The Signal Peptide of Staphylococcus aureus Panton Valentine Leukocidin LukS Component Mediates Increased Adhesion to Heparan Sulfates

Staphylococcus aureus necrotizing pneumonia is a severe disease caused by S. aureus strains carrying the Panton Valentine leukocidin (PVL) genes (lukS-PV & lukF-PV) encoded on various bacteriophages (such as phiSLT). Clinical PVL+ strains isolated from necrotizing pneumonia display an increased attachment to matrix molecules (type I and IV collagens and laminin), a phenotype that could play a role in bacterial adhesion to damaged airway epithelium during the early stages of necrotizing pneumonia (J Infect Dis 2004; 190: 1506–15). To investigate the basis of the observed adhesion of S. aureus PVL+ strains, we compared the ability of PVL+ and their isogenic PVL− strains to attach to various immobilized matrix molecules. The expression of recombinant fragments of the PVL subunits and the addition of synthetic peptides indicated that the processed LukS-PV signal peptide (LukS-PV SP) was sufficient to significantly enhance the ability of S. aureus to attach to extracellular matrix (ECM) components. Furthermore, we showed that adhesion to ECM components was inhibited by heparin and heparan sulfates (HS) suggesting that in vivo, HS could function as a molecular bridge between the matrix and S. aureus expressing the LukS-PV signal peptide. Site directed mutagenesis, biochemical and structural analyses of the LukS-PV signal peptide indicate that this peptide is present at the S. aureus surface, binds to HS in solid phase assay, and mediates the enhanced S. aureus matrix component adhesion. Our data suggests that after its cleavage by signal peptidase, the signal peptide is released from the membrane and associates to the cell wall through its unique C-terminus sequence, while its highly positively charged N-terminus is exposed on the bacterial surface, allowing its interaction with extracellular matrix-associated HS. This mechanism may provide a molecular bridge that enhances the attachment of the S. aureus PVL+ strains to ECM components exposed at damaged epithelial sites

Community-associated Methicillin-resistant Staphylococcus aureus, Switzerland

Two case-control studies evaluated the prevalence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) carriage at hospital admission and characteristics of patients with CA-MRSA. Among 14,253 patients, CA-MRSA prevalence was 0.9/1,000 admissions. Although 5 CA-MRSA isolates contained Panton-Valentine leukocidin, only 1 patient had a previous skin infection. No easily modifiable risk factor for CA-MRSA was identified

Rapid detection of Staphylococcus aureus Panton-Valentine leukocidin in clinical specimens by enzyme-linked immunosorbent assay and immunochromatographic tests

10.1128/JCM.02274-09Journal of Clinical Microbiology4841384-139

Keep an ear out for Francisella tularensis: otomastoiditis Cases after Canyoneering

We report here three unusual cases of otomastoiditis due to Francisella tularensis, complicated by cervical abscesses and persistent hearing loss, plus facial paralysis for one patient. Intriguingly, the three patients had practiced canyoneering independently in the same French river, between 2009 and 2014, several days before clinical symptoms onset. The results point out that fresh water exposure may be a potential contamination route for tularemia. Besides, due to the frequent complications and sequelae, we believe that F. tularensis should be considered as a possible etiology in case of otitis media, failure of the conventional antibiotic treatment, and suspicious exposure of the bacteria

Origin and Evolution of European Community-Acquired Methicillin- Resistant Staphylococcus aureus

ABSTRACT Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations. IMPORTANCE With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA

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

Multiple-Locus Variable Number Tandem Repeat Analysis of Staphylococcus Aureus: Comparison with Pulsed-Field Gel Electrophoresis and spa-Typing

(MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution.-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness. that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology

Cartography of Methicillin-Resistant S. aureus Transcripts: Detection, Orientation and Temporal Expression during Growth Phase and Stress Conditions

BACKGROUND: Staphylococcus aureus is a versatile bacterial opportunist responsible for a wide spectrum of infections. The severity of these infections is highly variable and depends on multiple parameters including the genome content of the bacterium as well as the condition of the infected host. Clinically and epidemiologically, S. aureus shows a particular capacity to survive and adapt to drastic environmental changes including the presence of numerous antimicrobial agents. Mechanisms triggering this adaptation remain largely unknown despite important research efforts. Most studies evaluating gene content have so far neglected to analyze the so-called intergenic regions as well as potential antisense RNA molecules. PRINCIPAL FINDINGS: Using high-throughput sequencing technology, we performed an inventory of the whole transcriptome of S. aureus strain N315. In addition to the annotated transcription units, we identified more than 195 small transcribed regions, in the chromosome and the plasmid of S. aureus strain N315. The coding strand of each transcript was identified and structural analysis enabled classification of all discovered transcripts. RNA purified at four time-points during the growth phase of the bacterium allowed us to define the temporal expression of such transcripts. A selection of 26 transcripts of interest dispersed along the intergenic regions was assessed for expression changes in the presence of various stress conditions including pH, temperature, oxidative shocks and growth in a stringent medium. Most of these transcripts showed expression patterns specific for the defined stress conditions that we tested. CONCLUSIONS: These RNA molecules potentially represent important effectors of S. aureus adaptation and more generally could support some of the epidemiological characteristics of the bacterium

A Field Guide to Pandemic, Epidemic and Sporadic Clones of Methicillin-Resistant Staphylococcus aureus

In recent years, methicillin-resistant Staphylococcus aureus (MRSA) have become a truly global challenge. In addition to the long-known healthcare-associated clones, novel strains have also emerged outside of the hospital settings, in the community as well as in livestock. The emergence and spread of virulent clones expressing Panton-Valentine leukocidin (PVL) is an additional cause for concern. In order to provide an overview of pandemic, epidemic and sporadic strains, more than 3,000 clinical and veterinary isolates of MRSA mainly from Germany, the United Kingdom, Ireland, France, Malta, Abu Dhabi, Hong Kong, Australia, Trinidad & Tobago as well as some reference strains from the United States have been genotyped by DNA microarray analysis. This technique allowed the assignment of the MRSA isolates to 34 distinct lineages which can be clearly defined based on non-mobile genes. The results were in accordance with data from multilocus sequence typing. More than 100 different strains were distinguished based on affiliation to these lineages, SCCmec type and the presence or absence of PVL. These strains are described here mainly with regard to clinically relevant antimicrobial resistance- and virulence-associated markers, but also in relation to epidemiology and geographic distribution. The findings of the study show a high level of biodiversity among MRSA, especially among strains harbouring SCCmec IV and V elements. The data also indicate a high rate of genetic recombination in MRSA involving SCC elements, bacteriophages or other mobile genetic elements and large-scale chromosomal replacements

A Field Guide to Pandemic, Epidemic and Sporadic Clones of Methicillin-Resistant Staphylococcus aureus

In recent years, methicillin-resistant Staphylococcus aureus (MRSA) have become a truly global challenge. In addition to the long-known healthcare-associated clones, novel strains have also emerged outside of the hospital settings, in the community as well as in livestock. The emergence and spread of virulent clones expressing Panton-Valentine leukocidin (PVL) is an additional cause for concern. In order to provide an overview of pandemic, epidemic and sporadic strains, more than 3,000 clinical and veterinary isolates of MRSA mainly from Germany, the United Kingdom, Ireland, France, Malta, Abu Dhabi, Hong Kong, Australia, Trinidad & Tobago as well as some reference strains from the United States have been genotyped by DNA microarray analysis. This technique allowed the assignment of the MRSA isolates to 34 distinct lineages which can be clearly defined based on non-mobile genes. The results were in accordance with data from multilocus sequence typing. More than 100 different strains were distinguished based on affiliation to these lineages, SCCmec type and the presence or absence of PVL. These strains are described here mainly with regard to clinically relevant antimicrobial resistance- and virulence-associated markers, but also in relation to epidemiology and geographic distribution. The findings of the study show a high level of biodiversity among MRSA, especially among strains harbouring SCCmec IV and V elements. The data also indicate a high rate of genetic recombination in MRSA involving SCC elements, bacteriophages or other mobile genetic elements and large-scale chromosomal replacements