Staphylococcus aureus isolates from chronic osteomyelitis are characterized by high host cell invasion and intracellular adaptation,but still induce inflammation

Osteomyelitis is a severe inflammatory disease of the bone that is mainly caused by Staphylococcus aureus. Particularly, bone infections are difficult to treat and can develop into a chronic course with a high relapsing rate despite of antimicrobial treatments. The complex interaction of staphylococci with osseous tissue and the bacterial ability to invade host cells are thought to determine the severity of infection. Yet, defined bacterial virulence factors responsible for the pathogenesis of osteomyelitis have not been clearly identified.The aim of this study was to detect S. aureus virulence factors that are associated with osteomyelitis and contribute to a chronic course of infection. To this purpose, we collected 41 S. aureus isolates, each 11 from acute osteomyelitis (infection period less than 2 months), 10 from chronic osteomyelitis (infection period more than 12 months), 10 from sepsis and 10 from nasal colonization. All isolates were analyzed for gene expression and in functional in-vitro systems. Adhesion assays to bone matrix revealed that all isolates equally bound to matrix structures, but invasion assays in human osteoblasts showed a high invasive capacity of chronic osteomyelitis isolates. The high invasion rate could not be explained by defined adhesins, as all infecting strains expressed a multitude of adhesins that act together and determine the level of adhesion. Following host cell invasion isolates from chronic osteomyelitis induced less cytotoxicity than all other isolates and a higher percentage of Small-colony-variant (SCV)-formation, which represents an adaptation mechanism during long-term persistence. Isolates from acute and chronic osteomyelitis strongly produced biofilm and highly expressed agr and sarA that regulate secreted virulence factors and induced an inflammatory response in osteoblasts. In conclusion, chronic osteomyelitis isolates were characterized by a high host cell invasion rate, low cytotoxicity and the ability to persist and adapt within osteoblasts. Furthermore, isolates from both acute and chronic osteomyelitis strongly produced biofilm and induced high levels of host cell inflammation, which may explain tissue destruction and bone deformation observed as typical complications of long-lasting bone infections.     

Aspergillus candidus eumycetoma with review of literature

ObjectiveMycetoma is a slowly progressive, chronic granulomatous infection of skin and subcutaneous tissues with involvement of underlying fasciae and bones, usually affecting extremities. In India, among mycetoma, the eumycetoma constitutes about 35% of cases. Hereby, we report the first case of eumycetoma caused by uncommon fungus, Aspergillus candidus.Case reportA 61 year old female presented to the Department of Dermatology with history of swelling of right foot associated with multiple firm nodules of approx. 1.5 × 1.5 cm each over dorsum of foot with discharging sinus containing white color granules. Biopsy of lesion on right foot on direct KOH examination revealed septate hyphae. Histopathology examination showed a histiocytic granuloma with fungal elements. Culture on Sabouraud Dextrose Agar grew white mycelial colonies which were identified to be Aspergillus species phenotypically. Genetic sequencing using Internal transcribed spacer gene, beta tubulin gene and Calmodulin gene was done and the isolate was identified to be Aspergillus candidus. Lesion was excised and patient was started on itraconazole.ConclusionTimely identification and starting of antifungal treatment can help in reducing morbidity due to eumycetoma to a large extent. As newer and newer species of fungi are emerging as significant causative agents of human infections, it is pertinent to report such findings from epidemiological point of view.     

Mechanisms of promoting the differentiation and bone resorption function of osteoclasts by Staphylococcus aureus infection

Bone infection is a common and serious complication in the field of orthopedics, which frequently leads to excessive bone destruction and fracture nonunion. Staphylococcus aureus (S. aureus) infection affects bone cell function which, in turn, causes bone destruction. Bone is mainly regulated by osteoblasts and osteoclasts. Osteoclasts are the only cell type with bone resorptive function. Their over-activation is closely associated with excessive bone loss. Understanding how S. aureus changes the functional state of osteoclasts is the key to effective treatment. By reviewing the literature, this paper summarizes several mechanisms of bone destruction caused by S. aureus influencing osteoclasts, thereby stimulating new ideas for the treatment of bone infection.     

Botryomycosis ('bacterial ball') of the sinonasal tract caused by Pseudomonas aeruginosa

BACKGROUND: Botryomycosis is a chronic bacterial infection that typically presents as a cutaneous lesion. Visceral involvement may occur, but mucosal disease is uncommon. We report two cases of sinonasal tract botryomycosis that clinically simulated a neoplasm. METHODS: Two cases of sinonasal tract botryomycosis were identified from the Otolaryngic Tumor Registry at the Armed Forces Institute of Pathology, Washington, DC. The clinical records, slides, and paraffin blocks were available for both cases. Histochemical stains, including Brown and Hopps, Gomori's methenamine-silver, acid-fast bacilli, mucicarmine, periodic acid-Schiff, and Warthin-Starry, were performed. RESULTS: The patients were an 81-year-old man and a 43-year-old woman. The man presented with acute ethmoiditis and a bulging eye. Radiographic studies showed a soft tissue mass in his left maxillary antrum with osseous erosion of adjacent anatomic sites. The woman presented with persistent headaches of more than 1 year's duration, with increasing severity in the months prior to presentation. An expansile soft tissue mass was identified in her right maxillary and ethmoid sinuses. Surgery was performed on both patients. The histology included amorphous, acellular material and separate, rounded eosinophilic granules associated with a neutrophilic infiltrate. A Splendore-Hoeppli phenomenon was seen. Filamentous gram-negative bacilli, identifiable only by histochemical staining, were morphologically compatible with Pseudomonas aeruginosa. Cultures of samples taken from both patients intraoperatively confirmed the organisms as P. aeruginosa. CONCLUSIONS: Sinonasal botryomycosis is a rare localized disease that may be mistaken clinically for an aggressive neoplasm. Complete surgical evacuation is curative.     

Primary cutaneous actinomycosis: a rare soft tissue infection

Actinomycosis caused by Actinomyces spp. is a chronic and suppurative infection caused by an endogenous gram positive bacterium. The unusual sites of infection are the head and neck, thorax and abdomen and are almost always endogenous in origin. Primary cutaneous actinomycosis is very rare and is usually associated with external trauma and local ischemia. We report a case of a primary cutaneous actinomycosis of the thigh in a 30-year-old man. The patient acquired the infection through an injection wound which progressed to multiple discharging sinuses. Clinical material from the wound demonstrated the presence of Actinomyces in smears and cultures. The patient was diagnosed and successfully treated with surgical resection and combined antibiotic therapy.     

First report of the predominance of clonal complex 398 Staphylococcus aureus strains in osteomyelitis complicating diabetic foot ulcers: a national French study

Staphylococcus aureus is the most common pathogen cultured from diabetic foot infection including diabetic foot osteomyelitis. This French multicentre study determined the genetic content of S. aureus isolated from 157 consecutive cases admitted to 12 diabetic foot centres between 2008 and 2011. We describe for the first time the emergence of the CC398 methicillin-susceptible S. aureus clone, the main clone in diabetic foot osteomyelitis, and its tropism for bone. This clone spreads to humans from an animal source through its intrinsic virulence. This adaptation of S. aureus isolates looks to be a worrisome problem and should be carefully monitored.     

Methicillin-resistant Staphylococcus aureus nasal carriage and infection among patients with diabetic foot ulcer

PurposeTo evaluate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage in patients with diabetic foot ulcer (DFU) in Taiwan, and to assess the concordance between colonizing and clinical MRSA isolates from the patients.MethodA total of 354 nasal specimens were collected from 112 to 242 diabetic patients with and without foot ulcer, respectively. MRSA clinical isolates from DFU wound cultures were collected for comparison.ResultsNasal carriage rate of S. aureus and MRSA was similar between diabetic patients with and without foot ulcer (15.2% vs. 16.9% for S. aureus and 5.4% vs. 1.7% for MRSA). Nasal S. aureus colonization was an independent predictor for wound S. aureus infection (Odds ratio [OR]: 5.33, 95% confidence interval [CI]: 1.61–17.59), so did nasal MRSA colonization (OR: 19.09, 95% CI: 2.12–171.91). The levels of glycated hemoglobin, and the usage with immunosuppressant agent were associated with S. aureus nasal colonization while oral hypoglycemic agent usage a protective factor. Sequence type 59/staphylococcal chromosome cassette mec IV or V, the local endemic community-associated clone, accounted for 42% and 70% of the clinical and colonizing isolates, respectively. Six of 10 patients with paired colonizing and clinical isolates, either MRSA or methicillin-sensitive S. aureus, had a genetically identical strain from a single patient.ConclusionLess than one-fifth of patients with DFU have nasal S. aureus, including MRSA, colonization; however, the colonization is significantly associated with S. aureus diabetic foot infection. Screening for S. aureus colonizing status in DFU patients might have a potential clinical implication.     

Four-Component Staphylococcus aureus Vaccine 4C-Staph Enhances Fcγ Receptor Expression in Neutrophils and Monocytes and Mitigates S. aureus Infection in Neutropenic Mice

Staphylococcus aureus is a human bacterial pathogen causing a variety of diseases. The occurrence of multidrug-resistant strains of Staphylococcus aureus underlines the need for a vaccine. Defining immune correlates of protection may support the design of an effective vaccine. We used a murine Staphylococcus aureus infection model, in which bacteria were inoculated in an air pouch generated on the back of the animal. Analysis of the air-pouch content in mice immunized or not with an adjuvanted multiantigen vaccine formulation, four-component S. aureus vaccine (4C-Staph), prior to infection allowed us to measure bacteria, cytokines, and 4C-Staph-specific antibodies and to analyze host immune cells recruited to the infection site. Immunization with 4C-Staph resulted in accumulation of antigen-specific antibodies in the pouch and mitigated the infection. Neutrophils were the most abundant cells in the pouch, and they showed the upregulation of Fcγ receptor (FcγR) following immunization with 4C-Staph. Reduction of the infection was also obtained in mice immunized with 4C-Staph and depleted of neutrophils; these mice showed an increase in monocytes and macrophages. Upregulation of the FcγR and the presence of antigen-specific antibodies induced by immunization with 4C-Staph may contribute to increase bacterial opsonophagocytosis. Protection in neutropenic mice indicated that an effective vaccine could activate alternative protection mechanisms compensating for neutropenia, a condition often occurring in S. aureus-infected patients.     

Are clindamycin and ciprofloxacin appropriate for the empirical treatment of diabetic foot infections?

The objective of this study was to determine if specific species of microorganisms are associated with severe infection and amputation in patients with a diabetic foot infection and to evaluate the effectiveness of clindamycin and ciprofloxacin in this population. A retrospective cohort study was performed at the University Medical Center Utrecht, The Netherlands, for the period January 1st 2005 to January 1st 2010. Patients with ICD-9 codes ‘diabetic foot’ were selected from the hospital database. We evaluated the association of Staphylococcus aureus and Gram-negative species (GNS) with severity of infection, number of amputations, and healing rates. No significant association was found between the different microorganisms and infection severity. Coinfections of GNS and S. aureus were significantly associated with amputation compared to infections with only S. aureus [p = 0.016, odds ratio (OR) 4.9, confidence interval (CI) 1.4–16.9]. The empiric antibiotic regimen of clindamycin and ciprofloxacin covered only 85 % of S. aureus and 78 % of GNS diabetic foot infections. In mild diabetic foot infection in the studied population, where methicillin-sensitive S. aureus and streptococci are the most likely pathogens, a beta-lactam antibiotic such as flucloxacillin would likely be more effective than clindamycin. In moderate and severe infections, where GNS as a causative organism cannot be safely excluded, broad-spectrum antibiotic therapy may be more adequate.     

Clinical Implications of Oral Candidiasis: Host Tissue Damage and Disseminated Bacterial Disease

The clinical significance of polymicrobial interactions, particularly those between commensal species with high pathogenic potential, remains largely understudied. Although the dimorphic fungal species Candida albicans and the bacterium Staphylococcus aureus are common cocolonizers of humans, they are considered leading opportunistic pathogens. Oral candidiasis specifically, characterized by hyphal invasion of oral mucosal tissue, is the most common opportunistic infection in HIV⁺ and immunocompromised individuals. In this study, building on our previous findings, a mouse model was developed to investigate whether the onset of oral candidiasis predisposes the host to secondary staphylococcal infection. The findings demonstrated that in mice with oral candidiasis, subsequent exposure to S. aureus resulted in systemic bacterial infection with high morbidity and mortality. Histopathology and scanning electron microscopy of tongue tissue from moribund animals revealed massive C. albicans hyphal invasion coupled with S. aureus deep tissue infiltration. The crucial role of hyphae in the process was demonstrated using a non-hypha-producing and a noninvasive hypha-producing mutant strains of C. albicans. Further, in contrast to previous findings, S. aureus dissemination was aided but not contingent upon the presence of the Als3p hypha-specific adhesion. Importantly, impeding development of mucosal C. albicans infection by administering antifungal fluconazole therapy protected the animals from systemic bacterial disease. The combined findings from this study demonstrate that oral candidiasis may constitute a risk factor for disseminated bacterial disease warranting awareness in terms of therapeutic management of immunocompromised individuals.     

Bringing together what belongs together: Optimizing murine infection models by using mouse-adapted Staphylococcus aureus strains

Staphylococcus (S.) aureus is a leading cause of bacterial infection world-wide, and currently no vaccine is available for humans. Vaccine development relies heavily on clinically relevant infection models. However, the suitability of mice for S. aureus infection models has often been questioned, because experimental infection of mice with human-adapted S. aureus requires very high infection doses. Moreover, mice were not considered to be natural hosts of S. aureus. The latter has been disproven by our recent findings, showing that both laboratory mice, as well as wild small mammals including mice, voles, and shrews, are naturally colonized with S. aureus. Here, we investigated whether mouse-and vole-derived S. aureus strains show an enhanced virulence in mice as compared to the human-adapted strain Newman. Using a step-wise approach based on the bacterial genotype and in vitro assays for host adaptation, we selected the most promising candidates for murine infection models out of a total of 254 S. aureus isolates from laboratory mice as well as wild rodents and shrews. Four strains representing the clonal complexes (CC) 8, 49, and 88 (n?=?2) were selected and compared to the human-adapted S. aureus strain Newman (CC8) in murine pneumonia and bacteremia models. Notably, a bank vole-derived CC49 strain, named DIP, was highly virulent in BALB/c mice in pneumonia and bacteremia models, whereas the other murine and vole strains showed virulence similar to or lower than that of Newman. At one tenth of the standard infection dose DIP induced disease severity, bacterial load and host cytokine and chemokine responses in the murine bacteremia model similar to that of Newman. In the pneumonia model, DIP was also more virulent than Newman but the effect was less pronounced. Whole genome sequencing data analysis identified a pore-forming toxin gene, lukF-PV(P83)/lukM, in DIP but not in the other tested S. aureus isolates. To conclude, the mouse-adapted S. aureus strain DIP allows a significant reduction of the inoculation dose in mice and is hence a promising tool to develop clinically more relevant infection models.     

Persistent Staphylococcus aureus Isolates from Two Independent Cases of Bacteremia Display Increased Bacterial Fitness and Novel Immune Evasion Phenotypes

Staphylococcus aureus bacteremia cases are complicated by bacterial persistence and treatment failure despite the confirmed in vitro susceptibility of the infecting strain to administered antibiotics. A high incidence of methicillin-resistant S. aureus (MRSA) bacteremia cases are classified as persistent and are associated with poorer patient outcomes. It is still unclear how S. aureus evades the host immune system and resists antibiotic treatment for the prolonged duration of a persistent infection. In this study, the genetic changes and associated phenotypic traits specific to S. aureus persistent bacteremia were identified by comparing temporally dispersed isolates from persistent infections (persistent isolates) originating from two independent persistent S. aureus bacteremia cases with the initial infection isolates and with three resolved S. aureus bacteremia isolates from the same genetic background. Several novel traits were associated specifically with both independent sets of persistent S. aureus isolates compared to both the initial isolates and the isolates from resolved infections (resolved isolates). These traits included (i) increased growth under nutrient-poor conditions; (ii) increased tolerance of iron toxicity; (iii) higher expression of cell surface proteins involved in immune evasion and stress responses; and (iv) attenuated virulence in a Galleria mellonella larva infection model that was not associated with small-colony variation or metabolic dormancy such as had been seen previously. Whole-genome sequence analysis identified different single nucleotide mutations within the mprF genes of all the isolates with the adaptive persistence traits from both independent cases. Overall, our data indicate a novel role for MprF function during development of S. aureus persistence by increasing bacterial fitness and immune evasion.     

Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections.     

The role of serum proteins in Staphylococcus aureus adhesion to ethylene glycol coated surfaces

Bacterial adhesion on implants is a first step in the development of chronic foreign body associated infections. Finding strategies to minimize bacterial adhesion may contribute to minimize such infections. It is known that surfaces with oligo-ethylene-glycol (EG₃OMe) or poly-ethylene-glycol (PEG2k) terminations decrease unspecific protein adsorption and bacterial adhesion. However, little is known about the influence of serum and its components on bacterial adhesion. We therefore prepared two coatings on gold surface with HS-(CH₂)₁₁EG₃OMe (EG₃OMe) and PEG2k-thiol and studied the role of bovine serum albumin (BSA), γ-globulins, and serum on Staphylococcus aureus adhesion. While BSA and lysozyme showed no adherence even when applied at very high concentrations (100 mg/ml), γ-globulins adsorbed already from 10 mg/ml on. The adsorption of γ-globulins was, however, significantly decreased when it was mixed with BSA in a ratio of 3:1, as it is in the serum. Pretreatment of EG₃OMe and PEG2k coatings with γ-globulins or serum strongly promoted adherence of S. aureus when resuspended in buffer, suggesting that γ-globulins play a pivotal role in promoting S. aureus adhesion by its IgG binding proteins; the finding that a spa-deletion mutant, lacking the IgG binding protein A, showed decreased adherence corroborated this. Similarly, when S. aureus was pretreated with serum or γ-globulins its adherence was also significantly decreased. Our findings show that particularly γ-globulins bind to the coated surfaces thus mediating adherence of S. aureus via its protein A. As pretreatment of S. aureus with serum or γ-globulins significantly decreased adherence, treatment of patients with γ-globulins before implant surgery might lower the risk of implant-associated infections.     

Innate and adaptive immune responses against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> skin infections

Staphylococcus aureus is an important human pathogen that is responsible for the vast majority of bacterial skin and soft tissue infections in humans. S. aureus can also become more invasive and cause life-threatening infections such as bacteremia, pneumonia, abscesses of various organs, meningitis, osteomyelitis, endocarditis, and sepsis. These infections represent a major public health threat due to the enormous numbers of these infections and the widespread emergence of methicillin-resistant S. aureus (MRSA) strains. MSRA is endemic in hospitals worldwide and is rapidly spreading throughout the normal human population in the community. The increasing frequency of MRSA infections has complicated treatment as these strains are more virulent and are increasingly becoming resistant to multiple different classes of antibiotics. The important role of the immune response against S. aureus infections cannot be overemphasized as humans with certain genetic and acquired immunodeficiency disorders are at an increased risk for infection. Understanding the cutaneous immune responses against S. aureus is essential as most of these infections occur or originate from a site of infection or colonization of the skin and mucosa. This review will summarize the innate immune responses against S. aureus skin infections, including antimicrobial peptides that have direct antimicrobial activity against S. aureus as well as pattern recognition receptors and proinflammatory cytokines that promote neutrophil abscess formation in the skin, which is required for bacterial clearance. Finally, we will discuss the recent discoveries involving IL-17-mediated responses, which provide a key link between cutaneous innate and adaptive immune responses against S. aureus skin infections.     

Direct translocation of staphylococci from the skin surface to deep organs

BackgroundStaphylococcus aureus can invade the bloodstream and cause bacteremic infections, but this organism frequently produces serious deep infections without bacteremia or an identifiable portal of entry.MethodsWe used experimental cutaneous S. aureus infections in mice to determine if the bacteria could reach deep organs without travel through the bloodstream.ResultsAfter skin surface application the bacteria rapidly distributed to lymph nodes, spleen, kidneys and other organs. In these animals, blood cultures were negative, dissemination was more efficient after surface application than injection near dermal blood vessels, and kidney bacterial localization sites were unlike those of bacteremic infections. Whereas normal mice eventually cleared bacteria from the deep sites, those with prolonged immunosuppression became moribund from these infections; they also had negative blood cultures and kidney localization not consistent with hematogenous dissemination. Bacteria were also found in the intervening abdominal wall outside the spleen and kidney sites, suggesting direct movement of the organisms from the skin surface through connecting tissues.ConclusionsAlthough capable of hematogenous dissemination, S. aureus can also spread from skin to deep organs by a non-bacteremic process. In this case the bacteria appear to migrate directly from the skin surface to the deep organs below.     

Escherichia coli and Staphylococcus aureus Elicit Differential Innate Immune Responses following Intramammary Infection

Staphylococcus aureus and Escherichia coli are among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that induce clinical mastitis. The innate immune system comprises the immediate host defense mechanisms to protect against infection and contributes to the initial detection of and proinflammatory response to infectious pathogens. The objective of the present study was to characterize the different innate immune responses to experimental intramammary infection with E. coli and S. aureus during clinical mastitis. The cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP), two proteins that contribute to host recognition of bacterial cell wall products, were studied. Intramammary infection with either E. coli or S. aureus elicited systemic changes, including decreased milk output, a febrile response, and induction of the acute-phase synthesis of LBP. Infection with either bacterium resulted in increased levels of interleukin 1β (IL-1β), gamma interferon, IL-12, sCD14, and LBP in milk. High levels of the complement cleavage product C5a and the anti-inflammatory cytokine IL-10 were detected at several time points following E. coli infection, whereas S. aureus infection elicited a slight but detectable increase in these mediators at a single time point. Increases in IL-8 and tumor necrosis factor alpha were observed only in quarters infected with E. coli. Together, these data demonstrate the variability of the host innate immune response to E. coli and S. aureus and suggest that the limited cytokine response to S. aureus may contribute to the well-known ability of the bacterium to establish chronic intramammary infection.     

Inside job: Staphylococcus aureus host-pathogen interactions

Staphylococcus aureus is a notorious opportunistic pathogen causing a plethora of diseases. Recent research established that once phagocytosed by neutrophils and macrophages, a certain percentage of S. aureus is able to survive within these phagocytes which thereby even may contribute to dissemination of the pathogen. S. aureus further induces its uptake by otherwise non-phagocytic cells and the ensuing intracellular cytotoxicity is suggested to lead to tissue destruction, whereas bacterial persistence within cells is thought to lead to immune evasion and chronicity of infections. We here review recent work on the S. aureus host pathogen interactions with a focus on the intracellular survival of the pathogen.