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Epidemiology

Dissemination of New Methicillin-Resistant Staphylococcus aureus Clones in the Community

Keiko Okuma, Kozue Iwakawa, John D. Turnidge, Warren B. Grubb, Jan M. Bell, Frances G. O'Brien, Geoffrey W. Coombs, John W. Pearman, Fred C. Tenover, Maria Kapi, Chuntima Tiensasitorn, Teruyo Ito, Keiichi Hiramatsu
Keiko Okuma
1Department of Bacteriology, Juntendo University, Tokyo, Japan
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Kozue Iwakawa
1Department of Bacteriology, Juntendo University, Tokyo, Japan
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John D. Turnidge
2Department of Microbiology and Infectious Diseases, Women's and Children's Hospital, North Adelaide
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Warren B. Grubb
3Molecular Genetics Research Unit, School of Biomedical Sciences, Curtin University of Technology
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Jan M. Bell
2Department of Microbiology and Infectious Diseases, Women's and Children's Hospital, North Adelaide
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Frances G. O'Brien
3Molecular Genetics Research Unit, School of Biomedical Sciences, Curtin University of Technology
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Geoffrey W. Coombs
4Department of Microbiology and Infectious Diseases, Royal Perth Hospital, Perth, Australia
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John W. Pearman
4Department of Microbiology and Infectious Diseases, Royal Perth Hospital, Perth, Australia
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Fred C. Tenover
5National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333
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Maria Kapi
1Department of Bacteriology, Juntendo University, Tokyo, Japan
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Chuntima Tiensasitorn
1Department of Bacteriology, Juntendo University, Tokyo, Japan
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Teruyo Ito
1Department of Bacteriology, Juntendo University, Tokyo, Japan
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Keiichi Hiramatsu
1Department of Bacteriology, Juntendo University, Tokyo, Japan
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  • For correspondence: hiram@med.juntendo.ac.jp
DOI: 10.1128/JCM.40.11.4289-4294.2002
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  • FIG. 1.
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    FIG. 1.

    C-MRSA strain shows heterogeneous phenotypic expression of methicillin resistance. Analysis of resistant subpopulations of the C-MRSA strain MW2, the related MSSA strain 476, and strain Mu3, with heterogeneous resistance to vancomycin, was performed with oxacillin (A), ceftriaxone (B), and imipenem (C) as described previously (13). Ceftriaxone was the antibiotic used in vain to treat the patient infected with MW2 (3). MW2 is an American Midwest MRSA strain representing the major C-MRSA (see the text). Strain 476 is an MSSA strain sharing its MLST allotype with MW2 (see Table 1). Mu3 is a representative H-MRSA strain with heterogeneous resistance to vancomycin (13). It is noted that MW2 contains subpopulations resistant to each of the three beta-lactam antibiotics.

  • FIG. 2.
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    FIG. 2.

    Illustrative representation of various types of SCCmec. SCCmec type is defined by the combination of the type of ccr gene complex and the class of mec gene complex. Type I SCCmec is defined by the combination of type 1 ccr and class B mec (IS1272-ΔmecR1-mecA); type II is defined by type 2 ccr and class A mec (mecI-mecR1-mecA); type III is defined by type 3 ccr and class A mec; and type IV is defined by type 2 ccr and class B mec. Type IV SCCmec is further classified into subtypes (type IVa and type IVb) based on the sequence difference in the J1 region of SCCmec (J stands for “junkyard”). Positions of primers used in this study to identify and type SCCmec are shown (see Table 2 for the nucleotide sequence of each primer). The allelic class of mec gene complex is determined by PCR detection of the presence or absence of IS1272, mecI, and mecR1 in two domains (PB, penicillin-binding domain; and MS, membrane-spanning domain), mecA, and IS431mec. PCR amplification was performed using 2.5 U of Ex Taq (Takara Shuzo Co., Ltd., Kyoto, Japan) in 50 μl of reaction mixture. Thermal cycling was set at 30 cycles (30 s for denaturation at 94°C, 1 min for annealing at 50°C, and 2 min for elongation at 72°C) using the Gene Amp PCR system 9600 (Perkin-Elmer, Wellesley, Mass.). For the detection of mecA-IS431mec, a long-range PCR method was used, set at 10 cycles (15 s for denaturation at 94°C, 30 s for annealing at 50°C, and 8 min for elongation at 68°C) followed by 20 cycles (15 s for denaturation at 94°C, 30 s for annealing at 50°C, and 12 min for elongation at 68°C). Note that this study identified a new type of SCCmec for three C-MRSA strains that carried the class C2 mec gene complex (21). The sequencing of the entire SCCmec is now ongoing.

Tables

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  • TABLE 1.

    Genotyping and antibiogram of tested MRSA strains

    CategoryaSourcebStrain nameCoagulase isotypeMIC (mg/liter) ofc:SCCmec typedPFGE patternMLSTluk-PVgDoubling time (min)hReference(s)
    ERYKANTOBGENTETNOROXACZXIMPST, allelic profileeCCf
    CMN, USC199800037070.580.50.5 32 1 4 512 0.25IVaJ41, 1-1-1-1-1-1-11+27.89 3, 24
    CND, USC199900052970.540.50.250.51 8 >512 0.5IVaJ41, 1-1-1-1-1-1-11+26.38 3, 24
    CMN, USC199900019370.580.50.50.51 4 >512 0.25IVaJ31, 1-1-1-1-1-1-11+26.73 3, 24
    CND, USMW270.540.250.250.51 8 >512 0.5IVaJ41, 1-1-1-1-1-1-11+28.67 2, 3, 24
    CMN, USC20010012017 >512 80.250.250.51 8 >512 0.5IVaJ41, 1-1-1-1-1-1-11+27.4 3, 24
    CMN, USC200100010170.580.50.50.51 4 >512 0.25IVaJ41, 1-1-1-1-1-1-11+27.25 3, 24
    CMN, USC2001008187 >512 80.50.250.51 16 >512 0.25IVaJ51, 1-1-1-1-1-1-11+27.72 3, 24
    CWoo, AUA80 335540.540.250.250.52 16 >512 0.125IVaH130, 2-2-2-2-6-3-230+26.22 25
    CWoo, AUA82 354940.520.250.250.52 16 >512 0.125IVaH330, 2-2-2-2-6-3-230+28.09 25
    CWoo, AUA83 052840.540.250.250.52 16 >512 ≤0.063IVaH130, 2-2-2-2-6-3-230+27.08 25
    CWoo, AUB82 655940.520.250.250.52 16 512 ≤0.063IVaH130, 2-2-2-2-6-3-230+27.57 25
    CWoo, AUD82 155240.520.250.250.52 16 >512 0.125IVaH130, 2-2-2-2-6-3-230+61.03 25
    CWoo, AUE80 253740.540.250.250.52 8 >512 ≤0.063IVaH230, 2-2-2-2-6-3-230+26.97 25
    CWoo, AUF81 053940.540.250.250.52 16 >512 ≤0.063IVaH130, 2-2-2-2-6-3-230+26.44 25
    CWoo, AU180 255240.520.250.250.52 16 512 0.125IVaH330, 2-2-2-2-6-3-230+26.86 25
    CPer, AUiM9N3 >512 40.250.250.52 16 512 0.125IVaI178, 22-1-14-23-12-53-31298−26.75 26
    CPer, AUiM33T3 >512 40.250.250.52 16 512 0.125IVaI278, 22-1-14-23-12-53-31298−26.44 26
    CPer, AUiW1S10.520.50.250.50.52 32 0.063NewE245, 10-14-8-6-10-3-245−27.74 26
    CPer, AUiC7N7 >512 40.50.50.52 16 512 0.25IVaJ61, 1-1-1-1-1-1-11−30.26 26
    CTN, USj0021570.2540.50.250.50.5 64 >512 2IVaE145, 10-14-8-6-10-3-245−27.71 28
    CMS, USk010833 64 >512 0.50.5 32 16 8 512 0.125IVaA38, 3-3-1-1-4-4-38+27.78 4
    CMS, USk010935 16 512 0.50.50.52 16 >512 0.125IVaF72, 1-4-1-8-4-4-38−25.94 4
    CMS, USk0110230.2540.50.50.51 8 >512 0.25IVbA28, 3-3-1-1-4-4-38+28.29 4
    NAde, AU81 034230.2540.50.50.5 8 216≤0.063NewA18, 3-3-1-1-4-4-38−26.86This study
    NAde, AU91 257270.540.250.250.51 4 >512 0.25IVaJ71, 1-1-1-1-1-1-11−27.15This study
    NAde, AU91 261970.580.50.50.52 64 >512 2IVaJ776, 1-63-1-1-1-1-11−27.47This study
    NAde, AUWCH37970.520.250.50.51 16 >512 4IVaJ81, 1-1-1-1-1-1-11−33.31This study
    NAde, AU91 257470.2510.250.250.5 128 8 256 ≤0.063NewG22, 7-6-1-5-8-8-622−29.52This study
    NAde, AUSAP2602 >512 40.50.250.50.5 4 >512 0.25IVaB73, 1-4-27-4-12-1-105−26.64This study
    NPer, AU81 093770.520.250.50.51 4 >512 0.5IVaJ11, 1-1-1-1-1-1-11−25.29This study
    NPer, AU91 212570.540.250.50.50.5 16 >512 0.5IVaJ21, 1-1-1-1-1-1-11−30.25This study
    NPer, AU91 170330.2520.1250.25 8 1 16 512 0.125IVaA28, 3-3-1-1-4-4-38−32.0This study
    NBri, AU81 123870.520.250.50.51 8 >512 0.5IVaJ11, 1-1-1-1-1-1-11−26.06This study
    NBri, AU91 266670.520.50.250.50.52 512 0.125IVaJ21, 1-1-1-1-1-1-11−26.65This study
    NDar, AUSAP4116≤0.12540.250.5 32 1 4 128 0.125IVaN75, 36-3-43-34-39-52-49S−27.68This study
    HAde, AU81 05084 >512 256 256 0.50.25 512 512 >512 128 IIM236, 2-2-2-2-3-3-230−32.22This study
    HAde, AU91 22314 >512 20.250.5 32 32 128 >512 16 IIIK4239, 2-3-1-1-4-4-38−35.8This study
    HBri, AU91 15734 >512 128 64 8 16 1 64 >512 4IIIK2239, 2-3-1-1-4-4-38−41.82This study
    HBri, AU91 15754 >512 512 16 32 1 32 256 >512 32 IIIK6239, 2-3-1-1-4-4-38−50.46This study
    HBri, AU91 21454 >512 512 16 32 32 32 64 >512 16 IIIK1239, 2-3-1-1-4-4-38−50.64This study
    HDar, AUSAP3444 >512 40.250.5 32 32 256 >512 32 IIIK3239, 2-3-1-1-4-4-38−34.51This study
    HPer, AU91 21184 >512 512 16 32 32 256 512 >512 64 IIIL239, 2-3-1-1-4-4-38−38.45This study
    HUKNCTC1044230.12510.1250.5 128 1 256 >512 16 ID250, 3-3-1-1-4-4-168−36.44 19
    HJPN3152 >512 >512 512 0.50.1252 16 161IIC5, 1-4-1-4-12-1-105−34.28 19
    HNZ85/20824 512 >512 8 64 128 2 32 >512 0.5IIIK5239, 2-3-1-1-4-4-38−43.53 19
    HUKStrain 252l4 >512 128 128 0.250.5 >512 512 >512 64 IIM136, 2-2-2-2-3-3-230−29.79 20, —m
    HUKMSSA47670.540.50.5110.58≤0.063J11, 1-1-1-1-1-1-11−27.66—m
    • ↵ a C, C-MRSA; N, NORSA; H, H-MRSA.

    • ↵ b MN, Minnesota; US, United States; ND, North Dakota; Woo, Wooloongabba, Australia, AU, Australia; Per, Perth, Australia; TN, Tennessee; MS, Mississippi; Ade, Adelaide, Australia; Bri, Brisbane, Australia; Dar, Darwin, Australia; UK, United Kingdom; JP, Japan; NZ, New Zealand.

    • ↵ c MICs were determined by the NCCLS-based plate dilution method. Antibiotics: ERY, erythromycin; KAN, kanamycin; TOB, tobramycin; GEN, gentamicin; TET, tetracycline; NOR, norfloxacin; OXA, oxacillin; CZX, ceftizoxime; IMP, imipenem. Values in bold signify resistance to these antibiotics.

    • ↵ d New, new type of SCCmec possessing class C2 mec gene complex (see Fig. 2).

    • ↵ e ST, sequence type.

    • ↵ f Clonal complex, based on BURST (based upon related sequence types). S, singleton (not assigned to any clonal complex).

    • ↵ g luk-PV genes encode Panton-Valentin leucocidin proteins.

    • ↵ h Doubling time during exponential growth phase (optical density of 0.05 to ∼1.0 at 660 nm) measured by using TN-2612 (Advantec Toyo Kaisha, Ltd., Tokyo, Japan) as previously described (8).

    • ↵ i Isolated from aborigine.

    • ↵ j Isolated from food-poisoning strain.

    • ↵ k Isolated from state prison.

    • ↵ l E-MRSA16.

    • ↵ m —, http://www.sanger.ac.uk/Projects/S.aureus/ .

  • TABLE 2.

    Primer sets used for identifying SCCmec

    Primer (previous name) for detection of:Nucleotide sequenceExpected size of product (gene[s] reactive to the primer)Reference
    ccr gene complexa
        βc (β2)5′-ATTGCCTTGATAATAGCCITCT-3′(all types of ccrB) 19
        αc5′-ATCTATTTCAAAAATGAACCA-3′560 bp, βc (all types of ccrA)This study
        α1 (α2)5′-AACCTATATCATCAATCAGTACGT-3′700 bp, βc (type 1 ccrA) 19
        α2 (α3)5′-TAAAGGCATCAATGCACAAACACT-3′1 kbp, βc (type 2 ccrA) 19
        α3 (α4)5′-AGCTCAAAAGCAAGCAATAGAAT-3′1.6 kbp, βc (type 3 ccrA) 19
    mec gene complex (all types)
        mecR1 (MS domain)
            mcR45′-GTCGTTCATTAAGATATGACG-3′(mecR1—MS domain)This study
            mcR35′-GTCTCCACGTTAATTCCATT-3′310 bp, mcR4 (mecR1—MS domain) 21
        mecA
            mA15′-TGCTATCCACCCTCAAACAGG-3′(mecA) 15
            mA25′-AACGTTGTAACCACCCCAAGA-3′200 bp, mA1 (mecA) 15
        mecA -IS431mec
            IS2 (iS-2)5′-TGAGGTTATTCAGATATTTCGATGT-3′4 kbp, mA1 (IS431mec) 21
        Class C mec gene complex
            mA25′-AACGTTGTAACCACCCCAAGA-3′<3 kbp, IS2 (mecA) 21
        Class B mec gene complex
            IS55′-AACGCCACTCATAACATATGGAA-3′(IS1272)This study
            mA65′-TATACCAAACCCGACAAC-3′2 kbp, IS5 (mecA) 21
        Class A mec gene complex
            mecI
                mI45′-CAAGTGAATTGAAACCGCCT-3′(mecI)This study
                mI35′-CAAAAGGACTGGACTGGAGTCCAAA-3′180 bp, mI4 (mecI)This study
            mecR1 (PB domain)
                mcR25′-CGCTCAGAAATTTGTTGTGC-3′(mecR1—PB domain) 21
                mcR55′-CAGGGAATGAAAATTATTGGA-3′320 bp, mcR2 (mecR1—PB domain)This study
    SCCmec subtype IVa
        4a15′-TTTGAATGCCCTCCATGAATAAAAT-3′(J1 region of IVa)This study
        4a25′-AGAAAAGATAGAAGTTCGAAAGA-3′450 bp, 4a1 (J1 region of IVa)This study
    SCCmec subtype IVb
        4b15′-AGTACATTTTATCTTTGCGTA-3′(J1 region of IVb)This study
        4b25′-AGTCATCTTCAATATCGAGAAAGTA-3′1 kbp, 4b1 (J1 region of IVb)This study
    • ↵ a ccr type is determined by PCR using primer βc (the common primer for three types of ccrB) and either one of the three types of ccrA, α1 (ccrA1), α2 (ccrA2), and α3 (ccrA3). This typing actually reflects the allotype of ccrA.

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Dissemination of New Methicillin-Resistant Staphylococcus aureus Clones in the Community
Keiko Okuma, Kozue Iwakawa, John D. Turnidge, Warren B. Grubb, Jan M. Bell, Frances G. O'Brien, Geoffrey W. Coombs, John W. Pearman, Fred C. Tenover, Maria Kapi, Chuntima Tiensasitorn, Teruyo Ito, Keiichi Hiramatsu
Journal of Clinical Microbiology Nov 2002, 40 (11) 4289-4294; DOI: 10.1128/JCM.40.11.4289-4294.2002

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Dissemination of New Methicillin-Resistant Staphylococcus aureus Clones in the Community
Keiko Okuma, Kozue Iwakawa, John D. Turnidge, Warren B. Grubb, Jan M. Bell, Frances G. O'Brien, Geoffrey W. Coombs, John W. Pearman, Fred C. Tenover, Maria Kapi, Chuntima Tiensasitorn, Teruyo Ito, Keiichi Hiramatsu
Journal of Clinical Microbiology Nov 2002, 40 (11) 4289-4294; DOI: 10.1128/JCM.40.11.4289-4294.2002
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KEYWORDS

community-acquired infections
methicillin resistance
Staphylococcal Infections
Staphylococcus aureus

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