Current methods for differentiating isolates of predominant lineages of pathogenic bacteria often do not provide sufficient resolution to define precise relationships. Here, we describe a high-throughput genomics approach that provides a high-resolution view of the epidemiology and microevolution of a dominant strain of methicillin-resistant Staphylococcus aureus (MRSA). This approach reveals the global geographic structure within the lineage, its intercontinental transmission through four decades, and the potential to trace person-to-person transmission within a hospital environment. The ability to interrogate and resolve bacterial populations is applicable to a range of infectious diseases, as well as microbial ecology.

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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 ...

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.

Tn551 inactivation has identified several determinants--fem or auxiliary genes--that, in addition to the mecA gene, are also critical for the expression of high-level and homogeneous resistance to methicillin. Genetic and/or biochemical analysis has shown that of the nearly dozen aux mutations described so far most are in genes involved in cell wall synthesis (murE, pbp2, glmM, glnR, femA/B, llm, etc.) or in complex regulatory functions (sigmaB), suggesting that optimal expression of resistance may involve the cooperative functioning of a number of genes in cell wall metabolism as well as stress response. The exact mechanism of these functions is not known. In an attempt to explore this unusual aspect of methicillin resistance more fully, a Tn551 transposon library, constructed in the background of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL, was screened for all independent insertional mutants in which the level of methicillin resistance of the parental strain (MIC, 1,600 microg/ml) was reduced by at least 15-fold and up to 500-fold. We now describe the sequencing of 21 Tn551-inactivated genes and their vicinities in 23 new auxiliary mutants that have been studied before. Using the inverted polymerase chain reaction (IPCR), we amplified fragments corresponding to the right and left junction of the Tn551 insertions, which were then sequenced by primer walking. The two largest groups of these new auxiliary genes encoded either proteins of unknown functions (6 genes) or showed homology with genes encoding proteins involved with putative sensory/regulatory activities (7 genes: protein kinases, ABC transporters, and a catabolite control protein). Sequencing upstream and downstream allowed the identification of a number of additional open reading frames, some of which may also include functions relevant for the expression of antibiotic resistance.

Background: The recently isolated MRSA LGA251 has low resistance and carries a new mecA homolog.Results: PBP2ALGA, the protein product of the new mecA, showed a “preference” for penicillins and instability at 37 °C. mecALGA251 introduced into susceptible S. aureus allowed expression of high-level resistance.Significance: This study provides insights into the relationship between structure and function of PBP2A-like proteins.

Mycobacterium tuberculosis kills more people than any other bacterial pathogen and is becoming increasingly untreatable due to the emergence of resistance. Verapamil, an FDA-approved calcium channel blocker, potentiates the effect of several antituberculosis (anti-TB) drugs in vitro and in vivo. This potentiation is widely attributed to inhibition of the efflux pumps of M. tuberculosis, resulting in intrabacterial drug accumulation. Here, we confirmed and quantified verapamil's synergy with several anti-TB drugs, including bedaquiline (BDQ) and clofazimine (CFZ), but found that the effect is not due to increased intrabacterial drug accumulation. We show that, consistent with its in vitro potentiating effects on anti-TB drugs that target or require oxidative phosphorylation, the cationic amphiphile verapamil disrupts membrane function and induces a membrane stress response similar to those seen with other membrane-active agents. We recapitulated these activities in vitro using inverted mycobacterial membrane vesicles, indicating a direct effect of verapamil on membrane energetics. We observed bactericidal activity against nonreplicating “persister” M. tuberculosis that was consistent with such a mechanism of action. In addition, we demonstrated a pharmacokinetic interaction whereby human-equivalent doses of verapamil caused a boost of rifampin exposure in mice, providing a potential explanation for the observed treatment-shortening effect of verapamil in mice receiving first-line drugs. Our findings thus elucidate the mechanistic basis for verapamil's potentiation of anti-TB drugs in vitro and in vivo and highlight a previously unrecognized role for the membrane of M. tuberculosis as a pharmacologic target.

A carboxy-terminal fragment of murF was used to construct and insert a suicide plasmid into the chromosomal copy of the gene in the highly and homogeneously methicillin-resistant Staphylococcus aureus (MRSA) strain COL by Campbell type integration. The plasmid insertion generated a mutant in which the MIC value for oxacillin was reduced from 400 microg/ml of the parental strain to 0.75 microg/ml in 90% of the cells of the mutant cultures that were heterogeneous: they contained subpopulations of bacteria with a frequency of 10(-3) that were capable of expressing resistance at nearly the parental level. The impact of the murF mutation on antibiotic resistance was selective for beta-lactam antibiotics: there was no change in the susceptibility of the mutant to D-cycloserine, fosfomycin, beta-D-chloro-alanine, moenomycin, bacitracin, or vancomycin. Analysis of the mutant peptidoglycan showed decrease in the percentage of oligomeric components in rough proportion to the accumulation of several abnormal muropeptide components, which were identified as structural variants of the disaccharide tripeptide monomer. An abnormal cell wall precursor identified as UDP MurNac tripeptide was also detected in the cytoplasmic pool of the mutant strain. A normal proportion of oligomers and a greatly reduced representation of the disaccharide tripeptide were demonstrated in the cell wall of the murF mutant's subpopulation that has retained the parental level of resistance. Northern analysis demonstrated a drastic reduction in the transcription rate of mecA in mutant F9 whereas mecA transcription increased in the subpopulation of bacteria that retained high-level resistance.