Molecular Characteristics of Community-Acquired Methicillin-Resistant Staphylococcus aureus in Hokkaido,Northern Main Island of Japan: Identification of Sequence Types 6 and 59 Panton-Valentine Leucocidin–Positive Community-Acquired Methicillin-Resistant Staphylococcus aureus

Abstract

Prevalence and molecular characteristics of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) were studied in Hokkaido, the main northern island of Japan. Among the 1,015 S. aureus isolates derived from clinical specimens of outpatients collected in 2009, methicillin resistance gene mecA was detected in 189 isolates (18.6%). The most frequent staphylococcal cassette chromosome mec (SCCmec) type in MRSA was II (83.1%), followed by IV (6.9%) and V (3.2%). MRSA with type II-SCCmec showed multiple drug resistance and harbored various toxin and virulence factor genes except for Panton-Valentine leucocidin (PVL) gene. These isolates were mostly classified into sequence type 5 (ST5) (or other STs in CC5) and coagulase genotype II and were thus genetically similar to hospital-acquired MRSA, which have been predominating in Japan (New York/Japan clone). PVL gene was detected in three MRSA strains belonging to ST6 (two strains) and ST59 (one strain), having type IVa- and Vt-SCCmec, respectively, and also in two methicillin-susceptible S. aureus ST121 and ST188. The arcA gene within the arginine catabolic mobile element (ACME) was detected in the two PVL-negative ST5 MRSA strains, which had type IIa- or V-SCCmec. The PVL gene-positive ST6 and ST59 CA-MRSA strains were susceptible to more antimicrobials and had less virulence factor genes than the PVL-negative ST5 MRSA, including the ACME-arcA–positive strains. In the present study, ST6 was identified as a lineage of PVL-positive CA-MRSA, the ACME-arcA was first detected in ST5 MRSA with type V-SCCmec, and ST59 Taiwanese CA-MRSA strain was isolated in Hokkaido for the first time. These findings suggest a potential spread of these emerging CA-MRSA clones in Japan.

Introduction

S taphylococcus aureus is the most common bacterial pathogen in humans, causing a wide range of infectious diseases. Since the 1960s, methicillin-resistant Staphylococcus aureus (MRSA) has been recognized as a life-threatening multidrug-resistant bacterium worldwide and has been a major cause of nosocomial infections; therefore, this MRSA is alternatively called hospital acquired- (or healthcare-associated) (HA-) MRSA.¹⁶ It is believed that MRSA is generated from methicillin-susceptible S. aureus (MSSA) by acquisition in its chromosome of staphylococcal cassette chromosome mec (SCCmec), a genetic element containing mecA, which is critical to methicillin resistance. SCCmec elements are diverse in their structural organization and genetic content and have been classified into several types and subtypes.²⁵ Three types of SCCmec elements (types I, II, and III) are carried mostly by HA-MRSA strains throughout the world. It has been presumed that a limited number of pandemic HA-MRSA clones with different SCCmec types have been spread worldwide.⁴³

In addition to the HA-MRSA, community-acquired MRSA (CA-MRSA) has also emerged as a major concern worldwide since the late 1990s.⁹ It has been reported that the CA-MRSA is associated with skin and soft-tissue infections and severe systemic infections such as sepsis and necrotizing pneumonia in healthy children or adolescents, otherwise healthy individuals in the community. The CA-MRSA has typically type IV- or V-SCCmec and often produces Panton-Valentine leucocidine (PVL), which has been considered as one of the factors related to severe symptoms of CA-MRSA infection.^26,43 In contrast to a limited number of HA-MRSA clones, CA-MRSA includes a variety of clones, in terms of sequence type (ST) defined by multilocus sequence typing (MLST).⁴³ Globally or regionally predominant CA-MRSA include ST1 and ST8 clones (corresponding to pulsed-field gel electrophoresis [PFGE] types USA400 and USA300, respectively) mostly distributed to the United States and Canada, ST80 in Europe, ST30 spreading worldwide, and ST59 in Taiwan.⁹ The USA300 clone (ST8) possesses the arginine catabolic mobile element (ACME), which is linked to SCCmec type IV and associated with enhanced ability of colonization and spread, and considered as a key role in the persistence of USA300 in the community.¹²

In Japan, it has been reported that most of the MRSA from inpatients and outpatients are the New York/Japan clone belonging to ST5, and most of CA-MRSA isolates are PVL negative.^6,33,40,43 Recently, detection of only a few PVL-positive ST30, ST59 CA-MRSA clones, and USA300 clone was reported.^17,18,37 However, information of CA-MRSA in Japan, that is, prevalence and their genetic characteristics, is still limited. Especially, the status of the spread of ST1 (USA400) and ST8 (USA300) has not yet been well investigated. In the present study, we analyzed the prevalence of CA-MRSA in the S. aureus isolates derived from outpatients in Hokkaido, a northern major island of Japan, to know the epidemiological trend, emergence, and spread of CA-MRSA clones in the community. The CA-MRSA isolates were analyzed genetically in terms of molecular epidemiologic markers, including SCCmec types and coagulase genotypes. Particularly, MRSA isolates having PVL or arcA gene were characterized in detail to understand their relatedness to the CA-MRSA strains reported to date.

Materials and Methods

Bacterial isolates

A total of 1,015 S. aureus isolates were analyzed. These bacterial strains were derived from outpatients who visited various hospitals and clinics in Hokkaido, Japan, during periods of January–February and June–July in 2009. Bacterial isolation from clinical specimens and identification were done in the Sapporo Clinical Laboratory (Inc.), Sapporo, Japan. Approximately 50% of the isolates were derived from urine, followed by other specimens such as pus, sputum, otorrhea, nasal discharge, eye discharge, and skin. Individual bacterial strains were stored in Microbank (Pro-Lab Diagnostics, Richmond Hill, ON, Canada) at −80°C and recovered when they were analyzed. In the present study, all the MRSA causing infections among outpatients were considered as CA-MRSA.⁹

Antimicrobial susceptibility testing

Minimum inhibitory concentrations (MICs) of antimicrobial agents were measured by broth microdilution test and interpreted as sensitive or resistant based on the National Clinical and Laboratory Standards Institute guidelines.⁷ MIC was defined as the lowest concentration of the antimicrobial agent that inhibited visible growth at 35°C after overnight incubation. The antimicrobial agents tested were oxacillin, ampicillin, cefoxitin, vancomycin, gentamycin, kanamycin, fosfomycin, tetracycline, minocycline, ciprofloxacin, clindamycin, and erythromycin.

Multiplex PCR assays for CA-MRSA, SCCmec typing, and coagulase gene typing

For all the S. aureus isolates, presence of staphylococcal 16s rRNA gene, nuc, mecA, PVL gene (lukS-PV/lukF-PV), ACME gene (arcA), and MW756 and MW1409 genes, which are carried with PVL gene mostly by USA400 clone, was investigated by multiplex PCR assay as described by Zhang et al.⁴⁶ Presence of ACME-arcA gene was further confirmed by PCR using primers described by Diep et al.¹² SCCmec type of MRSA was determined by PCR as previously described.²⁵ SCCmec type II and IV elements were further analyzed for their subtypes by PCR with the use of primers previously reported.^20,45 Presence of SCCmec type Vt, an SCCmec variant typically found in Taiwanese CA-MRSA strains,⁴ was confirmed by PCR using primers ccrC2-F2 and ccrC2-R2 as described by Takano et al.³⁹ Coagulase genotypes, including subtypes of IV and V, were determined by multiplex PCR assay as previously described.¹⁹ Subtypes of coagulase genotypes VI and VII were determined by PCR and direct sequencing of coagulase gene D1 region, as previously described.¹⁹

Detection of virulence factor and drug resistance genes

Virulence factor and drug resistance genes were detected by PCR. The target genes of virulence factors included five hemolysin genes (hla, hlb, hlg, hlg-v, and hld), 20 Staphylococcal enterotoxin or Staphylococcal enterotoxin-like genes (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, selq, ser, ses, set, and selu), three exfoliative toxin genes (eta, etb and etd), and tsst-1, edin-A, and edin-B genes. Although most PCR primers employed in this study have been previously reported,^{3,21–23,31,42} for detection of hlb, hld, ses, and set, primers were newly designed as follows: hlBn2 (5′-TTGGGGGCAATATAAACGCGCTG-3′) and hlBn4 (5′-TGTTCTGGTTTGCCATTAGGGT-3′) for hlb, hld1 (5′-TGTTCACTGTGTCGATAAT CC-3′) and hld4 (5′-CCACTCTCCTCACTGTCATTATACG-3′) for hld, ses1 (5′-TCATGAAGTGTTATTCCGCCA-3′) and ses2 (5′-CTAGACCTAAAATAGAAAGCCT-3′) for ses, and set1 (5′- CTTTTTCGGGTGTTACTTCTG-3′) and set2 (5′-AAATGCTAAGTCTGA TTCTCGTG-3′) for set. Genes conferring resistance to penicillin (blaZ), tetracycline (tet(K), tet(L), and tet(M)), macrolide (ermA, ermB, ermC, and msrA), and 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) were detected by monoplex or multiplex PCR using the primers previously reported.^27,28,30

MLST and accessory gene regulator typing

For selected strains of CA-MRSA and MSSA, ST was determined by MLST as previously described.¹⁴ Partial gene sequences of seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) were determined by PCR and direct sequencing, and their allelic profile (allele no.) and ST were obtained from the MLST website. ST data were further analyzed by eBURST to determine their clonal complex. In addition, the accessory gene regulator (agr) typing (subgroups I, II, III, and IV) was performed as previously described.^32,38

Results

Among the 1,015 isolates collected during the study period, 189 isolates (18.6%) were MRSA (mecA-positive). The most common specimen from which MRSA were isolated was urine (23.3%; 44/189), followed by nasal discharge (18.5%), pus (14.8%), otorrhea (14.8%), sputum (14.8%), eye discharge (5.8%), and others (8%). No MRSA was isolated from blood. Coagulase genotyping was performed for all the S. aureus isolates. Most MRSA isolates belonged to coagulase genotype IIa (82.5%), whereas MSSA isolates were assigned to various genotypes including some dominant types IVa (18.6%), Va (12.6%), Xa (10.9%), and VIIa (10.5%) (Table 1). The most frequent SCCmec type in MRSA was II (83.1%), followed by IV (6.9%) and V (3.2%) (Table 2). MRSA isolates having type II-SCCmec mostly belonged to coagulase genotype IIa, whereas MRSA isolates with type IV SCCmec were assigned to various coagulase genotypes (Table 2).

Table 1.

Frequency of Staphylocoagulase Genotypes in Staphylococcus aureus Isolates from Outpatients in Hokkaido, Japan

	No. of isolates (%)
Coagulase genotype	MRSA	MSSA	Total
Ia	12 (6.3)	16 (1.9)	28 (2.8)
IIa	156 (82.5)	75 (9.1)	231 (22.8)
IIIa	8 (4.2)	73 (8.8)	81 (8.0)
IVa	1 (0.5)	154 (18.6)	155 (15.3)
IVb	2 (1.1)	14 (1.7)	16 (1.6)
Va	2 (1.1)	104 (12.6)	106 (10.4)
Vb	0	81 (9.8)	81 (8.0)
VIa	0	2 (0.2)	2 (0.2)
VIc	0	7 (0.8)	7 (0.7)
VIIa	2 (1.1)	87 (10.5)	89 (8.8)
VIIb	5 (2.6)	79 (9.6)	84 (8.3)
VIIIa	1 (0.5)	41 (5.0)	42 (4.1)
Xa	0	90 (10.9)	90 (8.9)
XIa	0	1 (0.1)	1 (0.1)
Not determined	0	2 (0.2)	2 (0.2)
Total	189 (100)	826 (100)	1,015 (100)

Table 2.

Frequency of Staphylococcal Cassette Chromosome mec Types and Coagulase Genotypes in Methicillin-Resistant Staphylococcus aureus

	SCCmec type
Coagulase genotype	I	II	IV	V	Not determined	Total
Ia	0	6	3	0	3	12
IIa	1	148 (1)^a	1	1 (1)^a	5	156
IIIa	3	2	3	0	0	8
IVa	0	0	1	0	0	1
IVb	0	0	2 (2)^b	0	0	2
Va	0	0	0	2	0	2
VIIa	0	0	1	1	0	2
VIIb	0	1	2	2 (1)^b	0	5
VIIIa	1	0	0	0	0	1
Total	5	157	13	6	8	189
(%)	(2.6)	(83.1)	(6.9)	(3.2)	(4.2)	(100)

Number of ACME-arcA (+) isolates.

Number of PVL (+) isolates.

SCCmec, staphylococcal cassette chromosome mec.

PVL gene was identified in three MRSA strains (1.6% in MRSA) and two MSSA strains (0.2% in MSSA) without MW1409 gene locus, whereas ACME gene (arcA) was detected in only two MRSA strains without PVL gene (1.1% in MRSA) (Table 3). The PVL-positive MRSA strains were derived from pus or otorrhea, and had types IVa- or Vt-SCCmec, and belonged to coagulase genotype IVb or VIIb and agr-type I or IV, respectively. PVL gene-positive MRSA belonged to ST6 (strains SR581 and SR1018) or ST59 (SR560), whereas ACME-arcA–positive MRSA strains (SR338 and SR141) and most of PVL-negative MRSA examined were assigned to ST5 or other STs in CC5. Two PVL-positive MSSA strains were grouped into ST188 and ST121. The two ACME-arcA–positive MRSA strains belonged to coagulase genotype IIa and agr type II and had type IIa- or V-SCCmec. MW1409 gene locus was detected in 65.1% of MRSA (123/189) and 5.6% of MSSA (46/826), including an ACME-arcA–positive strain SR388, but none of these isolates harbored PVL gene simultaneously, indicating that the MRSA strains with MW1409 were not USA400 clone. The MW756 gene locus was detected in only one MSSA strain without MW1409.

Table 3.

Toxin and Virulence Factor Genes in Representative Methicillin-Resistant Staphylococcus aureus and Methicillin-Susceptible Staphylococcus aureus Strains in Hokkaido, Japan

	S. aureus strains
Type and gene	SR581	SR1018	SR560	SR388	SR141	SR733	SR950	SR219	SR193	SR710	SR865	SR111	SR802	SR635	SR772	SR853	SR834
Specimen	Pus	Pus	Otorrhea	Nasal discharge	Pus	Pus	Pus	Pus	Urine	Pus	Pus	Otorrhea	Urine	Otorrhea	Nasal discharge	Otorrhea	Skin
Age	19	47	57	1	39	56	33	39	77	31	75	73	71	40	9	61	44
Sex	Male	Female	Male	Male	Female	Female	Male	Male	Male	Female	Female	Female	Male	Female	Female	Female	Male
Types/genes
ST	6	6	59	5	5	188	121	746	764	512	764	512	5	5	5	1783	121
CC	6	6	59	5	5	1	121	97	5	5	5	5	5	5	5	1	121
mecA	+	+	+	+	+	−	−	+	+	+	+	+	+	+	+	−	−
SCCmec type	IVa	IVa	V_T	IIa	V			IIa	IIa	IIa	IIa	IIa	IIa	II^a	IVb
lukF-PV-lukS-PV	+	+	+	−	−	+	+	−	−	−	−	−	−	−	−	−	−
ACME-arcA	−	−	−	+	+	−	−	−	−	−	−	−	−	−	−	−	−
MW 1409	−	−	−	+	−	−	−	+	−	+	+	−	−	−	−	−	−
Coagulase genotype	IV b	IV b	VII b	II a	II a	V b	V a	II a	II a	II a	II a	II a	II a	II a	II a	VII a	V a
agr type^b	I	I	IV	II	II	I	IV
Toxin/virulence factor genes
Leukocidins
lukE-lukD	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
lukM	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
Hemolysins
hla	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
hlb	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
hlg	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
hlg-v	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
hld	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
Enterotoxins
sea	+	+	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−
seb	−	−	+	−	+	−	−	+	+	−	+	−	−	−	−	−	−
sec	−	−	−	+	−	−	−	+	+	+	+	+	−	−	−	−	−
sed	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
see	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
seg	−	−	−	+	+	−	+	+	+	+	+	+	+	+	+	−	+
seh	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−
sei	−	−	−	+	+	−	+	+	+	+	+	+	+	+	+	−	+
selj	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−	−
selk	−	−	+	−	+	−	−	−	−	−	−	−	−	−	−	−	−
sell	−	−	−	+	−	−	−	+	−	+	+	+	+	−	−	−	−
selm	−	−	−	+	+	−	+	+	+	+	+	+	+	+	+	−	+
seln	−	−	−	+	+	−	+	+	+	+	+	+	+	+	+	−	+
selo	−	−	−	+	+	−	+	+	+	+	+	+	+	+	+	−	+
selp	−	−	−	+	+	−	−	−	−	−	−	−	−	+	−	−	−
selq	−	−	+	−	+	−	−	−	−	−	−	−	−	−	−	−	−
ser	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−	−
ses	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
set	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
selu	−	−	−	+	+	−	+	+	+	+	+	+	+	+	+	−	+
Exfoliative toxins
eta	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+
etb	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
etd	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
Others
tsst-1	−	−	−	+	−	−	−	+	−	+	+	+	+	−	−	−	−
edin-A	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
edin-B	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−

Subtype could not be determined.

agr type was determined for six strains with PVL genes or ACME-arcA.

Presence of various toxin or virulence factor genes in representative CA-MRSA and MSSA isolates is shown in Table 3. Most of the PVL-negative, ACME-arcA–negative MRSA strains were grouped into ST5 or other STs in CC5 and had type IIa-SCCmec. These PVL(−)/ACME-arcA(−) MRSA strains and two ACME-arcA–positive MRSA had the enterotoxin gene cluster (seg, sei, sem, sen, seo, selu) and also mostly sec and tsst-1. In contrast, two ST6 PVL-positive strains harbored only sea, and an ST59 PVL-positive strain had only seb, sek, and seq.

The PVL-positive MRSA strains were more susceptible to most of antimicrobials examined and had less numbers of drug resistance genes, than PVL-negative MRSA strains as well as ACME-arcA–positive MRSA (Table 4). Although all the mecA-positive strains were resistant to oxacillin, oxacillin MICs of PVL-positive MRSA and an ACME-arcA–positive MRSA strain SR141 were considerably lower (8–64 μg/ml) than those of most PVL/arcA-negative MRSA strains. Like some of the PVL-negative MRSA strains, ACME-arcA–positive MRSA strains possessed ermA, ermB, aac(6′)-Ie-aph(2")-Ia, and ant(9)-Ia and exhibited resistance to fosfomycin and ciprofloxacin and high-level resistance to gentamicin. Except for ST59 PVL-positive MRSA strain SR388 having tet(K), PVL-positive or ACME-arcA–positive strains had no tetracycline resistance gene, whereas six PVL/arcA-negative MRSA strains harbored tet(M), mostly showing resistance to tetracyclines.

Table 4.

Minimum Inhibitory Concentrations Against Antimicrobials and Prevalence of Drug Resistance Genes in Representative Methicillin-Resistant Staphylococcus aureus and Methicillin-Susceptible Staphylococcus aureus Strains in Hokkaido, Japan

	S. aureus strains
Type, gene, or resistance	SR581	SR1018	SR560	SR388	SR141	SR733	SR950	SR219	SR193	SR710	SR865	SR111	SR802	SR635	SR772	SR853	SR834
Types/genes
ST	6	6	59	5	5	188	121	746	764	512	764	512	5	5	5	1783	121
CC	6	6	59	5	5	1	121	97	5	5	5	5	5	5	5	1	121
mecA	+	+	+	+	+	−	−	+	+	+	+	+	+	+	+	−	−
SCCmec type	IVa	IVa	V_T	IIa	V			IIa	IIa	IIa	IIa	IIa	IIa	II^a	IVb
lukF-PV-lukS-PV	+	+	+	−	−	+	+	−	−	−	−	−	−	−	−	−	−
ACME-arcA	−	−	−	+	+	−	−	−	−	−	−	−	−	−	−	−	−
MW1409	−	−	−	+	−	−	−	+	+	+	+	−	−	−	−	−	−
MIC (μg/ml)
Oxacillin	16	32	8	512	64	≤1	≤1	512	512	512	512	512	256	128	8	≤1	≤1
Ampicillin	64	64	8	64	64	32	4	64	64	128	64	64	64	64	8	≤1	4
Cefoxitin	32	32	16	512	64	4	4	512	512	1,024	512	512	128	128	32	4	8
Vancomycin	≤1	≤1	≤1	≤1	≤1	≤1	≤1	≤1	≤1	2	≤1	2	≤1	≤1	≤1	≤1	≤1
Gentamycin	2	2	≤1	>1,024	512	1,024	≤1	>1,024	128	≤1	1,024	2	1,024	≤1	≤1	≤1	256
Kanamycin	8	16	>1,024	>1,024	>1,024	1,024	4	>1,024	1,024	512	>1,024	>1,024	>1,024	512	4	4	>1,024
Fosfomycin	2	16	≤1	1,024	1,024	2	256	>1,024	>1,024	>1,024	>1,024	>1,024	>1,024	32	8	4	16
Tetracycline	1	≤0.5	32	≤0.5	1	≤0.5	≤0.5	128	64	256	8	64	64	4	1	≤0.5	≤0.5
Minocycline	≤0.5	≤0.5	≤0.5	≤0.5	≤0.5	≤0.5	≤0.5	32	32	64	2	8	16	1	≤0.5	≤0.5	≤0.5
Ciprofloxacin	≤0.5	≤0.5	≤0.5	256	128	32	≤0.5	256	256	256	256	512	256	4	1	4	≤0.5
Clindamycin	≤0.5	≤0.5	>512	512	≤0.5	512	≤0.5	>512	1,024	>512	>512	>1,024	512	≤0.5	≤0.5	≤0.5	≤0.5
Erythromycin	0.5	1	>128	>128	>128	>128	0.5	>128	>128	>128	>128	>128	>128	>128	>128	≤0.5	>128
Resistance genes
blaZ	+	+	+	+	+	+	+	+	+	+	+	−	+	+	+	+	+
tet(K)	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−
tet(L)	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
tet(M)	−	−	−	−	−	−	−	+	+	+	+	+	+	−	−	−	−
ermA	−	−	−	+	+	−	+	+	+	+	+	+	+	+	+	−	−
ermB	−	−	+	+	+	+	−	+	+	+	+	+	+	+	+	−	−
ermC	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+
msrA	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
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	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
aph(3')-IIIa	−	−	+	−	+	−	−	−	−	−	−	−	−	+	−	−	−

Subtype could not be determined.

Discussion

In Japan, the New York/Japan clone (ST5, SCCmec II) has been most predominating in HA-MRSA^6,24 and also predominant in community-onset MRSA isolates from outpatients.^33,40 CA-MRSA isolates reported were mostly PVL negative and belonged to ST8, ST89, or ST91, associated with bullous impetigo.⁴⁰ Although isolation rate was extremely low, PVL-positive ST30 CA-MRSA, which has been spreading worldwide, was detected also in Japan since the 1980s.⁴⁴ Single-locus variants of ST30 (ST765, ST1335) and ST22 were also described as genotypes of PVL-positive MRSA.^18,44 Recently, PVL-positive CA-MRSA USA300 clone (ST8, SCCmec IVa) was reported in two patients who moved to Japan from the United States^17,37 and PVL-positive ST59 CA-MRSA (SCCmec V), which has been predominant in Taiwan, was isolated in a Japanese boy.¹⁸

In the present study, most MRSA isolates from outpatients had the same characteristics (SCCmec II, ST5, coagulase genotype II, PVL negative) as those of New York/Japan clone, that is, predominant HA-MRSA strains in Japan. This finding suggests that outpatients with MRSA infection or colonization may have association with their preceding hospitalization or medical treatment in healthcare facilities, although history of medical treatment for each patient was not investigated in our study. It is also conceivable that the ST5 HA-MRSA may be prevalent in the community, even temporarily, whereas SCCmec II is not considered a typical trait of CA-MRSA.

Three PVL-positive CA-MRSA strains, two ST6 strains, and an ST59 strain were isolated in the present study. ST6 MRSA has been rarely isolated to date, and one report described the ST6-MRSA clone with type IVc-SCCmec in Lebanon, although the ST6 clone did not harbor PVL gene.⁴¹ The ST6 CA-MRSA strains isolated in the present study had SCCmec type IVa and showed low-level resistance to oxacillin, which were similar to USA400 clone.^9,44 However, ST6 CA-MRSA strains belonged to coagulase genotype IVb and had less numbers of toxin genes (only sea), whereas USA400 clone belongs to ST1 and coagulase type VII, having several toxin genes. Further, it was evident in the present study that the ST6 CA-MRSA does not have the same prophage carrying PVL gene and pathogenicity island as that of USA400. Accordingly, genomic background of ST6 CA-MRSA is considered to be quite distinct from that of ST1 CA-MRSA (USA400 clone).

The ST59 CA-MRSA is the most prevalent in Taiwan and has been detected as a sporadic isolate in China, Singapore, Australia, The Netherlands, Denmark, England, and the United States (PFGE type: USA1000).^8,9,11,39 In Japan, ST59 Taiwanese clone has been reported only once for an isolate from pediatric cellulitis in the main island (Honshu) in 2007.¹⁸ Therefore, isolation of the strain SR560 in the present study is the second report of ST59 PVL-positive CA-MRSA in Japan and also the first case in the Hokkaido island. The ST59 PVL-positive CA-MRSA have diverse genotypes and several SCCmec types including types IV, V, and Vt, among which the type Vt SCCmec is highly prevalent.³⁹ The strain SR560 isolated in the present study belonged to coagulase genotype VIIb and had type Vt-SCCmec and seb, sek, seq (SaPI3), tet(K), aph(3′)-IIIa, and ermB. These characteristics were the same as those of ST59 CA-MRSA strains in Taiwan.³⁹ However, strain SR560 belonged to agr type IV and showed low-level resistance to oxacillin (MIC; 8 μg/ml), which are distinguished from Taiwanese predominant ST59 clone. Therefore, the strain SR560 may be one of the PVL-positive ST59 variants described also among the Taiwanese CA-MRSA isolates.³⁹

The ACME was first identified in the genomic sequence of USA300 MRSA and was found to be important for its growth and survival, resulting in extensive dissemination.¹² To date, ACME-arcA gene have been identified in ST1-MRSA-SCCmecIVa, ST5-MRSA-SCCmecII, ST8-MSSA, ST8-MRSA-SCCmecIV, ST59-MRSA-SCCmecII, and ST97-MRSA-SCCmecV.^10,11,13,15 In Japan, the ACME-arcA gene in MRSA had been detected only in the two ST8-USA300 strains to date.^17,37 Therefore, our present study is the first report to detect the ACME-arcA gene in ST5-SCCmec V MRSA and identify the arcA in ST5-SCCmec II strain in Japan. The ACME-arcA–positive ST5-SCCmecII MRSA has been detected only in the United States (PFGE type: USA100), although ST8 MRSA was far more frequently isolated as those harboring the arcA.¹⁵ Therefore, it is conceivable that ACME-arcA–positive ST5-MRSA, a subset of the New York/Japan HA-MRSA clones, might have spread from the United States to Japan. However, it is also possible that the arcA-positive MRSA has emerged in Japan among the predominant ST5-MRSA through acquisition of ACME from certain coagulase-negative staphylococci, because ACME-arcA–positive ST5 MRSA strains isolated in the present study showed generally similar resistance (gene) and toxin gene profiles to those of the ACME-arcA–negative ST5 MRSA strains.

Predominant lineages of PVL-positive MSSA were described as ST1, ST5, ST30, ST80, and ST121, by investigation of isolates from mostly United States and European countries.^29,34 Although only limited information is available for isolates in Asian countries, the reported STs in PVL-positive MSSA are ST25, ST88, ST121, ST188 (CC1), ST573 (CC1), and ST772 (CC1).^1,2,35,36 The ST121, which was identified for PVL-positive MSSA in the present study, had been also reported in an isolate in Honshu Island, Japan.³⁵ Thus, it is suggested that this lineage may be distributed worldwide as a PVL-positive S. aureus. Particularly, ST121 was reported to be most frequent among PVL-positive MSSA, including a clone that caused fatal community-acquired pneumonia in Vladivostok, Russia, located in far-east Asia.² In a recent report in Cambodia, ST121 PVL-positive MRSA strains were suggested to be arisen locally from MSSA.⁵ Accordingly, it seems to be of significance to survey ST121 PVL-positive MSSA in the community as well as PVL-positive MRSA.

In conclusion, in the present study, ST6 was identified as a lineage of PVL-positive CA-MRSA, and ACME-arcA was identified in ST5-SCCmec V MRSA and in ST5-SCCmec II MRSA for the first time in Japan. ST59 PVL-positive CA-MRSA predominating in Taiwan was isolated in Hokkaido for the first time. These strains are considered as emerging CA-MRSA clones in Hokkaido, northern region of Japan. Because the epidemiology of MRSA is constantly changing, further surveillance is needed for their prevalence in the community.

Footnotes

Acknowledgment

This study was supported by a grant-in-aid for scientific research (no. 20590608) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

Disclosure Statement

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

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