Recent developments for Staphylococcus aureus vaccines: clinical and basic science challenges.

Bacterial vaccines have made dramatic impacts upon morbidity and mortality caused by a number of common pathogens, but a vaccine to prevent Staphylococcus aureus infections has proven to be illusive. With successful bacterial vaccines, the organisms are all part of the transient flora, whereas, S. aureus is part of the normal human flora. This means that S. aureus has had a prolonged time to adapt to the host milieu and its defences. The failure of several staphylococcal antigens to protect humans from infection in vaccine clinical trials using active or passive immunisation has stimulated a re-examination of the fundamental assumptions about staphylococcal immunity in humans vs. animals, especially rodents. This has spurred an active debate about the appropriate models for vaccine development and an examination of our current understanding of the protective immunity in humans. A major factor in the development of previous bacterial vaccines was a biomarker that predicted human protection, e.g., antibodies to tetanus toxoid or to pneumococcal polysaccharide. While antibodies against a number of staphylococcal antigens have proven to be an excellent biomarker for protection in rodents, these have not been translated to human infections. Thus, while much work remains, there is a growing consensus that T-cell immunity plays an important role in protecting humans. Moreover, the presence of anti-staphylococcal toxin antibodies correlates with reduced disease severity in humans. The most important recent advances concerning potential biomarkers, and the role of pre-existing immune status of vaccines in vaccine-associated mortality are considered in this review.

Where does a Staphylococcus aureus vaccine stand?

In this review, we examine the current status of Staphylococcus aureus vaccine development and the prospects for future vaccines. Examination of the clinical trials to date show that murine models have not predicted success in humans for active or passive immunization. A key factor in the failure to develop a vaccine to prevent S. aureus infections comes from our relatively limited knowledge of human protective immunity. More recent reports on the elements of the human immune response to staphylococci are analysed. In addition, there is some controversy concerning the role of antibodies for protecting humans, and these data are reviewed. From a review of the current state of understanding of staphylococcal immunity, a working model is proposed. Some new work has provided some initial candidate biomarker(s) to predict outcomes of invasive infections and to predict the efficacy of antibiotic therapy in humans. We conclude by looking to the future through the perspective of lessons gleaned from the clinical vaccine trials.

Pharmacodynamic evaluation of the activity of antibiotics against hemin- and menadione-dependent small-colony variants of Staphylococcus aureus in models of extracellular (broth) and intracellular (THP-1 monocytes) infections.

Staphylococcus aureus small-colony variants (SCVs) persist intracellularly, which may contribute to persistence/recurrence of infections and antibiotic failure. We have studied the intracellular fate of menD and hemB mutants (corresponding to menadione- and hemin-dependent SCVs, respectively) of the COL methicillin-resistant S. aureus (MRSA) strain and the antibiotic pharmacodynamic profile against extracellular (broth) and intracellular (human THP-1 monocytes) bacteria. Compared to the parental strain, SCVs showed slower extracellular growth (restored upon medium supplementation with menadione or hemin), reduced phagocytosis, and, for the menD SCV, lower intracellular counts at 24 h postinfection. Against extracellular bacteria, daptomycin, gentamicin, rifampin, moxifloxacin, and oritavancin showed similar profiles of activity against all strains, with a static effect obtained at concentrations close to their MICs and complete eradication as maximal effect. In contrast, vancomycin was not bactericidal against SCVs. Against intracellular bacteria, concentration-effect curves fitted sigmoidal regressions for vancomycin, daptomycin, gentamicin, and rifampin (with maximal effects lower than a 2-log decrease in CFU) but biphasic regressions (with a maximal effect greater than a 3-log decrease in CFU) for moxifloxacin and oritavancin, suggesting a dual mode of action against intracellular bacteria. For all antibiotics, these curves were indistinguishable between the strains investigated, except for the menD mutant, which systematically showed a lower amplitude of the concentration-effect response, with markedly reduced minimal efficacy (due to slower growth) but no change in maximal efficacy. The data therefore show that the maximal efficacies of antibiotics are similar against normal-phenotype and menadione- and hemin-dependent strains despite their different intracellular fates, with oritavancin, and to some extent moxifloxacin, being the most effective.

Two diarylurea electron transport inhibitors reduce Staphylococcus aureus hemolytic activity and protect cultured endothelial cells from lysis.

Reduction in electron transport is associated with decreased production in alpha-toxin despite the fact that Staphylococcus aureus is able to grow from 1 CFU to >10(7) CFU. Similarly, under anaerobic conditions, S. aureus does not produce alpha-toxin. Although the pathways that connect oxidative metabolism and toxin production are unknown, agents are available that exhibit greater inhibition of plant versus mammalian electron transport. Herbicides block electron transport in plants by inhibiting the formation of phosphoquinol (QH(2)) in plants. Commercial use in farming is possible because these compounds are much less active against the quinones found mammalian mitochondria. Because bacterial electron transport systems are closer to plant than mammalian systems, we hypothesized that inhibitors of respiration might be able to reduce S. aureus electron transport and block the production of alpha-toxin. We studied two compounds and found that the effective dose for the inhibition of bacterial respiration was 50 to >3,500 times lower than the concentration required to cause similar inhibition of rat mitochondrial respiration. Compounds I and II also reduced toxin production in S. aureus without causing overt toxicity to cultured endothelial cells. Finally, the compounds reduced the damage caused by S. aureus when cocultured with the endothelial cells. This raises the possibility that compounds that inhibit bacterial respiration might be prove valuable for the prevention of toxin production in S. aureus.

Coagulase-negative staphylococci. Pathogens have major role in nosocomial infections.

Coagulase-negative staphylococci live naturally on the skin and mucous membranes of humans and are therefore often found in clinical specimens. Distinguishing clinically significant, pathogenic strains from contaminant strains is one of the major challenges facing clinical microbiology laboratories. S epidermidis and other novobiocin-susceptible coagulase-negative staphylococci have emerged as a major cause of nosocomial infections, particularly of nosocomial bacteremia in immunocompromised patients. S epidermidis also is common in injecting drug users, who are particularly susceptible to right-sided endocarditis, and--most important--in patients with such indwelling foreign bodies as intravenous catheters. Depending on the kind of device and its insertion site, different infection syndromes generate a variety of clinical presentations. In these patients, the host defense mechanisms often seem unable to handle the infection and, in particular, to eliminate the staphylococci from the infected device because of a biofilm on the foreign body surface. S saprophyticus, the most commonly isolated bacterium of the novobiocin-resistant coagulase-negative staphylococci, is a common pathogen of the urogenital tract; it generally infects immunocompetent patients, particularly young, sexually active men and women.

Cutting edge: the nucleotide receptor P2X7 contains multiple protein- and lipid-interaction motifs including a potential binding site for bacterial lipopolysaccharide.

The nucleotide receptor P2X7 has been shown to modulate LPS-induced macrophage production of numerous inflammatory mediators. Although the C-terminal portion of P2X7 is thought to be essential for multiple receptor functions, little is known regarding the structural motifs that lie within this region. We show here that the P2X7 C-terminal domain contains several apparent protein-protein and protein-lipid interaction motifs with potential importance to macrophage signaling and LPS action. Surprisingly, P2X7 also contains a conserved LPS-binding domain. In this report, we demonstrate that peptides derived from this P2X7 sequence bind LPS in vitro. Moreover, these peptides neutralize the ability of LPS to activate the extracellular signal-regulated kinases (ERK1, ERK2) and to promote the degradation of the inhibitor of kappaB-alpha isoform (IkappaB-alpha) in RAW 264.7 macrophages. Collectively, these data suggest that the C-terminal domain of P2X7 may directly coordinate several signal transduction events related to macrophage function and LPS action.

Small colony variants of Staphylococci: a link to persistent infections.

In prospective studies, Staphylococcus aureus small-colony variants (SCVs) have been linked to persistent and recurrent infections. SCVs are a naturally occurring subpopulation often defective in electron transport which may be identified in the microbiological laboratory as nonpigmented, nonhemolytic, slow-growing pinpoint colonies after incubation on rabbit blood agar. In addition, the often relatively unstable SCVs demonstrate a number of other characteristics that are atypical for S. aureus including reduced alpha-toxin production and delayed coagulase activity. A site-directed hemB mutant with a stable SCV phenotype provided strong evidence for the link between these electron transport defective strains and persistent infections. The hemB mutant was phagocytized by cultured endothelial cells, but did not lyse these cells, because the mutant produced very little alpha-toxin. Thus, SCVs can hide within the host cell, then revert to the highly virulent rapidly growing form and lyse the host cell, once the host immune response has abated and antibiotic therapy is completed. The intracellular position shields SCVs from host defenses and decreases exposure to antibiotics. This review discusses what is known of the biology of SCVs and describes the recovery and significance of Staphylococcus SCVs in clinical specimen.

Identification, cloning, and initial characterization of rot, a locus encoding a regulator of virulence factor expression in Staphylococcus aureus.

A chromosomal insertion of transposon Tn917 partially restores the expression of protease and alpha-toxin activities to PM466, a genetically defined agr-null derivative of the wild-type Staphylococcus aureus strain RN6390. In co-transduction experiments, transposon-encoded erythromycin resistance and a protease- and alpha-toxin-positive phenotype are transferred at high frequency from mutant strains to agr-null strains of S. aureus. Southern analysis of chromosomal DNA and sequence analysis of DNA flanking the Tn917 insertion site in mutant strains revealed that the transposon interrupted a 498-bp open reading frame (ORF). Similarity searches using a conceptual translation of the ORF identified a region of homology to the known staphylococcal global regulators AgrA and SarA. To verify that the mutant allele conferred the observed phenotype, a wild-type allele of the mutant gene was introduced into the genome of a mutant strain by homologous recombination. The resulting isolates had a restored agr-null phenotype. Virulence factor gene expression in mutant, restored mutant, and wild-type strains was quantified by measuring alpha-toxin activity in culture supernatant fluids and by Northern analysis of the alpha-toxin transcript. We named this ORF rot (for repressor of toxins) (GenBank accession no. AF189239) because of the activity associated with rot::Tn917 mutant strains.

Staphylococcus aureus small colony variants, electron transport and persistent infections.

Staphylococcus aureus can mutate to form a sub-population of bacteria known as small colony variants (SCVs). These bacteria have a characteristic phenotype defined by slow growth, the lack of pigment, an altered pattern of carbohydrate utilization, and a reduction in toxin production. This complex phenotype can be explained by deficiencies in electron transport. In clinical isolates, the most common mutations that affect electron transport are in the operons encoding menaquinone or heme biosynthesis. These isolates are responsible for persistent antibiotic resistant infections. The clinical presentation of these infections is readily explained by a reduction electron transport. SCVs survive within host cells, increasing the instances of recurrent infections and have a novel mechanism of resistance based upon their altered trans-membrane potential. Additionally, SCVs provide a connection between energy metabolism and toxin production. This link may operate through the bacteria responding to altered levels of NADH and ATP. A more complete understanding of these signaling pathways may provide new targets for the development of drugs to ameliorate staphylococcal virulence and disease.

Bacterial colonization of functionalized polyurethanes.

A protocol was developed for studying the growth of bacteria upon polyurethanes subsequent to the establishment of an adherent bacterial population. An inocula of approximately 10(5) cfu S. aureus were spread on functionalized polyurethanes which included Pellethane, sulfonated Pellethane, phosphonated Pellethane, quaternized amine polyurethanes, and a zwitterionic phosphonated polyurethane. After 24 h incubation, Pellethane, sulfonated Pellethane, and phosphonated Pellethane showed bacterial growth by at least a factor of 10. In contrast, the zwitterionic phosphonated polyurethane showed a factor of 10 decrease in bacteria after 24 h and the quaternized amine polyurethanes reduced the bacteria to only a few hundred after only 1 h. When treated with bovine serum albumin, Pellethane, sulfonated Pellethane, and phosphonated Pellethane again showed bacterial growth by as much as a factor of 10 over 24 h. The quaternized amine polyurethanes and the zwitterionic phosphonated polyurethane still exhibited bactericidal abilities even when coated with bovine serum albumin, with the zwitterionic material reducing bacteria by more than a factor of 10 over 24 h and the quaternized amine polyurethane reducing the bacteria to only a few hundred after only 1 h. A zone of inhibition study suggested that the bactericidal activity of the zwitterionic phosphonated polyurethane was due to the leaching of cadmium ions. A quaternized amine polyurethane which contained chloride instead of iodide as the counterion to the amine moiety was less bactericidal than the iodide-containing polymer when treated with albumin. Thus, bacteria were able to colonize Pellethane, phosphonated sulfonated Pellethane, and phosphonated Pellethane, but the iodide-containing quaternized amine polyurethane and the zwitterionic polyurethane prevented colonization.

Staphylococcus aureus small colony variants: formation and clinical impact.

S. aureus small-colony variants are a naturally occurring subpopulation which grow slowly and produce small colonies on routine media. They also demonstrate a number of other characteristics that are atypical for S. aureus including reduced alpha-toxin production and delayed coagulase activity. The connection of S. aureus SCVs with persistent and relapsing infections has been defined over the past decade, especially in patients with chronic osteomyelitis and in cystic fibrosis patients as demonstrated by prospective studies. While the studies with clinical isolates of SCVs suggested a link between electron transport-defective strains and persistent infections, a defined hemB mutant with the SCV phenotype provided strong additional evidence for these connections. The hemB mutant was phagocytosed by cultured endothelial cells, but did not lyse these cells, because the mutant produced very little alpha-toxin. The intracellular location may shield the SCVs from host defenses and antibiotics, thus providing one explanation for the difficulty in clearing S. aureus SCVs from host tissues.

Bloodstream infections caused by small-colony variants of coagulase-negative staphylococci following pacemaker implantation.

Small-colony variants (SCVs) of Staphylococcus aureus cause persistent and relapsing infections. Relatively little is known regarding infections caused by SCVs of coagulase-negative staphylococci. We report two cases of pacemaker electrode infections due to SCVs of Staphylococcus epidermidis and Staphylococcus capitis. Sequence analysis of a portion of the 16S rRNA gene (16S rDNA) confirmed the identity of the staphylococcal species as S. capitis and S. epidermidis. Isolates from cultures of blood obtained over at least a 2-week interval were compared by pulsed-field gel electrophoresis and found to be clonal even though the colony morphology was very different. Analysis for auxotrophism revealed hemin dependencies for all isolated SCVs. The two cases have several clinical and laboratory characteristics (which are also seen with S. aureus SCV infections) and strongly suggest that SCVs of coagulase-negative staphylococci must be actively sought, because they grow very slowly and can be easily missed.

Bacterial adhesion to functionalized polyurethanes.

The effect of fibrinogen and high molecular weight kininogen on bacterial adhesion to functionalized polyurethanes was studied. Glass slides were coated with different polyurethanes, including Pellethane, sulfonated Pellethane, phosphonated Pellethane, a zwitterionic phosphonated polyurethane, and quaternized amine polyurethanes. The polymer-coated glass squares were exposed to radiolabelled S. aureus. When comparing adhesion to bare polyurethanes, it was found that adhesion was lowest on the phosphonated Pellethane and the zwitterionic phosphonated polyurethane while highest on the methyl quaternized polyurethanes. Fibrinogen-mediated adhesion was studied by first exposing the polymers to increasing concentrations of canine fibrinogen before incubating them with S. aureus. All the polymers except the quaternized amine polyurethanes exhibited at least ten-fold increases in bacterial adhesion as the fibrinogen treatment concentration was increased from 0.0 to 10.0 microg ml(-1). The quaternized amine polyurethanes maintained their relatively high amount of bacterial adhesion regardless of the fibrinogen concentration. The effect of two-chain high molecular weight kininogen (TCHMWK) on fibrinogen-mediated bacterial adhesion was assessed by exposing the polymers to 1.0 microg ml(-1) fibrinogen followed by two different concentrations of TCHMWK. Decreases in bacterial adhesion were observed on all the polymers except the quaternized amine polyurethanes, which again retained their relatively high amount of bacterial adhesion.