Immunological response to a Staphylococcus aureus fibronectin-binding protein.

A protein (gal-FnBP), constructed by fusion of the genes encoding beta-galactosidase of Escherichia coli and the binding domains of fibronectin-binding protein (FnBP) of Staphylococcus aureus was used. FnBP is a surface protein responsible for attachment of bacteria to extracellular matrix of various host tissues. Gal-FnBP is more stable and can be produced in larger quantities than native FnBP. The binding specificity of this fusion protein was established in a Western blot analysis. Treatment of gal-FnBP with formalin inactivated the binding capacity of the protein but immunogenicity was retained. Immunisation of mice with formalin-treated gal-FnBP resulted in high antibody titres against the fibronectin-binding part of this fusion protein. These antibodies were measured by their ability to block the specific binding of fibronectin to gal-FnBP in a blocking assay. Sera raised against formalin-treated gal-FnBP and non-treated gal-FnBP blocked this binding to 40 and 25% respectively, thereby indicating the usefulness of gal-FnBP as a vaccine component.     

Immunological control of methicillin-resistant Staphylococcus aureus (MRSA) infection in an immunodeficient murine model of thermal injuries

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is a major cause of sepsis in patients who are immunosuppressed by their burns. In this study, an immunological regulation of MRSA infection was attempted in a mouse model of thermal injury. SCIDbg mice were resistant to MRSA infection, while SCIDbgMN mice (SCIDbg mice depleted of neutrophils and macrophages (Mphi)) were susceptible to the same infection. Also, thermally injured SCIDbg mice were shown to be susceptible to MRSA infection. On the other hand, the resistance of SCIDbgMN mice to the infection was completely recovered after an inoculation with Mphi from normal mice. However, anti-MRSA resistance was not shown in SCIDbgMN mice inoculated with Mphi from thermally injured mice. Mphi from MRSA-infected thermally injured mice were identified as alternatively activated Mphi, and Mphi from MRSA-infected unburned mice were characterized as classically activated Mphi. Mphi from thermally injured SCIDbg mice previously treated with 2-carboxyethylgermanium sesquioxide (Ge-132) protected SCIDbgMN mice against MRSA infection. Ge-132 has been described as an inhibitor of alternatively activated Mphi generation. These results suggest that MRSA infection in thermally injured patients is controlled immunologically through the induction of anti-MRSA effector cells and elimination of burn-associated alternatively activated Mphi, which are cells that inhibit the generation of classically activated Mphi.     

Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks.

Staphylococcus aureus has long been recognized as an important pathogen in human disease. Due to an increasing number of infections caused by methicillin-resistant S. aureus (MRSA) strains, therapy has become problematic. Therefore, prevention of staphylococcal infections has become more important. Carriage of S. aureus appears to play a key role in the epidemiology and pathogenesis of infection. The ecological niches of S. aureus are the anterior nares. In healthy subjects, over time, three patterns of carriage can be distinguished: about 20% of people are persistent carriers, 60% are intermittent carriers, and approximately 20% almost never carry S. aureus. The molecular basis of the carrier state remains to be elucidated. In patients who repeatedly puncture the skin (e.g., hemodialysis or continuous ambulatory peritoneal dialysis [CAPD] patients and intravenous drug addicts) and patients with human immunodeficiency virus (HIV) infection, increased carriage rates are found. Carriage has been identified as an important risk factor for infection in patients undergoing surgery, those on hemodialysis or CAPD, those with HIV infection and AIDS, those with intravascular devices, and those colonized with MRSA. Elimination of carriage has been found to reduce the infection rates in surgical patients and those on hemodialysis and CAPD. Elimination of carriage appears to be an attractive preventive strategy in patients at risk. Further studies are needed to optimize this strategy and to define the groups at risk.     

Cytomegalovirus reactivation enhances the virulence of Staphylococcus aureus pneumonia in a mouse model

Objectives

Cytomegalovirus (CMV) reactivation in intensive care unit patients may increase mortality and favour bacterial pneumonia. We developed a murine model to compare the severity of staphylococcal pneumonia after CMV reactivation and in CMV-negative mice.

Virulence of Staphylococcus aureus small colony variants in the Caenorhabditis elegans infection model

Small colony variants (SCVs) of Staphylococcus aureus are slow-growing morphological variants that have been implicated in persistent, relapsing, and antibiotic-resistant infections. The altered phenotype of SCVs in most strains has been attributed to defects in electron transport due to mutations in hemin or menadione biosynthesis. The pathogenic capacity of SCVs compared to phenotypically normal strains is variable depending on the attribute examined, with some studies showing reduced virulence of SCVs and others demonstrating normal or heightened virulence. Recently, the nematode Caenorhabditis elegans has been successfully employed as an alternative host to investigate virulence mechanisms of a variety of bacterial pathogens, including S. aureus. In this study, we show that clinical SCVs as well as hemB- and menD-deficient mutants of S. aureus are greatly reduced in virulence in the C. elegans infection model.     

Vitamin A deficiency predisposes to Staphylococcus aureus infection.

We have investigated the consequences of vitamin A deficiency in a rat model of T-cell-dependent and superantigen-mediated Staphylococcus aureus arthritis. After intravenous inoculation of enterotoxin A-producing staphylococci, the vitamin-A-deficient rats showed a decreased weight gain compared with the paired fed controls despite equal food consumption. The control rats developed arthritis in the first few days after bacterial inoculation, with a peak frequency at day 5, and then gradually recovered; however, the frequency of arthritis 18 days after bacterial inoculation was 86% among the vitamin A-deficient rats and 44% among the control rats. During this period, 3 of 10 deficient rats and 1 of 10 control rats died. Further in vitro analysis revealed that T-cell responses to S. aureus were significantly higher in the vitamin A-deficient rats than in the control animals. In contrast, B-cell reactivity, measured as immunoglobulin levels, autoantibody levels, and specific antibacterial antibody levels in serum, did not differ between the groups. Interestingly, the innate host defense mechanisms against S. aureus were also profoundly affected by vitamin A deficiency. Thus, despite a larger number of circulating phagocytic cells in the vitamin-A-deficient group, the capacity to phagocytize and exert intracellular killing of S. aureus was significantly decreased in comparison with the control rats. Furthermore, serum from the vitamin A-deficient rats inoculated with Staphylococcus aureus displayed decreased complement lysis activity. Our results suggest that the increased susceptibility to S. aureus infection observed in the vitamin-A-deficient rats is due to a concerted action of antigen-specific T-cell hyperactivity, impaired function of the phagocytes, and decreased complement activity.     

Developmental mechanisms underlying polydactyly in the mouse mutant Doublefoot

The pre-axial polydactylous mouse mutant Doublefoot has 6-9 digits per limb but lacks anteroposterior polarity (there is no biphalangeal digit 1). It differs from other polydactylous mutants in showing normal Shh expression, but polarizing activity (shown by mouse-chick grafting experiments) and hedgehog signalling activity (shown by expression of Ptc1) are present throughout the distal mesenchyme. The Dbf mutation has not yet been identified. Here we review current understanding of this mutant, and briefly report new results indicating (1) that limb bud expansion is concomitant with ectopic lhh expression and with extension of the posterior high cell proliferation rate into the anterior region, and (2) that the Dbf mutation is epistatic to Shh in the limb.     

Protective immunization against Staphylococcus aureus infection in a novel experimental wound model in mice

A novel murine experimental wound infection model was used to assess the efficacy of multi‐component immunization against Staphylococcus aureus infection. Necrotic lesions were induced in mice with venom from Bothrops asper and infected with a low inoculum, 1 × 10² CFU. The wound infection model therefore more resembles a clinical case of S. aureus infection compared with conventional infection models where far more bacteria are required. Before infection, mice were immunized with four recombinant S.aureus proteins expressed from Escherichia coli: (i) domains 1–3 of Extracellular adherence protein (Eap), (ii) Efb – D (fusion protein combining Extracellular fibrinogen binding protein (Efb) and a fibronectin binding domain (D) of the fibronectin binding protein (FnBP) and (iii) clumping factor A (ClfA). In the immunized group, lower bacterial colonization, undisturbed crust formation and significantly faster wound healing were found compared with the unimmunized control group. Efb and Eap have previously been found to impair wound healing and neutralization of these proteins by antibodies restores a more natural wound healing process. This effect is further also enhanced by the proposed opsonic activity of antibodies against ClfA and FnBP.     

New epidemiology of Staphylococcus aureus infection in Africa

Research on African Staphylococcus aureus has been largely neglected in the past, despite the cultural and geographical diversity in Africa, which has a significant impact on the epidemiology of this pathogen. The polarity between developed urban societies and remote rural populations (e.g. Pygmies), combined with close contact with animals (e.g. livestock and domestic animals, and wildlife), makes the epidemiology of S. aureus on the African continent unique and fascinating. Here, we try to draw an epidemiological picture of S. aureus colonization and infection in Africa, and focus on the wide spread of Panton–Valentine leukocidin-positive isolates, the emergence of the hypervirulent methicillin-resistant S. aureus (MRSA) clone USA300, and the dissemination of the typical African clone MRSA sequence type 88.     

New epidemiology of Staphylococcus aureus infection in Asia

Not only is Asia the most populous region in the world, but inappropriate therapy, including seif-medication with over-the-counter antimicrobial agents, is a common response to infectious diseases. The high antibiotic selective pressure among the overcrowded inhabitants creates an environment that is suitable for the rapid development and efficient spread of numerous multidrug-resistant pathogens. Indeed, Asia is among the regions with the highest prevalence rates of healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) and community-associated methicillin-resistant S. aureus (CA-MRSA) in the world. Most hospitals in Asia are endemic for multidrug-resistant methicillin-resistant S. aureus (MRSA), with an estimated proportion from 28% (in Hong Kong and Indonesia) to >70% (in Korea) among all clinical S. aureus isolates in the early 2010s. Isolates with reduced susceptibility or a high level of resistance to glycopeptides have also been increasingly identified in the past few years. In contrast, the proportion of MRSA among community-associated S. aureus infections in Asian countries varies markedly, from <5% to >35%. Two pandemic HA-MRSA clones, namely multilocus sequence type (ST) 239 and ST5, are disseminated internationally in Asia, whereas the molecular epidemiology of CA-MRSA in Asia is characterized by clonal heterogeneity, similar to that in Europe. In this review, the epidemiology of S. aureus in both healthcare facilities and communities in Asia is addressed, with an emphasis on the prevalence, clonal structure and antibiotic resistant profiles of the MRSA strains. The novel MRSA strains from livestock animals have been considered to constitute a public health threat in western countries. The emerging livestock-associated MRSA strains in Asia are also included in this review.     

Life-threatening community-acquired methicillin-resistant Staphylococcus aureus infection in Australia

Eight patients with invasive bacteremic community-acquired methicillin-resistant Staphylococcus aureus infection in southeast Queensland, Australia, are reported. One patient died of septic shock. Haematogenous seeding to lungs, bone, and other sites was common. All isolates carried the virulence factor Panton-Valentine leukocidin and were either the southwest Pacific clone or the newly described Queensland clone. Clinicians should consider community-acquired methicillin-resistant Staphylococcus aureus infection in any patient presenting to hospital with severe staphylococcal sepsis or pneumonia.     

Community-associated methicillin-resistant Staphylococcus aureus in the patient with HIV infection

The prevalence of community-associated methicillin-resistant Staphylococus aureus (CA-MRSA) has increased alarmingly in both the general population and the HIV-infected community. We look at the background of MRSA including the mechanisms of resistance, genetics, and trends in the individual with HIV infection. Numerous studies have investigated the risk factors for CA-MRSA. Other studies have further characterized the incidence of and risk factors for MRSA infections in the HIV community. Although one might not readily associate advanced HIV infection with increased susceptibility to bacterial pathogens, a number of studies have explained the mechanisms of this B-cell-mediated susceptibility. Invasive MRSA infections have spread into communities, are increasingly prevalent, and pose a public health challenge for their containment, prevention, and treatment.     

Emerging pathogenetic mechanisms of the implant-related osteomyelitis by Staphylococcus aureus

Implant-related osteomyelitis is a severe and deep infection of bone that arises and develops all around an implant. Staphylococcus aureus is the first cause of osteomyelitis, whether implant-related or not. Bone is an optimal substratum for S. aureus, since this bacterium expresses various adhesins by which can adhere to bone proteins and to the biomaterial surfaces coated with the proteins of the host extracellular matrix. S. aureus is able not only to colonize bone tissues, but also to invade and disrupt them by entering bone cells and inducing cell death and osteolysis. Here we illustrate the pathogenetic mechanisms that can explain how the osteomyelitis sets in and develops around an implant.     

Discovery of potential anti-infectives against Staphylococcus aureus using a Caenorhabditis elegans infection model

Background

The limited antibiotic options for effective control of methicillin-resistant Staphylococcus aureus infections has led to calls for new therapeutic approaches to combat this human pathogen. An alternative approach to control MRSA is through the use of anti-infective agents that selectively disrupt virulence-mediated pathways without affecting microbial cell viability or by modulating the host natural immune defenses to combat the pathogen.

Methods

We established a C. elegans – S. aureus liquid-based assay to screen for potential anti-infectives against S. aureus. The assay was utilized to screen 37 natural extracts and 29 synthetic compounds for the ability to extend the lifespan of infected nematodes. Disc diffusion and MIC microdilution tests were used to evaluate the anti-microbial properties of these natural extracts and synthetic compounds whilst in vivo bacterial CFU within the C. elegans gut were also enumerated.

Results

We screened a total of 37 natural extracts and 29 synthetic compounds for anti-infective properties. The screen successfully revealed 14 natural extracts from six plants (Nypa fruticans, Swietenia macrophylla, Curcuma longa, Eurycoma longifolia, Orthosiphon stamineus and Silybum eburneum) and one marine sample (Faunus ater) that improved the survival of S. aureus-infected worms by at least 2.8-fold as well as 14 synthetic compounds that prolonged the survival of S. aureus-infected nematodes by 4-fold or greater. An anti-microbial screen of all positive hits demonstrated that 8/28 hits had no effect on S. aureus growth. Of these 8 candidates, 5 of them also protected the worms from MRSA infection. We also noted that worms exposed to N. fruticans root and O. stamineus leaf extracts showed reduced intestinal colonization by live S. aureus. This suggests that these extracts could possibly activate host immunity to eliminate the bacteria or interfere with factor/s that prevents pathogen accumulation.

Conclusion

We have successfully demonstrated the utility of this liquid-based screen to identify anti-infective substances that prolong S. aureus-infected host survival without affecting bacterial cell viability.     

Association between susceptibility to photodynamic oxidation and the genetic background of Staphylococcus aureus

Multidrug resistant strains of Staphylococcus aureus are a major cause of skin and soft tissue infections requiring the development of novel and alternative therapeutic options. Photodynamic oxidation is the cornerstone of antimicrobial photodynamic therapy (aPDT) involving the combined use of light and a photosensitizer, which, in the presence of oxygen, originates cytotoxic species capable of oxidizing biological molecules and leads to inactivation of target cells. We have previously shown that susceptibility to aPDT differs significantly across S. aureus isolates and could be associated with several genetic elements. However, the effect of the photodynamic process regarding the S. aureus genetic background has never been reported. We have compared the genetic backgrounds of the strains (SCCmec types, spa types and main clonal complexes) with respect to their susceptibility to protoporphyrin IX-mediated photodynamic inactivation. SCCmec typing revealed no differences in response to photoinactivation. However, detection of spa types and clonal complexes clustered the studied population of MRSA strains according to their response to photodynamic oxidation. Clonal complex 1 (CC1) accounted for elevated resistance and CC30 (ST36) for susceptibility to photoinactivation. Moreover, spa typing identified isolates resistant (t032) and susceptible to photodynamic oxidation (t051, t015). The very tight association between clonal lineages and response to photodynamic inactivation indicates the important role of genetic background for aPDT efficacy. These results make a case for the development of a diagnostic tool with the predictive value of aPDT efficacy according to an identified genetic background of S. aureus isolates.     

Staphylococcus aureus requires less virulence to establish an infection in diabetic hosts

Staphylococcus aureus is the most frequent pathogen causing diabetic foot infections. Here, we investigated the degree of bacterial virulence required to establish invasive tissue infections in diabetic organisms. Staphylococcal isolates from diabetic and non-diabetic foot ulcers were tested for their virulence in in vitro functional assays of host cell invasion and cytotoxicity. Isolates from diabetes mellitus type I/II patients exhibited less virulence than isolates from non-diabetic patients, but were nevertheless able to establish severe infections. In some cases, non-invasive isolates were detected deep within diabetic wounds, even though the strains were non-pathogenic in cell culture models. Testing of defined isolates in murine footpad injection models revealed that both low- and high-virulent bacterial strains persisted in higher numbers in diabetic compared to non-diabetic hosts, suggesting that hyperglycemia favors bacterial survival. Additionally, the bacterial load was higher in NOD mice, which have a compromised immune system, compared to C57Bl/6 mice. Our results reveal that high as well as low-virulent staphylococcal strains are able to cause soft tissue infections and to persist in diabetic humans and mice, suggesting a reason for the frequent and endangering infections in patients with diabetes.     

The ability of biofilm formation does not influence virulence of Staphylococcus aureus and host response in a mouse tissue cage infection model

The virulence of Staphylococcus aureus Sa113 (SA113) and an isogenic ica deletion mutant (ica-), deficient in the production of polysaccharide intercellular adhesin (PIA), which is crucial for biofilm formation, was compared in a mouse tissue cage infection model. The minimal infective doses for the induction of persistent tissue infections in C57BL/6 mice were 10(3) CFU for both SA113 and the ica- mutant. Bacterial growth, initial adherence to surfaces within the implants and the course of inflammation including growth-dependent host TNF and MIP-2 release, influx of phagocytes and an accumulation of dead leukocytes were similar as well. Since SA113 expressed PIA in vivo, we could demonstrate that PIA and the lack of biofilm formation did not influence the capacity of S. aureus to induce persistent infections and did not modulate host responses in the mouse tissue cage model.