Levels of alpha-toxin correlate with distinct phenotypic response profiles of blood mononuclear cells and with agr background of community-associated Staphylococcus aureus isolates

Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of α-toxin than did the proliferative supernatants. Addition of α-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, δ-toxin or phenol soluble modulin α-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes

Novel Rearrangements in the Staphylococcal Cassette Chromosome <i>Mec</i> Type V Elements of Indian ST772 and ST672 Methicillin Resistant <i>Staphylococcus aureus</i> Strains

<div><p><i>Staphylococcus aureus</i> is a commensal gram positive bacteria which causes severe and non severe infections in humans and livestock. In India, ST772 is a dominant and ST672 is an emerging clone of <i>Staphylococcus aureus</i>. Both cause serious human diseases, and carry type V SCC<i>mec</i> elements. The objective of this study was to characterize SCC<i>mec</i> type V elements of ST772 and ST672 because the usual PCR methods did not amplify all primers specific to the type. Whole genome sequencing analysis of seven ST772 and one ST672 <i>S. aureus</i> isolates revealed that the SCC<i>mec</i> elements of six of the ST772 isolates were the smallest of the extant type V elements and in addition have several other novel features. Only one ST772 isolate and the ST672 isolate carried bigger SCC<i>mec</i> cassettes which were composites carrying multiple <i>ccrC</i> genes. These cassettes had some similarities to type V SCC<i>mec</i> element from M013 isolate (ST59) from Taiwan in certain aspects. SCC<i>mec</i> elements of all Indian isolates had an inversion of the <i>mec</i> complex, similar to the bovine SCC<i>mec</i> type X. This study reveals that six out of seven ST772 <i>S. aureus</i> isolates have a novel type V (5C2) SCC<i>mec</i> element while one each of ST772 and ST672 isolates have a composite SCC<i>mec</i> type V element (5C2&5) formed by the integration of type V SCC<i>mec</i> into a MSSA carrying a SCC element, in addition to the <i>mec</i> gene complex inversions and extensive recombinations.</p></div

Comparison of orfs from SCC<i>mec</i> elements of S. <i>aureus</i> 3957 and corresponding orthologs of other ST772, ST672 and reference strains.

a<p>Identity of the amino acid sequence to each ortholog (orf);</p>b<p>Corresponding Ortholog in the reference strain,</p>C<p>Hypothetical Protein;</p>d<p>ugpQ: glycerophosphoryl diester phosphodiesterase;</p>e<p>PhnB-like proteins adopting structural fold similar to bleomycin resistance proteins;</p>f<p>No Identity.</p

Clinical history, molecular characterization and accession numbers of sequenced and reference isolates.

a<p>: Whole genome sequenced contigs;</p>b<p>: Annotations for the SCC<i>mec</i> region;</p>c<p>Huang TW, Chen F, Miu WC, Liao TL, Lin AC, et al. (2012) J. Bacteriol 194:1256-1257;</p>d<p>: O'Brien FG, Coombs GW, Pearson JC, Christiansen KJ, Grubb WB. (2005) Antimicrob. Agents Chemother 49: 5129-5132;</p>e:<p>:Li S, Skov RL, Han X, Larsen AR, Larsen J, et al. (2011) Antimicrob. Agents Chemother 55: 3046-3050;</p>f<p>: Ito, T., Y. Katayama, K. Asada, N. Mori, K. Tsutsumimoto, C. Tiensasitorn, and K. Hiramatsu. 2001. Antimicrob. Agents Chemother. 45: 1323-1336.</p

Molecular Basis for the Role of Staphylococcus aureus Penicillin Binding Protein 4 in Antimicrobial Resistance▿

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the d-Ala-d-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala. β-Lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a β-lactamase and is not trapped as an acyl intermediate with β-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance

Influence of the AgrC-AgrA Complex on the Response Time of Staphylococcus aureus Quorum Sensing

The Staphylococcus aureus agr quorum-sensing system plays a major role in the transition from the persistent to the virulent phenotype. S. aureus agr type I to IV strains are characterized by mutations in the sensor domain of the histidine kinase AgrC and differences in the sequences of the secreted autoinducing peptides (AIP). Here we demonstrate that interactions between the cytosolic domain of AgrC (AgrC(Cyto)) and the response regulator domain of AgrA (AgrA(RR)) dictate the spontaneity of the cellular response to AIP stimuli. The crystal structure of AgrC(Cyto) provided a basis for a mechanistic model of AgrC-AgrA interactions. This model enabled an analysis of the biochemical and biophysical parameters of AgrC-AgrA interactions in the context of the conformational features of the AgrC-AgrA complex. This analysis revealed distinct sequence and conformational features that determine the affinity, specificity, and kinetics of the phosphotransfer reaction. This step, which governs the response time for transcriptional reengineering triggered by an AIP stimulus, is independent of the agr type and similar for agonist and antagonist stimuli. These experimental data could serve as a basis on which to validate simulations of the quorum-sensing response and for strategies that employ the agr quorum-sensing system to combat biofilm formation in S. aureus infections

Characterization and Acceptor Preference of a Soluble Meningococcal Group C Polysialyltransferase▿ †

Vaccines against Neisseria meningitidis group C are based on its α-2,9-linked polysialic acid capsular polysaccharide. This polysialic acid expressed on the surface of N. meningitidis and in the absence of specific antibody serves to evade host defense mechanisms. The polysialyltransferase (PST) that forms the group C polysialic acid (NmC PST) is located in the cytoplasmic membrane. Until recently, detailed characterization of bacterial polysialyltransferases has been hampered by a lack of availability of soluble enzyme preparations. We have constructed chimeras of the group C polysialyltransferase that catalyzes the formation α-2,9-polysialic acid as a soluble enzyme. We used site-directed mutagenesis to determine the region of the enzyme necessary for synthesis of the α-2,9 linkage. A chimera of NmB and NmC PSTs containing only amino acids 1 to 107 of the NmB polysialyltransferase catalyzed the synthesis of α-2,8-polysialic acid. The NmC polysialyltransferase requires an exogenous acceptor for catalytic activity. While it requires a minimum of a disialylated oligosaccharide to catalyze transfer, it can form high-molecular-weight α-2,9-polysialic acid in a nonprocessive fashion when initiated with an α-2,8-polysialic acid acceptor. De novo synthesis in vivo requires an endogenous acceptor. We attempted to reconstitute de novo activity of the soluble group C polysialyltransferase with membrane components. We found that an acapsular mutant with a defect in the polysialyltransferase produces outer membrane vesicles containing an acceptor for the α-2,9-polysialyltransferase. This acceptor is an amphipathic molecule and can be elongated to produce polysialic acid that is reactive with group C-specific antibody

Genotyping of Methicillin-Resistant Staphylococcus aureus Strains from Two Hospitals in Bangalore, South India

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen in India, and up to 70% methicillin resistance has been reported from hospitals in various parts of India. Hospitals use phenotyping for the most part, and molecular genotyping is not done. Here we report on the genotyping of 82 single-patient isolates from two hospitals in Bangalore, South India, for the first time. Most of the strains possessed type III or IIIA staphylococcal cassette chromosome (SCCmec) cassettes, and we did not detect strains with type I, IA, or II cassettes. Most isolates also contained the type III cassette chromosome recombinase (ccr) AB region. Multilocus sequence typing (MLST) and staphylococcal protein A (spa) typing of a selected number of isolates have been carried out. Although most isolates that were chosen for MLST and spa typing had the same patterns, they were quite diverse in their pulsed-field gel electrophoresis (PFGE) patterns. PFGE, MLST, and spa typing of the Indian strains revealed that they are related to the previously described Hungarian and Brazilian clones

al complexes and virulence factors of Staphylococcus aureus from several cities in India.

International audienceABSTRACT: BACKGROUND: Diseases from Staphylococcus aureus are a major problem in Indian hospitals and recent studies point to infiltration of community associated methicillin resistant S. aureus (CA-MRSA) into hospitals. Although CA-MRSA are genetically different from nosocomial MRSA, the distinction between the two groups is blurring as CA-MRSA are showing multidrug resistance and are endemic in many hospitals. Our survey of samples collected from Indian hospitals between 2004 and 2006 had shown mainly hospital associated methicillin resistant Staphylococcus aureus (HA-MRSA) carrying staphylococcal cassette chromosome mec (SCCmec) type III and IIIA. But S. aureus isolates collected from 2007 onwards from community and hospital settings in India have shown SCCmec type IV and V cassettes while several variations of type IV SCCmec cassettes from IVa to IVj have been found in other parts of the world. In the present study, we have collected nasal swabs from rural and urban healthy carriers and pus, blood etc from in patients from hospitals to study the distribution of SCCmec types and sequence types (ST) in the community and hospital environment. We performed molecular characterization of all the isolates to determine their lineage and microarray of select isolates from each sequence type to analyze their toxins, virulence and immune-evasion factors. RESULTS: Molecular analyses of 68 S. aureus isolates from in and around Bengaluru and three other Indian cities have been carried out. Our strains fall into fifteen ST with all major clonal complexes (CC) present along with some minor ones. The dominant MRSA clones are ST22 and ST772 among healthy carriers and patients. We are reporting two methicillin sensitive S. aureus (MSSA) isolates belonging to ST291 (related to ST398 which is live stock associated), ST1208 (CC8), and ST672 as emerging MRSA clones in this study for the first time. Sixty nine percent of isolates carry Panton- Valentine Leucocidin genes (PVL) along with many other toxins. There is more diversity of ST among methicillin sensitive S. aureus than resistant ones. Microarray analysis of isolates belonging to different ST for the first time gives an insight into major toxins, virulence factors, adhesion and immune evasion factors present among the isolates in various parts of India. CONCLUSIONS: S. aureus isolates reported in this study belong to a highly diverse group of ST and CC and we are reporting several new ST which have not been reported earlier along with factors influencing virulence and host pathogen interactions