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Staphylococcus pseudintermedius is a frequent cause of infections in dogs. Infectious isolates of this coagulase-positive staphylococcal species are often methicillin- and multidrug-resistant, which complicates therapy. In staphylococci, methicillin resistance is encoded by determinants found on mobile genetic elements called Staphylococcal Chromosome Cassette mec (SCCmec), which, in addition to methicillin resistance factors, sometimes encode additional genes, such as further resistance factors and, rarely, virulence determinants. In this study, we analyzed SCCmec in a collection of infectious methicillin-resistant S. pseudintermedius (MRSP) isolates from predominant lineages in the United States. We found that several lineages characteristically have specific types of SCCmec elements and Agr types and harbor additional factors in their SCCmec elements that may promote virulence or affect DNA uptake. All isolates had SCCmec-encoded restriction–modification (R-M) systems of types I or II, and sequence types (STs) ST84 and ST64 had one type II and one type I R-M system, although the latter lacked a complete methylation enzyme gene. ST68 isolates also had an SCCmec-encoded CRISPR system. ST71 isolates had a psm-mec gene, which, in all but apparently Agr-dysfunctional isolates, produced a PSM-mec peptide toxin, albeit at relatively small amounts. This study gives detailed insight into the composition of SCCmec elements in infectious isolates of S. pseudintermedius and lays the genetic foundation for further efforts directed at elucidating the contribution of identified accessory SCCmec factors in impacting SCCmec-encoded and thus methicillin resistance-associated virulence and resistance to DNA uptake in this leading canine pathogen. [ABSTRACT FROM AUTHOR]

In contrast to the virulent human skin commensal Staphylococcus aureus, which secretes a plethora of toxins, other staphylococci have much reduced virulence. In these species, commonly the only toxins are those of the phenolsoluble modulin (PSM) family. PSMs are species-specific and have only been characterized in a limited number of species. S. xylosus is a usually innocuous commensal on the skin of mice and other mammals. Prompted by reports on the involvement of PSMs in atopic dermatitis (AD) and the isolation of S. xylosus from mice with AD-like symptoms, we here identified and characterized PSMs of S. xylosus with a focus on a potential involvement in AD phenotypes. We found that most clinical S. xylosus strains produce two PSMs, one of the shorter a- and one of the longer b-type, which were responsible for almost the entire lytic and pro-inflammatory capacities of S. xylosus. Importantly, PSMa of S. xylosus caused lysis and degranulation of mast cells at degrees higher than that of S. aureus d-toxin, the main PSM previously associated with AD. However, S. xylosus did not produce significant AD symptoms in wild-type mice as opposed to S. aureus, indicating that promotion of AD by S. xylosus likely requires a predisposed host. Our study indicates that non-specific cytolytic potency rather than specific interaction underlies PSM-mediated mast cell degranulation and suggest that the previously reported exceptional potency of d-toxin of S. aureus is due to its high-level production. Furthermore, they suggest that species that produce cytolytic PSMs, such as S. xylosus, all have the capacity to promote AD, but a high combined level of PSM cytolytic potency is required to cause AD in a non-predisposed host. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is one of the most frequent worldwide causes of morbidity and mortality due to an infectious agent. This pathogen can cause a wide variety of diseases, ranging from moderately severe skin infections to fatal pneumonia and sepsis. Treatment of S. aureus infections is complicated by antibiotic resistance and a working vaccine is not available. There has been ongoing and increasing interest in the extraordinarily high number of toxins and other virulence determinants that S. aureus produces and how they impact disease. In this review, we will give an overview of how S. aureus initiates and maintains infection and discuss the main determinants involved. A more in-depth understanding of the function and contribution of S. aureus virulence determinants to S. aureus infection will enable us to develop anti-virulence strategies to counteract the lack of an anti-S. aureus vaccine and the ever-increasing shortage of working antibiotics against this important pathogen. [ABSTRACT FROM AUTHOR]

Staphylococcal enterotoxin B (SEB), which is produced by the major human pathogen, Staphylococcus aureus, represents a powerful superantigenic toxin and is considered a bioweapon. However, the contribution of SEB to S. aureus pathogenesis has never been directly demonstrated with genetically defined mutants in clinically relevant strains. Many isolates of the predominant Asian community-associated methicillin-resistant S. aureus lineage sequence type (ST) 59 harbor seb, implying a significant role of SEB in the observed hypervirulence of this lineage. We created an isogenic seb mutant in a representative ST59 isolate and assessed its virulence potential in mouse infection models. We detected a significant contribution of seb to systemic ST59 infection that was associated with a cytokine storm. Our results directly demonstrate that seb contributes to S. aureus pathogenesis, suggesting the value of including SEB as a target in multipronged antistaphylococcal drug development strategies. Furthermore, they indicate that seb contributes to fatal exacerbation of community-associated methicillin-resistant S. aureus infection. [ABSTRACT FROM AUTHOR]

Bacterial sepsis is a major killer in hospitalized patients. Coagulase-negative staphylococci (CNS) with the leading species Staphylococcus epidermidis are the most frequent causes of nosocomial sepsis, with most infectious isolates being methicillin-resistant. However, which bacterial factors underlie the pathogenesis of CNS sepsis is unknown. While it has been commonly believed that invariant structures on the surface of CNS trigger sepsis by causing an over-reaction of the immune system, we show here that sepsis caused by methicillin-resistant S. epidermidis is to a large extent mediated by the methicillin resistance island-encoded peptide toxin, PSM-mec. PSM-mec contributed to bacterial survival in whole human blood and resistance to neutrophil-mediated killing, and caused significantly increased mortality and cytokine expression in a mouse sepsis model. Furthermore, we show that the PSM-mec peptide itself, rather than the regulatory RNA in which its gene is embedded, is responsible for the observed virulence phenotype. This finding is of particular importance given the contrasting roles of the psm-mec locus that have been reported in S. aureus strains, inasmuch as our findings suggest that the psm-mec locus may exert effects in the background of S. aureus strains that differ from its original role in the CNS environment due to originally “unintended” interferences. Notably, while toxins have never been clearly implied in CNS infections, our tissue culture and mouse infection model data indicate that an important type of infection caused by the predominant CNS species is mediated to a large extent by a toxin. These findings suggest that CNS infections may be amenable to virulence-targeted drug development approaches. [ABSTRACT FROM AUTHOR]

Abstract: A comparison was made of the cytotoxic activity and secondary structural features of four recombinant forms of adenylate cyclase toxin (CyaA). These forms were fully functional CyaA, CyaA lacking adenylate cyclase enzymatic activity (CyaA*), and non-acylated forms of these toxins, proCyaA and proCyaA*. At a toxin concentration >1μg/ml, CyaA* was as cytotoxic towards J774.2 cells as CyaA and mediated cell killing at a faster rate than CyaA. At concentrations <0.5μg/ml, CyaA* was less cytotoxic than CyaA and, at <0.1μg/ml of CyaA*, no activity was detected. CyaA, but not CyaA*, was able to induce caspase 3/7 activity, a measure of apoptosis. ProCyaA and proCyaA* had no detectable cytotoxic or apoptotic activity. CyaA caused 50% inhibition of the zymosan-stimulated oxidative burst at 0.003μg/ml, whereas a ∼500-fold greater toxin concentration of CyaA* or proCyaA was needed for 50% inhibition. ProCyaA* was inactive. CyaA is a calcium-binding protein and far UV circular dichroism (CD), near UV CD and fluorescence spectra analyses showed that all the forms of CyaA had similar overall structures at different calcium concentrations up to 5.0mM. At 7.5mM CaCl2, the far UV spectrum of CyaA altered significantly, indicating a change in secondary structure associated with high β-sheet content or a β-aggregated state, whereas the spectrum of CyaA* showed only a slight alteration at this calcium concentration. Near UV CD and fluorescence studies were consistent with a rearrangement of secondary structural elements in the presence of CaCl2 for all CyaA forms. There was a marked dependence on protein concentration of the far UV spectra of these CyaA forms, implying an interaction between individual molecules at higher protein concentrations. [Copyright &y& Elsevier]

Phenol-soluble modulins (PSMs) are amphipathic peptides produced by staphylococci that have multiple functions in pathogenesis. For example, they may function as cytotoxins and pro-inflammatory agents. Additionally, in a recent study we demonstrated that Staphylococcus aureus PSMs structure biofilms and cause dissemination during biofilm infection. Based on those results suggesting a surfactant-like mechanism by which PSMs work, we here propose that all PSM functions in pathogenesis arose from an original role in non-pathogenic surface colonization. This original role may have included overcoming surface tension in environments of strongly varying hydrophobicity and emulsification of hydrophobic molecules for use as food sources. [ABSTRACT FROM AUTHOR]