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Journal of Clinical Microbiology, July 2005, p. 3356-3363, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3356-3363.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Panton-Valentine Leucocidin (PVL)-Positive Community-Acquired Methicillin-Resistant Staphylococcus aureus (MRSA) Strain, Another Such Strain Carrying a Multiple-Drug Resistance Plasmid, and Other More-Typical PVL-Negative MRSA Strains Found in Japan

Yoko Takizawa,1 Ikue Taneike,1 Saori Nakagawa,1 Tomohiro Oishi,2 Yoshiyuki Nitahara,1 Nobuhiro Iwakura,1 Kyoko Ozaki,1 Misao Takano,1 Teruko Nakayama,1 and Tatsuo Yamamoto1*

Department of Infectious Disease Control and International Medicine, Division of Bacteriology, Niigata University Graduate School of Medical and Dental Sciences,1 Department of Pediatrics, Joetsu General Hospital, Niigata, Japan2

Received 29 August 2004/ Returned for modification 20 January 2005/ Accepted 29 March 2005


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ABSTRACT
 
Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was collected from children with bullous impetigo in 2003 and 2004. One strain collected in 2003 was Panton-Valentine leucocidin (PVL) positive. In 2004, a multiple-drug-resistant PVL+ CA-MRSA strain was isolated from an athlete with a cutaneous abscess. These strains were analyzed by multilocus sequence typing, spa typing, agr typing, coagulase typing, staphylococcal cassette chromosome mec (SCCmec) typing, PCR assay for 30 virulence genes, drug susceptibility testing, pulsed-field gel electrophoresis, and for plasmids. The two Japanese PVL+ CA-MRSA strains belonged to the globally extant ("pandemic") sequence type 30 (ST30) with SCCmec IV. A transmissible, multiple-drug resistance plasmid emerged in such ST30 strains. The PVL– CA-MRSA strains ("domestic" CA-MRSA) accumulated for bullous impetigo, exhibiting new genotypes. Hospital-acquired MRSA of ST91 (but not pandemic ST5) shared common features with the PVL– CA-MRSA strain.


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INTRODUCTION
 
Staphylococcus aureus causes skin and soft tissue infections such as bullous impetigo, abscesses, furunculosis, and staphylococcal scalded skin syndrome (SSSS); life-threatening infections such as pneumonia and toxic shock syndrome (TSS); or neonatal TSS-like exanthematous disease (NTED) (36, 37). Since at least 1960, methicillin-resistant S. aureus (MRSA) has become a common nosocomial pathogen (2), such that MRSA has come to be referred to as hospital-acquired MRSA (HA-MRSA).

Recently, community-acquired MRSA (CA-MRSA), which is found among members of a particular community who do not otherwise exhibit established risk factors for HA-MRSA infections (2, 26, 36, 38), has become a major concern worldwide (9, 10, 26, 38). CA-MRSA infection, which can cause fatal necrotizing pneumonia in children (4), transmits, e.g., through skin-to-skin contact. Common features of these CA-MRSA strains are the presence of the Panton-Valentine leucocidin (PVL) gene and the methicillin-resistance locus (staphylococcal cassette chromosome mec [SCCmec] IV) (38). CA-MRSA distributed to various multilocus sequence types (STs), such as ST1 (specific to the United States) (25, 38), ST80 (more specific to Europe) (38), and ST30 (probably world-spreading type) (9, 11, 32, 38, 39).

No PVL+ CA-MRSA has been previously reported in Japan. In this study, we isolated PVL+ and PVL– CA-MRSA strains from Japan and characterized their molecular characteristics, in comparison with the previously reported PVL+ CA-MRSA strain from outside Japan and with HA-MRSA strains reported in Japan.


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MATERIALS AND METHODS
 
Bacterial strains. Fifty-four S. aureus strains were isolated from 54 children (7 months to 10 years of age) with impetigo contagiosa (bullous impetigo) in Niigata in 2003. Of those, 11 strains (20.4%; strains NN1 to NN11), all from children 7 months to 6 years of age, were CA-MRSA; PLV+ strain NN1 was isolated from an infant (female) aged 11 months. The remaining 43 strains were methicillin-susceptible S. aureus (MSSA). In 2004, 30 S. aureus strains were isolated from 30 children (2 months to 8 years of age) with bullous impetigo, and 5 strains (16.7%; strains NN13 to NN17), from children 1 to 6 years of age, were CA-MRSA. The remaining 25 strains were MSSA. In 2004, CA-MRSA (PVL+ strain NN12) was also isolated from an 18-year-old female high school basketball player with an abscess in her bilateral gluteal regions.

HA-MRSA strains included seven NTED-associated MRSA strains, isolated from neonates in a neonatal intensive care unit (NICU). Of those, five strains (strains 3812, 3908-1, 4082, 4382, and 4410-1), belonging to pulsed-field gel electrophoresis (PFGE) types A2, A3, A10, A1, and A11, respectively (21), were isolated in Tokyo, and two strains (strains E6 and E7) were isolated in Niigata. SSSS-associated MRSA strains (strains H5 and H51) were isolated from neonates in a NICU in Niigata. TSS-associated MRSA strains (strains I6 and I8) were isolated from hospitalized patients in Niigata.

In this study, CA-MRSA was defined as MRSA isolated from outpatients who had no history of hospitalization within the past 1 year and presented no other established risk factors for MRSA infections, such as surgery, residence in a long-term care facility, dialysis, or indwelling percutaneous medical devices and catheters (2, 26, 36, 38). HA-MRSA was defined as MRSA isolated from hospitalized patients (e.g., those in the NICU), who were MRSA negative at the beginning of hospitalization.

ST30 CA-MRSA strains USA1100 from the United States (provided by L. K. McDougal and L. L. McDonald), HT20030336 from The Netherlands, and HT20010466 from Australia (provided by J. Etienne) were used as reference strains.

PFGE and computer analysis. For PFGE analysis, total bacterial DNA was extracted from CA-MRSA and HA-MRSA and digested with SmaI (13). The digested DNA was applied on a PFGE gel (1.2% agarose). Computer-assisted analysis of the PFGE patterns was performed using a program called Molecular Analyst Finger Printing PLUS (Bio-Rad, Tokyo, Japan), according to the unweighted-pair group method using average linkages clustering algorithm (28).

Molecular typing. Multilocus sequence typing was performed using seven housekeeping genes, as previously described (11). An allelic profile (allele number) was obtained from the multilocus sequence typing website (http://www.mlst.net/), and the ST data were further analyzed using eBURST software (12) to determine the clonal complex (CC) to which each ST belongs.

The spa (staphylococcal protein A gene) typing was performed as previously described (34). The spa type was determined using a public spa type database.

Detection of the accessory gene regulator (agr) allele group was done by PCR assay with the reported primers, as previously described (35).

The SCCmec types (I to IV) were analyzed by PCR assay as previously described (29) using reference strains. In the case of SCCmec IV, 3 subtypes (IVa, IVb, and IVc) were further analyzed by PCR assay with the reported primers, as previously described (16). In the case of SCCmec II, the subtype IIa was further analyzed by PCR assay (16).

Virulence gene analysis. Thirty staphylococcal virulence genes were detected by PCR assay using the previously reported primers. The argeted genes were 3 leucocidin genes (18), 5 hemolysin genes (18), 16 staphylococcal enterotoxin genes (1, 15, 18, 19, 31, 45), one putative staphylococcal enterotoxin gene (24), 3 exfoliative toxin (ET) genes (1, 43), the exotoxin-like gene cluster (40), and the epidermal cell differentiation inhibitor gene (18).

Coagulase typing. The coagulase type of the MRSA strains was examined using a staphylococcal coagulase antiserum kit (Denka Seiken, Tokyo, Japan) in accordance with the manufacturer's instructions.

Susceptibility testing. Susceptibility testing of bacterial strains was done by the agar dilution method with Mueller-Hinton agar according to previously published procedures (27). The final concentrations of antimicrobial agents ranged from 0.008 to 128 µg/ml.

Filter mating. Strain NN12 (donor) was mated with S. aureus RN2677 (recipient) on membrane filters, as previously described (44). Transconjugants were selected for both the donor resistance marker (gentamicin at 10 µg/ml) and the recipient resistance marker (novobiocin at 5 µg/ml).

Plasmid DNA analysis. Plasmid DNA was isolated by using the QIAGEN plasmid preparation kit (QIAGEN, Hilden, Germany) and lysostaphin (Wako Pure Chemicals, Osaka, Japan) according to the instructions of the manufacturer and analyzed by agarose gel electrophoresis.


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RESULTS
 
Characterization of CA-MRSA. The characteristics of a total of 17 Japanese CA-MRSA strains are summarized in Table 1. from bullous impetigo in 2003, and strain NN12, isolated from an abscess in 2004, carried the PVL genes. Other CA-MRSA strains were PVL–, and all of the MSSA strains derived from bullous impetigo (43 strains in 2003 and 25 strains in 2004) were also PVL–.


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  		TABLE 1. Characteristics of the Japanese CA- and HA-MRSA strains and PVL+ ST30 CA-MRSA reference strainsa

NN1 and NN12 strains shared the same genotypes. That is, these strains exhibited ST30, spa19, agr3, coagulase IV, and SCCmec IVc and carried additional leucocidin genes (lukE-lukD), 3 hemolysin genes (hla, hlg, hld), and the enterotoxin gene cluster (egc) consisting of 5 genes (seg, sei, sem, sen, and seo). They lacked exfoliative toxin genes and were resistant to oxacillin and cadmium. These characteristics were shared by the ST30 strains from the United States, The Netherlands, and Australia, although there existed some divergence: spa19 versus spa (new) and SCCmec IVa versus SCCmec IVc (Table 1). One particular point of interest is that strain NN12 exhibited multiple-drug resistance (to gentamicin, kanamycin, and tetracycline).

For the PVL– CA-MRSA (15 strains), a marked divergence existed. The most common ST was ST89 (66.7%), followed by ST8 (20%) and ST91 (13.3%). ST89 and ST91 belonged to the same CC (CC89). Of the 10 ST89 strains, 9 (90%) exhibited spa416 and 1 (10%) exhibited spa (new). The three ST8 strains exhibited both different and new spa types (spa604, spa605, and spa606), and the two ST91 strains exhibited different spa types (spa416 and spa604). With the exception of the ST8 strains which were of agr1, all strains were of agr type 3 (as reported for CA-MRSA) (10, 26, 38). Of the 10 ST89 strains, 7 (70%) exhibited coagulase I, 2 (20%) exhibited type III, and 1 (10%) exhibited type V. All three ST8 strains exhibited type III, and the two ST91 strains exhibited types I and III.

Additionally, very extensive variations existed in the SCCmec types of the PVL– CA-MRSA. Of the 10 ST89 strains, only 1 strain (10%) exhibited a known type (IVa), and the remaining 9 strains exhibited one of two unknown types: 5 strains (50%) of one type (x1; non-I, -II, -III, -IV) and 4 strains (40%) of another type (x2). In the three ST8 strains, one exhibited type IVa, while the remaining two exhibited an unknown subtype (non-a, -b, -c) of type IV (IVx). The ST91 strains exhibited type IVa.

Regarding the virulence genes of the PVL– CA-MRSA, all of the ST89 and ST91 strains (12 strains) lacked leucocidin genes. These strains carried hla (for {alpha}-hemolysin) at 91.7%, hlb (for ß-hemolysin) at 100%, hlg (for {gamma}-hemolysin) at 83.3%, hlg-v (for {gamma}-hemolysin variant) at 8.3%, and hld (for {delta}-hemolysin) at 100%. The strains also carried enterotoxin genes sem and seo at 100% and carried etb (for ETB) at 66.7%. One strain (8.3%) carried both eta and etb (for ETA and ETB); eta was found only in this strain. eta is carried by a phage (41), and etb is carried by a plasmid (42). No such combination of eta and etb has been reported previously. In the case of the ST8 strains, all three strains were positive for the LukE-LukD genes, the five hemolysin genes, and the enterotoxin genes tst (for toxic shock syndrome toxin 1) and sec (for SEC). One of the three was also positive for etb.

Regarding resistance, the PVL– CA-MRSA strains (15 strains) manifested resistance to kanamycin at 100%, gentamicin at 93.3%, erythromycin at 86.7%, clindamycin at 60%, and fosfomycin at 20%. Resistance to fosfomycin was found only in ST89 strains. Ethidium bromide resistance was found in one of the ST8 strains.

For drug resistance of the CA-MRSA from bullous impetigo, MIC90s (in µg/ml) of oxacillin and ceftazidime were 32 and 64, respectively. These resistance levels were relatively low compared with the data for HA-MRSA strains (described below). Those of ampicillin, gentamicin, kanamycin, and erythromycin were 16, 64, ≥256, and ≥256 µg/ml, respectively. The high frequency of gentamicin resistance is likely explained by the fact that gentamicin is commonly employed as the primary treatment for skin infections of this type in Japan.

Next, the HA-MRSA strains were examined (Table 1). No PVL genes were detected. Characteristics of the NTED and TSS strains are uniform and exhibited ST5 and SCCmec IIa, corresponding to the pandemic New York/Japan clone harboring ST5 and SCCmec II (30). In addition, all strains exhibited agr2 and coagulase type II and were positive for the lukE-lukD genes, all 5 hemolysin genes (hla, hlb, hlg, hlg-v, and hld) (with an exception, described below), and enterotoxin genes (tst, sec, and egc). Therefore, although toxic shock syndrome toxin 1 (encoded by tst) is a major toxin associated with NTED and TSS (36, 37), it may be significant that the above toxins were found in common.

Characterization of HA-MRSA. There also existed divergence for spa types, hlg-v, and sea (Table 1); of the spa types of the NTED and TSS strains, spa29 of strain 3812 (PFGE type A2) is different from those of pandemic New York/Japan or pandemic pediatric clones (30). SEA (encoded by sea) is not only associated with TSS (in combination with SEC) (7) but also with food poisoning and postantibiotic diarrhea (in combination with LukE-LukD) (14).

In contrast, the SSSS strains were remarkably divergent from the NTED and TSS strains. They exhibited ST91, spa416, agr3, coagulase I, and SCCmec IVa (Table 1). SCCmec IV has also frequently been detected in HA-MRSA strains in both Europe and the United States (3, 6, 38). The different ST (30) indicates that the SSSS strains do not correspond to the pandemic SCCmec IV pediatric clone.

The SSSS strains were positive for etb (and also for lukE-lukD, hlb, hlg, hld, sem, and seo); ET is associated with SSSS (23). All three ST91 strains were etb positive, regardless of HA-MRSA and CA-MRSA, and showed an extensive similarity (Table 1). However, there is a possibility that a combination of lukE-lukD and etb is associated with SSSS and etb alone is associated with bullous impetigo (Table 1).

For drug resistance of the HA-MRSA strains, MIC90s (in µg/ml) of oxacillin and ceftazidime were ≥256 (resistance levels were higher than those for the CA-MRSA strains). And, most HA-MRSA strains manifested resistance to more antimicrobial agents (Table 1). However, there was an exception that the ST91 and SCCmec IVa SSSS strains manifested lower levels of resistance to oxacillin (MIC, 8 µg/ml) and ceftazidime (MIC, 16 µg/ml), as did the ST91 and SCCmec IVa CA-MRSA strains.

Analysis of PFGE patterns. A computer-assisted comparison of PFGE patterns obtained with the CA-MRSA and HA-MRSA strains is shown in Fig. 1. As expected, PVL+ ST30 CA-MRSA strains comprised one cluster (including divergent branches), while ST89 and ST91 CA-MRSA strains comprised a much bigger cluster consisting of more-divergent branches and included the ST91 HA-MRSA strain and one ST8 CA-MRSA strain (ST91 CA- and HA-MRSA comprised a small cluster). The remaining two ST8 CA-MRSA strains were located far from the ST89-ST91 cluster and relatively close to the NTED and TSS clusters. In the NTED HA-MRSA, the Niigata strains were slightly divergent from the Tokyo strains.



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  		FIG. 1. PFGE analysis of the Japanese CA- and HA-MRSA strains and reference PVL+ ST30 CA-MRSA strains (right side) and dendrogram constructed by computer-assisted comparison (left side). For PFGE analysis, bacterial DNA was digested with SmaI. The strain numbers and STs (in parentheses) are shown at the right side of dendrogram (left side of PFGE patterns). In the dendrogram, the cluster consisting of the PVL+ ST30 CA-MRSA strains is marked with red, and those for the three HA-MRSA groups (those from NTED, TSS, and SSSS) are shaded. Clusters without a mark represent those for the PVL– CA-MRSA. Of the NTED strains, E6 and E7 are Niigata strains and 4382, 4410-1, 4082, 3812, and 3908-1 are Tokyo strains (PFGE type strains with A1, A11, A10, A2, and A3, respectively). Of the PVL+ ST30 strains, USA1100, HT20030336, and HT20010466 are from the United States, The Netherlands, and Australia, respectively. The molecular characteristics of each bacterial strain are listed in Table 1.

Drug resistance plasmid. The transfer of a drug resistance plasmid to S. aureus RN 2677 was performed with the multiple-drug-resistant PVL+ ST30 CA-MRSA strain (strain NN12). Gentamicin-resistant transconjugants were obtained at a transfer frequency (number of selected transconjugants per donor) of 1.8 x l0–6.

Strain NN12 carried two plasmids with molecular sizes of 27.5 kb and 41 kb, while strain NN1 carried only the 27.5-kb plasmid. As expected, the gentamicin-resistant transconjugant carried the 41-kb plasmid (designated pGKT1), as shown in Fig. 2.



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  		FIG. 2. Plasmid analysis of the PVL+ ST30 CA-MRSA strains NN1 and NN12 and the transconjugants obtained by filter mating. Plasmid DNA from each bacterial strain was electrophoresed in 0.7% agarose. Lanes: 1, strain NN1; 2, multiple-drug-resistant strain NN12; 3, transconjugants (RN2677 carrying pGKT1) obtained by mating between NN12 (donor) and RN2677 (recipient); 4, RN2677 (plasmid-less strain). Plasmid sizes were determined by using reference plasmids with known molecular sizes (the plasmid sizes thus determined are indicated to the left of the gel).

The transconjugants (RN2677 carrying pGKT1) manifested resistance to gentamicin (MIC, 128 µg/ml), kanamycin (MIC, ≥256 µg/ml), and tetracycline (MIC, 8 µg/ml). MICs (in µg/ml) for RN2667 were 0.25, 2, and 0.25, respectively; and those for strain NN12 were 128, ≥256, and 8, respectively. The data clearly conclude that pGKT1 is a transmissible plasmid encoding resistance to gentamicin, kanamycin, and tetracycline. Since strain NN1 was isolated in 2003 and strain NN12 was isolated in 2004, it is therefore conceivable that strain NN12 emerged from strain NN1 by acquiring pGKT1.


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DISCUSSION
 
In this study, the authors isolated a PVL+ CA-MRSA strain. To the best of our knowledge, this PVL+ CA-MRSA strain is the first case of isolation and characterization in Japan. PVL is associated with cutaneous abscess and furunculosis (10, 17). Indeed, the PVL+ CA-MRSA strain (NN12) was isolated from a patient with an abscess. Isolation of MRSA from abscesses and furunculoses is very rare in Japan, and only one strain was available for this study in 2004. This patient was an 18-year-old female high school student and a basketball player. As athletic activity is a risk factor for CA-MRSA infection (5), this case is a typical example of CA-MRSA infection in Japan. No MRSA infection was, however, confirmed among her teammates, classmates, or members of her family.

This patient was an outpatient with an abscess in the bilateral gluteal regions, with subsequent recurrent lesions, and gentamicin ointment was applied. Most probably, during this period, a gentamicin-susceptible MRSA strain (strain NN1?) acquired pGKT1 to become multiple-drug resistant. The origin of strain NN1 is also not known, although it was determined that there were no other patients among the relatives or close contacts of "NN1 patient," and thus, PVL+ MRSA could not been screened. Because ST30 has also been isolated in Australia, the United States, and Europe (9, 11, 32, 38, 39), ST30 may be a potentially "pandemic ST," but no route to emergence in Japan is currently known.

PVL+ CA-MRSA harboring various STs has increasingly been noted (11, 38, 39). Notably, ST8 SCCmec IV CA-MRSA has been isolated with very high frequency in the United States and Europe (3, 6). Although SCCmec IV is associated with CA-MRSA (26, 38), SCCmec IV is also distributed among HA-MRSA strains. For instance, SCCmec IV HA-MRSA was found in this study and found among HA-MRSA isolates from European countries and the United States with high frequency (3, 6, 8, 33).

To provide a firm denominator for total PVL+ CA-MRSA in Japan, national MRSA surveillance or analysis of a greater number of MRSA strains not only from minors but also from other patient groups (focusing on strains from abscesses and furunculoses) are required.

In conclusion, CA-MRSA (mostly PVL–) was found in approximately 17 to 20% of S. aureus from bullous impetigo. Those PVL– CA-MRSA strains ("domestic" CA-MRSA) showed very extensive variation with new genotypes, such as combination of eta and etb, tst and etb, or new spa types. They were divided into three STs (89, 8, and 91), including a uniform spa type or complicated spa and SCCmec types. The etb+ ST91 distributed to both CA-MRSA and HA-MRSA strains. It is possible that the ST91 HA-MRSA strain originated from the CA-MRSA strain or that it is, in fact, a CA-MRSA. A potentially "pandemic" ST30 PVL+ CA-MRSA strain emerged in Japan, associated with bullous impetigo and cutaneous abscess, accompanied by a transmissible, multiple-drug resistance plasmid.


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ACKNOWLEDGMENTS
 
We thank Jerome Etienne for encouragement and for supplying ST30 CA-MRSA strains, Helene Meugnier for assistance with eBURST, Sophie Jarraud for supplying agr type strains, L. K. McDougal and L. L. McDonald for supplying ST30 CA-MRSA strains, Ken Kikuchi for supplying strains of NTED MRSA, and Keiichi Hiramatsu for supplying SCCmec type strains.

This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Bacteriology, Department of Infectious Disease Control and International Medicine, Niigata University Graduate School of Medical and Dental Sciences, 757 Ichibanchou, Asahimachidori, Niigata, Japan. Phone: 81 25 227 2050. Fax: 81 25 227 0762. E-mail: tatsuoy{at}med.niigata-u.ac.jp. Back


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Journal of Clinical Microbiology, July 2005, p. 3356-3363, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3356-3363.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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