Host and Bacterial Determinants of Staphylococcus aureus Nasal Colonization in Humans

Staphylococcus aureus (SA), an opportunistic pathogen colonizing the anterior nares in approximately 30% of the human population, causes severe hospital-associated and community-acquired infections. SA nasal carriage 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 among a diverse population and determining factors responsible for SA nasal carriage have become major imperatives. Here, we report on an extensive longitudinal monitoring of SA nasal carriage in 109 healthy individuals over a period of up to three years to assess nasal carriage dynamics. Phylogenetic analyses of SA housekeeping genes and hypervariable virulence genes revealed that not only were SA strains colonizing intermittent and persistent nasal carriers genetically similar, but no preferential colonization of specific SA strains in these carriers was observed over time. These results indicated that other non-SA factors could be involved in determining specific carriage states. Therefore, to elucidate host responses during SA nasal carriage, we performed human SA nasal recolonization in a subset of SA nasal carriers within our cohort. In these studies, SA colonization levels were determined, and nasal secretions were collected and analyzed for host immune factors responsible for SA nasal carriage. Interestingly, we observed that stimulation of host immune responses lead to clearance of SA while sustained SA colonization was observed in hosts that did not mount a response during carriage. Further, analysis of nasal secretions from hosts revealed that proinflammatory cytokines and chemokines were significantly induced during SA nasal clearance suggesting that innate immune effectors influence carriage. SA utilizes a repertoire of surface and secreted proteins to evade host immune response and successfully colonize the nose. Analysis of the most abundant immunoevasive proteins in the exoproteome of SA nasal carrier strains revealed that expression levels of Staphylococcal protein A (SPA) produced by SA nasal carrier strains in vitro corresponded to the level of persistence of SA in the human nose. To determine if SPA is involved in modulating the host\u27s response to SA colonization, a subset of participants in our cohort was nasally recolonized with equal concentrations of both wild-type (WT) and spa-disrupted (?spa) autologous strains of SA. Interestingly, ?spa strains were eliminated from the nares significantly faster than WT when the host mounted an immune response, suggesting that the immunoevasive role of SPA is a determinant of carriage persistence. Collectively, this report augments our understanding of SA nasal carriage dynamics, in addition to identifying important host and microbial determinants that influence SA nasal colonization in humans. Better understanding of this phenomenon can lead to improved preventative strategies to thwart carriage-associated SA infections

Phylogenetic relationships among Staphylococcus species and refinement of cluster groups based on multilocus data

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

Longitudinal genetic analyses of Staphylococcus aureus nasal carriage dynamics in a diverse population

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

Evidence of Neutralizing and Non-Neutralizing Anti-Glucosaminidase Antibodies in Patients With S. Aureus Osteomyelitis and Their Association With Clinical Outcome Following Surgery in a Clinical Pilot

Staphylococcus aureus osteomyelitis remains a very challenging condition; recent clinical studies have shown infection control rates following surgery/antibiotics to be ~60%. Additionally, prior efforts to produce an effective S. aureus vaccine have failed, in part due to lack of knowledge of protective immunity. Previously, we demonstrated that anti-glucosaminidase (Gmd) antibodies are protective in animal models but found that only 6.7% of culture-confirmed S. aureus osteomyelitis patients in the AO Clinical Priority Program (AO-CPP) Registry had basal serum levels (>10 ng/ml) of anti-Gmd at the time of surgery (baseline). We identified a small subset of patients with high levels of anti-Gmd antibodies and adverse outcomes following surgery, not explained by Ig class switching to non-functional isotypes. Here, we aimed to test the hypothesis that clinical cure following surgery is associated with anti-Gmd neutralizing antibodies in serum. Therefore, we first optimized an in vitro assay that quantifies recombinant Gmd lysis of the M. luteus cell wall and used it to demonstrate the 50% neutralizing concentration (NC50) of a humanized anti-Gmd mAb (TPH-101) to be ~15.6 μg/ml. We also demonstrated that human serum deficient in anti-Gmd antibodies can be complemented by TPH-101 to achieve the same dose-dependent Gmd neutralizing activity as purified TPH-101. Finally, we assessed the anti-Gmd physical titer and neutralizing activity in sera from 11 patients in the AO-CPP Registry, who were characterized into four groups post-hoc. Group 1 patients (n=3) had high anti-Gmd physical and neutralizing titers at baseline that decreased with clinical cure of the infection over time. Group 2 patients (n=3) had undetectable anti-Gmd antibodies throughout the study and adverse outcomes. Group 3 (n=3) had high titers +/− neutralizing anti-Gmd at baseline with adverse outcomes. Group 4 (n=2) had low titers of non-neutralizing anti-Gmd at baseline with delayed high titers and adverse outcomes. Collectively, these findings demonstrate that both neutralizing and non-neutralizing anti-Gmd antibodies exist in S. aureus osteomyelitis patients and that screening for these antibodies could have a value for identifying patients in need of passive immunization prior to surgery. Future prospective studies to test the prognostic value of anti-Gmd antibodies to assess the potential of passive immunization with TPH-101 are warranted

Evolutionary Analyses of Staphylococcus aureus Identify Genetic Relationships between Nasal Carriage and Clinical Isolates

Nasal carriage of Staphylococcus aureus has long been hypothesized to be a major vector for the transmission of virulent strains throughout the community. To address this hypothesis, we have analyzed the relatedness between a cohort of nasal carriage strains and clinical isolates to understand better the genetic conformity therein. To assess the relatedness between nasal carriage and clinical isolates of S. aureus, a genetic association study was conducted using multilocus sequence typing (MLST) and typing of the hypervariable regions of clumping factor and fibronectin binding protein genes. At all loci analyzed, genetic associations between both nasal carriage and clinical isolates were observed. Computational analyses of MLST data indicate that nasal carriage and clinical isolates belong to the same genetic clusters (clades), despite differences in sequence type assignments. Genetic analyses of the hypervariable regions from the clumping factor and fibronectin binding protein genes revealed that not only do clinically relevant strains belong to identical genetic lineages as the nasal carriage isolates within our cohort, but they also exhibit 100% sequence similarity within these regions. The findings of this report indicate that strains of S. aureus being carried asymptomatically throughout the community via nasal colonization are genetically related to those responsible for high levels of morbidity and mortality

Humanized Mouse Models of Bacterial Infections

Bacterial infections continue to represent a significant healthcare burden worldwide, causing considerable mortality and morbidity every year. The emergence of multidrug-resistant bacterial strains continues to rise, posing serious risks to controlling global disease outbreaks. To develop novel and more effective treatment and vaccination programs, there is a need for clinically relevant small animal models. Since multiple bacterial species have human-specific tropism for numerous virulence factors and toxins, conventional mouse models do not fully represent human disease. Several human disease characteristic phenotypes, such as lung granulomas in the case of Mycobacterium tuberculosis infections, are absent in standard mouse models. Alternatively, certain pathogens, such as Salmonella enterica serovar typhi and Staphylococcus aureus, can be well tolerated in mice and cleared quickly. To address this, multiple groups have developed humanized mouse models and observed enhanced susceptibility to infection and a more faithful recapitulation of human disease. In the last two decades, multiple humanized mouse models have been developed to attempt to recapitulate the human immune system in a small animal model. In this review, we first discuss the history of immunodeficient mice that has enabled the engraftment of human tissue and the engraftment methods currently used in the field. We then highlight how humanized mouse models successfully uncovered critical human immune responses to various bacterial infections, including Salmonella enterica serovar Typhi, Mycobacterium tuberculosis, and Staphylococcus aureus

Interleukin-27 and Its Diverse Effects on Bacterial Infections

Innate and adaptive immune responses against pathogens are known to be carefully orchestrated by specific cytokines that initiate and down regulate immune cell functions from the initial infection through tissue repair and homeostasis. However, some cytokines, including interleukin-27, are expressed at multiple phases of the infection, such that their pro and anti-inflammatory functions have been difficult to interpret. As elucidation of specific cytokine functions throughout infection is central to our understanding of protective vs. susceptible immunity and return to homeostasis vs. prolonged inflammation leading to septic shock, here we review the literature on IL-27 signaling and the various functions of this heterodimeric ligand member of the IL-12 cytokine family. Canonically, IL-27 is produced by antigen-presenting cells, and is thought of as an immunostimulatory cytokine due to its capacity to induce Th1 differentiation. However, many studies have also identified various immunosuppressive effects of IL-27 signaling, including suppression of Th17 differentiation and induction of co-inhibitory receptors on T cells. Thus, the exact role of IL-27 in the context of infectious diseases remains a topic of debate and active research. Additionally, as recent interest has focused on clinical management of acute vs. chronic infections, and life-threatening “cytokine storm” from sepsis, we propose a hypothetical model to explain the biphasic role of IL-27 during the early and late phases of immune responses to reconcile its known pro and anti-inflammatory functions, which could be therapeutically regulated to improve patient outcomes of infection.</jats:p

Genetic Assessment Of Staphylococcus Aureus In An Underreported Locality: Ambulatory Care Clinic

Background: Staphylococcus aureus has strong association with anthropogenic environments. This association has not been well supported by use of genetic tools. The aim of this study was to phylogenetically relate numerous isolates from three environments — NCBI samples from hospitals, a community, and a previously unexplored healthcare environment: an ambulatory care clinic (ACC). Methods: This study incorporated hospital samples from NCBI, a community database from the University of Central Florida (UCF), and newly added samples taken from employees of an ambulatory care clinic located at UCF. Samples were collected from nasal swabs of employees, and positive samples were cultured, extracted, and sequenced at seven MLST loci and one virulence locus (spa). MLST sequences were used in eBURST and TCS population structure analyses and all sequences were incorporated into a phylogenetic reconstruction of relationships. Results: A total of 185 samples were incorporated in this study (15 NCBI sequences from hospital infections, 29 from the ACC, and 141 from the community). In both phylogenetic and population genetics analyses, samples proved to be panmixic, with samples not segregating monophyletically based on sample origin. Conclusion: Samples isolated from ambulatory care clinics are not significantly differentiated from either community or hospital samples at the representative loci chosen. These results strengthen previous conclusions that S. aureus may exhibit high genetic similarity across anthropogenic environments

Probiotic induced synthesis of microbiota polyamine as a nutraceutical for metabolic syndrome and obesity-related type 2 diabetes

The gut microbiota regulates multiple facets of host metabolism and immunity through the production of signaling metabolites, such as polyamines which are small organic compounds that are essential to host cell growth and lymphocyte activation. Polyamines are most abundant in the intestinal lumen, where their synthesis by the gut microbiota is influenced by microbiome composition and host diet. Disruption of the host gut microbiome in metabolic syndrome and obesity-related type 2 diabetes (obesity/T2D) results in potential dysregulation of polyamine synthesis. A growing body of evidence suggests that restoration of the dysbiotic gut microbiota and polyamine synthesis is effective in ameliorating metabolic syndrome and strengthening the impaired immune responses of obesity/T2D. In this review, we discuss existing studies on gut microbiome determinants of polyamine synthesis, polyamine production in obesity/T2D, and evidence that demonstrates the potential of polyamines as a nutraceutical in obesity/T2D hosts.</jats:p