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Mechanisms of Resistance

Mutation of RNA Polymerase β-Subunit Gene Promotes Heterogeneous-to-Homogeneous Conversion of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus

Yoshifumi Aiba, Yuki Katayama, Tomomi Hishinuma, Hiroko Murakami-Kuroda, Longzhu Cui, Keiichi Hiramatsu
Yoshifumi Aiba
Department of Infection Control Science, Graduate School of Medicine, Juntendo University, Tokyo, Japana
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Yuki Katayama
Department of Bacteriology, Faculty of Medicine, Juntendo University,b Tokyo, Japan
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Tomomi Hishinuma
Department of Bacteriology, Faculty of Medicine, Juntendo University,b Tokyo, Japan
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Hiroko Murakami-Kuroda
Department of Bacteriology, Faculty of Medicine, Juntendo University,b Tokyo, Japan
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Longzhu Cui
Department of Bacteriology, Faculty of Medicine, Juntendo University,b Tokyo, Japan
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Keiichi Hiramatsu
Department of Infection Control Science, Graduate School of Medicine, Juntendo University, Tokyo, Japana
Department of Bacteriology, Faculty of Medicine, Juntendo University,b Tokyo, Japan
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DOI: 10.1128/AAC.00720-13
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  • Fig 1
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    Fig 1

    Population analysis of N315 and its derivatives with exposure to imipenem (A) and methicillin (B). Pre-MRSA strain N315, hetero-type strain N315ΔIP, typical Eagle-type strain N315h4, and homo-type strain ΔIP-H5 were used as control strains. Note that N315 rpoB(N967I) showed an Eagle-type phenotype similar to that of N315h4.

  • Fig 2
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    Fig 2

    Investigation of Eagle-type phenotype for methicillin in N315 rpoB(N967I) by Etest. The hetero-type strain (pre-MRSA) N315, homo-type strain ΔIP-H5, and Eagle-type strain N315h4 were used as parent or control strains. The control strains N315, N315ΔIP, ΔIP-H5, and ΔIP-H14 and constructed mutant strain H5 rpoB(I967N) were inoculated with 108 CFU according to the manufacturer's recommendation. N315h4 and N315 rpoB(N967I) strains were inoculated with 1011 CFU. The reason for the inoculation with 1011 CFU was that the double inhibitory zone could be detected more clearly. There is no difference between MIC values with inoculum sizes of 1011 CFU and 108 CFU. An Eagle-type MRSA strain grows better on agar plates containing high concentrations of methicillin than on those with low concentrations of methicillin. The single inhibitory zone was visible with the pre-MRSA N315 and hetero-type MRSA N315ΔIP strains but not, however, with the homo-type MRSA strains.

  • Fig 3
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    Fig 3

    Effect of rpoB on methicillin-induced killing. The tested strains were hetero-type strains N315, N315ΔIP, and H5 rpoB(I967N), Eagle-type strains N315h4 and N315 rpoB(N967I), and homo-type strain ΔIP-H5. The killing assay was performed in the presence of methicillin at concentrations of 0 (○), 1 (▲), 8 (X), and 128 (●) mg/liter. The viable cell counts were done at 0, 1, 2, and 4 h after the start of culture.

  • Fig 4
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    Fig 4

    Western blot analysis for expression of mecA after 1 h of induction with methicillin. PBP2′ production induced by methicillin at 0, 1, 8, and 128 mg/liter is shown in lanes 1 to 4, respectively, for each strain. The pre-MRSA N315 and Eagle-type MRSA strains N315h4 and N315 rpoB(N967I) carried intact mecI repressor genes of mecA. In the N315 and two Eagle-type strains, the amount of PBP2′ was increased under the condition of induction with methicillin at 8 and 128 mg/liter (lanes 3 and 4) compared to levels with methicillin at 0 and 1 mg/liter (lanes 1 and 2), respectively.

  • Fig 5
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    Fig 5

    Triton X-100 (0.05%)-stimulated autolysis of three wild-type rpoB strains, N315, N315ΔIP, H5 rpoB(N967I), and their three rpoB mutant strains, N315 rpoB(N967I), N315h4 and ΔIP-H5. Autolysis was measured as the decline in optical density versus time and is expressed as the percentage of the initial optical density. The data presented are representative of three independent experiments.

  • Fig 6
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    Fig 6

    Schematic diagram of methicillin phenotypic conversion by population analysis. Two pathways of acquisition of homo-type methicillin resistance from pre-MRSA strain N315. Two genetic alterations, mecI inactivation and mutated rpoB [rpoB(N967I) or rpoB(R644H), indicated on the figure as rpoB*] as a chr* mutation, are required for N315 to achieve homo-type methicillin resistance. Eagle-type methicillin resistance was reproduced by mutated rpoB under the presence of mecI activation. conc, concentration.

Tables

  • Figures
  • Table 1

    Bacterial strains and plasmids used in this study

    Strain or plasmidDescriptionaβ-Lactam resistance phenotypeReference(s)
    Strains
        N315Japanese clinical pre-MRSA strain carrying a functional mecI encoding mecA gene repressorPre-MRSA11
        N315ΔIPmecI null mutant derived from N315P, an N315-derivative strain with the penicillinase plasmid eliminatedHetero-type12
        ΔIP-H5Mutant derived from N315ΔIP by selection with 8 mg/liter of imipenem, carrying rpoB(N967I)Homo-type30
        ΔIP-H14Mutant derived from N315ΔIP by selection with 8 mg/liter of imipenem, carrying vraS(S329L)Homo-type20, 30
        N315h4Mutant derived from N315 by selection with 128 mg/liter of methicillinEagle-type10
        H5 rpoB(I967N)ΔIP-H5 in which rpoB(I967) is replaced by the wild-type rpoBHetero-typeThis study
        N315 rpoB(N967I)N315 whose wild-type rpoB is replaced by rpoB(967I) mutationEagle-typeThis study
    Plasmids
        pKOR1E. coli-S. aureus shuttle vector for construction of allelic replacement26
        pKOR1-rpoB(967I)pKOR1 harboring the 1,820-bp PCR product of mutated rpoB carrying amino acid residue Ile at position 967This study
        pKOR1-rpoB(967N)pKOR1 harboring the 1,820-bp PCR product of wild-type rpoB carrying amino acid residue Asn at position 967This study
    • ↵a Strains obtained by antibiotic selection are denoted as mutant.

  • Table 2

    Doubling time and antibiotic susceptibility profiles by Etest among N315 and its derived strains

    StrainAmino acid and/or substitution in RpoBDoubling time (min)cMIC (mg/liter)a
    METOXAIPMbRIFVANDAPLZD
    N315N967 (wild type)25.1 ± 0.7140.1250.0080.751.51
    N315ΔIPN967 (wild type)26.7 ± 0.51280.250.0080.751.51
    ΔIP-H5N967I30.3 ± 0.9*>256>256>320.0040.7510.75
    ΔIP-H14N967 (wild type)28.3 ± 1.3>256>256>320.00421.750.75
    H5 rpoB(I967N)N967 (wild type)d25.4 ± 1.0660.750.0080.7510.75
    N315 rpoB(N967I)N967I30.2 ± 0.3*>256 (2–12)b>256>32,(0.05–0.5)0.0040.51.50.75
    N315h4R644H31.6 ± 2.4*>256 (0.75–8)b>256>32,(0.02–1.0)0.0040.7510.02
    • ↵a MET, methicillin; OXA, oxacillin; IPM, imipenem; RIF, rifampin; VAN, vancomycin; DAP, daptomycin; LZD, linezolid.

    • ↵b Eagle-type resistance was observed as the growth of the strain was inhibited at the drug concentrations shown in parentheses (Fig. 2).

    • ↵c The level of significance was determined by a two sided Student t test (*, P < 0.012) in the comparison of rpoB mutants and wild-type rpoB strains.

    • ↵d Revertant strain.

  • Table 3

    Alterations of transcription for ΔIP-H5 versus N315ΔIP and N315 rpoB(N967I) versus N315

    Function and ORFaGeneProductbRatio of signal intensity (mutant to parent strain)
    ΔIP-H5/N315ΔIPN315rpoB(N967I)/N315
    Environmental information processing
        Autolysin-associated genes and regulators
            SA0252lrgAHolin-like protein; murein hydrolase regulator LrgA2.536.57
            SA0253lrgBAntiholin-like protein LrgB2.086.60
            SA2327cidCRegulatory protein for murein hydrolase activity0.440.26
            SA2328cidBRegulatory protein for murein hydrolase activity0.480.27
            SA2329cidARegulatory protein for murein hydrolase activity0.420.25
            SA1898sceD-likeSimilar to SceD protein, predicted to be a lytic transglycosylase2.213.26
        Other signal transduction and regulators
            SA2090rsrRepressor of sarR and agr genes0.300.38
            SA2091sarYStaphylococcal accessory regulator Y0.220.31
            SA2092Similar to transcription regulator AraC type regulator0.200.35
    Metabolism
        Cell-wall biosynthesis and metabolism
            SA0124Similar to glycosyltransferase TuaA0.390.43
            SA0125Similar to exopolysaccharide G (EpsG)0.350.40
            SA0523Similar to poly(glycerol-phosphate) alpha-glucosyltransferase2.032.33
            SA1964fmtB (mrp)Cell surface protein; inactivation reduces MET resistance0.240.29
        Cell envelope biogenesis
            SA0126Similar to capsular polysaccharide synthesis protein 14H0.390.43
            SA0127Similar to capsular polysaccharide synthesis protein 14L0.470.50
        Carbon hydrate metabolism
        Nitrogen metabolism
        Energy metabolism
        Nucleotide transport and metabolism
            SA1047pyrFOrotidine-5-phosphate decarboxylase0.340.48
            SA1172Similar to GMP reductase0.320.47
        Sugar transport and metabolism
            SA0837Similar to 2-isopropylmalate synthase2.952.86
            SA1991lacG6-Phospho-beta-galactosidase0.290.23
            SA1992lacEPTS system, lactose-specific IIBC component0.290.25
            SA1993lacFPTS system, lactose-specific IIA component0.340.19
            SA1994lacDTagatose-1,6-diphosphate aldolase0.360.21
            SA1995lacCTagatose-6-phosphate kinase0.420.21
            SA1996lacBGalactose-6-phosphate isomerase LacB subunit0.490.25
            SA24862-Oxoglutarate/malate translocator homolog2.132.03
        Amino acid transport and metabolism
            SA0822argGArgininosuccinate synthase0.400.48
            SA0850Similar to oligopeptide ABC transporter oligopeptide-binding protein0.270.44
        Terpenoid metabolism
            SA1304Similar to component A of hexaprenyl diphosphate synthase2.242.19
        Secretion and other transport system
            SA0956Similar to Mn2+ transport protein0.200.32
            SA2203mdeASimilar to multidrug resistance protein2.362.14
            SA2442secA2Preprotein translocase secA homolog2.312.18
            SA2443asp3Accessory secretory protein Asp33.103.20
            SA2444asp2Accessory secretory protein Asp23.893.36
            SA2445asp1Accessory secretory protein Asp15.934.04
            SA2446secY2Similar to preprotein translocase secY5.514.01
    Genetic information processing
        SA0706Similar to comF operon protein 32.343.16
        SA1899Similar to single-strand DNA binding protein2.412.56
    Virulence factor
        SA0393ssl11Exotoxin 150.310.23
        SA0519sdrCSer-Asp-rich fibrinogen-binding, bone sialoprotein-binding protein2.593.48
        SA0520sdrDSer-Asp-rich fibrinogen-binding, bone sialoprotein-binding protein2.263.45
        SA0521sdrESer-Asp-rich fibrinogen-binding, bone sialoprotein-binding protein4.454.60
        SA1267ebhAExtracellular matrix-binding protein EbhA0.220.11
        SA1577sasCS. aureus surface protein C0.360.40
        SA2423clfBClumping factor B4.582.49
        SA2430aurZinc metalloproteinase aureolysin0.403.50
    Hypothetical protein
        SA0262Hypothetical protein2.182.35
        SA0266Conserved hypothetical protein2.172.16
        SA0267Hypothetical protein2.102.21
        SA0268Hypothetical protein2.072.74
        SA0269Hypothetical protein2.002.30
        SA0271Conserved hypothetical protein2.413.90
        SA0275Conserved hypothetical protein2.182.16
        SA0331Conserved hypothetical protein0.310.33
        SA0332Conserved hypothetical protein0.230.35
        SA0333Conserved hypothetical protein0.260.35
        SA0394Hypothetical protein0.300.24
        SA0663Hypothetical protein0.320.48
        SA0737Hypothetical protein2.902.28
        SA0738Hypothetical protein2.552.37
        SA0739Conserved hypothetical protein2.865.11
        SA0741Conserved hypothetical protein3.934.14
        SA0742Hypothetical protein3.743.29
        SA0761Conserved hypothetical protein2.546.06
        SA0830Conserved hypothetical protein2.492.63
        SA1002Hypothetical protein2.403.25
        SA1049Hypothetical protein0.410.37
        SA1222Hypothetical protein2.042.49
        SA1268Hypothetical protein0.360.25
        SA1664Conserved hypothetical protein2.222.23
        SA1755Hypothetical protein (bacteriophage ϕN315)0.250.188
        SA2299Conserved hypothetical protein2.702.04
        SA2491Conserved hypothetical protein2.492.55
    • ↵a Locus tags are based on S. aureus strain N315. ORF, open reading frame.

    • ↵b MET, methicillin; PTS, phosphotransferase system.

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Mutation of RNA Polymerase β-Subunit Gene Promotes Heterogeneous-to-Homogeneous Conversion of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus
Yoshifumi Aiba, Yuki Katayama, Tomomi Hishinuma, Hiroko Murakami-Kuroda, Longzhu Cui, Keiichi Hiramatsu
Antimicrobial Agents and Chemotherapy Sep 2013, 57 (10) 4861-4871; DOI: 10.1128/AAC.00720-13

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Mutation of RNA Polymerase β-Subunit Gene Promotes Heterogeneous-to-Homogeneous Conversion of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus
Yoshifumi Aiba, Yuki Katayama, Tomomi Hishinuma, Hiroko Murakami-Kuroda, Longzhu Cui, Keiichi Hiramatsu
Antimicrobial Agents and Chemotherapy Sep 2013, 57 (10) 4861-4871; DOI: 10.1128/AAC.00720-13
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