Received 12 July 2006/Returned for modification 17 August 2006/Accepted 4 October 2006

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2006, p Vol. 50, No /06/$ doi: /aac Copyright 2006, American Society for Microbiology. All Rights Reserved. Molecular Characteristics and In Vitro Susceptibility to Antimicrobial Agents, Including the Des-Fluoro(6) Quinolone DX-619, of Panton-Valentine Leucocidin-Positive Methicillin-Resistant Staphylococcus aureus Isolates from the Community and Hospitals Tatsuo Yamamoto, 1 * Soshi Dohmae, 1 Kohei Saito, 1 Taketo Otsuka, 1 Tomomi Takano, 1 Megumi Chiba, 2 Katsuko Fujikawa, 2 and Mayumi Tanaka 2 Division of Bacteriology, Department of Infectious Disease Control and International Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, 1 and Daiichi Pharmaceutical Co., Ltd., Tokyo, 2 Japan Received 12 July 2006/Returned for modification 17 August 2006/Accepted 4 October 2006 Highly virulent, community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strains with Panton-Valentine leucocidin (PVL) have been found increasingly worldwide. Among a total of 2,101 MRSA strains isolated from patients in hospitals in Japan, two were positive for PVL. One strain was identified as a community-acquired MRSA strain with genotype sequence type 30 (ST30) and spa (staphylococcal protein A gene) type 19 from Japan and was resistant only to -lactam antimicrobial agents. The other strain was closely related to PVL multidrug-resistant, hospital-acquired MRSA strains (ST30, spa type 43) derived from nosocomial outbreaks in the 1980s to 1990s in Japan but with a divergent sequence type, ST765 (a single-locus variant of ST30). Twenty-two PVL MRSA strains, including those from Japan and those from other countries with various sequence types (ST1, ST8, ST30, ST59, and ST80) and genotypes, were examined for susceptibility to 31 antimicrobial agents. Among the agents, DX-619, a des-fluoro(6) quinolone, showed the greatest activity, followed by rifampin and sitafloxacin, a fluoroquinolone. The data suggest that DX-619 exhibits a superior activity against PVL MRSA strains with various virulence genetic traits from the community as well as from hospitals. Methicillin-resistant Staphylococcus aureus (MRSA), alternatively called hospital-acquired MRSA (HA-MRSA), has been a major cause of nosocomial infections since 1961 (5). Besides this, since 1997 to 1999 (6), infection due to new highly virulent MRSA, called community-acquired MRSA (CA- MRSA), has become a major concern worldwide (52, 56). CA-MRSA infection is associated with skin and soft tissue infection in young, otherwise healthy people (33, 52, 56) and with more severe diseases, such as osteomyelitis or necrotizing pneumonia (preceded by influenza or influenza-like prodromes) (11, 16, 56). CA-MRSA carries for Panton-Valentine leucocidin (PVL) (52, 56), which causes apoptosis and necrosis in human polymorphonuclear cells (19), and possesses limited types of methicillin resistance locus (staphylococcal cassette chromosome mec [SCCmec]), such as SCCmecIV or SCCmecV (10, 52, 56). There are geographically two types of CA-MRSA: one being rather continent specific (e.g., multilocus sequence type 1 [ST1] and ST8, found mostly in the United States, and ST80, found mostly in Europe [30, 52]) and the other worldwide * 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) Fax: (81) (25) Published ahead of print on 16 October (ST30, found in the United States, Europe, Oceania, Japan, and Brazil [12, 31, 41, 48, 52]). CA-MRSA is also isolated from hospitals (2, 37, 44). In Japan, MRSA strains at the time of nosocomial outbreaks ( hospital MRSA panics ) in the late 1980s and early 1990s were largely PVL (49). In this study, we surveyed and characterized PVL strains of MRSA isolated from patients in hospitals. We also examined the in vitro susceptibilities of PVL MRSA strains, including those isolated in this study and (with various sequence types) from other countries, to 31 antimicrobial agents, including DX-619, a des-fluoro(6) [des- F(6)] quinolone, and sitafloxacin, a fluoroquinolone. MATERIALS AND METHODS Bacterial strains. A total of 2,101 MRSA strains isolated from patients in hospitals in Japan from 1996 through 2002 by a levofloxacin surveillance group (led by Keizo Yamaguchi) were examined for PVL in this study. CA- MRSA ST30 strains used included SCCmecIVc strains NN1, NN12, NN31A, and NN31D isolated from Japan (48) and HT isolated from The Netherlands and SCCmecIVa strains USA1100 isolated from the United States (30) and HT isolated from Australia. PVL MRSA ST30 strains 80s-1 (SCCmecIVx; x, subtype unknown), 80s-2 (SCCmecIVc), 80s-3 (SCCmecIVa), 80s-4 (SCCmecIVa), 90s-1 (SCCmecIVx), and 90s-2 (SCCmecIVc) were isolated from hospital MRSA panics in the 1980s or 1990s in Japan (49). PVL-positive CA-MRSA strains with sequence types other than ST30 used included strains USA400 (MW2; ST1, SCCmecIVa), USA300 (ST8, SCCmecIVa), and USA1000 (ST59, SCCmecIV) from the United States; strains SSF17 (ST59, SCCmecIV) and Ce7 (ST59) from Taiwan; strain HT (ST80) from The Netherlands; and strain HT (ST80) from France. Strains HT , HT , HT , and HT were kindly provided by Jerome Etienne; strains 4077

2 4078 YAMAMOTO ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Characteristics of PVL MRSA strains isolated in this study compared with characteristics of nosocomial-outbreak-derived PVL MRSA ST30 strains and PVL CA-MRSA ST30 strains from Japan and CA-MRSA ST30 strains from other countries a Type, gene, or resistance Result for PVL HA-MRSA strains isolated in: Result for PVL 1980s 1990s CA-MRSA strains isolated in this study Result for PVL CA-MRSA strains isolated in 2000s 80s-1 80s-2 80s-3 80s-4 90s-1 90s-2 DB00319 EB00449 NN1 NN12 Result for PVL MRSA strains isolated from: United States (USA1100) The Netherlands (HT ) Australia (HT ) Types CC ST b spa agr SCCmec IVx IVc IVa IVa IVx IVc IVx IVc IVc IVc IVa IVc IVa Coagulase IV IV IV IV IV IV IV IV IV IV IV IV IV Presence/absence of toxin Leucocidin luk PV SF luke-lukd lukm Hemolysin hla hlb hlg hlg-v hld Staphylococcal enterotoxin tst sea seb sec sed see seg seh sei sej sek sem sen seo sep seq seu Exfoliative toxin eta etb etd

3 VOL. 50, 2006 CHARACTERISTICS AND SUSCEPTIBILITIES OF PVL MRSA 4079 Other set edin Adhesin functions/ targets and presence/ absence of Biofilm formation icaa icad Collagen cna Laminin eno Fibronectin fnba fnbb Elastin ebps Fibrinogen clfa clfb fib sdrc sdrd sdre Bone sialoprotein bbp Drug resistance Penicillins OXA, OXA, AMP c AMP Aminoglycosides GEN, KAN c, STR c GEN, KAN OXA, OXA, OXA, OXA, OXA, OXA, OXA, OXA, AMP c AMP c AMP c AMP c AMP c AMP c AMP c GEN, KAN c, STR c GEN, KAN c, STR c GEN, KAN c, STR c KAN c, GEN, STR c KAN c, STR c Macrolide ERY ERY c ERY c ERY c ERY c ERY c Lincosamide CLI c CLI c CLI c CLI c Tetracycline(s) TET, TET TET TET TET d MINO Phenicol CHL OXA, AMP c OXA, AMP c OXA, AMP c GEN d, KAN d Presence of PCase (37) (42) (44) (44) (29) (44) f (32) g (32) (32) (29) (29) (29) plasmid (kb) e Continued on following page

4 4080 YAMAMOTO ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 1 Continued Result for PVL MRSA strains isolated from: Result for PVL CA-MRSA strains isolated in 2000s Result for PVL HA-MRSA strains isolated in: Result for PVL CA-MRSA strains 1980s 1990s isolated in this study Type, gene, or resistance Australia (HT ) The Netherlands (HT ) United States (USA1100) 80s-1 80s-2 80s-3 80s-4 90s-1 90s-2 DB00319 EB00449 NN1 NN12 Presence/absence of drug-resistant on PCase plasmid h blaz aph(3 )-IIIa aade ermb a Symbols/abbreviations:, present;, absent; OXA, oxacillin; AMP, ampicillin; GEN, gentamicin; KAN, kanamycin; STR, streptomycin; ERY, erythromycin; CLI, clindamycin; TET, tetracycline; MINO, minocycline; CHL, chloramphenicol. b A single-locus variant (2,2,2,2,6,104,2) of ST30 (2,2,2,2,6,3,2), according to the MLST website; the mutation is T3C intpi. c Drug resistance encoded by on PCase plasmid. d Drug resistance encoded by a conjugative drug resistance plasmid (pgkt1) (48). e PCase plasmid was identified previously (49), except for DB00319 and EB It codes for ampicillin resistance (blaz) and cadmium resistance. PCase plasmid of PVL HA-MRSA strains isolated in the 1980s and 1990s conferred resistance even to additional drugs. f Multidrug-resistant PCase plasmid (pac00s7), coding for ampicillin resistance (blaz), cadmium resistance, and resistance to kanamycin, streptomycin, erythromycin, and clindamycin, was identified in this study. g PCase plasmid (pac00s8), coding for ampicillin resistance (blaz) and cadmium resistance, was identified in this study. h Genes blaz, aph(3 )-IIIa, aade, and ermb code for resistance to ampicillin, kanamycin, streptomycin, and erythromycin-clindamycin, respectively. US1100, USA400, USA300, and USA1000 by L. K. McDougal and L. L. Mc- Donald; and strains SSF17 and Ce7 by Ma Ling and Leung K. Siu as strains representative of different sequence types. Media and bacterial growth. For bacterial growth, we used LB broth (Difco, Sparks, MD), which was inoculated and incubated at 37 C to log phase with agitation, as the liquid medium. Nutrient agar (Eiken Chemical, Tokyo) was used as the solid medium. Molecular typing. Multilocus sequence typing (MLST) was performed using seven housekeeping, as previously described (13). An allelic profile (allele number) was obtained from the MLST website ( and the ST data were further analyzed using eburst software (14) to determine a clonal complex to which each ST belonged. spa (staphylococcal protein A gene) typing was performed as previously described (46). The spa type was determined using a public spa type database ( Detection of the accessory gene regulator (agr) allele group was done by PCR with the reported primers, as previously described (47). The SCCmec types (I to V) were analyzed by PCR as previously described (38) by use of reference strains. In the case of SCCmecIV, three subtypes (IVa, IVb, and IVc) were further analyzed by PCR with the reported primers, as previously described (24). Virulence gene analysis. Forty-one staphylococcal virulence were detected by PCR using the previously reported primers. The targeted were three leucocidin (26, 34), five hemolysin (26), 16 staphylococcal enterotoxin (3, 23, 25, 39, 57), one putative staphylococcal enterotoxin gene (28), three exfoliative toxin (3, 55), an exotoxin-like gene cluster (54), the epidermal cell differentiation inhibitor gene (26), and 14 adhesin (32, 40, 50, 51, 53). Drug resistance gene analysis. Resistance were detected by PCR. They included for penicillin resistance (29), aminoglycoside resistance (7, 8, 29), and macrolide and lincosamide resistance (29). Coagulase typing. MRSA and methicillin-susceptible S. aureus strains were examined for coagulase type by use of a staphylococcal coagulase antiserum kit (Denka Seiken, Tokyo, Japan) in accordance with the manufacturer s instructions. PFGE and computer analysis. For pulsed-field gel electrophoresis (PFGE) analysis, total bacterial DNA was extracted from MRSA or methicillin-susceptible S. aureus strains and digested with SmaI (21). The digested DNA was applied on PFGE (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 a clustering algorithm for the unweighted-pair group method using average linkages (36). Plasmid analysis. Plasmid DNA was isolated using a QIAGEN Plasmid Midi kit (QIAGEN, Hilden, Germany) and lysostaphin (Wako Pure Chemicals, Osaka, Japan) according to the instructions of the manufacturer. Plasmid DNA was then introduced into S. aureus RN2677 (49) by electroporation using a Gene Pulser II electroporator (Bio-Rad) according to the manufacturer s instructions. Briefly, RN2677 cells were grown at 37 C in brain heart infusion broth (Difco) to log phase (optical density at 600 nm of 0.3). Bacterial cells from 100 ml of culture were harvested, washed with 40 ml of cold sterilized water twice, and suspended in 10 ml of cold 10% (vol/vol) glycerol and finally in 0.5 ml of cold 10% (vol/vol) glycerol. Competent cells (50 l) thus made were mixed with 1 l of plasmid DNA, and the mixture was subjected to electroporation in a 0.2-cm electrode spacing cuvette (Bio-Rad) (setting, 100, 2.5 kv, 25 F). One milliliter of brain heart infusion broth was then added, and the mixture was incubated for 90 min at 37 C. Resistance plasmid-containing clones were selected on agar plates containing cadmium acetate at 10 g/ml, kanamycin at 10 g/ml, or erythromycin at 10 g/ml. Plasmid was isolated using a QIAGEN Plasmid Midi kit (QIAGEN) and lysostaphin (Wako Pure Chemicals) according to the instructions of the manufacturer and analyzed by agarose gel electrophoresis. Antimicrobial agents. DX-619, a novel des-f(6) quinolone (17), and sitafloxacin, a novel fluoroquinolone (1), were described previously. Other antimicrobial agents were gifts from their manufacturers. Susceptibility testing. Susceptibility testing of bacterial strains was done by agar dilution method with Mueller-Hinton agar (Difco) according to previously described procedures (9, 35). The final concentrations of antimicrobial agents ranged from to 128 g/ml. RESULTS Isolation of PVL MRSA. Among a total of 2,101 MRSA strains examined, two were PVL (0.1%). One strain (strain EB00449) was isolated from the pus of an outpatient (male) aged 27 years in The other strain (strain DB00319) was

5 VOL. 50, 2006 CHARACTERISTICS AND SUSCEPTIBILITIES OF PVL MRSA 4081 FIG. 1. Plasmid analysis of PVL MRSA strains DB00319 and EB00449 and S. aureus RN2677 carrying a PCase plasmid. Plasmid DNA from each bacterial strain was electrophoresed in 0.7% agarose. Lanes: 1, strain DB00319; 2, RN2677 carrying a PCase plasmid (pac00s7) from strain DB00319; 3, strain EB00449; 4, RN2677 carrying a PCase plasmid (pac00s8) from strain EB00449; 5, RN2677 (strain without plasmid). 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. isolated from centesis of an inpatient (male) aged 61 years in Characteristics of PVL MRSA. To investigate the evolutionary relationships and the characteristics of virulence and drug resistance, strains EB00449 and DB00319 were examined for genotypes, virulence, penicillinase (PCase) plasmids, and drug resistance (Table 1). Strain EB00449 belonged to ST30, while strain DB00319 possessed a novel sequence type (ST765, a single-locus variant of the founder [ST30] of clonal complex 30 [CC30]). The two strains were also distinguishable from each other in terms of spa type, i.e., spa type 19 for EB00449 and spa type 43 for DB In addition, EB00449 was negative for hlb (which codes for -hemolysin), sea (which codes for staphylococcal enterotoxin A), and icaa (which codes for biofilm formation), while DB00319 was positive for hlb, sea, and icaa. Moreover, EB00449 was resistant only to -lactam antimicrobial agents and carried a 32-kb simple PCase plasmid coding for resistance to ampicillin (and penicillin G) and cadmium (Table 1; Fig. 1); EB00449 carried an additional, 46-kb plasmid of unknown function (Fig. 1). In contrast, DB00319 was resistant to multiple drugs and carried a 44-kb multidrug-resistant PCase plasmid coding for resistance not only to ampicillin (and penicillin G) and cadmium but also to kanamycin, streptomycin, erythromycin, and clindamycin (Table 1; Fig. 1); DB00319 carried an additional, 5.4-kb plasmid of unknown function (Fig. 1). The data for strains EB00449 and DB00319 were compared with data for PVL MRSA strains (strains 80s-1 to -4 and 90s-1 and -2) isolated during the nosocomial outbreaks in the 1980s and 1990s, recent PVL CA-MRSA strains (strains NN1 and NN12) from Japan, and PVL CA-MRSA ST30 strains from other countries (Table 1). Strain EB00449 was very similar to PVL CA-MRSA strain NN1 from Japan; there was a divergence in that EB00449 carried an additional 46-kb plasmid compared with NN1, which carried only PCase plasmid. Moreover, DB00319 closely resembled nosocomial-outbreakderived PVL HA-MRSA strains, especially strain 90s-1, except for the sequence type. FIG. 2. PFGE analysis of PVL MRSA strains (DB00319 and EB00449) isolated in this study, nosocomial-outbreak-derived PVL MRSA ST30 strains (80s-1 to -4 and 90s-1 to -2) isolated in the 1980s and 1990s, PVL CA-MRSA ST30 strains (NN1 and NN12) from Japan, and PVL CA-MRSA ST30 strains (USA1100, HT , and HT ) from other countries (right side) and dendrogram constructed by computerassisted comparison (left side). The characteristics of each bacterial strain are listed in Table 1.

6 4082 YAMAMOTO ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Characteristics of global PVL MRSA strains not of ST30 Result for PVL CA-MRSA strains isolated from a : Type, gene, or resistance United States Taiwan The Netherlands France USA400 USA300 USA1000 SSF17 Ce7 HT HT Types CC ST spa ND ND 70 agr ND 3 SCCmec IVa IVa IVx IVx ND IVc IVc Coagulase VII III ND VII VII VIII I Presence/absence of toxin Leucocidin luk PV SF luke-lukd lukm Hemolysin hla hlb hlg hlg-v hld Staphylococcal enterotoxin tst sea seb sec sed see seg seh sei sej sek sem sen seo sep seq seu Exfoliative toxin eta etb etd Other set edin Adhesin functions/ targets and presence/ absence of Biofilm formation icaa icad Continued on facing page

7 VOL. 50, 2006 CHARACTERISTICS AND SUSCEPTIBILITIES OF PVL MRSA 4083 TABLE 2 Continued Result for PVL CA-MRSA strains isolated from a : Type, gene, or resistance United States Taiwan The Netherlands France USA400 USA300 USA1000 SSF17 Ce7 HT HT Collagen cna Laminin eno Fibronectin fnba fnbb Elastin ebps Fibrinogen clfa clfb fib sdrc sdrd sdre Bone sialoprotein bbp Drug resistance Penicillin(s) OXA, AMP OXA, AMP OXA, AMP AMP OXA, AMP OXA, AMP OXA, AMP Aminoglycoside(s) KAN KAN, STR KAN, STR KAN, STR KAN, STR Macrolide ERY ERY ERY ERY Lincosamide CLI CLI Tetracycline TET TET TET TET TET TET Others CHL CHL FUS FUS a Symbols/abbreviations:, presence;, absence; ND, not determined; OXA, oxacillin; AMP, ampicillin; KAN, kanamycin; STR, streptomycin; ERY, erythromycin; CLI, clindamycin; TET, tetracycline; CHL, chloramphenicol; FUS, fusidic acid. Cluster analysis of PFGE patterns. A computer-assisted comparison of PFGE patterns obtained with PVL HA- MRSA strains (80s-1 to -4 and 90s-1 and -2) isolated during the nosocomial outbreaks in the 1980s and 1990s, strains DB00319 and EB00449, recent PVL CA-MRSA strains (NN1 and NN12) from Japan, and PVL CA-MRSA ST30 strains from other countries is shown in Fig. 2. Consistent with the spa typing, PVL HA-MRSA and DB00319 (spa type 43) strains constituted subclusters, which were distinguished from subclusters of PVL CA-MRSA and EB00449 (spa type 19 or 654) strains. Characteristics of PVL MRSA strains not of ST30. Characteristics of global PVL MRSA strains belonging to U.S. sequence types (ST1 and ST8), a Taiwanese (or U.S.) sequence type (ST59), and a European sequence type (ST80) are summarized in Table 2. A remarkable difference in adhesive properties () was observed between strains of ST30 and strains not of ST30. In contrast to ST30 strains, strains not of ST30 were negative for bbp (which codes for bone sialoprotein adhesin). Moreover, many strains not of ST30 (except for the U.S. ST1 strain) were negative for cna (which codes for collagen adhesin). Regarding toxin, the U.S. strains (ST1 and ST8) were positive for sea, just like nosocomial-outbreak-derived PVL HA-MRSA strains from Japan. U.S. ST1 and European ST80 strains, but not ST8, ST30, or ST59 strains, were positive for seh. In vitro susceptibility. Next, PVL MRSA strains (22 strains) were examined for susceptibility to 31 antimicrobial agents. They included the two strains DB00319 (ST765) and EB00449 (ST30), six PVL HA-MRSA (ST30) strains isolated from nosocomial outbreaks in the 1980s and 1990s, four PVL CA-MRSA (ST30) strains from Japan, and 10 PVL CA-MRSA (ST1, ST8, ST30, ST59, and ST80) strains from other countries. The data are summarized in Table 3. DX-619 showed the greatest activity among the antimicrobial agents tested (MIC 50, g/ml; MIC 90, g/ml). This activity was followed by those of rifampin (MIC 50, g/ml; MIC 90, g/ml) and sitafloxacin or tosufloxacin (MIC 50, g/ml; MIC 90, g/ml). Sitafloxacin showed a better MIC range than tosufloxacin (0.016 to g/ml versus to 0.25 g/ml, respectively). Gentamicin resistance (MICs, 8 to 256 g/ml) was found only in Japanese PVL MRSA ST30 strains (in two of four PVL CA-MRSA strains, in five of six nosocomial-outbreakderived PVL HA-MRSA strains, and in one [strain DB00319]

8 4084 YAMAMOTO ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. In vitro activities of DX-619 and other antimicrobial agents against PVL MRSA strains a Antimicrobial agent MIC ( g/ml) 50% 90% Range % Susceptible b Penicillins Penicillin G Ampicillin Oxacillin Cephems Cefazolin Cefaclor Cefotiam Ceftazidime Cefpirome Glycopetides Vancomycin Teicoplanin Oxazolidinone Linezolid Aminoglycosides Gentamicin Kanamycin Streptomycin Arbekacin Tetracyclines Tetracycline Doxycycline Minocycline Macrolides Erythromycin Clarithromycin Azithromycin Lincosamide Clindamycin Quinolones Nalidixic acid Norfloxacin Ciprofloxacin Ofloxacin Levofloxacin Tosufloxacin Sitafloxacin DX Phenicol Chloramphenicol Others Fosfomycin Rifampin Fusidic acid a The number of strains tested was 22. They included the 13 strains with characteristics outlined in Table 1 and the 7 strains with characteristics outlined in Table 2. The remaining two strains (NN31A and NN31D) were recent isolates from Niigata, Japan, which resembled PVL CA-MRSA strain NN12 (data not shown). b Based on CLSI breakpoints. of two PVL MRSA strains isolated in this study) (Tables 1, Table 2, and Table 3). Minocycline resistance (MIC, 8 g/ml) was also found only in Japanese PVL MRSA ST30 strains (nosocomial-outbreak-derived PVL HA-MRSA strain 80s-1) (Table 1,Table 2 and Table 3). Fusidic acid resistance (MIC, 8 g/ml) was found only in European PVL CA-MRSA ST80 strains (Table 1, Table 2, and Table 3). Resistance to some other drugs was distributed among different sequence types: kanamycin resistance (MICs, 256 g/ ml) among ST8, ST30, ST59, and ST80; streptomycin resistance (MICs, 128 to 256 g/ml) among ST30, ST59, and ST80; erythromycin resistance (MICs, 256 g/ml) among ST8, ST30, and ST59; clindamycin resistance (MICs, 256 g/ml) among ST30 and ST59; and tetracycline resistance (MICs, 16 g/ml) among ST1, ST8, ST30, ST59, and ST80. DISCUSSION PVL CA-MRSA strains in Japan are of a worldwide sequence type (ST30) (48). No cases of ST1 and ST8, found mainly in the United States (30, 52), ST80, found mainly in Europe (52), or ST59, found mainly in Taiwan and the United States (4, 12), have been found in Japan. There were nosocomial MRSA outbreaks ( hospital MRSA panics ) in Japan in the late 1980s and early 1990s. At that time, decubitus, pneumonia, bacteremia, and postoperative MRSA enteritis were found frequently (18, 27, 45), but they have drastically decreased recently. Interestingly, MRSA strains isolated during the nosocomial outbreaks were largely PVL (49). Although these PVL HA-MRSA strains belonged to ST30, similarly to present PVL CA-MRSA strains, they had unique features (49), e.g., they exhibited spa type 43, possessed hlb, sea (which is associated with severity of infection [sepsis and shock] [15]), and icaa, were multidrug resistant, and carried a multidrug-resistant PCase plasmid, in contrast to present PVL CA-MRSA ST30 strains (spa type 19). The possibility of high virulence of hlb sea multidrug-resistant PVL HA-MRSA strains remains to be clarified. In this study, we further surveyed PVL MRSA strains from patients in hospitals, and two PVL MRSA strains were studied. One PVL MRSA strain, strain DB00319, from an inpatient aged 61 years, shared common features (CC30, spa type 43, hlb, sea, icaa, and multidrug-resistant PCase plasmid) with multidrug-resistant PVL HA-MRSA strains from the hospital MRSA panics but with a genetic divergence in sequence type (novel ST765). ST765 is a single-locus variant (single base substitution in tpi) of the founder type, ST30, of CC30. This suggests that a hospital MRSA panic clone continues to exist in hospitals in Japan, with a slight divergence. The other PVL MRSA strain, strain EB00449, from an outpatient aged 27 years, was a PVL CA-MRSA ST30 strain. EB00449 was, however, slightly divergent in terms of plasmid profile from previously characterized PVL CA-MRSA ST30 strains, i.e., strain NN1, isolated from an infant (aged 11 months) with bullous impetigo (48), and strain NN12, carrying a conjugative drug resistance plasmid, isolated from a high school student (a basketball player aged 18 years) with bacteremia and osteomyelitis (40, 48). Thus far, PVL CA-MRSA ST30 strains found in Japan have been distributed among individuals below 30 years of age. Both types of PVL MRSA strains of CC30 present in Japan, PVL hlb sea HA-MRSA ST30/765 spa type 43 strains (continuing from the 1980s) and PVL CA-MRSA spa type 19 strains (emerged in the community recently), are highly adhesive (bbp and cna ). bbp codes for bone sialoprotein

9 VOL. 50, 2006 CHARACTERISTICS AND SUSCEPTIBILITIES OF PVL MRSA 4085 adhesin, which leads to hematogenously spread osteomyelitis and arthritis (20, 42, 43). cna codes for collagen adhesin, which is correlated with pulmonary manifestations (22). Such highly adhesive characteristics may be a cause of continuous infection in hospitals and in the community in Japan. Twenty-two PVL MRSA strains, including PVL HA- MRSA ST30/765 strains and recent PVL CA-MRSA ST30 strains from Japan and PVL CA-MRSA strains with various global sequence types (ST1, ST8, ST30, ST59, and ST80) from other countries, were examined for their susceptibilities to 31 antimicrobial agents. Some drug resistance is associated with sequence type, e.g., fusidic acid resistance with ST80 (52). In Japan, gentamicin has been widely used not only in hospitals but also in the community as an ointment for skin infection, and due to this, gentamicin-resistant PVL MRSA ST30/765 strains are notable. Antimicrobial agents tested in this study included DX-619, a des-f(6) quinolone, which has shown an in vitro activity against gram-positive bacteria (17), and sitafloxacin, a fluoroquinolone (1). DX-619 showed the greatest activity against PVL MRSA among the antimicrobial agents tested (MIC 90, g/ml; MIC 50, g/ml). This activity was eightfold greater than that of sitafloxacin (MIC 90, g/ml), 32-fold greater than that of levofloxacin (MIC 90, 0.25 g/ml), and 125-fold greater than that of vancomycin (MIC 90,1 g/ml). It was even twofold greater than that of rifampin (MIC 90, g/ml). Among fluoroquinolones tested, sitafloxacin showed the greatest activity, e.g., it showed a better MIC range than tosufloxacin (0.016 to g/ml versus to 0.25 g/ml, respectively). The effects of DX-619 (or sitafloxacin) on severe diseases due to PVL CA-MRSA strains (such as bacteremia, osteomyelitis, or necrotizing pneumonia) remain to be investigated. In conclusion, there exist two types of highly adhesive (bbp cna ) PVL MRSA strains in Japan: hlb sea, multidrugresistant HA-MRSA ST30/765 strains (spa type 43) (originating from hospital MRSA panics ) and CA-MRSA ST30 strains (spa type 19) carrying or not carrying the conjugative drug resistance plasmid (emerged recently in the community). DX-619 showed higher activity than other anti-mrsa agents against those PVL MRSA strains as well as global PVL CA-MRSA strains with ST1, ST8, ST30, ST59, and ST80. Further studies are necessary to evaluate the anti-pvl MRSA activity of DX-619 (or sitafloxacin) in vivo, such as in animal infection models. ACKNOWLEDGMENTS We thank Jerome Etienne, L. K. McDougal, L. L. McDonald, Ma Ling, and Leung K. Siu for PVL MRSA strains. This study was supported by a grant from Daiichi Pharmaceutical Co., Ltd., Tokyo, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. REFERENCES 1. 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