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Evolution of resistance mechanisms and biological characteristics of rifampicin-resistant Staphylococcus aureus strains selected in vitro.

ABSTRACT: BACKGROUND:We aimed to determine the evolutionary pathways of rifampicin resistance in Staphylococcus aureus, and the impact of resistance mutations in the rpoB gene on fitness. METHODS:Three clinical strains and one reference strain were used to select for rifampicin-resistant S. aureus variants. The mutations responsible for rifampicin resistance in all of the selected isolates in vitro were investigated by polymerase chain reaction (PCR) and DNA sequencing. To compare the fitness cost of rpoB mutations against their corresponding original isolates, we performed bacterial growth curve assays, static biofilm assays, in vitro competition experiments and an infection model of Galleria mellonella larvae. RESULTS:We obtained four rifampicin-resistant S. aureus isolates that showed high levels of resistance to rifampicin with a minimal inhibitory concentration (MIC) of 128?mg/L, and all isolates had a mutation at position 481 (H481F/Y) in RpoB. A broth microdilution assay indicated that mutation of H481F/Y did not affect susceptibility to common antibacterial drugs but slightly increased the vancomycin MIC. To identify the pathways involved in the development of rifampicin resistance, 32 variants (eight mutants for each strain) and four original isolates were selected for gene sequencing. Different generations of isolates were found to harbor various mutations sites. Compared with the corresponding original isolates, an in vitro fitness assay of the variant isolates showed that growth and virulence were reduced, with a statistically significantly decreased fitness, whereas the capacity for biofilm formation was elevated. CONCLUSIONS:Our findings suggested that the acquisition of rifampicin resistance in S. aureus was dynamic and was associated with a significant fitness cost.

SUBMITTER: Wang C

PROVIDER: S-EPMC6751903 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Evolution of resistance mechanisms and biological characteristics of rifampicin-resistant Staphylococcus aureus strains selected in vitro.

Wang Chong C Fang Renchi R Zhou Beibei B Tian Xuebin X Zhang Xiucai X Zheng Xiangkuo X Zhang Siqin S Dong Guofeng G Cao Jianming J Zhou Tieli T

BMC microbiology 20190918 1

<h4>Background</h4>We aimed to determine the evolutionary pathways of rifampicin resistance in Staphylococcus aureus, and the impact of resistance mutations in the rpoB gene on fitness.<h4>Methods</h4>Three clinical strains and one reference strain were used to select for rifampicin-resistant S. aureus variants. The mutations responsible for rifampicin resistance in all of the selected isolates in vitro were investigated by polymerase chain reaction (PCR) and DNA sequencing. To compare the fitne ...[more]

PMID: 31533633

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Project description:Resistance to antibiotics is an increasing problem and necessitates novel antibacterial therapies. The polyketide antibiotics cervimycin A to D are natural products of Streptomyces tendae HKI 0179 with promising activity against multidrug-resistant staphylococci and vancomycin-resistant enterococci. To initiate mode of action studies, we selected cervimycin C- and D-resistant (CmR) Staphylococcus aureus strains. Genome sequencing of CmR mutants revealed amino acid exchanges in the essential histidine kinase WalK, the Clp protease proteolytic subunit ClpP or the Clp ATPase ClpC, and the heat shock protein DnaK. Interestingly, all characterized CmR mutants harbored a combination of mutations in walK and clpP or clpC. In vitro and in vivo analyses showed that the mutations in the Clp proteins abolished ClpP or ClpC activity, and the deletion of clpP rendered S. aureus but not all Bacillus subtilis strains cervimycin-resistant. The essential gene walK was the second mutational hotspot in the CmR S. aureus strains, which decreased WalK activity in vitro and generated a vancomycin-intermediate resistant phenotype, with a thickened cell wall, a lower growth rate, and reduced cell lysis. Transcriptomic and proteomic analyses revealed massive alterations in the CmR strains compared to the parent strain S. aureus SG511, with major shifts in the heat shock regulon, the metal ion homeostasis, and the carbohydrate metabolism. Taken together, mutations in the heat shock genes clpP, clpC, and dnaK, and the walK kinase gene in CmR mutants induced a vancomycin-intermediate resistant phenotype in S. aureus, suggesting cell wall metabolism or the Clp protease system as primary target of cervimycin. IMPORTANCE Staphylococcus aureus is a frequent cause of infections in both the community and hospital setting. Resistance development of S. aureus to various antibiotics is a severe problem for the treatment of this pathogen worldwide. New powerful antimicrobial agents against Gram-positives are needed, since antibiotics like vancomycin fail to cure vancomycin-intermediate resistant S. aureus (VISA) and vancomycin-resistant enterococci (VRE) infections. One candidate substance with promising activity against these organisms is cervimycin, which is an antibiotic complex with a yet unknown mode of action. In our study, we provide first insights into the mode of action of cervimycins. By characterizing cervimycin-resistant S. aureus strains, we revealed the Clp system and the essential kinase WalK as mutational hotspots for cervimycin resistance in S. aureus. It further emerged that cervimycin-resistant S. aureus strains show a VISA phenotype, indicating a role of cervimycin in perturbing the bacterial cell envelope.

Project description:UNLABELLED:Overexpression of the Staphylococcus aureus multidrug efflux pump MepA confers resistance to a wide variety of antimicrobials. mepA expression is controlled by MarR family member MepR, which represses mepA and autorepresses its own production. Mutations in mepR are a primary cause of mepA overexpression in clinical isolates of multidrug-resistant S. aureus. Here, we report crystal structures of three multidrug-resistant MepR variants, which contain the single-amino-acid substitution A103V, F27L, or Q18P, and wild-type MepR in its DNA-bound conformation. Although each mutation impairs MepR function by decreasing its DNA binding affinity, none is located in the DNA binding domain. Rather, all are found in the linker region connecting the dimerization and DNA binding domains. Specifically, the A103V substitution impinges on F27, which resolves potential steric clashes via displacement of the DNA binding winged-helix-turn-helix motifs that lead to a 27-fold reduction in DNA binding affinity. The F27L substitution forces F104 into an alternative rotamer, which kinks helix 5, thereby interfering with the positioning of the DNA binding domains and decreasing mepR operator affinity by 35-fold. The Q18P mutation affects the MepR structure and function most significantly by either creating kinks in the middle of helix 1 or completely unfolding its C terminus. In addition, helix 5 of Q18P is either bent or completely dissected into two smaller helices. Consequently, DNA binding is diminished by 2,000-fold. Our structural studies reveal heretofore-unobserved allosteric mechanisms that affect repressor function of a MarR family member and result in multidrug-resistant Staphylococcus aureus. IMPORTANCE:Staphylococcus aureus is a major health threat to immunocompromised patients. S. aureus multidrug-resistant variants that overexpress the multidrug efflux pump mepA emerge frequently due to point mutations in MarR family member MepR, the mepA transcription repressor. Significantly, the majority of MepR mutations identified in these S. aureus clinical isolates are found not in the DNA binding domain but rather in a linker region, connecting the dimerization and DNA binding domains. The location of these mutants underscores the critical importance of a properly functioning allosteric mechanism that regulates MepR function. Understanding the dysregulation of such allosteric MepR mutants underlies this study. The high-resolution structures of three such allosteric MepR mutants reveal unpredictable conformational consequences, all of which preclude cognate DNA binding, while biochemical studies emphasize their debilitating effects on DNA binding affinity. Hence, mutations in the linker region of MepR and their structural consequences are key generators of multidrug-resistant Staphylococcus aureus.

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Evolution of resistance mechanisms and biological characteristics of rifampicin-resistant Staphylococcus aureus strains selected in vitro.

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Evolution of resistance mechanisms and biological characteristics of rifampicin-resistant Staphylococcus aureus strains selected in vitro.

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