Microbiology, Faculty of Medicine, Khon Kaen University, Thailand;

Transcription

1 JCM Accepts, published online ahead of print on 29 April 2009 J. Clin. Microbiol. doi: /jcm Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 The First Vancomycin-intermediate Staphylococcus aureus (VISA) in Thailand Aroonlug Lulitanond 1, Chulapan Engchanil 2, Prajuab Chaimanee 3, Malai Vorachit 4, Teruyo Ito 5,6 and Keiichi Hiramatsu 5,6* 1 Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Thailand; 2 Research and Diagnostic Center of Emerging Infectious Disease, Department of Clinical Microbiology, Faculty of Medicine, Khon Kaen University, Thailand; 3 Clinical Microbiology Unit, Srinagarind Hospital, Khon Kaen University, Thailand; 4 Faculty of Medical Technology, Mahidol University, Thailand; 5 Department of Infection Control Science, Graduate School of Medicine, Juntendo University, Tokyo, Japan; 6 Department of Bacteriology, Graduate School of Medicine, Juntendo University, Tokyo, Japan * Corresponding author. Mailing address: Department of Bacteriology, Graduate School of Medicine, Juntendo University, Hongo Bunkyo-ku, Tokyo, Japan, Phone: 81-(0) Fax: 81-(0) khiram06@juntendo.ac.jp

2 Abstract We screened 533 and 361 MRSA strains isolated in a university hospital in and , respectively, and identified 4 (0.8%) and 8 (2.2%) hetero-visa strains in the first and second group, respectively, and 3 (0.8%) VISA strains in the second group. This is the first report of VISA strains from Thailand. Methicillin-resistant Staphylococcus aureus (MRSA) was first reported nearly fifty years ago (20) and it is now an increasingly common pathogen associated with nosocomial and community-acquired infections (1, 2, 29), such as surgical-site infection, bloodstream infection and pneumonia. Vancomycin which has been available for more than 50 years was not initially widely used because of oto- and nephrotoxicity caused by impurities in the earlier preparations (11). Since the late 1970s, new methods of purification have resulted in an improved vancomycin with less oto- or nephro-toxicity, in animal models (3, 7); consequently, with the worldwide emergence of methicillin-resistant Staphylococcus in the 1970s, interest in vancomycin has been rekindled. Since the 1980s, vancomycin has been used widely in many countries for the treatment of serious infection caused by Gram-positive bacteria. Its frequent use for MRSA infection gradually selected MRSA strains with reduced susceptibility to glycopeptides antibiotics, which led to the discovery in Japan in 1997 of MRSA with intermediate resistance to vancomycin (VISA) (13). Although the resistant strains are designated as intermediate, they are practically resistant to the concentrations of vancomycin used in clinical chemotherapy (4, 9, 28). Most recently, several highly vancomycin-resistant strains of S. aureus carrying the enterococcal 2

3 43 44 vana transposon were recovered from patients co-infected with MRSA and vancomycin-resistant enterococci (VRE) (21, 26, 27) VISA is not easily generated from vancomycin-susceptible MRSA (VSSA) but it is frequently generated from certain MRSA strains which are heterogeneously resistant to vancomycin (hetero-visa) (10). The vancomycin MIC of hetero-visa is within the susceptible range (<4 g/ml), but these strains contain subpopulations that exhibit reduced susceptibility to vancomycin (15). These organisms are clinically important, because they persist latently when they are disseminated in the hospital environment, and are able to generate VISA during vancomycin therapy in patients infected with hetero-visa (23, 34). In Thailand, MRSA is an important pathogen causing serious nosocomial infections. A study in 2001 reported 3 hetero-visa strains among 155 MRSA isolates from various hospitals in Thailand (32). Later, a preliminary report announced identification of 7 hetero-visa strains in the 246 MRSA strains isolated from patients at a university hospital (24). We therefore considered it important to continue surveillance for vancomycin susceptibility of MRSA clinical strains in hospitals where MRSA is isolated at high frequency and where relatively generous use of vancomycin is inevitable. This communication thus reports the prevalence of MRSA isolates with reduced susceptibility to vancomycin among patients of a university hospital in Northeast Thailand, screened in two time periods. We present the first evidence of the isolation of VISA in Thailand A total of 894 MRSA isolates were recovered and studied from patients at the 842-bed Srinagarind (university) Hospital in Khon Kaen, Northeast Thailand. Five hundred and thirty- three isolates were collected between August 2002 and April 2003, and 361 isolates between 3

4 November 2006 and December All of the isolates were identified by conventional coagulase tube test and DNase test, then were stored in skimmed milk supplemented with 30% glycerol at -70 C. The references strains used for the vancomycin-susceptibility tests were: the hetero-visa strain Mu3 (ATCC ) (13), the VISA strain Mu50 (ATCC ) (14) and the vancomycin-susceptible S. aureus (ATCC 29213). All of the isolates were screened for reduced susceptibility to vancomycin by one-point population analysis (13). The overnight culture in brain heart infusion (BHI) broth was adjusted to an optical density of 578 nm at 0.3 (about 10 8 CFU/mL). One hundred microliters of the cell suspension was evenly spread on a BHI agar plate containing 4 µg/ml of vancomycin. The plates were then incubated at 37 C for 48 h, and cell growth was inspected at 24 and 48 h. Strains were suspected of VISA if more than 1000 colonies grew within 48 h, and hetero- VISA if the grown colonies were less than All of the isolates that grew on the screening agar plate were subjected to further confirmation tests by population analysis (PAP). Briefly, colonies from culture grown overnight on BHI agar were inoculated into BHI broth. After 24 h incubation, the culture was adjusted to an optical density of 578 nm at 0.3 then a ten-fold serial dilution was prepared in Normal saline solution from 10-1 to One hundred microliters aliquots of each dilution were spread evenly on each of the BHI agar plates containing 0, 1, 2, 3, 4, 5, 6, 7, 8 µg/ml of vancomycin (0, 0.5, 1, 2, 4, 8,16, 32, 64, 128 µg/ml in the case of teicoplanin). The plates were then incubated at 37 C for 48 h before the colonies were counted, and the log of CFU/mL plotted against the antibiotic concentration For each screening-positive isolate, MICs were determined by the agar dilution method, according to the CLSI guidelines (5) for 7 antimicrobial agents (Sigma Chemical, St. Louis, 4

5 USA, except the teicoplanin was purchased from Sanofi-Aventis, Tokyo, Japan): vancomycin ( µg/ml), cefazolin ( µg/ml), oxacillin ( µg/ml), ofloxacin ( µg/ml), tetracycline ( µg/ml), erythromycin ( µg/ml), gentamicin ( µg/ml) and teicoplanin ( µg/ml) Representative isolates from each group of the strains with different antibiotic susceptibility patterns were determined for their SCCmec types by multiplex PCR. Chromosomal DNA was extracted using the Isoplant II Kit (Wako Chemical, Tokyo, Japan) as per the manufacturer s instructions. Multiplex PCR for meca, ccr gene complex type, mec gene complex class were performed using primer sets and conditions (22). A 286 bp fragment of the meca gene was amplified using primers ma1 and ma2. Simultaneously, the ccr gene complex type was identified using primers α1, α2 and α3 with primer βc to amplify the respective 695, 937 or 1791 bp fragment of ccra1, ccra2 or ccra3. Either the 1287 bp fragment of ccr4 or the 518 bp fragment of ccrc was amplified using primers α 4.2 with β 4.2 and primers γ R with γ F, respectively. The mec gene complex class of the isolates were identified by using the primers mi6, IS7, IS2 with ma7 to amplify the 1963, 2827 or 804 bp fragment of mec class A, B or C, respectively. Multilocus sequence typing (MLST) of the representative isolates was performed (6). The alleles of 7 loci were determined by comparing the sequences to the corresponding loci in the S. aureus MLST database ( Sequence types were determined according to the combined pattern of the seven alleles, and clonal complexes by the BURST (based upon related sequence types) program, available on the MLST website. Agr typing was performed (30). PFGE of SmaI-digested chromosomal DNA was performed with a CHEF Mapper system 5

6 according to the manufacturer s instructions (Bio-Rad, USA.) and a 48.5 Kb ladder (Bio-Rad) was used as the DNA size marker. After running for 22 h. the gel was then stained with ethidium bromide and photo recorded. The band patterns were compared visually and were classified as indistinguishable (clonal), closely related (clonal variants, a three-band difference or less), possibly related (a 4 6 band difference) and unrelated, according to previously described criteria (31). Among the 533 MRSA strains isolated between August 2002 and April 2003, 19 strains (3.6%) generated colonies on the BHI agar plates with 4 µg/ml of vancomycin, whereas 26 (7.2%) from the 361 strains isolated between November 2006 and December 2007, formed colonies on the screening agar plate. These strains were isolated from various specimens from the patients ranging from 1 to 93 years of age (Table 1). All of the 45 screening-positive strains were further tested by a population analysis profile. Of the 19 screening positive isolates in the 1 st group, four hetero-visa strains were identified by the confirmatory PAP tests. In comparison, eight hetero-visa and three VISA strains were confirmed of the 26 strains in the 2 nd group. The population analysis profiles of the representative strains against vancomycin and teicoplanin are shown in Figures 1 and 2. Statistic analysis, using the Fisher s exact test, showed a significant increase in screen-positive strains between the two sampling periods (p=0.02, risk-ratio CI [0.25, 0.94]) and a marginally significant increase for the confirmed hetero-visa (p=0.08, risk-ratio CI [0.07, 1.29]) Regarding the 19 screening-positive strains from the 1 st group, all were susceptible to vancomycin (MIC µg/ml) but resistant to cefazolin, oxacillin, ofloxacin, tetracycline, erythromycin and gentamicin. The exceptions were: one strain susceptible to cefazolin, 2 strains 6

7 susceptible to ofloxacin, and 4 strains susceptible to erythromycin. Of the 26 screening-positive isolates from the 2 nd group, all were resistant to cefazolin, oxacillin, ofloxacin, tetracycline, erythromycin and gentamicin. The exceptions were 4 strains that were susceptible to gentamicin. The vancomycin MIC of this group of strains was between 2-3 µg/ml. The MIC range of the screening-positive strains are shown in Table 1. The vancomycin MICs for the 12 PAPconfirmed hetero-visa strains were 2 µg/ml, and 3 µg/ml (equivalent to 4 µg/ml using regular two-fold dilution system for MIC determination) for the 3 VISA strains. The teicoplanin MICs of the hetero-visa strains were between 2-8 µg/ml whereas that of the VISA strains were between 8-16 µg/ml (Table 2). By using the paired-wilcoxon rank test, a significant increase in teicoplanin MICs was confirmed between hetero-visa and VISA strains. It was found that the genotype of the 6 hetero-visa strains (arbitrarily chosen from 12 strains) were all ST239-SCCmec type III. Two of the three VISA strains, strain JCSC7193 and JCSC7203, had the genotype ST239-SCCmec type III, whereas the third one, JCSC7195, possessed ST5-SCCmec type II, which was the same with Mu3 and Mu50. The agr type of strains JCSC7193 and JCSC7203 were type I whereas that of JCSC7195 was type II (Table II). PFGE analysis of these isolates revealed that 1 of the 12 hetero-visa (strain JCSC6905) was identical to a hospital-associated MRSA (HA-MRSA) from the same hospital. The remaining 11 hetero-visa and 2 VISA strains, JCSC7193 and JCSC7203, were closely related to the HA- MRSA strain. In contrast, the VISA strain JCSC7195 had a similar banding pattern to that of Mu3 and Mu50 (Figure 3) We found 3 VISA strains in 3 patients in different wards during the second period of the surveillance. The first strain (JCSC 7193) was isolated from a 12-year-old boy in October of 7

8 with underlying acute leukemia, who had a fractured leg and ruptured spleen after a motorcycle accident. After having been hospitalized and undergone open reduction and internal fixation (ORIF) in a provincial hospital, he developed chronic osteomyelitis. He was referred to our tertiary hospital in September with septic shock caused by extended spectrum ß-lactamaseproducing Escherichia coli and chronic osteomyelitis from MRSA. Between October 2007 and March 2008, he was treated periodically with vancomycin combined with either aminoglycoside or fluoroquinolone or carbapenem. The second strain (JCSC 7195) was found in a 56-year-old man with underlying triple vessel anomaly and chronic renal failure. After undergoing a coronary artery bypass graft, he underwent continuous ambulatory peritoneal dialysis and became infected with MRSA. Vancomycin was used to treat him. The third strain (JCSC 7203) was found in a 9-year-old girl with underlying osteofibroma displasia and persistent abscesses due to reconstructive surgery. The patient achieved good recovery after receiving cefazolin for 5 days. Our screening test detected 3.6% of MRSA isolates in the years 2002 and 2003 and 7.2% of MRSA in the years 2006 and 2007 were suspected to have reduced vancomycin susceptibility. After confirmation by PAP, the prevalence of hetero-visa was 0.8% in the first period versus 2.2% in the second. In addition, 0.8% of the strains from the second period exhibited VISA phenotype, implying that the reduction of vancomycin susceptibility was gradually increasing in our hospital over the previous half decade. These strains with reduced vancomycin susceptibility were associated with infection as well as colonization in the patients. Two of the three VISA strains caused serious infection refractory to vancomycin therapy, and prolonged hospitalization in two patients. The two VISA strains were genetically different, suggesting that reduced vancomycin susceptibility in our hospital occurred among different clones of MRSA. This 8

9 finding coincides with a previous report of reduced susceptibility to vancomycin emerging in various lineages of MRSA (17) The current prevalence of hetero-visa and VISA strains in our hospital was 2.2% and 0.8%, respectively, which is comparable to several previous reports, namely, 2.7% in Hong Kong (35, 36), 2.6% in Ireland (8) and 1.1% in Italy (25), but lower than those of university hospitals in Japan in 1996 (9.3-20%) (13). For patients who were colonized by a hetero-visa strain, subsequent infection by this organism may occur. It is reported that patients with MRSA colonization have 35% risk of invasive disease within a year (19). Moreover, the family members of the patients carrying MRSA may potentially be at risk of becoming colonized as well (12, 16). Though these strains were not characterized for the resistance phenotype by laboratory testing, they may nevertheless cause infection with increased risk of clinical failure (4). The strains with reduced susceptibility to vancomycin could not be recognized by a routine susceptibility test. Various screening methods for strains with reduced vancomycin susceptibility have been reported (33, 34); however, confirmation of the isolates screening positive by PAP is recommended (8, 18). This study revealed a slight increase in strains with reduced vancomycin susceptibility at a university hospital in Northeast Thailand. This is the first report of VISA strains in Thailand. Appropriate use of antimicrobial agents and effective control of transmission will help retard the emergence of these organisms. 9

10 Acknowledgements This work was supported by a Grant-in-Aid for 21st Century COE Research and Japan Society for the Promotion of Science. We thank the CMDL, Faculty of Associated Medical Sciences, Khon Kaen University, for support, Natthanan Hongsrichan, Boualay Norchaleun and the staff of the Clinical Microbiology Laboratory at Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, for collecting the clinical isolates and Mr. Bryan Roderick Hamman for assistance with the English-language presentation of the manuscript. References 1. Appelbaum, P.C MRSA the tip of the iceberg. Clin. Micribiol. Infect. 12 (Suppl 2): Beam, J.W., and B. Buckley Community-acquired methicillin-resistant Staphylococcus aureus : Prevalence and risk factors. J. Athl. Train. 41: Brummett, R.E Effects of antibiotic-diuretic interactions in the guinea pig model of ototoxicity. Rev. Infect. Dis. 3(Suppl):S Charles, P.G.P., P.B. Ward, D.P.R. Johnson, B.P. Howden, and M.L. Grayson Clinical features associated with bacteremia due to heterogeneous vancomycin-intermediate Staphylococcus aureus. Clin. Infect. Dis. 38: Clinical and Laboratory Standards Institute Performance standards for antimicrobial susceptibility testing, 16 th informational supplement M100-S16. CLSI, Wayne, PA, USA. 6. Enright, M.C., N.P. Day, C.E. Davies, S.J. Peacock, and B.G. Spratt Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible 10

11 clones of Staphylococcus aureus. J. Clin. Microbiol. 38: Farber, B.F., and R.C. Jr. Moellering Retrospective study of the toxicity of preparations of vancomycin from 1974 to Antimicrob. Agents Chemother. 23: Fitzgibbon, M.M., A.S. Rossney, and B. O'Connell Investigation of reduced susceptibility to glycopeptides among methicillin-resistant Staphylococcus aureus isolates from patients in Ireland and evaluation of agar screening methods for detection of heterogeneously glycopeptide-intermediate S. aureus. J. Clin. Microbiol. 45: Fridkin, S.K., J. Hageman, L.K. McDougal, J. Mohammed, W.R. Jarwis, T.M. Perl, and F.C. Tenover Epidemiological and microbiological characterization of infections caused by Staphylococcus aureus with reduced susceptibility to vancomycin, United States, Clin. Infect. Dis. 36: Gemmel, C.G Glycopeptide resistance in Staphylococcus aureus: is it a real threat? J. Infect. Chemother. 10: Griffith, R.S Introduction to vancomycin. Rev. Infect. Dis. 3(Suppl):S200-S Hicks, N.R., E.P. Moore, and E.W. Williams Carriage and community treatment of methicillin-resistant Staphylococcus aureus: what happens to colonized patients after discharge? J. Hosp. Infect. 19: Hiramatsu, K., N. Aritaka, H. Hanaki, S. Kawasaki, Y. Hosoda, S. Hori, Y. Fukuchi, and I. Kobayashi Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet. 350: Hiramatsu, K., H. Hanaki, T. Ino, K. Yabuta, T. Oguri, and F.C. Tenover Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J. Antimicrob. Chemother. 40:

12 Hiramatsu, K Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance. Lancet. Infect. Dis. 1: Hollis, R.J., J.L. Barr, B.N. Doebbeling, M.A. Pfaller, and R.P. Wenzel Familial carriage of methicillin-resistant Staphylococcus aureus and subsequent infection in a premature neonate. Clin. Infect. Dis. 21: Howe, R.A., A. Monk, M. Wootton, T.R Walsh, and M.C. Enright Vancomycin susceptibility within methicillin-resistant Staphylococcus aureus lineages. Emerg. Infect. Dis. 10: Howe, R.A., M. Wootton, T.R. Walsh, P.M. Bennett, and A.P. Macgowan Heterogeneous resistance to vancomycin in Staphylococcus aureus. J. Antimicrob. Chemother. 45: Huang, S.S., and R. Platt Risk of methicillin-resistant Staphylococcus aureus infection after previous infection or colonization. Clin. Infect. Dis. 36: Jevons, M.P 'Celbenin'-resistant staphylococci. Br. Med. J.1: Kaccia, M., and L.C. McDonald Vancomycin-resistant Staphylococcus aureus_new York. Morb. Mortal. Wkly. Rep. 53: Kondo, Y., T. Ito, X.X. Ma, S. Watanabe, B.N. Kreiswirth, J. Etienne, and K. Hiramatsu Combination of Multiplex PCRs for Staphylococcal Cassette Chromosome mec Type Assignment: Rapid Identification System for mec, ccr, and Major Differences in Junkyard Regions. Antimicrob. Agents Chemother. 51: Liu, C., and H.F. Chambers Staphylococcus aureus with heterogeneous resistance to vancomycin: epidemiology, clinical significance, and critical assessment of diagnostic methods. Antimicrob. Agents Chemother. 47:

13 Lulitanond, A., A. Chanawong, P. Sribenjalux, W. Kaewkes, N. Charoensri, D. Leumsai, and P. Monpou Occurrence of Methicillin-Resistant Staphylococcus aureus with reduced susceptibility to vancomycin in Srinagarind Hospital. J. Infect. Dis. Antimicrob. Agents. 22: Marchese, A., G. Balistreri, E. Tonoli, E.A. Debbia, and G.C. Schito Heterogeneous vancomycin resistance in methicillin-resistant Staphylococcus aureus strains isolated in a large Italian hospital. J. Clin. Microbiol. 38: Miller, D., V. Urdaneta, and A. Weltman Vancomycin-resistant Staphylococcus aureus_pennsylvania. Morb. Mortal. Wkly. Rep. 51: Saha, B., A.K. Singh, A. Ghosh, and M. Bal Identification and characterization of a vancomycin-resistant Staphylococcus aureus isolated from Kolkata (South Asia). J. Med. Microbiol. 57: Schwaber, M.J., S.B. Wright, Y. Carmeli, L. Venkataraman, P.C. DeGirolami, A. Gramatikova, T.M. Perl, G. Sakoulas, and H.S. Gold Clinical implications of varying degrees of vancomycin susceptibility in methicillin-resistant Staphylococcus aureus bacteremia. Emerg. Infect. Dis. 9: Shaw, B.E., T. Boswell, J.L. Byrne, C. Yates, and N.H. Russell Clinical impact of MRSA in a stem cell transplant unit: analysis before, during and after an MRSA outbreak. Bone Marrow Transplant. 39: Shopsin, B., B. Mathema, P. Alcabes, B. Said-Salim, G. Lina, A. Matsuka, J. Martinez, and B.N. Kreiswirth Prevalence of agr specificity groups among Staphylococcus aureus strains colonizing children and their guardians. J Clin Microbiol 41(1): Tenover, F.C., R.D. Arbeit, R.V. Goering, P.A. Mickelsen, B.E. Murry, D.H. Persing, 13

14 and B. Swaminathan Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33: Trakulsomboon, S., S. Danchaivijitr, Y. Rongrungruang, C. Dhiraputra, W. Susaemgrat, T. Ito, and K. Hiramatsu First report of methicillin-resistant Staphylococcus aureus with reduced susceptibility to vancomycin in Thailand. J. Clin. Microbiol. 39: Voss, A., J.W. Mouton, E.P. van Elzakker, R.G. Hendrix, W. Goessens, J.A. Kluytmans, P.F. Krabbe, H.J. de Neeling, J.H. Sloos, N. Oztoprak, R.A. Howe, and T.R. Walsh A multi-center blinded study on the efficiency of phenotypic screening methods to detect glycopeptide intermediately susceptible Staphylococcus aureus (GISA) and heterogeneous GISA (h-gisa). Ann. Clin. Microbiol. Antimicrob. 6: Walsh, T.R., A. Bolmström, A. Qwärnström, P. Ho, M. Wootton, R.A. Howe, A.P. MacGowan, and D. Diekema Evaluation of Current Methods for Detection of Staphylococci with Reduced Susceptibility to Glycopeptides. J. Clin. Microbiol. 39: Wong, S.S., P.L. Ho, P.C. Woo, and K.Y. Yuen Bacteremia caused by staphylococci with inducible vancomycin heteroresistance. Clin. Infect. Dis. 29: Wong, S.S., T.K. Ng, W.C. Yam, D.N. Tsang, P.C. Woo, S.K. Fung, and K.Y. Yuen Bacteremia due to Staphylococcus aureus with reduced susceptibility to vancomycin. Diagn. Microbiol. Infect. Dis. 36:

15 Table1 Number and Characteristics of positive screening isolates Group a Total tested No. of screening Range of age Sex Specimen Range of Minimum inhibitory concentration b (ug/ml) isolates positive (year) M F Sputum Tracheal Pus, Blood Body Van Cefaz Oxa Oflox Tetra Erythro Gen isolates aspirate wound fluid > > > > > 128 > > > > > > > 128 a Group 1, isolates recovered between year ; Group 2, isolates recovered between year b Van, vancomycin; Cefaz, cefazolin; Oxa, oxacillin; Oflox, ofloxacin; Tetra, tetracycline; Erythro, erythromycin; Gen, gentamicin. Downloaded from on April 6, 2019 by guest

16 Table2 Phenotypic and genotypic characteristics of hvisa and VISA isolates Group a / Type of resistance JCSC No. Specimen Date of isolation Ward b Sex Age (year) Infection or colonized Minimum inhibitory concentration c (ug/ml) Van Cefaz Oxa Oflox Tetra Erythro Gen Teico 1 / hvisa 6902 sputum C M 57 2 >128 > > >128 4 III ST / hvisa 6903 Wound E F >128 >128 >128 2 III 1 / hvisa 6904 sputum B F 74 2 > >128 >128 2 III 1 / hvisa 6905 sputum B F 75 C > >128 4 III ST / hvisa 7192 pus C F 66 I > III ST / hvisa 7194 fluid D F 12 I 2 >128 > >128 > / hvisa 7196 Wound A F 71 C > III ST / hvisa 7201 sputum B M 47 C 2 >128 > >128 >128 1 NT d III ST / hvisa 7204 blood F M 52 I 2 >128 > >128 >128 NT III ST / hvisa 7205 pus B M 67 2 > > >128 NT 2 / hvisa 7206 blood B F 71 C >128 > / hvisa 7207 pus Mk M 78 2 >128 > >128 >128 >128 NT 2 / VISA 7193 pus D M 12 I 3 >128 > > III ST 239 I 2 / VISA 7195 fluid B M 56 I 3 >128 > > II ST 5 II 2 / VISA 7203 wound C F 9 C >128 >128 8 III ST239 I a Group 1, isolates recovered between year ; Group 2, isolates recovered between year b 3A, surgical ward; 3B, surgical ward; 3C, surgical ward; 3D, paediatric ward; 3F, orthopedic ward; 4B, medical ward; 4C, medical ward; 5E, chemotherapeutic ward; Mk, ward for monks. c Van, vancomycin; Cefaz, cefazolin; Oxa, oxacillin; Oflox, ofloxacin; Tetra, tetracycline; Erythro, erythromycin; Gen, gentamicin; Teico, teicoplanin. d NT, not tested. SCCmec type MLST Agr type

17 Figure 1 Log CFU/mL Vancomycin concentration (µg/ml) Mu 3 Mu ATCC29213 Vancomycin PAPs of 3 VISA isolates, 1 hetero-visa isolate and control strains

18 Log CFU/mL Teicoplanin concentration ( µg/ml) Mu 3 Mu Figure 2 Teicoplanin PAP of 3 VISA isolates and control strains

19 M A B C D E F G H I J K L N O P Q R S M Figure 3. Pulsed-Field Gel Electrophoresis banding patterns of SmaI digested chromosomal DNAs. Lanes: M, Molecular size marker; A, JCSC 6903; B, JCSC 6904 ; C, JCSC 7194 ; D, JCSC 7204; E, JCSC 7206; F, JCSC 7207 ; G, JCSC 7192 ; H, JCSC 7196 ; I, JCSC 7201 ; J, JCSC 7205 ; K, JCSC 7193 ; L, JCSC 7203 ; N, JCSC 6902 ; O, JCSC 6905 ; P, JCSC 7195 ; Q, Mu3 ; R, Mu50 ; S, JCSC 6890 (a HA-MRSA from the same hospital).