Clonal Diversity and Genetic Characteristics of Methicillin-Resistant Staphylococcus aureus Isolates from a Tertiary Care Hospital in Japan

Abstract

Molecular epidemiological characteristics were investigated for 1,041 isolates of methicillin-resistant Staphylococcus aureus (MRSA) collected in a tertiary care hospital in northern Japan for a 4-year period (2011–2014). Genotypes (staphylococcal cassette chromosome mec [SCCmec], sequence type, spa, coa, etc.) and the presence of drug resistance/virulence factor genes in the isolates were analyzed by multiplex/uniplex PCR, and PCR-direct sequencing as needed. Among these MRSA, predominant SCCmec type was IIa (87.2%), followed by IV (10.1%) and V (1.2%). The SCCmec IIa-MRSA belonged to coagulase genotype (coa) IIa and ST5/ST764, which are known as major health care-associated-MRSA (HA-MRSA) in Japan (New York/Japan clone) and its variant. Panton–Valentine leucocidine (PVL) genes were detected in only five isolates (0.5%) with genotypes ST8-SCCmec IVa/spa-t008/coa-IIIa (USA300 clone), ST6-SCCmec IVb, and ST59-SCCmec V (Taiwanese clone). Arginine catabolic mobile element (ACME) type I and II′ were identified in three and five isolates of ST8-SCCmec IVa and ST764-SCCmec IIa MRSA, respectively. PVL−/ACME− isolates were classified into various STs/clonal complexes (CCs), with CC1, CC5, CC8, CC89, and CC121 being common. It was notable that SCCmec IVl was the most common among SCCmec IV subtypes, and was carried by almost half of coa-IIIa isolates (47%, 34/72) without PVL genes, which represented the novel ST8 MRSA clone spreading in Japan (i.e., “ST8/CA-MRSA/J”). Uncommon MRSA clones in Japan, ST72-SCCmec IV (South Korean clone), ST398 livestock-associated clone, and ST20 bovine-associated MRSA, were identified. Furthermore, we isolated PVL-negative ST8-SCCmec I/coa-IIIa and ST81-SCCmec V/coa-VIIa MRSA, which were considered presumptive novel clones. The present study revealed the genetic diversity of HA-MRSA, including potentially emerging clones of putative different origins.

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) has been recognized as a primary cause of nosocomial infections that acquired multiple drug resistance associated with its global spread since the 1960s. It still remains a leading cause of health care- as well as community-associated infections.¹ Methicillin resistance of MRSA is conferred by mecA gene, which encodes an alternative penicillin-binding protein (PBP2a) with low affinity to β-lactams, and is carried within a transmissible genome element, staphylococcal cassette chromosome mec (SCCmec). The SCCmec in MRSA is highly diverse in structural organization and classified into at least 13 types (I–XIII),^2,3 among which from types I through V are commonly reported in health care-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA).⁴

Symptoms caused by MRSA infections are associated with various virulence factors, including hemolysins, enterotoxin/superantigens, exfoliative toxins, and leucocidins.^4,5 Panton–Valentine leucocidin (PVL) is a bicomponent pore-forming protein encoded by prophage, primarily prevalent in a part of CA-MRSA strains associated with severe skin and soft tissue infections and necrotizing pneumonia, while it has also been detected in methicillin-susceptible S. aureus (MSSA) as well as in HA-MRSA.^4,5

MRSA clones with different genetic characteristics predominate in different geographical regions around the world.^4,6 For example, ST8-MRSA-SCCmec IVa known as USA300 clone has been dominating in the United States and spreading worldwide, and ST80 is prevalent in Europe.⁶ While ST30 is widely distributed to Asian countries, ST72 is a dominant CA-MRSA in South Korea, and ST59 is common mostly in Taiwan.⁷ In Japan, most of the MRSA isolated from inpatients and outpatients have been reported as New York/Japan clone belonging to ST5-SCCmec IIa.^8,9 However, recent epidemiological studies revealed changing trends in MRSA clone, that is, potential increase in SCCmec IIa-ST764 (single-locus variant of ST5)^10,11 and ST8-SCCmec IV, including PVL-positive USA300 clones.¹² Among the ST8-SCCmec IV MRSA, PVL-negative strains having a novel SCCmec subtype IVl were described as an emerging clone in Japan and designated CA-MRSA/J.¹³ These newly identified MRSA clones possess genetic traits that are relevant to enhancement of virulence and/or colonization on human host.

USA300 clone (PVL-positive, ST8/SCCmec IV) characteristically possesses arginine catabolic mobile element (ACME, type I) containing arc cluster, opp-3 cluster, and speG.¹⁴ ACME is a genomic island juxtaposed to the SCCmec and mediates the arginine deiminase pathway, resulting in enhanced fitness of bacterial cells to skin of the host. Spermine/spermidine N-acetyltransferase (SpeG) encoded by speG detoxifies polyamines that are bactericidal substances produced by all living organisms,¹⁵ allowing the USA300 clones to better potentiate its colonization on the human skin and infection. Type II ACME is an incomplete form lacking opp-3 cluster encoding oligopeptide permease operon found in mostly S. epidermidis,¹⁶ ACME type II′ is a truncated form of ACME II.¹⁷ Despite low prevalence, ACME II′ has been found in various lineages of MRSA. In our previous studies, ACME II′ was detected in ST5 and ST764 SCCmec IIa MRSA, and their potential increase was reported.^11,18 The ST8 CA-MRSA/J clone lacks PVL and ACME, nevertheless, they possess a novel cell wall-anchored surface protein (SasL or CWASP/W) encoded by sasL (spj) located within SCCmec IVl.¹⁹

The present study was conducted from 2011 to 2014 to survey genetic characteristics of MRSA from a university teaching hospital in northern Japan after the preceding study from 2008 to 2010.¹⁸ Molecular epidemiological analysis revealed the presence of genetically diverse MRSA, including putative novel clones and those with potentially increasing trend, as well as persistently predominant strains since before. While TSST-1 is an important virulence factor of S. aureus and genetically highly conserved, the genetic diversity of TSST-1 gene (tst-1) was found among human and animal S. aureus isolates.²⁰ Thus, we tried to find any genetic variants of tst-1, which might be related to S. aureus of animal origin or emerging MRSA clones. Furthermore, elastin binding protein genes (ebpS) in ST121 isolates were analyzed to confirm the presence of ebpS variant (ebpS-v) with internal 180-bp deletion, which was first identified in ST121 strains in Myanmar.²¹

Materials and Methods

Bacterial isolates and susceptibility testing

A total of 1,041 clinical isolates of MRSA were analyzed. These isolates were collected consecutively from patients with infections who visited the Sapporo Medical University Hospital, Hokkaido, northern main island of Japan, from 2011 to 2014. The Sapporo Medical University Hospital is one of the three advanced treatment hospitals in Hokkaido, having about 750 inpatients and 1,700 outpatients per day. The main specimens were sputum (22.2%, n = 231), followed by pus (11.7%, n = 122), urine (11.6%, n = 121), throat swab (9.1%, n = 95), skin (8.7%, n = 91), nasal discharge (7.1%, n = 74), ear discharge (4.5%, n = 47), stool (5.9%, n = 61), and blood (2.3%, n = 24). Age range of patients was 0–106 years, while the sex distribution (male/female) was 2.3 (727/314). Eighty six percent of isolates (890/1,041) were derived from hospitalized patients, while the remaining 14% (151/1,041) from outpatient specimens. While most of the isolates were regarded as HA-MRSA, we did not definitely distinguish between HA-MRSA and CA-MRSA in this study. Only one isolate per patient was included in the analysis. The clinical specimens were inoculated onto blood agar plates and incubated at 37°C for 24 hr aerobically. Isolates grown on the agar plate were tested for mannitol degradation on mannitol salt agar and coagulase production with latex agglutination slide-card test, and MRSA was phenotypically identified by the automated bacterial identification and susceptibility testing system (MicroScan^® WalkAway 96 plus system MicroScan; SIEMENS Healthcare Diagnostics). Isolates showing oxacillin MIC of ≥4 μg/mL or cefoxitin MIC of ≥8 μg/mL were judged as MRSA. Subsequently, the presence of mecA and nuc in these isolates was confirmed by multiplex PCR as described below. In the present study, MRSA was defined as mecA-positive S. aureus (n = 1,041). Individual bacterial isolates were stored in Microbank (Pro-Lab Diagnostics, Richmond Hill, Ontario, Canada) at −80°C and recovered when they were analyzed.

In the present study, all the 10 PVL- or ACME-positive isolates, and 46 ACME−/PVL− isolates belonging to different SCCmec types/subtypes and coa types, and derived from different clinical specimens, were selected for further genetic analysis, including multilocus sequencing typing (MLST). For these 56 isolates, antimicrobial susceptibility was measured by broth microdilution test using Dry Plate Eiken DP32 (Eiken, Tokyo, Japan). MICs within limited ranges were measured for 18 antimicrobial agents; oxacillin (OXA), ampicillin (AMP), cefazolin (CFZ), cefmetazole (CMZ), flomoxef (FMX), imipenem (IPM), gentamicin (GEN), arbekacin (ABK), erythromycin (ERY), clindamycin (CLI), vancomycin (VAN), teicoplanin (TEC), linezolid (LZD), minocycline (MIN), fosfomycin (FOF), levofloxacin (LVX), cefoxitin (FOX), and trimethoprim/sulfamethoxazole (SXT). Resistance was judged according to breakpoints mentioned in the Clinical Laboratory Standards Institute guidelines²² for most of the antimicrobial drugs examined. For antimicrobials whose breakpoints are not defined by CLSI guidelines, we used the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint for FOF (32 μg/mL, Staphylococcus spp.),²³ a unique breakpoint for ABK (4 μg/mL, which is higher than the 2 μg/mL defined by the Japanese Society of Chemotherapy for respiratory infection),²⁴ and a breakpoint of flomoxef (16 μg/mL) defined by the Japanese Society of Chemotherapy for urinary tract infection.²⁴

Multiplex PCR for MRSA, genotyping

The presence of staphylococcal 16S rRNA gene, nuc, mecA, PVL gene (pvl), ACME-arcA was investigated for all the 1,041 isolates by multiplex PCR assay as described previously.²⁵ SCCmec type and subtypes of SCCmec IV and SCCmec II were determined by multiplex PCR by using previously published primers and conditions,^26,27 and the newly described SCCmec IVl subtype was identified by using primers and condition reported previously.²⁸ PVL phage was typed by PCR assay as described previously.²⁹ For all the ACME arcA-positive strains, ACME types I, II, and II′ were classified by long-range PCR as described previously.¹⁷ Staphylocoagulase (coa) genotype was determined by multiplex PCR assay as described previously.³⁰ For the isolates of which coa-type was not classified by the multiplex PCR, partial coa gene sequences (D1, D2, and the central regions) were determined and their highly similar coa gene sequences were searched by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to assign their coa types.³¹ Accessory gene regulator (agr) group was assigned by the PCR with specific primers reported previously.³² Sequence type (ST) was determined for the 56 selected isolates, according to the scheme of MLST,³³ and the obtained ST data were further analyzed by eBURST to determine their clonal complex (CC). Sequence of protein A gene X-region (spa type) was determined by PCR and sequencing,³⁴ using Ridom SpaServer (http://spa.ridom.de/index.shtml).

Detection and characterization of virulence factor and drug resistance genes

For the 56 selected isolates, including those with ACME and/or PVL genes, MRSA with different coa genotypes, and SCCmec types/subtypes derived from various clinical specimens, the presence of 23 staphylococcal enterotoxin (-like) genes (sea-see, seg-selu, selx, sely, and selw), the TSST-1 gene (tsst-1) and exfoliative toxin genes (eta, etb, and etd), leucocidins (lukDE and lukM), hemolysins (hla, hlb, hld, hlg, and hlg2), adhesin genes (bbp, eno, cna, sdrC, sdrD, sdrE, fib, clfA, clfB, fnbA, fnbB, icaA, icaD, ednA, ednB, bap, and vWbp), and modulators of host defense (sak, chp, and scn) were analyzed by multiplex or uniplex PCRs.^21,35 Genes conferring resistance to penicillin (blaZ), tetracycline (tet(K), tet(L), and tet(M)), macrolide (ermA, ermB, ermC, and msrA), aminoglycoside (aac(6′)-Im, aac(6′)-Ie-aph(2”)-Ia, ant(3”)-Ia, ant(4′)-Ia, ant(6)-Ia, ant(9)-Ia, ant(9)-Ib, aph(2”)-Ib, aph(2”)-Ic, aph(2”)-Id, and aph(3′)-IIIa) and factor essential for methicillin resistance (femA/femB) were detected by uniplex or multiplex PCR using the primers previously reported.²¹ For all the 1,041 isolates, the presence of sasL (spj) gene encoding a novel cell wall-anchored surface protein (SasL or CWASP/W) typically found in SCCmec IVl, and speG gene, was examined by PCR using previously reported primers and conditions.^11,28 tst-1 genes detected were sequenced by PCR and direct sequencing with reported primers.²¹ Nucleotide sequences of putative variant of elastin-binding protein gene (ebpS-v) was determined as described in the previous studies,²¹ by direct sequencing with the PCR products, using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA) on an automated DNA sequencer (ABI PRISM 3100). Phylogenetic dendrogram of elastin-binding protein gene (ebpS/ebpS-v) was constructed by maximum likelihood method using the MEGA.6 software package. Multiple alignment of elastin-binding protein (EbpS) amino acid sequences determined in the present study and those retrieved from the GenBank database was performed by ClustalW program (http://clustalw.ddbj.nig.ac.jp/) available in the DDBJ website.

GenBank accession numbers

Full-length sequences of ebpS genes detected in ST121 isolates and tst-1 gene of ST5, ST764, and ST8 strains were deposited in the GenBank database and their accession numbers are shown in Supplementary Table S1.

Results

During the study period, the detection rate of MRSA among all the S. aureus isolates in our hospital was 33.8% (2011), 31.7% (2012), 31.3% (2013), and 29.7% (2014). Among a total of 1,041 MRSA isolates, dominant SCCmec type was IIa (87.2%, n = 908) followed by IVl (3.3%, n = 34), IVa (2.3%, n = 24), IVNT (2.2%, n = 23), IVd (1.3%, n = 14), V (1.2%, n = 12), and I (1.1%, n = 11) (Table 1). SCCmec IV, including all subtypes (a, b, c, d, g, l, and nontypable), accounted for 10.1%. The most common coagulase (coa) genotype was IIa (90.2%), followed by IIIa (6.9%), Va (0.7%), and VIIa (0.6%), and the remaining were Ia, IVa, IVb, Vb, VIIb, and VIIIa. All the SCCmec IIa isolates belonged to coa-IIa, while SCCmec IVl belonged to coa-IIIa. PVL genes and ACME-arcA were detected in five isolates (0.5%) and eight isolates (0.8%), respectively. During the 4-year period of this study, no apparent changing trend was observed for the prevalence of SCCmec types and coa types (Supplementary Table S2). SCCmec IIa/coa-IIa was the predominating genotype, and SCCmec IVl had been dominant among SCCmec IV subtypes.

Table 1.

Frequencies of PVL Genes and ACME Among Methicillin-Resistant Staphylococcus aureus Isolates with Different SCCmec Types and Coagulase Genotypes

PVL/ACME	No. of MRSA with SCCmec type											Total	ACME type
PVL/ACME	I	IIa	IVa	IVb	IVc	IVd	IVg	IVl	IVNT	V	NT	Total	I	II′
PVL+/ACME+			3									3	3
PVL+/ACME−			1							1		2
PVL−/ACME+		5										5		5
PVL−/ACME−	11	903	20	1	7	14	1	34	24	11	5	1,031

coa Genotype	No. of MRSA with SCCmec type (No. of PVL genes and/or ACME-positive isolates)											Total	ACME type
coa Genotype	I	IIa	IVa	IVb	IVc	IVd	IVg	IVl	IVNT	V	NT	Total	I	II′
Ia			2						1	2		5
IIa	1	908 (5)^a	7			14			6		3	939		5
IIIa	10		10 (3)^b	1	2			34	15			72	3
IVa									1		1	2
IVb			2 (1)^c									2
Va			1							6		7
Vb					3				0		1	4
VIIa			1		2				1	3 (1)^c		7
VIIb			1				1					2
VIIIa										1		1
Total	11	908	24	1	7	14	1	34	24	12	5	1041	3	5

Number of PVL−/ACME+ isolates.

Number of PVL+/ACME+ isolates.

Number of PVL+/ACME− isolates.

ACME, arginine catabolic mobile element; IVNT, SCCmec IV-subtype nontypable; MRSA, methicillin-resistant Staphylococcus aureus; NT, nontypable; PVL, Panton–Valetine leucocidin; SCCmec, staphylococcal cassette chromosome mec.

Genotypes, antimicrobial resistance, and presence of virulence factors were analyzed for the 10 PVL- and/or ACME-positive isolates (Table 2). Three PVL+/ACME+ isolates derived from skin or sputum had SCCmec IVa with PVL prophage ΦSa2usa and ACME I, and were typed as ST8/spa-t008/agr-I/coa-IIIa, which was typical of the USA300 clone. Two PVL+/ACME− isolates with PVL prophage ΦPVL had SCCmec IVa or V, and belonged to ST6/coa-IVb or ST59/coa-V, respectively. Five PVL−/ACME+ isolates had SCCmec IIa and type II′ ACME, and were classified into ST764/spa-t002/agr-II/coa-IIa, harboring seb and enterotoxin gene clusters (egc: seg-sei-sem-sen-seo-seu). These ACME-positive SCCmec IIa isolates had more toxin genes and drug resistance genes, showing resistance to more antimicrobial agents than PVL-positive isolates. Bone sialoprotein-binding protein gene (bbp) was detected in four PVL−/ACME+ isolates and three PVL-positive isolates. In addition to three PVL-positive ST8/USA300 isolates, three PVL−/ACME+ isolates harbored speG, while this gene was not detected in all the PVL−/ACME− isolates.

Table 2.

Genotypes, Virulence Factors, and Antimicrobial Resistance Profile of PVL/ACME-Positive Isolates

PVL/ACME (no. of isolates)	Isolate ID	Isolate source	Age/sex of patient	SCCmec	ST	coa Genotype	spa	agr	ACME type ^a	PVL phage	Leucocidins, hemolysins, enterotoxins, TSST-1 ^b,c	Adhesins/sasL (spj)^b,c	Modulators of host defense/speG	Antimicrobial resistance pattern ^d	Drug resistance gene ^e
PVL+/ACME+ (3)	SH1733	Skin	32/M	IVa	ST8	IIIa	t008	I	I	ΦSa2usa	sek, seq	sdrD, icaA, icaD, bbp	sak, chp, speG	OXA, FOX, AMP, ERY, GEN, LVX	msrB, aac(6′)-Ie-aph(2″)-Ia
	SH2029	Sputum	32/M	IVa	ST8	IIIa	t008	I	I	ΦSa2usa	sek, seq	sdrD, icaD	sak, scn, chp, speG	OXA, FOX, AMP, ERY, GEN, LVX	blaZ, msrB, aph(3′)-IIIa
	SH2195	Skin	45/M	IVa	ST8	IIIa	t008	I	I	ΦSa2usa	sek, seq	icaD	sak, scn, chp, speG	OXA, FOX, AMP, ERY, CLI, GEN, LVX	blaZ, msrB, aac(6′)-Ie-aph(2″)-Ia, aph(3′)-IIIa
PVL+/ACME− (2)	SH2149	Pharynx	19/F	IVa	ST6	IVb	t304	I		ΦPVL	sea	icaA, bbp	sak, scn, chp	OXA, FOX, AMP, ERY, CLI	blaZ
PVL+/ACME− (2)	SH1459	Pus	25/F	V	ST59	VIIa	t437	I		ΦPVL	seb, sek, seq, sely	icaA, icaD, bbp	sak, scn, chp	OXA, FOX, AMP	blaZ, tet(K), aph(3′)-IIIa, ant(6)-Ia
PVL−/ACME+ (5)	SH1521	Pus	74/M	IIa	ST764	IIa	t002	II	II′		seb, seg, sei, sem, sen, seo, seu	sdrD, icaA, icaD, bbp	scn, chp	OXA, FOX, AMP, CFZ, CMZ, FMX, GEN, IPM, MIN, ERY, CLI, LVX	blaZ, tet(M), erm(A), aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia
	SH1582	Urine	58/F	IIa	ST764	IIa	t002	II	II′		seb, seg, sei, sem, sen, seo, seu	sdrD, icaA, icaD	scn, chp, speG	OXA, FOX, AMP, CFZ, FMX, GEN, IPM, LVX	blaZ, tet(M), erm(A), aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia
	SH1853	Urine	24/M	IIa	ST764	IIa	t002	II	II′		seb, seg, sei, sem, sen, seo, seu	sdrD, icaA, icaD, bbp	scn, chp, speG	OXA, FOX, AMP, CFZ, FMX, GEN, IPM, ERY, CLI, LVX	blaZ, tet(M), erm(A), aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia
	SH1943	Urine	45/M	IIa	ST764	IIa	t002	II	II′		seb, seg, sei, sem, sen, seo, seu	sdrD, icaA, icaD, bbp	chp	OXA, FOX, AMP, CFZ, FMX, GEN, IPM, MIN, ERY, CLI, FOF, LVX	blaZ, tet(M), erm(A), aac(6′)-Ie-aph(2″)-Ia
	SH1947	Nasal swab	44/M	IIa	ST764	IIa	t002	II	II′		seb, seg, sei, sem, sen, seo, seu	sdrD, icaA, icaD, bbp	scn, speG	OXA, FOX, AMP, CFZ, FMX, GEN, IPM, MIN, ERY, CLI, FOF, LVX	blaZ, tet(M), erm(A), aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia

ACME type: I, arcA+/opp3AB+/copA+; I′, shorter bp in opp3AB+; II′, arcA+/opp3AB-/copA+.

The following genes were detected in all strains: selx, selw, lukDE, hla, hlb, hld, hlg, ebpS, sdrC, sdrE, fnbA, fnbB, fib, clfA, clfB, eno, vWbp.

The following genes were not detected in any strain: sed, see, seh, ser, ses, set, eta, etb, etd, lukM, bap, edn-A, edn-B.

None of the strains showed resistance to linezolid, teicoplanin, and vancomycin.

The following genes were undetectable in any strain: tet(L), erm(B), erm(C), msrA, aac(6′)-Im, ant(9)-Ia, ant(9)-Ib, ant(3″)-Ia, aph(2″)-Ib, aph(2″)-Ic, and aph(2″)-Id. femA/femB: factor essential for expression of methicillin resistance was detected in all the strains.

ABK, arbekacin; AMP, ampicillin; CFZ, cefazolin; CLI, clindamycin; CMZ, cefmetazole; ERY, erythromycin; FMX, flomoxef; FOF, fosfomycin; FOX, cefoxitin; GEN, gentamicin; IPM, imipenem; LVX, levofloxacin; LZD, linezolid; MIN, minocycline; OXA, oxacillin; ST, sequence type; SXT, sulfamethoxazole/trimethoprim; TEC, teicoplanin; VAN, vancomycin.

A total of 46 PVL−/ACME− isolates belonging to different SCCmec types/subtypes and coa-types were selected for further genetic characterizations and antimicrobial susceptibility test (Table 3). Four SCCmec I isolates belonged to ST8/coa-IIIa/agr-I and spa types t460, t1581, t1627, and t1767 with similar repeat profiles (Supplementary Table S3). Seven SCCmec IIa/coa-IIa isolates were assigned to ST5 or ST764, and typed as spa-t002/agr-II. These SCCmec I and IIa MRSA were resistant to more drugs (seven or more antimicrobials), having generally more numbers of resistance genes than all other MRSA isolates with SCCmec IV and V. tetK or tetM was detected in isolate SH1459 (Table 2) and SH1671, SH1425, SH1551, SH1627, and SH1827) (Table 3) even though these isolates were phenotypically sensitive to minocycline. The reason for this inconsistency was not analyzed, although it may be presumed that the expression level of tetK/tetM might be reduced in bacterial cells.

Table 3.

Genotypes, Virulence Factors, and Other Genetic and Phenotypic Characteristics of 46 PVL/ACME-Negative Isolates

Isolate ID	Isolate source	Age/sex of patient	Genotype					Leucocidins, hemolysins, enterotoxins, TSST-1 ^a	Adhesins/sasL (spj)^a	Modulators of host defense	Antimicrobial resistance profile	Drug resistance gene ^b
Isolate ID	Isolate source	Age/sex of patient	SCCmec	ST (CC)	coa	spa	agr	Leucocidins, hemolysins, enterotoxins, TSST-1 ^a	Adhesins/sasL (spj)^a	Modulators of host defense	Antimicrobial resistance profile	Drug resistance gene ^b
SH1551	Sputum	27/F	I	ST8 (CC8)	IIIa	t1767	I	sej, ser, selx	sdrC, sdrD, sdrE, eno, fnbA, fnbB, clfA, clfB	sak	OXA, FOX, AMP, CEZ, FOF, IPM, GEN, ERY, CLI, LVX	blaZ, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, erm(A), tet(M)
SH1627	Urine	50/M	I	ST8 (CC8)	IIIa	t1627	I	selx	sdrC, sdrD, sdrE, eno, fib, fnbA, fnbB, clfA, clfB	sak, chp	OXA, FOX, AMP, CEZ, CMZ, GEN, ERY, CLI, LVX	blaZ, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, erm(A), tet(M)
SH1827	Skin	51/F	I	ST8 (CC8)	IIIa	t1581	I	selx	sdrC, eno, fib, fnbA, fnbB, clfA, clfB		OXA, FOX, AMP, CEZ, GEN, ERY, LVX	blaZ, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, erm(A), tet(M)
SH1974	Ear swab	33/M	I	ST8 (CC8)	IIIa	t460	I	sej, ser, selx	sdrC, sdrD, sdrE, eno, fib, fnbA, fnbB, clfA, clfB	sak, chp	OXA, FOX, AMP, CEZ, CMZ, GEN, ERY, CLI, LVX	blaZ, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, erm(A), tet(M)
SH1417	Urine	46/F	IIa	ST5 (CC5)	IIa	t002	II	sec, seg, sei, sem, sen, seo, tst-1	sdrD, icaA	scn, chp	OXA, FOX, AMP, CFZ, CMZ, FMX, GEN, IPM, MIN, ERY, CLI, FOF, LVX	blaZ, tet(M), erm(A), msrB, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia
SH1426	Others	9/M	IIa	ST5 (CC5)	IIa	t002	II	sec, seg, sei, sem, sen, seo, sep, seu, tst-1	sdrD, icaA, icaD	scn	OXA, FOX, AMP, CFZ, CMZ, FMX, GEN, IPM, MIN, ERY, CLI, LVX	tet(M), erm(A), ant(4′)-Ia
SH1870	Stool	34/M	IIa	ST5 (CC5)	IIa	t002	II	sec, seg, sei, sem, seo, selx, selw, tst-1	sdrC, sdrD, sdrE, clfA	sak, chp	OXA, FOX, AMP, CEZ, FOF, IPM, GEN, ERY, CLI, LVX	blaZ, ant(4′)-Ia, erm(A)
SH2288	Trachea	18/M	IIa	ST5 (CC5)	IIa	t002	II	seg, sei, sem, sen, seo, selx, selw	sdrC, sdrD, sdrE, eno, fnbB, fib, clfA, clfB	sak, scn, chp	OXA, FOX, AMP, CEZ, GEN, ERY, CLI, LVX	aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, erm(A), tet(M)
SH1394	Nasal swab	4/M	IIa	ST764 (CC5)	IIa	t002	II	seb, sec, seg, sei, sem, sen, seo, seu, tst-1	sdrD, icaA, icaD, cna	sak	OXA, FOX, AMP, CFZ, MIN, ERY, CLI, LVX	blaZ, tet(M), erm(A), aac(6′)-Ie-aph(2″)-Ia, aph(3′)-IIIa
SH1416	Nasal swab	77/M	IIa	ST764 (CC5)	IIa	t002	II	sec, seg, sei, sek, sem, sen, seo, seu	sdrD, icaA, icaD	sak, scn, chp	OXA, FOX, AMP, CFZ, CMZ, FMX, GEN, IPM, MIN, ERY, CLI, FOF, LVX	blaZ, tet(M), erm(A), aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia
SH2299	Stool	14/M	IIa	ST764 (CC5)	IIa	t002	II	sec, seg, sei, sem, seo, selx, selw, tst-1	sdrC, sdrD, sdrE, eno, fib, clfA, clfB	sak, chp	OXA, FOX, AMP, CEZ, MIN, GEN, ERY, CLI, LVX	blaZ, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, erm(A), tet(M)
SH1538	Skin	42/F	IVa	ST89 (CC89)	Ia	t375	III	sem, seo, selx, selw	sdrC, sdrE, eno, cna, fnbA, fnbB, fib, clfA, clfB	sak, chp	OXA, FOX, AMP, GEN	blaZ, aac(6′)-Ie-aph(2″)-Ia, erm(A)
SH1756	Nasal swab	39/M	IVa	ST91 (CC89)	Ia	t375	III	sem, seo, selx, sely, selw	sdrC, sdrE, eno, cna, fnbA, fnbB, fib, clfA, clfB	sak, chp	OXA, FOX, AMP, GEN	blaZ, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia
SH2142	Trachea	23/M	IVa	ST1 (CC1)	IIa	t1784	III	sea, sek, seq, selx, selw	sdrC, sdrD, sdrE, eno, cna, fnbB, fib, clfA, clfB	sak, scn	OXA, FOX, AMP, ERY, LVX	blaZ, erm(A)
SH2310	Pharynx	7/M	IVa	ST2725 (CC1)	IIa	t1784	III	sea, sek, seq, selx, selw	sdrC, sdrD, sdrE, eno, fibB, fib, clfA, clfB	sak, scn, chp	OXA, FOX, AMP, ERY, LVX	blaZ, erm(A)
SH2162	Pharynx	9/M	IVa	ST8 (CC8)	IIIa	t4133	I	sei, selx, selw	sdrC, sdrD, sdrE, eno, fib, clfA, clfB	sak, scn, chp	OXA, FOX, AMP	blaZ
SH2298	Pus	33/M	IVa	ST2804 (CC6)	IVb	t304	I	sea, selx, selw	ednA, sdrC, sdrD, eno, cna, fib, clfA, clfB	sak, scn	OXA, FOX, AMP, GEN	blaZ, aac(6′)-Ie-aph(2″)-Ia
SH1506	Sputum	61/M	IVa	ST59 (CC59)	Va	t976	I	seb, sek, seq, selx, selw	sdrC, sdrE, eno, cna, fnbA, fnbB, fib, clfA, clfB	sak, chp	OXA, FOX, AMP, ERY	blaZ, msrA
SH1884	Pharynx	4/M	IVa	ST59 (CC59)	Va	t976	I	sea, sek, seq, selx, sely, selw	sdrC, sdrE, eno, fib, clfA, clfB	sak, scn, chp	OXA, FOX, AMP, ERY	blaZ, msrA
SH1671	Skin	16/F	IVa	ST398 (CC398)	VIIb	t571	I		sdrC, sdrE, cna, fnbB, clfA, clfB	sak	OXA, FOX, AMP, ERY, LVX	blaZ, tet(K), aac(6′)-Ie-aph(2″)-Ia, aph(3′)-IIIa, msrA
SH1887	Nasal swab	32/M	IVc	ST72 (CC8)	Vb	t148	I	seg, sei, sem, seo, selx, selw	sdrC, eno, fib, clfA, clfB	sak	OXA, FOX, AMP	blaZ, ant(4′)-Ia
SH2164	Oral swab	21/M	IVc	ST72 (CC8)	Vb	t324	I	seg, sei, sem, sen, seo, selx, selw	sdrC, sdrD, sdrE, eno, fib, clfA, clfB	sak, scn, chp	OXA, FOX, AMP, CEZ, GEN, ERY, CLI	blaZ, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia
SH2177	Pharynx	68/F	IVc	ST72 (CC8)	Vb	t324	I	seg, sei, sem, sen, seo, selx, selw	sdrC, sdrD, sdrE, eno, fib, clfA, clfB	sak	OXA, FOX, AMP, CEZ	blaZ, ant(4′)-Ia
SH1964	Oral swab	34/F	IVc	ST544 (CC8)	VIIa	t324	IV	seg, sei, sem, sen, seo, seu, selx, selw	sdrC, sdrD, sdrE, eno, fib, fnbA, fnbB, clfA, clfB	sak	OXA, FOX, AMP	blaZ, ant(4′)-Ia
SH2073	Trachea	36/F	IVc	ST1516 (CC8)	IIIa	t17634^c	I	selx	sdrC, sdrD, sdrE, fib, fnbA, fnbB, clfA, clfB	sak	OXA, FOX, AMP, GEN, LVX	blaZ, aac(6′)-Ie-aph(2″)-Ia
SH1425	Urine	55/M	IVc	ST4386 (CC8)	IIIa	t4133	I	selx	sdrC, sdrD, sdrE, fib, fnbA, fnbB, clfA, clfB	sak	OXA, FOX, AMP, GEN, LVX	blaZ, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, erm(A), tet(M)
SH1664	Urine	5/M	IVg	ST1891 (CC45)	VIIb	t065	I	sei, seo, sen	sdrE, eno, cna, fnbA, fnbB, clfA, clfB	sak, chp	OXA, FOX, AMP, ERY, CLI, LVX	blaZ, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia, erm(A)
SH1184	Skin	34/M	IVl	ST8 (CC8)	IIIa	t1767	I	sec, sel, sep, selx, selw, tst-1	edn-A, sdrC, sdrD, sdrE, sasL		OXA, FOX, AMP, GEN	blaZ, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia
SH1229	Sputum	27/F	IVl	ST8 (CC8)	IIIa	t5071	I	sec, sel, sep, selx, selw, tst-1	edn-A, sdrC, sdrD, sdrE, sasL	sak, scn	OXA, FOX, AMP, GEN, ERY	blaZ, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia
SH1251	Blood	5/M	IVl	ST8 (CC8)	IIIa	t16525^c	I	sec, sel, sep, selx, selw, tst-1	edn-A, sdrC, sdrD, sdrE, sasL, bbp	sak, scn, chp	OXA, FOX, AMP, GEN, ERY, CLI	blaZ, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia
SH1326	Urine	40/F	IVl	ST8 (CC8)	IIIa	t5071	I	sec, sel, sep, selx, selw, tst-1	sdrC, sdrD, sdrE, sasL	sak, chp	OXA, FOX, AMP, GEN	blaZ, aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia
SH1412	Pharynx	4/M	IVl	ST8 (CC8)	IIIa	t5071	I	sec, sel, sep, selx, selw, tst-1	edn-A, sdrC, sdrD, sdrE, sasL	scn	OXA, FOX, AMP, GEN	blaZ, aac(6′)-Ie-aph(2″)-Ia
SH1650	Trachea	42/M	IVNT	ST89 (CC89)	Ia	t375	III	sem, seo, selx, selw	sdrC, sdrE, eno, cna, fnbA, fnbB, clfA, clfB	sak, chp	OXA, FOX, AMP, GEN, ERY, CLI	blaZ, aac(6′)-Ie-aph(2″)-Ia, erm(A)
SH2235	Skin	89/F	IVNT	ST8 (CC8)	IIIa	t1581	I	sem, sep, selx	sdrC, sdrD, sdrE, eno, cna, fnbA, fnbB, fib, clfA, clfB	sak	OXA, FOX, AMP, ERY, CLI, LVX	blaZ, erm(A)
SH1966	Others	39/M	IVNT	ST81 (CC1)	VIIa	t127	III	sea, sei, sek, seq, selx, selw	sdrC, sdrD, sdrE, bap, eno, fnbB, fib, clfA, clfB	sak, chp	OXA, FOX, AMP, GEN, LVX	blaZ, aac(6′)-Ie-aph(2″)-Ia
SH1492	Pharynx	55/F	V	ST89 (CC89)	Ia	t375	III	sem, seo, seu, selx, selw	sdrC, sdrE, eno, cna, clfA, clfB		OXA, FOX, AMP, GEN	blaZ, aac(6′)-Ie-aph(2″)-Ia
SH1571	Bile	75/F	V	ST89 (CC89)	Ia	t375	III	sem, seo, selx, selw, etb	sdrC, sdrE, eno, cna, fnbA, fnbB, clfA, clfB	sak, chp	OXA, FOX, AMP, GEN	blaZ, aac(6′)-Ie-aph(2″)-Ia
SH2138	Pharynx	12/M	V	ST121 (CC121)	Va	t10641	IV	seg, sei, sem, sen, seo, seu, selx, sely, selw, eta	sdrE, bbp, eno, cna, fib, clfA, clfB, ebpS-v	sak, scn, chp	OXA, CEZ, GEN, ERY	blaZ, aac(6′)-Ie-aph(2″)-Ia
SH2146	Pharynx	19/F	V	ST121 (CC121)	Va	t10641	IV	seg, sei, sem, sen, seo, seu, selx, sely, selw, eta	sdrE, bbp, eno, cna, fib, clfA, clfB, ebpS-v	sak, scn, chp	FOX, AMP, GEN	blaZ, aac(6′)-Ie-aph(2″)-Ia
SH2250	Pharynx	12/M	V	ST121 (CC121)	Va	t10641	IV	seg, sei, sem, sen, seo, seu, selx, sely, selw	ednA, sdrE, bbp, eno, cna, fib, clfA, clfB, ebpS-v	sak, scn, chp	OXA, FOX, AMP, GEN, ERY	blaZ, aac(6′)-Ie-aph(2″)-Ia
SH2251	Sputum	58/F	V	ST121 (CC121)	Va	t10641	IV	seg, sei, sem, sen, seo, seu, selx, sely, selw	ednA, sdrE, bbp, eno, cna, fib, clfA, clfB, ebpS-v	sak, scn, chp	FOX, AMP, GEN, ERY	blaZ, aac(6′)-Ie-aph(2″)-Ia
SH2129	Pus	12/M	V	ST121 (CC121)	Va	t5110	IV	seg, sei, sem, sen, seo, seu, selx, sely, selw, eta	ednA, sdrE, bbp, eno, cna, fib, clfA, clfB, ebpS-v	sak, scn, chp	FOX, AMP, GEN, ERY	blaZ, aac(6′)-Ie-aph(2″)-Ia
SH2232	Pharynx	8/M	V	ST121 (CC121)	Va	t10641	IV	seg, sei, sem, sen, seo, seu, selx, sely, selw	sdrE, bbp, eno, cna, fib, clfA, clfB, ebpS-v	sak, scn, chp	FOX, AMP, ERY	blaZ, aac(6′)-Ie-aph(2″)-Ia
SH1547	HVS	67/F	V	ST81 (CC1)	VIIa	t127	III	sea, sek, seq, selx, selw	sdrC, sdrD, sdrE, eno, fib, clfA, clfB	sak, scn	OXA, FOX, AMP, CEZ, CMZ, FOF, IPM, GEN, ERY, CLI, LVX	blaZ, aac(6′)-Ie-aph(2″)-Ia
SH2069	Pus	35/M	V	ST81 (CC1)	VIIa	t127	III	selx, selw	sdrC, sdrD, eno, fib, clfA, clfB	sak, chp	OXA, FOX, AMP, LVX	blaZ
SH1893	Pharynx	10/F	V	ST20 (CC20)	VIIIa	t164	I	seg, sei, sem, sen, seo, selx, sely, selw	sdrC, sdrD, sdrE, eno, fnbB, fib, clfA, clfB	sak	OXA, FOX, AMP	blaZ

The following genes were detected in all the strains: lukDE, hla, hlb, hld, hlg, ebpS, icaA, icaD, vWbp. ebpS-v is a variant of ebpS with internal 180-bp deletion.²¹ The following genes were not detected in any strain: lukM, sed, see, seh, ses, set, eta, etb, etd, bap, ednB.

femA/femB: factor essential for expression of methicillin resistance was detected in all the strains. The following genes were undetectable in any strain: tet(L), erm(B), ierm(C), ant(6)-Ia, acc(6′)-Im, ant(9)-Ia, ant(9)-Ib, ant(3″)-Ia, aph(2″)-Ib, aph(2″)-Ic, and aph(2″)-Id.

New spa type was detected in this study (see Supplementary Table S3).

CC, clonal complex.

SCCmec IVa MRSA isolates were genetically divergent and classified into eight STs (ST1, ST8, ST59, ST89, ST91, ST398, ST2725, and ST2804) grouped into six CCs (CC1, CC6, CC8, CC59, CC89, and CC398). SCCmec IVc MRSA isolates were classified into four STs of CC8, among which ST72 isolates belonged to coa-Vb/agr-I, and spa-types t148 or t324, which are closely related, having selx, selw, and egc. The same toxin gene profile was found in an ST544 (SLV of ST72) isolate with spa-t324, while being typed as coa-VIIa/agr-IV. ST72 and ST544 MRSA were not resistant to cephalosporins, or resistant to only cefazolin.

Five isolates with SCCmec IVl from various specimens were typed as ST8/coa-IIIa/agr-I and spa-t5071 (or closely related types to t5071), and harbored sec, sel, sep, selx, selw, and tst-1. Among SCCmec V-MRSA, genotypes ST89/coa-Ia, ST121/coa-Va, ST81 (CC1)/coa-VIIa, and ST20/coa-VIIIa were detected. Six ST121 isolates harbored various virulence factors, including egc and other enterotoxin genes, eta, and adhesin genes including bbp, eno, and cna. Genotypes ST89 (CC89)/coa-Ia/agr-III and ST81 (CC1)/coa-VIIa/agr-III/spa-t127 were found in MRSA of both SCCmec types IV and V. ST20, ST81, and ST89-SCCmec V isolates had less number of toxin genes than ST121 isolates, and showed resistance to three to four antimicrobials, except for an ST81 isolate (SH1547) with multidrug resistance.

TSST-1 gene (tst-1) was detected in only SCCmec IIa/ST5 or ST764 and SCCmec IVl isolates (five isolates each). Nucleotide sequences of tst-1 from these isolates were identical to that reported for most of S. aureus strains, including NRS1, N315, and HOU1444-VR, while showing slight diversity with bovine strains (e.g., RF122) with 97–98% identity. All the six ST121 MRSA belonging to coa-Va/agr-IV represented by SH2529 possessed a variant of ebpS (ebpS-v) lacking internal 180-nucleotide sequence. Phylogenetically, ebpS-v of the six ST121 isolates clustered with those of other ST121 strains reported previously in Myanmar (Y12), Bangladesh (NZ111, etc.), and South Africa (93b_S9) in the clade III (Supplementary Fig. S1). Deduced amino acid (aa) sequences of EbpS revealed internal deletion of 60 aa and the presence of alanine at position 97 compared with intact EbpS, as found in strain Y12²¹ (Supplementary Fig. S2). EbpS variant of all the six ST121 isolates had an identical aa sequence, but different from those of other ST121 strains by only a single aa.

Discussion

The present study revealed molecular epidemiological traits of HA-MRSA from a university hospital in northern Japan from 2011 to 2014. One of the evident findings is the significant increase in the rate of SCCmec IV (10.1%) (p < 0.001), compared with our previous study (3.8%, 23/601) (2008–2010)¹⁸ in the same hospital, while SCCmec IIa has been still dominant. Similarly, in our studies for MRSA from outpatients in Hokkaido, northern main island of Japan, proportion of SCCmec IV apparently increased from 6.9% in 2009⁹ to 20% in 2013–2014.¹¹ Such trend agrees with a report for hospital isolates in Tokyo, showing a substantial rise of SCCmec IV rate reaching >50% in 2016.³⁶ Notably, our present study first elucidated that SCCmec IVl subtype was the most dominant among SCCmec IV subtypes, although type IVa had been dominant among MRSA in the community.¹⁸ The SCCmec IVl MRSA isolates in our present study are considered the CA-MRSA/J clone reported previously,^13,19 because they belonged to typical genotypes ST8/coa-IIIa/agr-I, having tsst-1, sec, sel, and sep, and sasL gene encoding a novel cell wall-anchored surface protein (SasL), lacking PVL genes. The ST8 CA-MRSA/J clone has been detected in central and western Japan, and Hong Kong, since 2003,¹³ and also identified among CA-MRSA in northern Japan.¹¹ As observed in the present study, the ST8-SCCmec IVl MRSA derived from different specimens showed almost the same profiles in virulence factors, resistance genes, and drug resistance, suggesting a spread of single clone in the hospital. Although these isolates do not appear to have acquired multidrug resistance, prevalence and genetic diversity of this clone should be carefully monitored.

PVL-positive ST8-SCCmec IVa (USA300 clone) was detected at low rate (0.3%, 3/1,041) in the present study, as seen in our previous study (0.3%, 2/601).¹⁸ Although a slightly increasing trend of PVL-positive rate in MRSA (0.6–3.1%) has been observed in Tokyo,¹² the incidence of USA300 clone still remains low in Japan since its first report in 2007,³⁷ suggesting that this clone has not spread among the Japanese population as efficiently as in the United States.³⁶ ACME II′-carrying ST5 or ST764 MRSA-SCCmec IIa isolates was identified in the present study with low incidence (0.5%, 5/1,041), as in our previous study for hospital isolates (1.2%)¹⁸ and community isolates (2.4%).¹¹ These MRSA are considered variants of ST5-MRSA-IIa (New York/Japan clone, a major HA-MRSA in Japan) that acquire ACME II′, and showed resistance to almost equal or more antimicrobials than ACME-negative ST5/ST764 isolates. Some of these clones with ACME II′ (three among five isolates in our study) have speG, which is also located in ACME I of USA300 clone and may potentiate their colonization on skin.^15,38 Therefore, ACME-positive ST5/ST764 MRSA-SCCmec IIa clone is suggested to have the potential to spread as hospital-associated pathogens.

In the present study, uncommon MRSA clones in Japan were detected, including ST20, ST72, ST81, and ST398 isolates. PVL-negative ST72-MRSA-IV is a major CA-MRSA clone in South Korea,^7,39 and its prevalence is mostly limited within the country. In Japan, there is only one report describing isolation of ST72-MRSA-IV from a patient with mediastinal abscess.⁴⁰ ST72/spa-t324/SCCmec IV, genotypes of MRSA identified in our study, have been repeatedly described also for MRSA clones from bovine milk and mastitis, pigs, and farmers in Korea.⁴¹ Accordingly, for the spread of ST72 MRSA, zoonotic potential should be considered as well as geographically close locations to Japan and Korea.

ST398 MRSA is known as a major livestock-associated (LA)-MRSA clone in Europe and North America, colonizing and causing infections in livestock animals represented by pigs,⁷ associated with SCCmec IV or V.⁴² The presence of ST398 MSSA in swine was evidenced in Japan,⁴³ and human infection with this clone is described in only one case report of bacteremia with PVL-positive isolate with SCCmec V.⁴⁴ While ST398 with SCCmec IV is a rare lineage in LA-MRSA, this lineage with spa-t571, which was identified in the present study, had been described in isolates from human infections in Europe⁴⁵ and China,⁴⁶ and also from breast milk in China.⁴⁷ ST20 and ST81 S. aureus identified in the present study are also related to livestock, and methicillin-susceptible isolates have been found in bovine milk,⁴⁸ and cats and dogs⁴⁹ in Japan. However, there are few reports of these STs in infections in humans; ST20 MSSA that caused infective endocarditis⁵⁰ and ST81-SCCmec IV from otitis media⁸ and HA-MRSA strains in Japan.⁵¹ Because these reported ST81 MRSA had SCCmec IV, ST81-MRSA-V is considered a novel MRSA clone. Detection of various lineages of LA-MRSA clones as described above may underscore the significance of MRSA in animals as the potential origin of infections in humans.

It is noteworthy that four PVL-negative ST8/SCCmecI/coa-IIIa, a putative novel MRSA clone, were isolated in the present study. CC8 MRSA with SCCmec I is known as early or ancestral MRSA represented by strain COL (ST250, SLV of ST8) in the United Kingdom,⁴² subsequently evolved into the epidemic clone ST247 MRSA-I (Iberian clone).⁵² While the isolation of ST250 or ST8-MRSA-I was reported in Ireland in the early 1980s and Italy before 2000, respectively,^53,54 thereafter, very little information was available. In only one study in Tokyo, Japan, detection of ST8-SCCmec I was described from 2012 to 2015.¹² Although it is not certain whether this clone occurred newly in Japan or not, ST8-MRSA-I in the present study has the same genotypes (coa-IIIa/spa-t1767/agr-I) as those of an ST8-SCCmec IV (CA-MRSA/J) clone, suggesting these ST8 clones were derived from the same origin. The ST8-SCCmec I MRSA were isolated from different specimens and showed multiple drug resistance in the present study, suggesting the potential of this clone to spread as HA-MRSA. Accordingly, to monitor this clone may be of significance in health care settings in Japan.

Elastin binding protein (EbpS) is a cell surface protein that binds to host cellular matrix factors, and it is produced by most MRSA. A variant of EbpS gene (ebpS-v) with internal 180-bp deletion as found in previous studies²¹ was detected in the six ST121 MRSA in the present study, and these ebpS-v genes were revealed to phylogenetically cluster with those of ST121 strains from other countries (Myanmar, Bangladesh, South Africa). This finding may suggest that ebpS-v is stably conserved as a genetic trait of ST121 S. aureus clone distributed globally, although the pathogenic role of the EbpS variant is yet to be determined.

In conclusion, the present study revealed the prevalence and genetic traits of HA-MRSA in northern Japan. Increasing trend of the locally emerging MRSA clone ST8/SCCmec IVl and detection of novel MRSA clones (ST8/SCCmec I, ST81/SCCmec V), and LA-MRSA (ST20, ST398) highlight the need for continuous surveillance and molecular epidemiological analysis in the hospital.

Footnotes

Acknowledgment

This study was supported, in part, by JSPS (Japan Society for the Promotion of Science) KAKENHI Grant No. 18K10054.

Disclosure Statement

The authors of this article have no commercial associations that might create a conflict of interest in connection with the submitted article.

Supplementary Material

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