Evolving MRSA: High-level β-lactam resistance in Staphylococcus aureus is associated with RNA Polymerase alterations and fine tuning of gene expression

Abstract

Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level β-lactam resistance (oxacillin MIC 2-4 μg/ml) due to the acquisition of a novel penicillin binding protein (PBP2A), encoded by mecA. However, strains can evolve high-level resistance (oxacillin MIC �256 μg/ml) by an unknown mechanism. Here we have developed a robust system to explore the basis of the evolution of high-level resistance by inserting mecA into the chromosome of the methicillin-sensitive S. aureus SH1000. Low-level mecA-dependent oxacillin resistance was associated with increased expression of anaerobic respiratory and fermentative genes. High-level resistant derivatives had acquired mutations in either rpoB (RNA polymerase subunit β) or rpoC (RNA polymerase subunit β') and these mutations were shown to be responsible for the observed resistance phenotype. Analysis of rpoB and rpoC mutants revealed decreased growth rates in the absence of antibiotic, and alterations to, transcription elongation. The rpoB and rpoC mutations resulted in decreased expression to parental levels, of anaerobic respiratory and fermentative genes and specific upregulation of 11 genes including mecA. There was however no direct correlation between resistance and the amount of PBP2A. A mutational analysis of the differentially expressed genes revealed that a member of the S. aureus Type VII secretion system is required for high level resistance. Interestingly, the genomes of two of the high level resistant evolved strains also contained missense mutations in this same locus. Finally, the set of genetically matched strains revealed that high level antibiotic resistance does not incur a significant fitness cost during pathogenesis. Our analysis demonstrates the complex interplay between antibiotic resistance mechanisms and core cell physiology, providing new insight into how such important resistance properties evolve.

Comprehensive Genomic Investigation of Adaptive Mutations Driving the Low-Level Oxacillin Resistance Phenotype in Staphylococcus aureus

“…First, we reconstructed the pbp3 -V613A mutation in the AUS0325 background (case 1, index isolate), but there was no change in the oxacillin MIC compared to AUS0325, indicating that the pbp3 mutation was not responsible for the increase in oxacillin MIC. As rpoB mutations have also been associated with reduced susceptibility to oxacillin ( 24 , 39 , 40 ), we also reconstructed the rpoB allele A477V I527T using the same background: the oxacillin MIC increased slightly from 0.5 to 1 mg/liter by broth microdilution and from 0.75 to 1 mg/liter by Etest. Overall, there was either no change or very limited increase in oxacillin MIC in pbp3 and rpoB mutants.…”

Modified Oxacillin Resistant Staphylococcus aureus detected in neonatal intensive care patients

“…However, functional PBP2 has an important role in the expression of resistance to methicillin [5,22], and the T552I substitution observed in the transpeptidase domain of PBP2 has previously been identified in mec -negative methicillin resistant S. aureus isolates [7]. There is also evidence to suggest that rpoB mutations may contribute to MRSA [23, 24]. Aiba et al demonstrated that introduction of an rpoB mutation was accompanied by tolerance to bactericidal concentrations of methicillin [23].…”

“…Aiba et al demonstrated that introduction of an rpoB mutation was accompanied by tolerance to bactericidal concentrations of methicillin [23]. Moreover, Panchal et al demonstrated that insertion of a mutant rpoB gene into a MSSA strain led to conversion to MRSA [24]. Thus, mutations in both genes may contribute to the MODSA phenotype observed in this strain.…”

Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

“…This was hypothesised to be caused by an increased production of host IL-1β [ 29 ]. MRSA strains also encode the non-native mecA gene, encoding PBP2A, responsible for low level β-lactam antibiotic resistance, or high level when present with specific rpoB and rpoC mutations [ 30 ]. S .…”