which was encoded on SA2 PM1-like phage. The nucleotide sequence of SA PM1 phage, originated from the S. aureus PM1 strain, showed 99.9% identity (data not shown).

Discussion
Here, for the first time in Poland, we present a detailed genomic characterization of a representative Polish variant of CA-MRSA ST338-SCCmecV T /CC59 PVL-positive clone, encoding the ermB gene cluster as a determinant of constitutive resistance to MLS-B antibiotics (cMLS-B). The main theme of this study, MRSA strain SO574/12, was isolated from an infected surgical wound in 2012. We have shown that it carries SCCmec type (subtype) Vb, also known as SCCmecV T (5C2&5), cassette chromosome recombinase ccr genes complex type ccrC1-allele-2, ccrC1-allele-8, and mec gene complex class C2. The isolate represents the spa-type t437, agr-type I [29], sequence type ST338, and clonal complex CC59 and exhibits the lukS/F-PVpositive genotype. It is positive for the chemotaxis inhibitory protein (chp gene) but does not contain sak and sep virulence factors mediating immune avoidance functions. Based on all such extensive genomic analysis, the strain has been classified as an epidemic CA-MRSA clone "Taiwan". ST338/CC59 is a single locus variant (within the gmk gene) derived from ST59/CC59 and has not been reported yet as a global pandemic clone. The first genome of ST338-SCCmecV/CC59 PVL-positive isolate has been published just recently in 2020 [5,30]. Representatives of this clone have so far been detected mainly in Taiwan, China, and several other Asia-Pacific countries, such as Japan, Vietnam, Singapore, and Australia [5,18,31,32]. Unfortunately, the patient's medical history does not mention any travel in the period preceding the transplant procedure. In Europe, single cases have been reported so far in England, Denmark, The Netherlands, Norway, Sweden, Hungary, Germany, and also in Poland [4,26,[33][34][35][36], which might be a more probable travel history. However, none of them were confirmed as the ermB-positive isolate.
Comparative analyses at the genomic DNA level, as well as analysis of the genetic structure and organization of the ermB-carrying region within the surrounding genetic environment, were carried out compared to the sequence of the most-detailed characterized strains CA-MRSA ZY05 and PM1, originating in China and Taiwan [17,30]. According to that analysis of whole genomic DNA, we have suggested that the Polish isolate of CA-MRSA ST338-SCCmecVb/CC59 PVL-positive strain is closely related to the Taiwan clone, represented by the CA-MRSA ZY05 strain from China [30]. The SO574/12 strain presents 98.50% genome homology compared to ZY05. However, the Polish strain had been isolated four years earlier than ZY05, which strongly suggests the SO574/12 MRSA had originated from other parts of the world, not from the Asian regions.
The strain SO574/12 expresses a multidrug-resistance phenotype, highly similar to the previously described PM1 ST59-SCCmecV T /CC59 strain from Taiwan and the ZY05 ST338-SCCmecV T /CC59 strain from China [17,30]. Meanwhile, 13 ST338/CC59 strains among MRSA isolates obtained in China between 2014-2019, from human blood, were resistant only to erythromycin, clindamycin, and oxacillin [37]. The SO574/12 isolate is an older variant of ST338/CC59 CA-MRSA, but it appears more resistant than the more recent Chinese ones. The genetic features, which are transferred in MGE, are unstable and metabolic-cost consuming but play an important role as an adaptative factor. In the case of the lack of selective factor (e.g., antibiotic), the MGE-carried genes can be lost from bacterial cells [38,39]. This may depend on the local antibiotic policy. The global whole-genome alignment with the use of the progressive Mauve algorithm needs to be performed for the SO574/12 genome and the 13 ST338/CC59 MRSA genomes described by Jin et al., to assess their phylogenetic relation. Unfortunately, the genomic sequences of the Chinese isolates were not provided for an independent verification.
The antimicrobial resistance profile of SO574/12 correlates with the presence of the mecA gene and blaZ/blaR1/blaI gene cluster, which are responsible for resistance to βlactams. Resistance to aminoglycosides is associated with the presence of aminoglycosidemodifying enzyme genes: aph(3 )-III and ant(6)-I. Resistance to tetracyclines is expressed due to MFS efflux pump genes: tetK and tet38 [20,40]. The cMLS-B phenotype correlates with the presence of ermB gene cluster consisting of the ermB gene, its leader peptide regulatory region ermBL and an additional ermB-AP CDS, which encodes a peptide of unknown function [41,42]. Until recently, the main determinants of ermA and ermC of the MLS-B phenotype have been dominant among Polish MRSA strains. The ermB gene has been most often found in Gram-negative rods, Streptococcus spp., and Enterococcus species [20]. This study is the first description of the ermB-positive MRSA strain from Poland.
We have shown that the CA-MRSA isolate SO574/12 has the ermB gene located on the chromosome within a mobile element structure, similar to the part of MES PM1 of the CA-MRSA PM1 ST59-SCCmecVb/CC59 strain from Taiwan [17]. This genetic region has been detected in contig 33 of the SO574/12 genome and was named MES PM1-like structure. It is a 14,690 bp composite transposon consisting of 19 CDSs and two 127 bp IS1216V-mediated direct repeats flanking both ends of the structure. Insertion sequence IS1216V, belonging to the IS6/IS26 family, is 809 bp in length with 18 inverted repeats. IS1216V is a typical enterococcal insertion sequence, rarely found in S. aureus, but up to five copies of IS1216V are located in MES PM1 and MES 6272-2 of ST59 S. aureus [43]. Multiple local alignment comparison of nucleotide sequences of four mobile genetic structures encoding the ermB gene cluster, MES PM1-like structure of CA-MRSA SO574/12 SCCmecVb/ST338/CC59 clinical isolate, MES PM1-like structure of CA-MRSA ZY05 SCCmecVb/ST338/CC59 reference Taiwan clone, MES PM1 structure of CA-MRSA PM1 SCCmecVb/ST59/CC59 reference clone, and Enterococcus faecalis N48 plasmid pEflis48, has shown that in the case of MES PM1 and two MES PM1-like structures in the Taiwan clones from Taiwan, China, and Poland, the identity and coverage is equal to 100%, without any mismatches. In turn, the identity and coverage of the homologous sequence in the pEflis48 plasmid are 99.99% and 99.15%, respectively, with only one discrepancy detected. It is most likely that ermB-carrying MES PM1-like structure had been transferred in a multi-stage evolutionary process between enterococci and S. aureus. Part of this process was by horizontal gene transfer from a strain similar to Enterococcus faecalis N48, via plasmid pEflis48-like. This plasmid is a mobile, self-replicating genetic element with a mosaic structure containing regions typical of both E. faecalis and S. aureus genomes. The other significant evolutionary stage was probably insertion sequence IS1216V-mediated bidirectional interspecies gene transfer via homologous recombination mechanism between plasmid-carried and chromosomeencoded gene clusters [43].
In our study, the ermB ORF is preceded by a 273 bp regulatory region that contains the regulatory 84 bp ermBL open reading frame, encoding the 27 amino acid leader peptides. The hypothesis of translation arrest on ermBL as a mechanism for ermB induction by erythromycin has been proven many times using an in vitro toe-printing assay. According to the literature, the ermB regulatory region contains one short leader peptide called ermBL with its ribosome binding site (RBS1), a non-translational loop-stem structure, and several ermB coding sequences, including its ribosome binding site (RBS2) [41,42,[45][46][47]. The latest research carried out by Wang et al. suggests that a new leader peptide, ermBL2, exists in the ermB regulatory region [48]. Based on the premature termination mutation and alanine-scanning mutagenesis of ermBL2, researchers have shown that the N-terminus of ermBL2 is essential for the expression of ermB-dependent resistance to MSL-B drugs [48]. Therefore, the detailed function of ermBL2 requires further investigations.
A total of 54 genes for antibiotic resistance were detected in the SO574/12 genome. The mere presence of some of these genetic factors does not correlate with the resistance phenotype (see Table S2). These are: tcaA/tcaB/tcaR associated with resistance to teicoplanin; clsA, gdpD, mprF, liaF/liaR/liaS associated with resistance to daptomycin; rpsJ (S10p) and mepA/mepR associated with resistance to tigecycline; iso-tRNA (ileS) and fusA genes associated with resistance to mupirocin and fusidic acid. Most of these genes are ubiquitous, internal, and species-specific, or regulator-dependent or drug-target genes. Their absence, derepression, or overexpression due to mutational changes indicate resistant phenotypes [2,23,49,50]. The lack of mutations correlates with susceptibility phenotypes (see Table 2).
The virulome of CA-MRSA isolates has been recognized as abundant, mainly due to presence of the genes, which are connected with their ability to produce a wide spectrum of exotoxins and superantigens (hemolysins, leukocidin PVL, and exfoliative toxin) [7]. Whereas, the virulome of HA-MRSA strains tends to contain more genes that determine their adhesive properties, immune avoidance factors, and exoenzymes [15]. In the analyzed genome of SO574/12 CA-MRSA, 65 virulence-associated genes have been detected. Of the seven functional groups, genes of toxins and superantigens are the most numerous and diverse. The lukS/F-PV genes, located on phage ΦSA2 PM1-like , determine the ability to produce Panton-Valentine leukocidin. The presence of these genes is typical among community-associated MRSA isolates [12,13]. The CA-MRSA analyzed in this study emerged from the hospital environment, which unexpectedly and significantly changes the local epidemiological situation in hospital wards, where, until recently, only healthcareacquired variants of MRSA were diagnosed. The adhesive properties of the SO574/12 strain are due to the presence of several genes encoding microbial surface components, recognizing the adhesive matrix molecules, MSCRAMM. The ability to synthesize the polysaccharide, poly-n-succinyl-β-1,6-glucosamine (PNSG), during infection is an important virulence factor based on the SO574/12 adhesive isolate. PNSG is critical for biofilm formation, allowing bacteria to adhere to one another and may also promote adherence to other molecules, such as extracellular matrix (ECM) components. This may act as an excellent environment for the formation of icaABCDR-dependent biofilm, a natural biological membrane. Due to the lack of the sdrD gene, the virulome of SO574/12 isolate (2012) shows similarity to ZY05 CA-MRSA (2016) but differs from the strains analyzed by Jin et al. (2014Jin et al. ( -2019, which were described as negative in clf A, clf B, eap, cna, sdrC, sdrD, and icaD genes [37].

Bacterial Strain
Methicillin-resistant Staphylococcus aureus strain SO574/12 was isolated from a male adult patient hospitalized in a surgical unit of the Infant Jesus Clinical Hospital of the Medical University of Warsaw (Poland) for a routine diagnostic procedure. The strain was recovered in February 2012 from the pus draining from an infected postoperative wound. Clinical sample was inoculated on Columbia Agar plate supplemented with 5% sheep blood (BioMerieux, Marcy-I'Etoile, France) and MRSA Chrom Agar plate (BioMerieux). Incubation was performed for 24 h at 37 • C under aerobic conditions. The identification of the strain was performed with the use of automatic system VITEK2, BioMerieux (GP cassettes). After preliminary tests, the SO574/12 S. aureus isolate was archived and stored deeply frozen at −70 • C.

Detecting Resistance to Other Antibiotics
Resistance to a set of other agents was assessed with the use of the disc diffusion (DD) method and the E-test method. The former (by Oxoid) was applied for penicillin (P); amikacin (AK); gentamycin (CN); ciprofloxacin (CIP); levofloxacin (LEV); mupirocin (MUP); fusidic acid (FUS); tetracycline (TET), and the latter (by BioMerieux) for ceftaroline (CPT); vancomycin (VA); teicoplanin (TP); linezolid (LZD); daptomycin (DPC); tigecycline (TGC); and spectinomycin (SC), according to the EUCAST guidelines [28,51] Pure MRSA strain SO574/12 colonies were revived by culturing on nutrient agar. Bacterial genomic DNA was isolated with use of commercial Genomic Mini Kit (A&A Biotechnology, Gdansk Poland), in accordance with the protocol of the manufacturer. Quality and quantity of DNA were assessed in Eppendorf BioSpectrometer ® fluorescence (Eppendorf, Wesseling Germany), with the use of Quant-iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen, CA, USA). DNA integrity was analyzed after 0.8% agarose gel electrophoresis. High-quality pure genomic DNA was stored at −20 • C until further analysis.

Detection of Antibiotic-Resistance Genetic Determinants-Targeted PCR Amplification
The presence of mecA and mecC genes was determined with PCR and appropriate primer pairs, according to the procedure described previously [52]. The cMLS-B phenotype was verified with PCR. The ermA, ermB, ermC, msrA, msrB, and linA/A' resistance determinants were detected, according to the method described previously [53].

SCCmec (staphylococcal chromosome cassettes mec) Assignment
The type/subtype of SCCmec, the type of ccr gene complex, and the class of mec gene complex were determined according to the procedure described by Okuma and Oliveira [54,55].

Multilocus Sequence Typing (MLST)
Conventional MLST was performed based on an evaluation of seven housekeeping gene sequences (arcC, aroE, glpF, gmk, pta, tpi, and yqiL), according to the procedure described by Enright [56]. The sequence type (ST) and clonal complex (CC) were determined by small amplicon sequence analysis in a database available at https://pubmlst.org/ bigsdb?db=pubmlst_saureus_seqdef, accessed on 1 June 2021. The evaluated isolate was classified as individual MRSA clone, based on the results of SCCmec, ST, and CC typing.

Whole-Genome Library Preparation and Sequencing
Based on the previously isolated pure genomic DNA, tagmentation was performed using the NEBNext Ultra II FS DNA Library Prep Kit (Illumina, CA USA), in accordance with the protocol of the manufacturer. Accurate quantitation of the library was performed with the NEBNext Library Quant Kit for Illumina.
The draft genome was obtained through short-read bacterial whole-genome sequencing (WGS) on an Illumina MiSeq platform (Illumina Inc., USA). Paired-end 300 base-pair sequencing was done with the use of MiSeq reagent kit 2 × 300 cycles, targeting at least 100× genome coverage.

Genome Annotation and Genomic Features Assignments
The genome strain of interest was annotated using the Genome Annotation Service (GAnS), which uses the RAST tool kit (RASTtk) to provide annotation of genomic features [63,64]. The GAnS uses the k-mer-based antibiotic-resistance genes (ARG) detection method, and assigns functional annotation to each ARG, broad mechanism of antibiotic resistance, drug class, and, in some cases, specific antibiotic it confers resistance to.

Comparative Genomics and Mobile ermB-Carrying Genetic-Structure Analysis
A comparative analysis was performed and phylogenetic relationships with genomes and mobile genetic structures of other strains of the respective species of interest were assessed using the Genome Alignment Service, according to the progressive Mauve algorithm [75] and Phylogenetic Tree Building Service according to RAxML algorithm, available at a livestream platform, PATRIC BBRC (v3.6.9) [76].
Graphical display for the multiple alignments of nucleotide sequences was created and visualized using the nucleotide Basic Local Alignment Search Tool, nBLAST, and NCBI Multiple Sequence Alignment Viewer (MSA) v1.20.1, available at https://www.ncbi.nlm. nih.gov/tools/msaviewer/, accessed on 1 August 2021.

Conclusions
In this study, for the first time in Poland, we introduce a detailed genomic characterization of a representative Polish variant of the CA-MRSA ST338-SCCmecV T /CC59 PVL-positive clone, known as the Taiwan clone, encoding the ermB gene cluster as a determinant of constitutive resistance to MLS-B antibiotics. The analyzed SO574/12 strain was reported as an extremely rare and significant microbiological material, unique in Poland.
The analyzed CA-MRSA isolate emerged in a hospital setting, which has unexpectedly and significantly changed the local epidemiological situation in wards, where until recently only healthcare-acquired variants of MRSA were identified.
We demonstrated that the ermB gene, unique among S. aureus, was located on a chromosome within the MES PM1-like structure, which also encoded aminoglycoside-and streptothricin-resistance genes. We also proved that the MES PM1-like structure was a composite transposon, contained a smaller Tn551, and was flanked by direct repeats of IS1216V insertion sequences, probably originated from Enterococcus sp.
The ermB is preceded by the 273 bp regulatory region that contains the regulatory 84 bp ermBL ORF, encoding the 27 amino acid leader peptides. The latest research suggests that a new leader peptide, ermBL2, also exists in the ermB regulatory region. Therefore, the detailed function of ermBL2 requires further investigations.