Antimicrobial resistance in Staphylococcus aureus is a major public health threat, compounded by emergence of strains with resistance to vancomycin and daptomycin, both last line antimicrobials. Here we have performed high throughput DNA sequencing and comparative genomics for five clinical pairs of vancomycin-susceptible (VSSA) and vancomycin-intermediate ST239 S. aureus (VISA); each pair isolated before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mutation among the VISA strains within the essential walKR two-component regulatory locus involved in control of cell wall metabolism. We then conducted bi-directional allelic exchange experiments in our clinical VSSA and VISA strains and showed that single nucleotide substitutions within either walK or walR lead to co-resistance to vancomycin and daptomycin, and caused the typical cell wall thickening observed in resistant clinical isolates. Ion Torrent genome sequencing confirmed no additional regulatory mutations had been introduced into either the walR or walK VISA mutants during the allelic exchange process. However, two potential compensatory mutations were detected within putative transport genes for the walK mutant. The minimal genetic changes in either walK or walR also attenuated virulence, reduced biofilm formation, and led to consistent transcriptional changes that suggest an important role for this regulator in control of central metabolism. This study highlights the dramatic impacts of single mutations that arise during persistent S. aureus infections and demonstrates the role played by walKR to increase drug resistance, control metabolism and alter the virulence potential of this pathogen.

Methicillin-resistant Staphylococcus aureus (MRSA), once restricted to hospitals, is spreading rapidly through the wider community. Resistance to vancomycin, the principal drug used to treat MRSA infections, has only recently emerged, is mainly low level, and characteristically appears during vancomycin therapy (vancomycin-intermediate S. aureus [VISA] and hetero-resistant VISA). This phenomenon suggests the adaptation of MRSA through mutation, although defining the mutations leading to resistance in clinical isolates has been difficult. We studied a vancomycin-susceptible clinical MRSA isolate (MIC of 1 g/ml) and compared it with an isogenic blood culture isolate from the same patient, despite 42 days of vancomycin treatment (MIC of 4 g/ml). A whole-genome sequencing approach allowed the nearly complete assembly of the genome sequences of the two isolates and revealed only six nucleotide substitutions in the VISA strain compared with the parent strain. One mutation occurred in graS, encoding a putative two-component regulatory sensor, leading to a change from a polar to a nonpolar amino acid (T136I) in the conserved histidine region of the predicted protein. Replacing the graS allele of the vancomycinsusceptible parent strain with the graS allele from the VISA derivative resulted in increased vancomycin resistance at a level between those of the vancomycin-susceptible S. aureus and VISA clinical isolates, confirming a role for graRS in VISA. Our study suggests that MRSA is able to develop clinically significant vancomycin resistance via a single point mutation, and the two-component regulatory system graRS is a key mediator of this resistance. However, additional mutations are likely required to express the full VISA phenotype.

Clostridium difficile binary toxin (CDT) is an actin-specific ADP-ribosyltransferase that is produced by various C. difficile isolates, including the "hypervirulent" NAP1/027 epidemic strains. In contrast to the two major toxins from C. difficile, toxin A and toxin B, little is known about the role of CDT in virulence or how C. difficile regulates its production. In this study we have shown that in addition to the cdtA and cdtB toxin structural genes, a functional cdt locus contains a third gene, here designated cdtR, which is predicted to encode a response regulator. By introducing functional binary toxin genes into cdtR ؉ and cdtR-negative strains of C. difficile, it was established that the CdtR protein was required for optimal expression of binary toxin. Significantly increased expression of functional binary toxin was observed in the presence of a functional cdtR gene; an internal deletion within cdtR resulted in a reduction in binary toxin production to basal levels. Strains that did not carry intact cdtAB genes or cdtAB pseudogenes also did not have cdtR, with the entire cdt locus, or CdtLoc, being replaced by a conserved 68-bp sequence. These studies have shown for the first time that binary toxin production is subject to strict regulatory control by the response regulator CdtR, which is a member of the LytTR family of response regulators and is related to the AgrA protein from Staphylococcus aureus.