The role of encapsulation in the pathogenesis of anaerobic gram-positive cocci

The pathogenicity of 22 anaerobic and facultative Gram-positive cocci (AFGPC) was investigated by inoculating them into mice and determining their ability to cause subcutaneous abscesses. Only 11 heavily encapsulated isolates (greater than 50% of the cells were encapsulated) induced abscesses. However, when the other 11 isolates were injected with Bacteroides sp. or facultative and aerobic bacteria, abscesses were formed in 8 of the 11 combinations. The AFGPC recovered from the mixed infections contained many encapsulated cells. Encapsulation also occurred in cocci injected with capsular material or with Formalin-killed cells of Klebsiella pneumoniae or capsule-positive Bacteroides sp. After acquisition of capsules, these strains could induce abscesses on reinoculation in mice.     

Identification and characterization of cell wall-cell division gene clusters in pathogenic gram-positive cocci.

Clusters of peptidoglycan biosynthesis and cell division genes (DCW genes) were identified and sequenced in two gram-positive cocci, Staphylococcus aureus and Enterococcus faecalis. The results indicated some similarities in organization compared with previously reported bacterial DCW gene clusters, including the presence of penicillin-binding proteins at the left ends and ftsA and ftsZ cell division genes at the right ends of the clusters. However, there were also some important differences, including the absence of several genes, the comparative sizes of the div1B and ftsQ genes, and a wide range of amino acid sequence similarities when the genes of the gram-positive cocci were translated and compared to bacterial homologs.     

In vitro activity of fosfomycin against 'problem' gram-positive cocci.

Fosfomycin was active in vitro against 54 of 60 'problem' Gram-positive cocci (20 methicillin-resistant Staphylococcus aureus, 20 coagulase-negative staphylococci and 20 enterococci). Its activity was significantly greater under anaerobic conditions, especially against coagulase-negative staphylococci. Mutants resistant to fosfomycin were readily demonstrated, but their growth was prevented by rifampicin or ciprofloxacin. The combinations rifampicin+fosfomycin and ciprofloxacin+fosfomycin showed MIC synergy. It is concluded that fosfomycin in an appropriate combination would be a valuable addition to the small and dwindling range of antibiotics active against problem Gram-positive cocci.     

Identification of Gram-positive anaerobic cocci by MALDI-TOF mass spectrometry

Gram-positive anaerobic cocci (GPAC) are part of the commensal microbiota of humans and are a phylogenetically heterogeneous group of organisms. To evaluate the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of GPAC, a database was constructed, using reference strains of commonly encountered GPAC and clinical isolates of which the sequence of the 16S rRNA gene was determined. Subsequently, the database was validated by identifying 107 clinical isolates of GPAC. Results were compared with the identifications obtained by 16S sequencing or fluorescent in situ hybridization (FISH). Strains belonging to the same species grouped together, in most cases, by MALDI-TOF MS analyses. Strains with sequence similarities less than 98% to their closest relatives, formed clusters distinct from recognized species in the MALDI-TOF MS dendrogram and, therefore could not be identified. These strains probably represent new species. Only three clinical isolates (2 strains of Finegoldia magna and 1 strain of Anaerococcus vaginalis) could not be identified. For all the other GPAC strains (96/107), reliable identifications were obtained. Therefore, we concluded that MALDI-TOF MS is an excellent tool for the identification of phylogenetically heterogeneous groups of micro-organisms such as GPAC.     

Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci

We report the identification of a previously unknown gene, inlA, which is necessary for the gram-positive intracellular pathogen Listeria monocytogenes to invade cultured epithelial cells. The inlA region was localized by transposon mutagenesis, cloned, and sequenced. inlA was introduced into Listeria innocua and shown to confer on this normally noninvasive species the ability to enter cells. Sequencing of inlA predicts an 80 kd protein, internalin. Two-thirds of internalin is made up of two regions of repeats, region A and region B, and the C-terminus of the molecule is similar to that of surface proteins from gram-positive cocci. Internalin has a high content of threonine and serine residues, and the repeat motif of region A has regularly spaced leucine residues. As evidenced by Southern blot analysis, inlA is part of a gene family. One of them is the gene situated directly downstream of inlA, called inlB, which also encodes a leucine-rich repeat protein.     

Engineering the gram-positive cell surface for construction of bacterial vaccine vectors.

A genetic system for surface display of heterologous proteins has been developed in Streptococcus gordonii, a gram-positive human oral commensal that is naturally competent for genetic transformation. Our approach is based on chromosomal integration downstream from a resident promoter and translational fusion to an M6 protein. Using this strategy a variety of proteins, of different origin and size, were displayed on the cell surface and were shown to be stably expressed both in vitro and in vivo. Animal models of mucosal colonization (oral and vaginal) and intragastric immunization with recombinant S. gordonii were developed and the local and systemic immune responses were studied. Here we report the techniques for the construction of recombinant bacteria, use of animal models, and analysis of the immune response.     

Reagents specific for cell surface components.

Mercury, diazonium ions and dyes which bind nucleic acids were covalently linked to dextrans using methods that resulted in non-hydrolyzable reagent-dextran bonds without impairing the binding abilities of the reagents, i.e. these dextran derivatives reacted with thiols, phenols/imidazoles and nucleic acids respectively. Since these dextran derivatives cannot penetrate into cells and since dextran itself does not bind to cells, these compounds represent reagents specific for the cell surface. They may be used both to evaluate cell surface constituents of intact cells and to affect viable cells via an interaction with those constituents. Mercury-dextran was found to bind to cells; the amount of mercury thus attached to the cells was about ten times smaller than when an equivalent concentration of free mercury ions was used. Mercury-dextran, bound to cells after a 30-min exposure at room temperature, was localized on the surface of these cells, as sodium borohydride reduced this complex giving rise to the intact cells, elementary mercury and free dextran which was released into medium. When cells were constantly exposed to the mercury-dextran, its toxic effects were comparable to that of free mercury ions. Diazonium-dextran, which also binds tightly to the cell surface, was also considerably toxic. Dextrans substituted with dyes which bind to nucleic acids were less toxic than the parent dyes themselves; it was shown that the attachment of such a dye to dextran decreased the binding of dye to cells under detection limits.     

In vitro activity of monoclonal and recombinant yeast killer toxin-like antibodies against antibiotic-resistant gram-positive cocci

BACKGROUND: Monoclonal (mAbKT) and recombinant single-chain (scFvKT) anti-idiotypic antibodies were produced to represent the internal image of a yeast killer toxin (KT) characterized by a wide spectrum of antimicrobial activity, including gram-positive cocci. Pathogenic eukaryotic and prokaryotic microorganisms, such as Candida albicans, Pneumocystis carinii, and a multidrug-resistant strain of Mycobacterium tuberculosis, presenting specific, although yet undefined, KT-cell wall receptors (KTR), have proven to be killed in vitro by mAbKT and scFvKT. mAbKT and scFvKT exert a therapeutic effect in vivo in experimental models of candidiasis and pneumocystosis by mimicking the functional activity of protective antibodies naturally produced in humans against KTR of infecting microorganisms. The swelling tide of concern over increasing bacterial resistance to antibiotic drugs gives the impetus to develop new therapeutic compounds against microbial threat. Thus, the in vitro bactericidal activity of mAbKT and scFvKT against gram-positive, drug-resistant cocci of major epidemiological interest was investigated. MATERIALS AND METHODS: mAbKT and scFvKT generated by hybridoma and DNA recombinant technology from the spleen lymphocytes of mice immunized with a KT-neutralizing monoclonal antibody (mAb KT4) were used in a conventional colony forming unit (CFU) assay to determine, from a qualitative point of view, their bactericidal activity against Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae strains. These bacterial strains are characterized by different patterns of resistance to antibiotics, including methicillin, vancomycin, and penicillin. RESULTS: According to the experimental conditions adopted, no bacterial isolate proved to be resistant to the activity of mAbKT and scFvKT. CONCLUSIONS: scFvKT exerted a microbicidal activity against multidrug resistant bacteria, which may represent the basis for the drug modeling of new antibiotics with broad antibacterial spectra to tackle the emergence of microbial resistance.     

BMAP-28 improves the efficacy of vancomycin in rat models of gram-positive cocci ureteral stent infection

An experimental study was performed to evaluate the efficacy of BMAP-28 alone and in combination with vancomycin in animal models ureteral stent infection due to Enterococcus faecalis and Staphylococcus aureus. Study included a control group without bacterial challenge to evaluate the sterility of surgical procedure, a challenged control group that did not receive any antibiotic prophylaxis and for each bacterial strain three challenged groups that received (a) 10 mg/kg vancomycin intraperitoneally, immediately after stent implantation, (b) BMAP-28-coated ureteral stents where 0.2-cm(2) sterile ureteral stents were incubated in 1mg/l BMAP-28 solution for 30 min immediately before implantation and (c) intraperitoneal vancomycin plus BMAP-28-coated ureteral stent at the above concentrations. Experiments were performed in duplicate. Ureteral stents were explanted at day 5 following implantation and biofilm bacteria enumerated. Our data showed that rats that received intraperitoneal vancomycin showed the lowest bacterial numbers. BMAP-28 combined with vancomycin showed efficacies higher than that of each single compound. These results highlight the potential usefulness of this combination in preventing ureteral stent-associated in gram-positive infections.