Background: Estimates of relationships among Staphylococcus species have been hampered by poor and inconsistent resolution of phylogenies based largely on single gene analyses incorporating only a limited taxon sample. As such, the evolutionary relationships and hierarchical classification schemes among species have not been confidently established. Here, we address these points through analyses of DNA sequence data from multiple loci (16S rRNA gene, dnaJ, rpoB, and tuf gene fragments) using multiple Bayesian and maximum likelihood phylogenetic approaches that incorporate nearly all recognized Staphylococcus taxa. Results: We estimated the phylogeny of fifty-seven Staphylococcus taxa using partitioned-model Bayesian and maximum likelihood analysis, as well as Bayesian gene-tree species-tree methods. Regardless of methodology, we found broad agreement among methods that the current cluster groups require revision, although there was some disagreement among methods in resolution of higher order relationships. Based on our phylogenetic estimates, we propose a refined classification for Staphylococcus with species being classified into 15 cluster groups (based on molecular data) that adhere to six species groups (based on phenotypic properties). Conclusions: Our findings are in general agreement with gene tree-based reports of the staphylococcal phylogeny, although we identify multiple previously unreported relationships among species. Our results support the general importance of such multilocus assessments as a standard in microbial studies to more robustly infer relationships among recognized and newly discovered lineages.

Bacterial vaginosis (BV) is the most commonly treated female reproductive tract affliction, characterized by the displacement of healthy lactobacilli by an overgrowth of pathogenic bacteria. BV can contribute to pathogenic inflammation, preterm birth, and susceptibility to sexually transmitted infections. As the bacteria responsible for BV pathogenicity and their interactions with host immunity are not understood, we sought to evaluate the effects of BV-associated bacteria on reproductive epithelia. Here we have characterized the interaction between BV-associated bacteria and the female reproductive tract by measuring cytokine and defensin induction in three types of FRT epithelial cells following bacterial inoculation. Four BV-associated bacteria were evaluated alongside six lactobacilli for a comparative assessment. While responses differed between epithelial cell types, our model showed good agreement with clinical BV trends. We observed a distinct cytokine and human β-defensin 2 response to BV-associated bacteria, especially Atopobium vaginae, compared to most lactobacilli. One lactobacillus species, Lactobacillus vaginalis, induced an immune response similar to that elicited by BV-associated bacteria, stimulating significantly higher levels of cytokines and human β-defensin 2 than other lactobacilli. These data provide an important prioritization of BV-associated bacteria and support further characterization of reproductive bacteria and their interactions with host epithelia. Additionally, they demonstrate the distinct immune response potentials of epithelial cells from different locations along the female reproductive tract.

Background: Staphylococcus aureus (SA) nasal colonization plays a critical role in the pathogenesis of staphylococcal infections and SA eradication from the nares has proven to be effective in reducing endogenous infections. To understand SA nasal colonization and its relation with consequent disease, assessment of nasal carriage dynamics and genotypic diversity among a diverse population is a necessity. Results: We have performed extensive longitudinal monitoring of SA nasal carriage isolates in 109 healthy individuals over a period of up to three years. Longitudinal sampling revealed that 24% of the individuals were persistent SA nasal carriers while 32% were intermittent. To assess the genetic relatedness between different SA isolates within our cohort, multi locus sequence typing (MLST) was performed. MLST revealed that not only were strains colonizing intermittent and persistent nasal carriers genetically similar, belonging to the same clonal complexes, but strain changes within the same host were also observed over time for both types of carriers. More highly discriminating genetic analyses using the hypervariable regions of staphylococcal protein A and clumping factor B virulence genes revealed no preferential colonization of specific SA strains in persistent or intermittent carriers. Moreover, we observed that a subset of persistent and intermittent carriers retained clinically relevant community-acquired methicillin-resistant SA (CA-MRSA) strains in their nares over time. Conclusions: The findings of this study provides added perspective on the nasal carriage dynamics between strains colonizing persistent and intermittent carriers; an area currently in need of assessment given that persistent carriers are at greater risk of autoinfection than intermittent carriers.

Human Staphylococcus aureus (SA) nasal carriage provides a reservoir for the dissemination of infectious strains; however, factors regulating the establishment and persistence of nasal colonization are mostly unknown. We measured carriage duration and nasal fluid inflammatory markers after nasally inoculating healthy participants with their previously isolated SA strains. Out of 15 studies, 10 resulted in rapid clearance (9±6 days) that corresponded with upregulated chemokines, growth factors, and predominantly Th1-type cytokines, but not interleukin (IL)-17. Nasal SA persistence corresponded with elevated baseline levels of macrophage inflammatory protein-1β, IL-1β, and IL-6, no induction of inflammatory factors after inoculation, and decreased IL-1 receptor antagonist/IL-1β ratio. SA-expressed staphylococcal protein A (SpA) levels correlated positively with carriage duration. Competitive inoculation studies revealed that isogenic SpA knockout (ΔSpA) strains were cleared faster than wild type only in participants with upregulated inflammatory markers after inoculation. The remaining participants did not mount an inflammatory response and did not clear either strain. ΔSpA strains demonstrated lower growth rates in carrier nasal fluids and lower survival rates when incubated with neutrophils. Collectively, the presented studies identify innate immune effectors that cooperatively modulate nasal carriage duration, and confirm SpA as a bacterial codeterminant of SA nasal carriage.

E-cigarette (e-cig) vapor has been shown to play a pathological role in oral health and alter the oral microbiota, providing growth advantages for opportunistic pathogens. Enrichment of Staphylococcus aureus, a commensal resident in the oral cavity, correlates with the progression of periodontal disease, suggesting a role as an opportunistic pathogen. Environmental conditions, such as cigarette smoke, are known to increase S. aureus virulence, yet the role of S. aureus in periodontitis and oral preneoplasia is unknown. We exposed oral epithelial cells to e-cig aerosols and showed a dose-dependent cell viability reduction, regardless of nicotine content, in a possible attempt to repair DNA damage, as measured by pH2AX. S. aureus attachment to oral epithelial cells and bacterial biofilm formation were enhanced upon e-cig exposure, indicating an increased capacity for oral colonization. Mechanistically, e-cig aerosol exposure resulted in an immunosuppression, as determined by a reduction in IL8, IL6, and IL1β secretion by oral epithelial cells during co-culture with S. aureus. Consistent with this, e-cig vape reduced the oral epithelial cell clearance of S. aureus. Furthermore, we observed an increased expression of the inflammatory regulator COX2. This work suggests that e-cigs promote S. aureus colonization and modulate the oral inflammatory response, possibly promoting oral periodontitis and preneoplasia.

Bacterial vaginosis (BV), a disorder of the female reproductive tract (FRT) in which a healthy Lactobacillus -dominant microflora is replaced by BV-associated bacteria (BVAB), can significantly increase the incidence of human immunodeficiency virus (HIV) acquisition. Discerning the effect of BV on the mucosal epithelium of the FRT may yield novel preventatives and therapeutics for HIV infection. Here, we investigated barrier dysfunction of the endocervix by host-derived factors, secreted in response to BV, as a potential cause of HIV infection.