Impact of accessory gene regulator (agr) dysfunction on vancomycin pharmacodynamics among Canadian community and health-care associated methicillin-resistant Staphylococcus aureus

<p>Abstract</p> <p>Background</p> <p>The accessory gene regulator (<it>agr</it>) is a quorum sensing cluster of genes which control colonization and virulence in <it>Staphylococcus aureus</it>. We evaluated <it>agr </it>function in community- (CA) and healthcare-associated (HA) MRSA, to compare the pharmacodynamics and bactericidal activity of vancomycin against <it>agr </it>functional and dysfunctional HA-MRSA and CA-MRSA.</p> <p>Methods</p> <p>40 clinical isolates of MRSA from the Canadian Nosocomial Infection Surveillance Program were evaluated for delta-haemolysin production, as a surrogate marker of <it>agr </it>function. Time kill experiments were performed for vancomycin at 0 to 64 times the MIC against an initial inoculum of 10⁶and 10⁸cfu/ml of <it>agr </it>functional and dysfunctional CA-MRSA and HA-MRSA and these data were fit to a hill-type pharmacodynamic model.</p> <p>Results</p> <p>15% isolates were <it>agr </it>dysfunctional, which was higher among HA-MRSA (26.3%) versus CA-MRSA (4.76%). Against a low initial inoculum of 10⁶cfu/ml of CA-MRSA, vancomycin pharmacodynamics were similar among <it>agr </it>functional and dysfunctional strains. However, against a high initial inoculum of 10⁸cfu/ml, killing activity was notably attenuated against <it>agr </it>dysfunctional CA-MRSA (USA400) and HA-MRSA (USA100). CA-MRSA displayed a 20.0 fold decrease in the maximal reduction in bacterial counts (Emax) which was 3.71 log₁₀CFU/ml for <it>agr </it>functional vs. 2.41 log₁₀CFU/ml for <it>agr </it>dysfunctional MRSA (p = 0.0007).</p> <p>Conclusions</p> <p>Dysfunction in <it>agr </it>was less common among CA-MRSA vs. HA-MRSA. <it>agr </it>dysfunction demonstrated an impact on vancomycin bactericidal activity and pharmacodynamics against a high initial inoculum of CA-MRSA and HA-MRSA, which may have implications for optimal antimicrobial therapy against persistent, difficult to treat MRSA infections.</p

Cloning and expression of two different genes from Streptococcus dysgalactiae encoding fibronectin receptors.

Binding of bacteria to fibronectin has been implicated as a mechanism of bacterial adhesion to the host tissue. In this report we have analyzed the binding of a strain of Streptococcus dysgalactiae to fibronectin. The cells bind to a site in the NH2-terminal domain of the protein via trypsin-sensitive cell surface components. Furthermore, a lysate prepared by sonication of streptococcal cells contained fibronectin-binding proteins that inhibit the binding of the ligand to intact bacteria. When the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to an Immobilon-P filter, and probed with 125I-labeled fibronectin, a 140-kDa fibronectin-binding protein was identified along with a number of smaller binding proteins. A genomic DNA library was constructed and screened for the expression of fibronectin-binding proteins. Two clones were isolated and shown to contain unrelated inserts by restriction mapping and cross-hybridization experiments. The two encoded proteins were also immunologically distinct although both bound to the same region of the fibronectin molecule, and both effectively inhibited the binding of 125I-fibronectin to bacterial cells. Immunological analyses showed that only one of the two proteins tentatively identified as fibronectin receptors was expressed in detectable quantities in the Streptococcus dysgalactiae strain under the culture conditions employed

Global analysis of community-associated methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro and during infection

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a threat to human health worldwide. Although progress has been made, mechanisms of CA-MRSA pathogenesis are poorly understood and a comprehensive analysis of CA-MRSA exoproteins has not been conducted. To address that deficiency, we used proteomics to identify exoproteins made by MW2 (USA400) and LAC (USA300) during growth in vitro. Two hundred and fifty unique exoproteins were identified by 2-dimensional gel electrophoresis coupled with automated direct infusion-tandem mass spectrometry (ADI-MS/MS) analysis. Eleven known virulence-related exoproteins differed in abundance between the strains, including alpha-haemolysin (Hla), collagen adhesin (Cna), staphylokinase (Sak), coagulase (Coa), lipase (Lip), enterotoxin C3 (Sec3), enterotoxin Q (Seq), V8 protease (SspA) and cysteine protease (SspB). Mice infected with MW2 or LAC produced antibodies specific for known or putative virulence factors, such as autolysin (Atl), Cna, Ear, ferritin (Ftn), Lip, 1-phosphatidylinositol phosphodiesterase (Plc), Sak, Sec3 and SspB, indicating the exoproteins are made during infection in vivo. We used confocal microscopy to demonstrate aureolysin (Aur), Hla, SspA and SspB are produced following phagocytosis by human neutrophils, thereby linking exoprotein production in vitro with that during host–pathogen interaction. We conclude that the exoproteins identified herein likely account in part for the success of CA-MRSA as a human pathogen

Chemical Synthesis of Staphyloferrin B Affords Insight into the Molecular Structure, Iron Chelation, and Biological Activity of a Polycarboxylate Siderophore Deployed by the Human Pathogen

Staphyloferrin B (SB) is a citrate-based polycarboxylate siderophore produced and utilized by the human pathogen Staphylococcus aureus for acquiring iron when colonizing the vertebrate host. The first chemical synthesis of SB is reported, which enables further molecular and biological characterization and provides access to structural analogues of the siderophore. Under conditions of iron limitation, addition of synthetic SB to bacterial growth medium recovered the growth of the antibiotic resistant community isolate S. aureus USA300 JE2. Two structural analogues of SB, epiSB and SBimide, were also synthesized and employed to investigate how epimerization of the citric acid moiety or imide formation influence its function as a siderophore. Epimerization of the citric acid stereocenter perturbed the iron-binding properties and siderophore function of SB as evidenced by experimental and computational modeling studies. Although epiSB provided growth recovery to S. aureus USA300 JE2 cultured in iron-deficient medium, the effect was attenuated relative to that of SB. Moreover, SB more effectively sequestered the Fe(III) bound to human holo-transferrin, an iron source of S. aureus, than epiSB. SBimide is an imide analogous to the imide forms of other citric acid siderophores that are often observed when these molecules are isolated from natural sources. Here, SBimide is shown to be unstable, converting to native SB at physiological pH. SB is considered to be a virulence factor of S. aureus, a pathogen that poses a particular threat to public health because of the number of drug-resistant strains emerging in hospital and community settings. Iron acquisition by S. aureus is important for its ability to colonize the human host and cause disease, and new chemical insights into the structure and function of SB will inform the search for new therapeutic strategies for combating S. aureus infections.Alfred Benzon Foundation (Postdoctoral fellowship)Pacific Southwest Regional Center of ExcellenceAlfred P. Sloan Foundatio

Diet rapidly and reproducibly alters the human gut microbiome

Long-term diet influences the structure and activity of the trillions of microorganisms residing in the human gut1–5, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here, we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila, and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale, and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals2, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi, and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids, and the outgrowth of microorganisms capable of triggering inflammatory bowel disease6. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles

Induction of the staphylococcal proteolytic cascade by antimicrobial fatty acids in community acquired methicillin resistant Staphylococcus aureus.

Community acquired methicillin resistant Staphylococcus aureus (CA-MRSA), and the USA300 strain of CA-MRSA in particular, are known for their rapid community transmission, and propensity to cause aggressive skin and soft tissue infections. To assess factors that contribute to these hallmark traits of CA-MRSA, we evaluated how growth of USA300 and production of secreted virulence factors was influenced on exposure to physiologic levels of unsaturated free fatty acids that would be encountered on the skin or anterior nares, which represent the first sites of contact with healthy human hosts. There was a sharp threshold between sub-inhibitory and inhibitory concentrations, such that 100 µM sapienic acid (C16∶1) and linoleic acid (C18∶1) were sufficient to prevent growth after 24 h incubation, while 25 µM allowed unrestricted growth, and 50 µM caused an approximate 10-12 h lag, followed by unimpeded exponential growth. Conversely, saturated palmitic or stearic acids did not affect growth at 100 µM. Although growth was not affected by 25 µM sapienic or linoleic acid, these and other unsaturated C16 and C18 fatty acids, but not their saturated counterparts, promoted robust production of secreted proteases comprising the Staphylococcal proteolytic cascade. This trait was also manifested to varying degrees in other CA-MRSA, and in genetically diverse methicillin susceptible S. aureus strains. Therefore, induction of the Staphylococcal proteolytic cascade by unsaturated fatty acids is another feature that should now be evaluated as a potential contributing factor in the aggressive nature of skin and soft tissue infections caused by USA300, and as a general virulence mechanism of S. aureus

Staphylococcus aureus Uses the GraXRS Regulatory System To Sense and Adapt to the Acidified Phagolysosome in Macrophages

Macrophages are critical to innate immunity due to their ability to phagocytose bacteria. The macrophage phagolysosome is a highly acidic organelle with potent antimicrobial properties, yet remarkably, ingested Staphylococcus aureus replicates within this niche. Herein we demonstrate that S. aureus requires the GraXRS regulatory system for growth within this niche, while the SaeRS and AgrAC two-component regulatory systems and the α-phenol soluble modulins are dispensable. Importantly, we find that it is exposure to acidic pH that is required for optimal growth of S. aureus inside fully acidified macrophage phagolysosomes. Exposure of S. aureus to acidic pH evokes GraS signaling, which in turn elicits an adaptive response that endows the bacteria with increased resistance to antimicrobial effectors, such as antimicrobial peptides, encountered inside macrophage phagolysosomes. Notably, pH-dependent induction of antimicrobial peptide resistance in S. aureus requires the GraS sensor kinase. GraS and MprF, a member of the GraS regulon, play an important role for bacterial survival in the acute stages of systemic infection, where in murine models of infection, S. aureus resides within liver-resident Kupffer cells. We conclude that GraXRS represents a vital regulatory system that functions to allow S. aureus to evade killing, prior to commencement of replication, within host antibacterial immune cells.S. aureus can infect any site of the body, including the microbicidal phagolysosome of the macrophage. The ability of S. aureus to infect diverse niches necessitates that the bacteria be highly adaptable. Here we show that S. aureus responds to phagolysosome acidification to evoke changes in gene expression that enable the bacteria to resist phagolysosomal killing and to promote replication. Toxin production is dispensable for this response; however, the bacteria require the sensor kinase GraS, which transduces signals in response to acidic pH. GraS is necessary for phagolysosomal replication and survival of S. aureus in the acute stage of systemic infection. Disruption of this S. aureus adaptation would render S. aureus susceptible to phagocyte restriction