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Volume 49, Number 3, June 2016

RNA polymerase B subunit gene mutations in biofilm-embedded methicillin-resistant Staphylococcus aureus following rifampin treatment 

Hung-Jen Tang, Chih-Cheng Lai, Po-Ren Hsueh, Chi-Chung Chen, Kuan-Ying Wu, Yi-Chung Lin, Chun-Cheng Zhang, Tzu-Chieh Weng, Yu-Hsin Chiu, Han-Siong Toh, Shyh-Ren Chiang, Wen-Liang Yu, Wen-Chien Ko, Yin-Ching Chuang


Corresponding author:

Corresponding author. Yin-Ching Chuang, Department of Medical Research, Chi Mei Medical Center, Number 901 Chung-Hwa Road, Yung-Kang District, 710 Tainan, Taiwan. 


Background and purpose: 

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.




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.




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%). 



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. 


Key words:

biofilm-embedded MRSA, mutations, rpoB gene