The impact of Staphylococcus aureus genomic variation on clinical phenotype of children with acute hematogenous osteomyelitis.
ABSTRACT: Background:Children with acute hematogenous osteomyelitis (AHO) have a broad spectrum of illness ranging from mild to severe. The purpose of this study is to evaluate the impact of genomic variation of Staphylococcus aureus on clinical phenotype of affected children and determine which virulence genes correlate with severity of illness. Methods:De novo whole genome sequencing was conducted for a strain of Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA), using PacBio Hierarchical Genome Assembly Process (HGAP) from 6 Single Molecule Real Time (SMRT) Cells, as a reference for DNA library assembly of 71 Staphylococcus aureus isolates from children with AHO. Virulence gene annotation was based on exhaustive literature review and genomic data in NCBI for Staphylococcus aureus. Clinical phenotype was assessed using a validated severity score. Kruskal-Wallis rank sum test determined association between clinical severity and virulence gene presence using False Discovery Rate (FDR), significance <0.01. Results:PacBio produced an assembled genome of 2,898,306 bp and 2054 Open Reading Frames (ORFs). Annotation confirmed 201 virulence genes. Statistical analysis of gene presence by clinical severity found 40 genes significantly associated with severity of illness (FDR ?0.009). MRSA isolates encoded a significantly greater number of virulence genes than did MSSA (p < 0.0001). Phylogenetic analysis by maximum likelihood (PAML) demonstrated the relatedness of genomic distance to clinical phenotype. Conclusions:The Staphylococcus aureus genome contains virulence genes which are significantly associated with severity of illness in children with osteomyelitis. This study introduces a novel reference strain and detailed annotation of Staphylococcus aureus virulence genes. While this study does not address bacterial gene expression, a platform is created for future transcriptome investigations to elucidate the complex mechanisms involved in childhood osteomyelitis.
Project description:BACKGROUND:Acute hematogenous osteomyelitis (AHO) occurs primarily in children and is believed to evolve from bacteremia followed by localization of infection to the metaphysis of bones. Currently, there is no consensus on the route and duration of antimicrobial therapy to treat AHO. METHODS:We conducted a systematic review of a short versus long course of treatment for AHO due primarily to Staphylococcus aureus in children aged 3 months to 16 years. We searched Medline, Embase and the Cochrane trials registry for controlled trials. Clinical cure rate at 6 months was the primary outcome variable, and groups receiving less than 7 days of intravenous therapy were compared with groups receiving one week or longer of intravenous antimicrobials. RESULTS:12 eligible prospective studies, one of which was randomized, were identified. The overall cure rate at 6 months for the short course of intravenous therapy was 95.2% (95% CI = 90.4 - 97.7) compared to 98.8% (95% CI = 93.6, 99.8) for the longer course of therapy. There was no significant difference in the duration of oral therapy between the two groups. CONCLUSIONS:Given the potential increased morbidity and cost associated with longer courses of intravenous therapy, this finding should be confirmed through a randomized controlled equivalence trial
Project description:Staphylococcus aureus is the most common cause of the difficult-to-treat osteomyelitis (OM). To better diagnose and manage S. aureus OM, especially for severe and long duration cases, indicators for risk prediction and severity evaluation are needed. Here, 139 clinical S. aureus isolates from orthopedic infections were divided into OM group (60 isolates from 60 OM patients) and non-OM group (79 isolates from 79 non-OM patients). Molecular types, antimicrobial susceptibility, and virulence factor profiles were evaluated and compared between the two groups to identify potential indicators associated with the prevalence of S. aureus OM. Clinical manifestations and laboratory data were analyzed to identify indicators affecting OM duration and severity. We found that some sequence types were specific to OM infection. The pvl, bbp, and ebps genes were associated with S. aureus OM prevalence. The pvl, bbp, and sei genes were associated with relatively longer OM duration. Panton-Valentine leucocidin (PVL)-positive S. aureus OM presented more serious inflammatory responses. Our results emphasize the significance of PVL in affecting the prevalence, duration, and severity of S. aureus OM. Diagnosing and monitoring PVL-related S. aureus OM may help direct better prognosis and treatment of these patients.
Project description:Staphylococcus aureus is a Gram-positive, commensal bacterium known to asymptomatically colonize the human skin, nares, and gastrointestinal tract. Colonized individuals are at increased risk for developing S. aureus infections, which range from mild skin and soft tissue infections to more severe diseases, such as endocarditis, bacteremia, sepsis, and osteomyelitis. Different virulence factors are required for S. aureus to infect different body sites. In this study, virulence gene expression was analyzed in two S. aureus isolates during nasal colonization, bacteremia and in the heart during sepsis. These models were chosen to represent the stepwise progression of S. aureus from an asymptomatic colonizer to an invasive pathogen. Expression of 23 putative S. aureus virulence determinants, representing protein and carbohydrate adhesins, secreted toxins, and proteins involved in metal cation acquisition and immune evasion were analyzed. Consistent upregulation of sdrC, fnbA, fhuD, sstD, and hla was observed in the shift between colonization and invasive pathogen, suggesting a prominent role for these genes in staphylococcal pathogenesis. Finally, gene expression data were correlated to the roles of the genes in pathogenesis by using knockout mutants in the animal models. These results provide insights into how S. aureus modifies virulence gene expression between commensal and invasive pathogens.Many bacteria, such as Staphylococcus aureus, asymptomatically colonize human skin and nasal passages but can also cause invasive diseases, such as bacteremia, pneumonia, sepsis, and osteomyelitis. The goal of this study was to analyze differences in the expression of selected S. aureus genes during a commensal lifestyle and as an invasive pathogen to gain insight into the commensal-to-pathogen transition and how a bacterial pathogen adapts to different environments within a host (e.g., from nasal colonization to invasive pathogen). The gene expression data were also used to select genes for which to construct knockout mutants to assess the role of several proteins in nasal colonization and lethal bacteremia. These results not only provide insight into the factors involved in S. aureus disease pathogenesis but also provide potential therapeutic targets.
Project description:Osteomyelitis is a common manifestation of invasive Staphylococcus aureus infection. Pathogen-induced bone destruction limits antimicrobial penetration to the infectious focus and compromises treatment of osteomyelitis. To investigate mechanisms of S. aureus-induced bone destruction, we developed a murine model of osteomyelitis. Microcomputed tomography of infected femurs revealed that S. aureus triggers profound alterations in bone turnover. The bacterial regulatory locus sae was found to be critical for osteomyelitis pathogenesis, as Sae-regulated factors promote pathologic bone remodeling and intraosseous bacterial survival. Exoproteome analyses revealed the Sae-regulated protease aureolysin as a major determinant of the S. aureus secretome and identified the phenol-soluble modulins as aureolysin-degraded, osteolytic peptides that trigger osteoblast cell death and bone destruction. These studies establish a murine model for pathogen-induced bone remodeling, define Sae as critical for osteomyelitis pathogenesis, and identify protease-dependent exoproteome remodeling as a major determinant of the staphylococcal virulence repertoire.
Project description:The clinical spectrum of Staphylococcus aureus infection ranges from asymptomatic nasal carriage to osteomyelitis, infective endocarditis (IE) and death. In this study, we evaluate potential association between the presence of specific genes in a collection of prospectively characterized S. aureus clinical isolates and clinical outcome.Two hundred thirty-nine S. aureus isolates (121 methicillin-resistant S. aureus [MRSA] and 118 methicillin-susceptible S. aureus [MSSA]) were screened by array comparative genomic hybridization (aCGH) to identify genes implicated in complicated infections. After adjustment for multiple tests, 226 genes were significantly associated with severity of infection. Of these 226 genes, 185 were not in the SCCmec element. Within the 185 non-SCCmec genes, 171 were less common and 14 more common in the complicated infection group. Among the 41 genes in the SCCmec element, 37 were more common and 4 were less common in the complicated group. A total of 51 of the 2014 sequences evaluated, 14 non-SCCmec and 37 SCCmec, were identified as genes of interest.Of the 171 genes less common in complicated infections, 152 are of unknown function and may contribute to attenuation of virulence. The 14 non-SCCmec genes more common in complicated infections include bacteriophage-encoded genes such as regulatory factors and autolysins with potential roles in tissue adhesion or biofilm formation.
Project description:Obesity and associated type 2 diabetes (T2D) are important risk factors for infection following orthopedic implant surgery. Staphylococcus aureus, the most common pathogen in bone infections, adapts to multiple environments to survive and evade host immune responses. Whether adaptation of S. aureus to the unique environment of the obese/T2D host accounts for its increased virulence and persistence in this population is unknown. Thus, we assessed implant-associated osteomyelitis in normal versus high-fat-diet obese/T2D mice and found that S. aureus infection was more severe, including increases in bone abscesses relative to nondiabetic controls. S. aureus isolated from bone of obese/T2D mice displayed marked upregulation of four adhesion genes (clfA, clfB, bbp, and sdrC), all with binding affinity for fibrin(ogen). Immunostaining of infected bone revealed increased fibrin deposition surrounding bacterial abscesses in obese/T2D mice. In vitro coagulation assays demonstrated a hypercoagulable state in obese/T2D mice that was comparable to that of diabetic patients. S. aureus with an inactivating mutation in clumping factor A (clfA) showed a reduction in bone infection severity that eliminated the effect of obesity/T2D, while infections in control mice were unchanged. In infected mice that overexpress plasminogen activator inhibitor-1 (PAI-1), S. aureusclfA expression and fibrin-encapsulated abscess communities in bone were also increased, further linking fibrin deposition to S. aureus expression of clfA and infection severity. Together, these results demonstrate an adaptation by S. aureus to obesity/T2D with increased expression of clfA that is associated with the hypercoagulable state of the host and increased virulence of S. aureus.
Project description:BACKGROUND:Staphylococcus aureus (S. aureus) infection accounts for more than 50% of the osteomyelitis cases. Currently, methicillin-resistant S. aureus (MRSA) strains present an urgent medical problem. The YycFG two-component regulatory system (TCS) can allow bacteria to rapidly adapt to physical, chemical, and biological stresses. To define the role of YycFG in modulation virulence of S. aureus in osteomyelitis, we isolated clinical MRSA strains and compared these with ATCC29213 methicillin-sensitive S. aureus (MSSA). METHODS:In the present study, 13 MRSA strains from chronic osteomyelitis tissues were isolated. The in-depth sequencing of 16S rRNA amplicons of the samples was conducted. Bacterial growth was monitored, and biofilm biomass was determined by crystal violet microtiter assay. Furthermore, quantitative RT-PCR analysis was adopted to identify the expression of yycF/G/H and icaA/D in MRSA and MSSA strains. Analysis of variance with one-way ANOVA was used for statistical analysis. RESULTS:The in-depth sequencing of 16S rRNA amplicons of the clinical samples indicated a polymicrobial infection, with the phylum Firmicutes made up 13% of the microbial population. The MRSA strains showed an accelerated growth rate compared to the MSSA strains. Of note, MRSA biofilms showed an accumulation of an intercellular polysaccharides matrix and enhanced biomass upon microscopic examination. Furthermore, MRSA strains had a higher expression of the yycF/G/H and icaA/D genes and adhesion force. CONCLUSIONS:These data suggested the roles of intercellular polysaccharide in S. aureus pathogenesis, indicating a possible association between YycFG pathways and MRSA strain virulence.
Project description:Staphylococcus aureus osteomyelitis is a common and debilitating invasive infection of bone. Treatment of osteomyelitis is confounded by widespread antimicrobial resistance and the propensity of bacteria to trigger pathological changes in bone remodeling that limit antimicrobial penetration to the infectious focus. Adjunctive therapies that limit pathogen-induced bone destruction could therefore limit morbidity and enhance traditional antimicrobial therapies. In this study, we evaluate the efficacy of the U.S. Food and Drug Administration-approved, nonsteroidal anti-inflammatory (NSAID) compound diflunisal in limiting S. aureus cytotoxicity toward skeletal cells and in preventing bone destruction during staphylococcal osteomyelitis. Diflunisal is known to inhibit S. aureus virulence factor production by the accessory gene regulator (agr) locus, and we have previously demonstrated that the Agr system plays a substantial role in pathological bone remodeling during staphylococcal osteomyelitis. Consistent with these observations, we find that diflunisal potently inhibits osteoblast cytotoxicity caused by S. aureus secreted toxins independently of effects on bacterial growth. Compared to commonly used NSAIDs, diflunisal is uniquely potent in the inhibition of skeletal cell death in vitro Moreover, local delivery of diflunisal by means of a drug-eluting, bioresorbable foam significantly limits bone destruction during S. aureus osteomyelitis in vivo Collectively, these data demonstrate that diflunisal potently inhibits skeletal cell death and bone destruction associated with S. aureus infection and may therefore be a useful adjunctive therapy for osteomyelitis.
Project description:Infection of foot ulcers is a common, often severe and costly complication in diabetes. Diabetic foot infections (DFI) are mainly polymicrobial, and Staphylococcus aureus is the most frequent pathogen isolated. The numerous virulence factors and toxins produced by S. aureus during an infection are well characterized. However, some particular features could be observed in DFI. The aim of this review is to describe the role of S. aureus in DFI and the implication of its toxins in the establishment of the infection. Studies on this issue have helped to distinguish two S. aureus populations in DFI: toxinogenic S. aureus strains (harboring exfoliatin-, EDIN-, PVL- or TSST-encoding genes) and non-toxinogenic strains. Toxinogenic strains are often present in infections with a more severe grade and systemic impact, whereas non-toxinogenic strains seem to remain localized in deep structures and bone involving diabetic foot osteomyelitis. Testing the virulence profile of bacteria seems to be a promising way to predict the behavior of S. aureus in the chronic wounds.
Project description:Osteomyelitis is a debilitating infection of bone that results in substantial morbidity. Staphylococcus aureus is the most commonly isolated pathogen causing bone infections and features an arsenal of virulence factors that contribute to bone destruction and counteract immune responses. We previously demonstrated that diflunisal, a nonsteroidal anti-inflammatory drug, decreases S. aureus-induced bone destruction during osteomyelitis when delivered locally from a resorbable drug delivery depot. However, local diflunisal therapy was complicated by bacterial colonization of the depot's surface, highlighting a common pitfall of devices for local drug delivery to infected tissue. It is, therefore, critical to develop an alternative drug delivery method for diflunisal to successfully repurpose this drug as an antivirulence therapy for osteomyelitis. We hypothesized that a nanoparticle-based parenteral delivery strategy would provide a method for delivering diflunisal to infected tissue while circumventing the complications associated with local delivery. In this study, we demonstrate that poly(propylene sulfide) (PPS) nanoparticles accumulate at the infectious focus in a murine model of staphylococcal osteomyelitis and are capable of efficaciously delivering diflunisal to infected bone. Moreover, diflunisal-loaded PPS nanoparticles effectively decrease S. aureus-mediated bone destruction, establishing the feasibility of systemic delivery of an antivirulence compound to mitigate bone pathology during osteomyelitis.