Article Figures & Data
Figures
- 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
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
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
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
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
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
- Table 1
Bacterial strains and plasmids used in this study
Strain or plasmid Descriptiona β-Lactam resistance phenotype Reference(s) Strains N315 Japanese clinical pre-MRSA strain carrying a functional mecI encoding mecA gene repressor Pre-MRSA 11 N315ΔIP mecI null mutant derived from N315P, an N315-derivative strain with the penicillinase plasmid eliminated Hetero-type 12 ΔIP-H5 Mutant derived from N315ΔIP by selection with 8 mg/liter of imipenem, carrying rpoB(N967I) Homo-type 30 ΔIP-H14 Mutant derived from N315ΔIP by selection with 8 mg/liter of imipenem, carrying vraS(S329L) Homo-type 20, 30 N315h4 Mutant derived from N315 by selection with 128 mg/liter of methicillin Eagle-type 10 H5 rpoB(I967N) ΔIP-H5 in which rpoB(I967) is replaced by the wild-type rpoB Hetero-type This study N315 rpoB(N967I) N315 whose wild-type rpoB is replaced by rpoB(967I) mutation Eagle-type This study Plasmids pKOR1 E. coli-S. aureus shuttle vector for construction of allelic replacement 26 pKOR1-rpoB(967I) pKOR1 harboring the 1,820-bp PCR product of mutated rpoB carrying amino acid residue Ile at position 967 This study pKOR1-rpoB(967N) pKOR1 harboring the 1,820-bp PCR product of wild-type rpoB carrying amino acid residue Asn at position 967 This 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
Strain Amino acid and/or substitution in RpoB Doubling time (min)c MIC (mg/liter)a MET OXA IPMb RIF VAN DAP LZD N315 N967 (wild type) 25.1 ± 0.7 1 4 0.125 0.008 0.75 1.5 1 N315ΔIP N967 (wild type) 26.7 ± 0.5 12 8 0.25 0.008 0.75 1.5 1 ΔIP-H5 N967I 30.3 ± 0.9* >256 >256 >32 0.004 0.75 1 0.75 ΔIP-H14 N967 (wild type) 28.3 ± 1.3 >256 >256 >32 0.004 2 1.75 0.75 H5 rpoB(I967N) N967 (wild type)d 25.4 ± 1.0 6 6 0.75 0.008 0.75 1 0.75 N315 rpoB(N967I) N967I 30.2 ± 0.3* >256 (2–12)b >256 >32,(0.05–0.5) 0.004 0.5 1.5 0.75 N315h4 R644H 31.6 ± 2.4* >256 (0.75–8)b >256 >32,(0.02–1.0) 0.004 0.75 1 0.02 ↵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 ORFa Gene Productb Ratio of signal intensity (mutant to parent strain) ΔIP-H5/N315ΔIP N315rpoB(N967I)/N315 Environmental information processing Autolysin-associated genes and regulators SA0252 lrgA Holin-like protein; murein hydrolase regulator LrgA 2.53 6.57 SA0253 lrgB Antiholin-like protein LrgB 2.08 6.60 SA2327 cidC Regulatory protein for murein hydrolase activity 0.44 0.26 SA2328 cidB Regulatory protein for murein hydrolase activity 0.48 0.27 SA2329 cidA Regulatory protein for murein hydrolase activity 0.42 0.25 SA1898 sceD-like Similar to SceD protein, predicted to be a lytic transglycosylase 2.21 3.26 Other signal transduction and regulators SA2090 rsr Repressor of sarR and agr genes 0.30 0.38 SA2091 sarY Staphylococcal accessory regulator Y 0.22 0.31 SA2092 Similar to transcription regulator AraC type regulator 0.20 0.35 Metabolism Cell-wall biosynthesis and metabolism SA0124 Similar to glycosyltransferase TuaA 0.39 0.43 SA0125 Similar to exopolysaccharide G (EpsG) 0.35 0.40 SA0523 Similar to poly(glycerol-phosphate) alpha-glucosyltransferase 2.03 2.33 SA1964 fmtB (mrp) Cell surface protein; inactivation reduces MET resistance 0.24 0.29 Cell envelope biogenesis SA0126 Similar to capsular polysaccharide synthesis protein 14H 0.39 0.43 SA0127 Similar to capsular polysaccharide synthesis protein 14L 0.47 0.50 Carbon hydrate metabolism Nitrogen metabolism Energy metabolism Nucleotide transport and metabolism SA1047 pyrF Orotidine-5-phosphate decarboxylase 0.34 0.48 SA1172 Similar to GMP reductase 0.32 0.47 Sugar transport and metabolism SA0837 Similar to 2-isopropylmalate synthase 2.95 2.86 SA1991 lacG 6-Phospho-beta-galactosidase 0.29 0.23 SA1992 lacE PTS system, lactose-specific IIBC component 0.29 0.25 SA1993 lacF PTS system, lactose-specific IIA component 0.34 0.19 SA1994 lacD Tagatose-1,6-diphosphate aldolase 0.36 0.21 SA1995 lacC Tagatose-6-phosphate kinase 0.42 0.21 SA1996 lacB Galactose-6-phosphate isomerase LacB subunit 0.49 0.25 SA2486 2-Oxoglutarate/malate translocator homolog 2.13 2.03 Amino acid transport and metabolism SA0822 argG Argininosuccinate synthase 0.40 0.48 SA0850 Similar to oligopeptide ABC transporter oligopeptide-binding protein 0.27 0.44 Terpenoid metabolism SA1304 Similar to component A of hexaprenyl diphosphate synthase 2.24 2.19 Secretion and other transport system SA0956 Similar to Mn2+ transport protein 0.20 0.32 SA2203 mdeA Similar to multidrug resistance protein 2.36 2.14 SA2442 secA2 Preprotein translocase secA homolog 2.31 2.18 SA2443 asp3 Accessory secretory protein Asp3 3.10 3.20 SA2444 asp2 Accessory secretory protein Asp2 3.89 3.36 SA2445 asp1 Accessory secretory protein Asp1 5.93 4.04 SA2446 secY2 Similar to preprotein translocase secY 5.51 4.01 Genetic information processing SA0706 Similar to comF operon protein 3 2.34 3.16 SA1899 Similar to single-strand DNA binding protein 2.41 2.56 Virulence factor SA0393 ssl11 Exotoxin 15 0.31 0.23 SA0519 sdrC Ser-Asp-rich fibrinogen-binding, bone sialoprotein-binding protein 2.59 3.48 SA0520 sdrD Ser-Asp-rich fibrinogen-binding, bone sialoprotein-binding protein 2.26 3.45 SA0521 sdrE Ser-Asp-rich fibrinogen-binding, bone sialoprotein-binding protein 4.45 4.60 SA1267 ebhA Extracellular matrix-binding protein EbhA 0.22 0.11 SA1577 sasC S. aureus surface protein C 0.36 0.40 SA2423 clfB Clumping factor B 4.58 2.49 SA2430 aur Zinc metalloproteinase aureolysin 0.40 3.50 Hypothetical protein SA0262 Hypothetical protein 2.18 2.35 SA0266 Conserved hypothetical protein 2.17 2.16 SA0267 Hypothetical protein 2.10 2.21 SA0268 Hypothetical protein 2.07 2.74 SA0269 Hypothetical protein 2.00 2.30 SA0271 Conserved hypothetical protein 2.41 3.90 SA0275 Conserved hypothetical protein 2.18 2.16 SA0331 Conserved hypothetical protein 0.31 0.33 SA0332 Conserved hypothetical protein 0.23 0.35 SA0333 Conserved hypothetical protein 0.26 0.35 SA0394 Hypothetical protein 0.30 0.24 SA0663 Hypothetical protein 0.32 0.48 SA0737 Hypothetical protein 2.90 2.28 SA0738 Hypothetical protein 2.55 2.37 SA0739 Conserved hypothetical protein 2.86 5.11 SA0741 Conserved hypothetical protein 3.93 4.14 SA0742 Hypothetical protein 3.74 3.29 SA0761 Conserved hypothetical protein 2.54 6.06 SA0830 Conserved hypothetical protein 2.49 2.63 SA1002 Hypothetical protein 2.40 3.25 SA1049 Hypothetical protein 0.41 0.37 SA1222 Hypothetical protein 2.04 2.49 SA1268 Hypothetical protein 0.36 0.25 SA1664 Conserved hypothetical protein 2.22 2.23 SA1755 Hypothetical protein (bacteriophage ϕN315) 0.25 0.188 SA2299 Conserved hypothetical protein 2.70 2.04 SA2491 Conserved hypothetical protein 2.49 2.55
