Genetic Pathway in Acquisition and Loss of Vancomycin Resistance in a Methicillin Resistant Staphylococcus aureus (MRSA) Strain of Clonal Type USA300

Abstract

An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible “parental” strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a “stealth” strategy to evade detection by the host immune system.

Author Summary

The extensive use of antibiotics has led to the selection of methicillin-resistant S. aureus (MRSA) strains that are resistant to most antimicrobial agents and a treatment of choice against such strains is vancomycin. However, during the last decade reports of treatment failure with vancomycin non-susceptible MRSA (e.g., vancomycin intermediate S. aureus, VISA) began to appear in the clinical setting. In this paper we analyze the mechanism of resistance in a VISA strain that belongs to the epidemic and highly virulent MRSA clone USA300. We had 3 isogenic isolates available for analysis: the vancomycin susceptible parental strain recovered from the patient before the onset of therapy; the VISA strain recovered at the time of clinical treatment failure and a susceptible revertant of the VISA strain acquired during in vitro passage. We identified genetic differences among the three strains through whole genome sequencing. In this strain, the key genetic change responsible for vancomycin resistance was in the functionally connected yvqF/vraSR - two component sensory regulatory system involved with the control of cell wall metabolism of the bacteria. The same genetic change also caused repression of virulence related properties which may help the resistant bacteria to evade the host immune system.