The advent of transcription profiling technologies has provided researchers with an unprecedented ability to study biological processes. Accordingly, a custom-made Affymetrix GeneChip, constituting >86% of the Staphylococcus aureus genome, was used to identify open reading frames that are regulated by agr and/or SarA, the two best-studied regulators of the organism's virulence response. RNA extracted from wild-type cells and agr, sarA, and agr sarA mutant cells in the early-, mid-, and late-log and stationary phases of growth was analyzed. Open reading frames with transcription patterns expected of genes either up-or downregulated in an agrand/or SarA-dependent manner were identified. Oligonucleotide microarray and Northern blot analyses confirmed that the transcription of several known virulence genes, including hla (alpha-toxin) and spa (protein A), is regulated by each effector and provided insights about the regulatory cascades involved in both alpha-hemolysin and protein A expression. Several putative virulence factors were also identified as regulated by agr and/or SarA. In addition, genes that are involved in several biological processes but which are difficult to reconcile as playing a direct role in the organism's pathogenesis also appeared to be regulated by each effector, suggesting that products of both the agr and the sarA locus are more-global transcription regulators than previously realized.Staphylococcus aureus is a major cause of human disease. The organism causes a variety of clinical manifestations, ranging from localized skin infections to severe sepsis, and is a leading cause of hospital-acquired infection (3). Despite advances in antibacterial chemotherapy, S. aureus strains have demonstrated resistance to all currently available antibiotics. Due in part to the immense clinical importance of this organism, an enormous amount of effort has been directed toward identifying the genes and regulatory mechanisms associated with S. aureus pathogenesis. Collectively, this work has demonstrated that the organism's pathogenesis can be attributed to its capacity to produce a variety of virulence factors (29).The identification of virulence factors and the regulatory networks that influence their expression has been facilitated by the observation(s) that many, if not most, virulence genes are expressed in laboratory cultures. While there is currently a substantial list of staphylococcal virulence factors, it is likely that this list is incomplete and is skewed by the limitations of the experiments used to identify them. Virulence factors that have already been identified generally include (i) bacterial surface proteins that are involved in processes such as adhesion and evasion of the host immune response and (ii) secreted exoproteins that degrade host tissue(s) and inactivate host defensive mechanisms (29).The genes encoding most virulence factors belong to an extensive regulon that is coordinately regulated in response to a variety of intra-and extracellular signals (1,5,21). Octapeptide signaling m...

The spread of multidrug-resistant Staphylococcus aureus (MRSA) strains in the clinical environment has begun to pose serious limits to treatment options. Yet virtually nothing is known about how resistance traits are acquired in vivo. Here, we apply the power of whole-genome sequencing to identify steps in the evolution of multidrug resistance in isogenic S. aureus isolates recovered periodically from the bloodstream of a patient undergoing chemotherapy with vancomycin and other antibiotics. After extensive therapy, the bacterium developed resistance, and treatment failed. Sequencing the first vancomycin susceptible isolate and the last vancomycin nonsusceptible isolate identified genome wide only 35 point mutations in 31 loci. These mutations appeared in a sequential order in isolates that were recovered at intermittent times during chemotherapy in parallel with increasing levels of resistance. The vancomycin nonsusceptible isolates also showed a 100-fold decrease in susceptibility to daptomycin, although this antibiotic was not used in the therapy. One of the mutated loci associated with decreasing vancomycin susceptibility (the vraR operon) was found to also carry mutations in six additional vancomycin nonsusceptible S. aureus isolates belonging to different genetic backgrounds and recovered from different geographic sites. As costs drop, whole-genome sequencing will become a useful tool in elucidating complex pathways of in vivo evolution in bacterial pathogens.

Custom-designed gene chips (Affymetrix) were used to determine genetic relatedness and gene expression profiles in Staphylococcus aureus isolates with increasing MICs of vancomycin that were recovered over a period of several weeks from the blood and heart valve of a patient undergoing extensive vancomycin therapy. The isolates were found to be isogenic as determined by the GeneChip based genotyping approach and thus represented a unique opportunity to study changes in gene expression that may contribute to the vancomycin resistance phenotype. No differences in gene expression were detected between the parent strain, JH1, and JH15, isolated from the nares of a patient contact. Few expression changes were observed between blood and heart valve isolates with identical vancomycin MICs. A large number of genes had altered expression in the late stage JH9 isolate (MIC ‫؍‬ 8 g/ml) compared to JH1 (MIC ‫؍‬ 1 g/ml). Most genes with altered expression were involved in housekeeping functions or cell wall biosynthesis and regulation. The sortase-encoding genes, srtA and srtB, as well as several surface protein-encoding genes were downregulated in JH9. Two hypothetical protein-encoding genes, SAS016 and SA2343, were dramatically overexpressed in JH9. Interestingly, 27 of the genes with altered expression in JH9 grown in drug-free medium were found to be also overexpressed when the parental strain JH1 was briefly exposed to inhibitory concentrations of vancomycin, and more than half (17 of 27) of the genes with altered expression belonged to determinants that were proposed to form part of a general cell wall stress stimulon (S. Utaida et al., Microbiology 149:2719-2732, 2003). Staphylococcus aureus strains with decreased susceptibility to vancomycin (so-called vancomycin-intermediate S. aureus[VISA] strains) have been identified in clinical specimens in several countries during the past decade (10,11,25). Although several abnormal physiological properties have been described in VISA isolates, the mechanisms of resistance and whether or not it involves acquisition of genes has remained unclear. A major problem in mechanistic studies has been the lack of availability of isogenic vancomycin-susceptible strains that could be considered the parental strains of the VISA isolates. Even when a VISA isolate shared a common multilocus sequence type with a fully sequenced vancomycin-susceptible strain, the clinical origins and times of isolation were far apart, excluding the possibility of attributing genetic and phenotypic differences to the antibiotic susceptibility phenotype.A recently described series of methicillin-resistant S. aureus (MRSA) isolates (JH1 through JH15) were recovered from a single patient during extensive chemotherapy with vancomycin (24). Bacterial isolates recovered at various times during therapy showed increasing vancomycin MICs which in isolates JH9 and JH14 eventually reached 8 g/ml, a value typical of many clinical VISA isolates. The first isolate chronologically, JH1, and all of the subsequent isolates sha...

Exposure of Staphylococcus aureus to cell wall inhibitors induces massive overexpression of a number of genes, provided that the VraSR two-component sensory regulatory system is intact. Inactivation of vraS blocks this transcriptional response and also causes a drastic reduction in the levels of resistance to beta-lactam antibiotics and vancomycin. We used an experimental system in which the essential cell wall synthesis gene of S. aureus, pbpB, was put under the control of an isopropyl-␤-D-thiogalactopyranoside-inducible promoter in order to induce reversible perturbations in cell wall synthesis without the use of any cell wall-active inhibitor. Changes in the level of transcription of pbpB were rapidly followed by parallel changes in the vraSR signal, and the abundance of the pbpB transcript was precisely mirrored by the abundance of the transcripts of vraSR and some additional genes that belong to the VraSR regulon. Beta-lactam resistance in S. aureus appears to involve a complex stress response in which VraSR performs the critical role of a sentinel system capable of sensing the perturbation of cell wall synthesis and allowing mobilization of genes that are essential for the generation of a highly resistant phenotype. One of the sites in cell wall synthesis "sensed" by the VraSR system appears to be a step catalyzed by PBP 2.Brief exposure of Staphylococcus aureus to inhibitors of cell wall synthesis invoke an immediate and massive change in the transcription of a unique set of genes, some of which-such as mgtB, murZ, and pbpB-are clearly involved with wall biosynthesis, while others have as yet undefined functions (14,16,31). It was proposed that these genes be referred to as members of a coordinately regulated "cell wall stimulon" (31). A particularly interesting member of this group of genes is vraSR, the DNA sequence of which shows features typical of a two-component sensory regulatory system (14). A careful study by Kuroda and colleagues (14) has demonstrated that transcription of vraSR is a specific response to inhibitors of various stages in wall synthesis: inhibitors of other cellular polymers and/or conditions of stress, such as shifts in temperature, pH, or osmotic pressure, did not alter the transcription of vraSR. Furthermore, and most importantly, the burst of transcription of genes following exposure to cell wall inhibitors was greatly reduced and/or annulled in bacteria in which vraSR was inactivated (14), indicating the essentiality of an intact vraSR system for this response. On the basis of these observations, it was proposed that VraSR functions as a sentinel system capable of detecting conditions that threaten to interrupt the synthesis of the bacterial cell wall. Nevertheless, the precise site(s) of perturbation of wall synthesis "sensed" by this system has just begun to be explored. Recent studies by Boyle-Vavra, Daum, and colleagues have allowed a better definition of the vraSR operon and its mode of induction by cell wall-active antibiotics (2, 3, 32).Kuroda and colleagues (14) also ...