title: RNA polymerase B subunit gene mutations in biofilm-embedded methicillin-resistant Staphylococcus aureus following rifampin treatment.
abstract: OBJECTIVE
  This study was conducted to compare the mutation rates of different rpoB sites and rifampin minimum inhibitory concentration (MIC) changes prior to and after rifampin therapy for biofilm-embedded methicillin-resistant Staphylococcus aureus (MRSA) isolates.
  
  
  METHODS
  The screening of rifampin-resistant MRSA isolates, from the biofilm at Day 5 with or without exposure to the susceptible breakpoint concentration of rifampin recommended by the Clinical and Laboratory Standards Institute (1 mg/L), was conducted using agar plates containing rifampin. A partial fragment of RNA polymerase B subunit gene (rpoB), including clusters I and II, was amplified and sequenced. The rifampin MIC values and mutation frequencies at different sites of rpoB were measured and evaluated in rifampicin-resistant isolates.
  
  
  RESULTS
  Rifampin-resistant mutants could be selected from all of 39 randomly selected rifampin-susceptible MRSA isolates in the biofilm model. The spontaneous mutation frequency ranged from 1.00 × 10(-4) to 3.85 × 10(-7). Mutation at codon 481 was most commonly found at 35 (89.7%) of 39 MRSA isolates. Without rifampin induction, the MIC ranged between 0.125 mg/L and1024 mg/L and mutation sites included cluster I 464, 466, 468, 471, 474, 477, 481, 484, 486 and cluster II 519, 527, 529 with the percentage of 471 (35.9%), 477 (33.3%), 481 (53.8%), and 484 (35.9%). Conversely, with the induction of rifampin, the MIC value ranged ∼256-1024 mg/L. The mutation sites that were more concentrated included 468 (17.9%), 477 (30.8%), 481 (89.7%), 484 (17.9%), and 486 (33.3%).
  
  
  CONCLUSIONS
  We documented high rifampin resistance induction activity when MRSA was engaged in biofilm with rifampin exposure. Monotherapy seems to be inadequate for MRSA in biofilm. There is an urgent need for developing effective combination therapies with less rifampin resistance-inducing activities for treating MRSA in biofilms.
authors: Department of Medicine, Chi Mei Medical Center, Tainan, Taiwan; Department of Health and Nutrition, Chia Nan University of Pharmacy and Science, Tainan, Taiwan.
authors: Hung-Jen
authors: HJ
authors: Tang
authors: Department of Intensive Care Medicine, Chi Mei Medical Center, Liou Ying, Tainan, Taiwan.
authors: Chih-Cheng
authors: CC
authors: Lai
authors: Department of Laboratory and Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan; National Taiwan University College of Medicine, Taipei, Taiwan.
authors: Po-Ren
authors: PR
authors: Hsueh
authors: Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan; Institute of Biotechnology, National Cheng Kung University Medical College and Hospital, Tainan, Taiwan.
authors: Chi-Chung
authors: CC
authors: Chen
authors: Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan.
authors: Kuan-Ying
authors: KY
authors: Wu
authors: Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan.
authors: Yi-Chung
authors: YC
authors: Lin
authors: Department of Medicine, Chi Mei Medical Center, Tainan, Taiwan.
authors: Chun-Cheng
authors: CC
authors: Zhang
authors: Department of Medicine, Chi Mei Medical Center, Tainan, Taiwan.
authors: Tzu-Chieh
authors: TC
authors: Weng
authors: Department of Internal Medicine, Chi Mei Medical Center, Liou Ying, Tainan, Taiwan.
authors: Yu-Hsin
authors: YH
authors: Chiu
authors: Department of Medicine, Chi Mei Medical Center, Tainan, Taiwan.
authors: Han-Siong
authors: HS
authors: Toh
authors: Department of Medicine, Chi Mei Medical Center, Tainan, Taiwan.
authors: Shyh-Ren
authors: SR
authors: Chiang
authors: Department of Medicine, Chi Mei Medical Center, Tainan, Taiwan; Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan.
authors: Wen-Liang
authors: WL
authors: Yu
authors: Department of Medicine, National Cheng Kung University Medical College and Hospital, Tainan, Taiwan. Electronic address: winston3415@gmail.com.
authors: Wen-Chien
authors: WC
authors: Ko
authors: Department of Medicine, Chi Mei Medical Center, Tainan, Taiwan; Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan; Department of Internal Medicine, Chi Mei Medical Center, Liou Ying, Tainan, Taiwan. Electronic address: chuangkenneth@hotmail.com.
authors: Yin-Ching
authors: YC
authors: Chuang
mesh_terms: D000900:Anti-Bacterial Agents / Q000627:therapeutic use*; D001483:Base Sequence; D018441:Biofilms / Q000187:drug effects*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D006801:Humans; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D012319:RNA Polymerase II / Q000235:genetics*; D012293:Rifampin / Q000627:therapeutic use*; D013203:Staphylococcal Infections / Q000188:drug therapy*
chemical_list: D000900:Anti-Bacterial Agents; D012319:RNA Polymerase II; D012293:Rifampin
keywords: biofilm-embedded MRSA; mutations; rpoB gene