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obtained from the MLST website (www.mlst.net), and the ST for each strain was ... sed, see, seg, seh, sei, sek, sem, sen, seo, sep, seq), a putative seu gene (14), ...

Microbiol. Immunol., 51(2), 171–176, 2007

Molecular Characterization of Methicillin-Resistant Staphylococcus aureus in Hospitals in Niigata, Japan: Divergence and Transmission Hassan Zaraket, Taketo Otsuka, Kohei Saito, Soshi Dohmae, Tomomi Takano, Wataru Higuchi, Takeshi Ohkubo, Kyoko Ozaki, Misao Takano, Ivan Reva, Tatiana Baranovich, and Tatsuo Yamamoto* Division of Bacteriology, Department of Infectious Disease Control and International Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Niigata 951–8510, Japan Received June 13, 2006; in revised form, October 31, 2006. Accepted November 2, 2006

Abstract: The major methicillin-resistant Staphylococcus aureus (MRSA) distributed among hospitals in Japan is New York/Japan clone [multilocus sequence type 5 (ST5), agr type 2 and methicillin resistance locus type (SCCmec) II] which possesses both the toxic shock syndrome toxin 1 gene (tst) and staphylococcal enterotoxin C gene (sec). In this study, we collected 245 MRSA strains from four hospitals during 2001 to 2005 in Niigata, Japan, and analyzed tst and sec genes and SCCmec type among them. A total of 13 strains were further examined for their genotypes, virulence gene patterns and drug resistance. Among the 245 strains four tst sec genes patterns were observed; tstsec strains represented a majority of 86.5% and 9.4% were tstsec. SCCmec typing revealed that 91.4% had type II, 4.1% type IV and 4.1% type I. Multilocus sequence typing (MLST) revealed that 10 of the 13 typed strains belonged to clonal complex 5 (7 had ST5 while 3 were single locus variants of ST5) with similar characteristics to the New York/Japan clone and possessed multi-drug resistance with high virulence gene content. The remaining 3 strains were ST8 (n2) and ST91 (n1). The ST91 strain had SCCmecIV and seemed to originate in the community, while ST8 strains exhibited SCCmec type I, which is distinct from community type IV. The data suggest that MRSA in hospitals in Niigata now mainly includes the New York/Japan clone (undergoing genomic divergence and clonal expansion) and other minor types (e.g. ST8) as well as the community type. Key words: Methicillin-resistant Staphylococcus aureus (MRSA), New York/Japan clone, Toxic shock syndrome toxin 1 gene (tst), Staphylococcal enterotoxin C gene (sec)

Methicillin-resistant Staphylococcus aureus (MRSA) has been a major cause of nosocomial infections since its emergence in 1961 (19). It has acquired resistance to the majority of available antimicrobial agents, leaving a limited choice for its eradication. Hospital-acquired MRSA (HA-MRSA) is associated with infections ranging from skin and soft tissue infections to systemic or fatal infections such as septicemia, pneumonia, endocarditis (6) and toxic shock syndrome (TSS) (15). In Japan, the incidence of HA-MRSA is assessed to be high (60–70%) (12). Recent HA-MRSA in Japan has been considered to

be the New York/Japan clone, which is one of (at least Abbreviations: agr, accessory gene regulator; bbp, bone sialoprotein-binding protein gene; c9ag, core nine adhesin genes; CAMRSA, community-acquired methicillin-resistant Staphylococcus aureus; CC, clonal complex; clfA, clfB, clumping factor A and B genes; cna, collagen-binding protein gene; ebpS, elastinbinding protein gene; egc, enterotoxin gene cluster; eno, lamininbinding protein gene; ET genes, exfoliative toxin genes (eta, etb, etd); fib, fibrinogen-binding protein gene; fnbA, fnbB, fibronectinbinding protein A and B genes; HA-MRSA, hospital-acquired methicillin-resistant Staphylococcus aureus; icaA, icaD, intracellular adhesin A and D genes; MDR, multi-drug resistance; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible Staphylococcus aureus; NICU, neonatal intensive care unit; PVL, Panton-Valentine leucocidin; SCCmec, methicillin resistance locus type; SE genes, staphylococcal enterotoxin genes (e.g. sea, seb, sec etc.); SLV, single locus variant; ST, sequence type; TSS, toxic shock syndrome; tst, toxic shock syndrome toxin 1 gene.

*Address correspondence to Dr. Tatsuo Yamamoto, Division of Bacteriology, Department of Infectious Disease Control and International Medicine, Niigata University Graduate School of Medical and Dental Sciences, 1–757 Asahimachidori, Niigata, Niigata 951–8510, Japan. Fax: 81–25–227–0762. E-mail: [email protected]

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seven) major worldwide MRSA clones (6, 19, 21). The New York/Japan clone is characterized by sequence type 5 (ST5), agr type 2, methicillin resistance locus (SCCmec) type II, coagulase type II, and tstsec (11, 21). MRSA spread in the community exhibits distinct genotypes from that in hospitals, such as ST1, 8, 30 or 80 and SCCmecIV or V, and possesses Panton-Valentine leucocidin (PVL) (24, 28). Such community-acquired MRSA (CA-MRSA) has also been isolated even in hospitals (31). In Japan, most of CA-MRSA is PVL and exhibits ST8, 81 or 91 (24). ST91 MRSA has also been isolated from a neonatal intensive care unit (NICU) (24). However, the present status of HA-MRSA in Japan has not been well understood at the molecular level, and ST data for HA-MRSA are not abundant. The purpose of this study was to investigate HA-MRSA in Niigata and compare the data with those previously reported from other areas in Japan (12, 21, 22). Thus, HA-MRSA strains isolated in hospitals in Niigata during 2001 through 2005 were molecularly typed, their virulence genes were analyzed and their genetic backgrounds were compared.

obtained from the MLST website (www.mlst.net), and the ST for each strain was determined. agr type (1 to 4) was assigned by PCR as previously described (23). Coagulase type (I to VIII) was determined using a coagulase typing kit (Denka Seiken, Tokyo) in accordance with the manufacturer’s instructions. Virulence gene analysis. The virulence genes were analyzed by PCR assay. These included 13 staphylococcal enterotoxin genes (2, 8–10, 20, 33) (sea, seb, sed, see, seg, seh, sei, sek, sem, sen, seo, sep, seq), a putative seu gene (14), 3 exfoliative toxin genes (2, 30) (eta, etb, etd) and 11 adhesin genes (16, 26, 27, 29) including intracellular adhesin A and D genes (icaA, icaD), the collagen-binding protein gene (cna), the laminin-binding protein gene (eno), fibronectin-binding protein A and B genes (fnbA, fnbB), the elastin-binding protein gene (ebpS), clumping factor A and B genes (clfA, clfB), fibrinogen-binding protein gene (fib) and the bone sialoprotein-binding protein gene (bbp). Susceptibility testing. In vitro susceptibility of MRSA strains to antimicrobial agents was tested by the agar dilution method using Mueller-Hinton agar according to CLSI procedures described previously (3, 17). Final concentrations of antimicrobial agents were from 0.002 to 128 µg/ml.

Materials and Methods Results Bacterial strains. A total of 245 clinical strains of MRSA isolated from hospitalized patients in four general hospitals in Niigata, Japan during a period of 5 years (2001–2005) were examined. These included 132 strains isolated from adult patients from sputum (n39), nasal mucosa (n7), blood (n5), pleural effusion (n29), eye discharge (n2), ear discharge (n2), craniospinal fluid (n2), urinary tract discharge (n4), feces (n6), bile (n2), catheter (n3), ascitis (n19), and others (n12), in addition to 113 strains from NICU patients from sputum (n60), nasal mucosa (n48), eye discharge (n3), and others (n2). In this study, HA-MRSA was defined as MRSA isolated from patients at least 48 hr after admission to a hospital. All MRSA isolates were stored at 80 C. Molecular typing. All 245 strains were first analyzed by PCR for staphylococcal enterotoxin C (sec) and TSS toxin 1 gene (tst) and SCCmec type was determined using multiplex PCR as previously described (10, 18, 32). Then 13 MRSA strains, chosen at random from different tst sec toxin gene patterns, were further typed. Multi-locus sequence typing (MLST) of the seven house-keeping genes (arcc, aroe, glpf, gmk, pta, tpi, yqil) was performed as previously described (7). The allelic profile of these genes (a seven digit number) was

Among the 245 HA-MRSA strains four groups were obtained according to the tst and sec genes pattern (Table 1). The tstsec group represented a majority of 86.5% followed by the tstsec group (9.4%). SCCmecII was detected in 91.4% of the strains; types I, IV, and V were also observed in 4.1%, 4.1% and 0.4% respectively. Of 13 strains selected at random, from the four tst sec gene patterns, 7 were ST5, 3 were single locus variants (SLV) of ST5 (2 of them, ST763 and 764, were novel types), 2 strains were ST8 and 1 was ST91 (Table 2). ST5 and its SLV strains (clonal complex, CC5) had SCCmecII and agr2. ST8 strains had SCCmecI and agr1, while ST91 strain had SCCmecIVa and agr3. All strains that belonged to CC5 possessed high toxin gene content (more than five genes) with some variation (Table 2). In contrast, ST8 and 91 strains had low toxin gene content (one of the ST8 strains did not possess any toxin gene). All the analyzed strains possessed nine adhesion genes (c9ag). ST91 was the only strain that had the cna adhesin gene plus c9ag. All CC5 and ST8 strains were multiple drug-resistant (MDR; Table 2). CC5 strains were resistant to fosfomycin while ST8 and ST91 strains were susceptible. The ST91 strain manifested only β-lactam agents and

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CHARACTERIZATION OF HA-MRSA IN NIIGATA, JAPAN Table 1. Distribution of 245 HA-MRSA isolates according to their tst, sec gene pattern and SCCmec type SCCmec type

tstsec n212 (86.5) 3 209 0 0 0 0 0 0

I II III IVa IVb IVc IVd V

tstsec n9 (3.7) 1 8 0 0 0 0 0 0

No. (%) of isolates tstsec n1 (0.4) 0 1 0 0 0 0 0 0

tstsec n23 (9.4) 6 6 0 10 0 0 0 1

Total 245 10 (4.1) 224 (91.4) 0 10 (4.1) 0 0 0 1 (0.4)

Table 2. Molecular characterization, and drug resistance of MRSA strains showing different tst and sec toxin gene patterns Toxin genes

Strain codee)

tst/sec

S387 S597 S512 S544 S592 S566 S577 S591 S485 S595 S276 S552 S351

tst/sec tst/sec tst/sec tst/sec tst/sec tst/sec tst/sec tst tst tst sec none none

b)

Other

egc egc egc egc sei, sem, sen, seo egc egc none egc egc seb, egc none sem, seo, etb

Adhesin genesc) c9ag c9ag c9ag c9ag c9ag c9ag c9ag c9ag c9ag c9ag c9ag c9ag c9ag, cna

MLST analysis ST

CC

5 5 5 5 148 763d) 5 8 5 5 764d) 8 91

5 5 5 5 5 5 5 8 5 5 5 8 509

SCCmec agr Coagulase type type type MIC of OXA µg/ml II II II II II II II I II II II I IVa

2 2 2 2 2 2 2 1 2 2 2 1 3

II II II II II II II III II II II III I

256 128 128 256 128 256 128 128 256 128 128 128 4

Drug resistancea) Non-β lactam antibiotics KAN, ERY, FOS TET, ERY, CLI, LVX, FOS, SMX KAN, TET, ERY, CLI, LVX, FOS KAN, TET, ERY, CLI, LVX, FOS KAN, TET, ERY, CLI, LVX, FOS, SMX KAN, ERY, CLI, LVX KAN, TET, ERY, CLI, LVX, FOS GEN, KAN, TET, ERY KAN, TET, ERY, FOS GEN, KAN, TET, ERY, CLI, LVX, FOS GEN, KAN, TET, ERY, CLI, LVX, FOS, SMX GEN, KAN, TET, ERY, LVX KAN

a)

Abbreviations: OXA: oxacillin, GEN: gentamicin, KAN: kanamycin, TET: tetracycline, ERY: erythromycin, CLI: clindamycin, LVX: levofloxacin, FOS: fosfomycin, SMX: sulfamethoxazole. No resistance was observed for streptomycin, arbekacin, doxycycline, minocycline, vancomycin, teicoplanin, trimethoprim or fusidic acid. b) egc, enterotoxin gene cluster (seg, sei, sem, sen, seo). c) c9ag, core 9 adhesin genes shared by all strains (icaA, icaD, eno, fnbA, fnbB, ebpS, clfA, clfB, fib). d) Novel ST types. e) The strains S591, S552 and S351were isolated from pus, blood and nasal cavity, respectivley.

kanamycin resistance. The MIC of oxacillin was very low (4 µg/ml) in the ST91 strain compared to that in the ST8 and CC5 strains (128–256 µg/ml). Discussion It has been considered that only a limited number of MRSA clones had emerged and such pandemic clones have spread worldwide, represented by the New York/Japan clone (ST5, SCCmecII), Pediatric clone (ST5, SCCmecIV), Iberian clone (ST247, SCCmecIA), Brazilian/Hungarian clone (ST239, SCCmecIII/IIIA), EMRSA-16 clone (ST36, SCCmecII), E-MRSA-15 clone (ST22, SCCmecIV) and Berlin clone (ST45, SCCmecIV) (6, 19, 21).

Saito et al. (22) reported that MRSA isolated in NICU from three different hospitals in Tochigi, Japan, belonged to a major single pulse type, and was widely tstsec. Similar data were reported from Tokyo by Kikuchi et al. (21); the majority of MRSA strains isolated in their hospital was SCCmecII. Our data were in correlation with previous reports, which suggests that the tstsec, SCCmecII clone (representing the New York/Japan clone) continues to successfully colonize hospitals in Japan; however, divergence within this clone was observed (SLVs). A minority of the strains possessed SCCmecI, IV or V. MRSA with SCCmecIV or V is considered to be of community origin (6), but in this study our cases were strictly hospital acquired. However, whether the patients with hospital-acquired

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infections were nasal carriers of MRSA with SCCmecIV, or they actually acquired it in the hospital is not known. Aires de Sousa et al. (1) reported that most MRSA isolates in a hospital in Tokyo had the same clonal type of that widely spread in hospitals in New York. Genome-analyzed strain Mu50 (ST5, SCCmecII) is a Japanese isolate of the New York/Japan clone (13), and well-characterized USA100 (ST5, SCCmecII) is an isolate from the United States (4, 25). However, there is a marked divergence between the two. While Mu50 is tstsec, USA100 is tstsec. In this study, ST5 (CC5), SCCmecII strains were all tst, sec and egc (enterotoxin gene cluster; seg, sei, sem, sen, seo) like Mu50 (13), except for two strains that lacked sec. A divergence was also noted in ST types. Two of seven tstsec CC5 strains were ST148 [showing truncated egc (sei, sem, sen, seo)] and the novel ST763 (egc). In addition, one tstsec CC5 strain was the novel ST764 (seb, egc). Similarly to the Japanese ST5, USA100 ST5 had some variation in its toxin gene content, e.g. sed, sej, and sep were not shared by the consensus USA100 ST5 (4). Evolution of ST5 clone has been previously described by Enright et al. (6). Similar characteristics shared by Japanese and USA ST5 strains suggest that they might have originated from the same ancestor. It is known that toxin genes are

carried on mobile genetic elements (15); this explains the variation in the toxin genes. Moreover, detection of single-locus variants (novel STs 763 and 764 and ST148) of the New York/Japan clone suggests that this clone is undergoing divergence (Fig. 1). We previously reported that PVL CA-MRSA of ST8 (CC8), ST89 (CC509; previously CC89) and ST91 (CC509; previously CC89) spread in the community (in association with bullous impetigo) in Japan (24). In this study, we detected MRSA with a community background (e.g. ST91, SCCmecIV; Table 3) in the hospital, suggesting that some CA-MRSA strains have been transmitted from the community to hospital. Enright et al. (5) reported that MRSA ST8 clones could have emerged by multiple independent acquisition of SCCmec into methicillin-susceptible S. aureus (MSSA) ST8 leading to MRSA ST8 with four SCCmec types. In our study hospital isolates of ST8 possessed SCCmec type I, which is different from that of MRSA (SCCmecIV) isolated in the community (Table 3). Thus ST8 SCCmecI could have originated from the same progenitor of the community ST8 and is emerging in the hospital (Fig. 1). In conclusion, HA-MRSA in Niigata, Japan, is largely tstsec, SCCmecII, in addition to other minor types including those of community origin. The New York/ Japan clone was found to be undergoing genomic diver-

Fig. 1. Genetic divergence of New York/Japan HA-MRSA clone and the relation between community and hospital types described in this study.

CHARACTERIZATION OF HA-MRSA IN NIIGATA, JAPAN

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Table 3. Comparison of ST8 or 91 HA-MRSA strains with previous CA-MRSA strains Isolation

Strain name

agr type

SCCmec type

Coagulase type

8 8 8 8 8

community community community hospital hospital

NN2 NN3 NN4 S591 S552

1 1 1 1 1

IVa IVxb) IVxb) I I

III III III III III

91 91 91

community community hospital

NN11 NN15 S351

3 3 3

IVa IVa IVa

I I I

ST

a) b)

Adhesin genesa)

Toxin genes

Drug resistancea)

Reference

tst, sec, sem, seo, etb tst, sec tst, sec tst none

c9ag c9ag c9ag c9ag c9ag

GEN, KAN, ERY KAN GEN, KAN GEN, KAN, TET, ERY GEN, KAN, TET, ERY, LVX

22 22 22 this study this study

sem, seo, etb sem, seo, etb sem, seo, etb

c9ag, cna c9ag, cna c9ag, cna

GEN, KAN, ERY GEN, KAN, ERY KAN

22 22 this study

Refer to Table 2 for abbreviations. Type IV with unknown subtype (other than IVa, IVb, IVc, or IVd).

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