Analysis of EDTA-soluble cell surface components of gram-positive anaerobic cocci

The protein content of EDTA extracts from 76 strains of Gram-positive anaerobic cocci was examined using SDS-PAGE. Strains of Peptostreptococcus anaerobius produced almost identical profiles; greater heterogeneity was observed within the species Peptococcus magnus, Peptococcus prevotii and Peptococcus asaccharolyticus, but several strains within each biotype produced similar patterns. Serological investigation of these extracts by ELISA revealed numerous cross-reactions among the different biotypes. Immunoblot transfers from polyacrylamide gels demonstrated two common antigens within strains of the species, Ps. anaerobius, but these were not species-specific.     

Fibronectin-binding proteins of gram-positive cocci

Cell wall-attached fibronectin-binding proteins are important multifunctional virulence factors of Staphylococcus aureus and Streptococcus pyogenes. This review describes recent advances in the understanding of the function of these proteins on a molecular level and of their role in infections.     

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.     

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.     

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.     

Enhanced killing of penicillin-treated gram-positive cocci by human granulocytes: role of bacterial autolysins, catalase, and granulocyte oxidative pathways

Staphylococci pretreated with subminimal inhibitory concentrations (subMIC) of cell-wall active antibiotics exhibit increased susceptibility to killing by human polymorphonuclear leukocytes (PMNs), even when phagosome information is impaired by the mold metabolite, cytochalasin B. To investigate the role of specific bacterial factors in the process, studies were carried out with organisms lacking catalase (streptococci) or cell-wall autolytic enzymes and compared to findings with Staphylococcus aureus 502A. Neutrophil factors were studied using inhibitors, oxygen radical scavengers, myeloperoxidase (MPO)-deficient PMNs, or PMNs from a patient with chronic granulomatous disease (CGD). Documentation of the enhanced susceptibility of the streptococcal strains to killing by PMNs following subMIC penicillin pretreatment required the use of cytochalasin B. Enhancement of killing occurred independent of the presence or absence of bacterial autolysins or catalase. SubMIC penicillin pretreatment of S. pneumoniae R36A specifically promoted the susceptibility of these organisms to killing by myeloperoxidase (MPO)-mediated mechanisms (enhancement lost using MPO-deficient or azide-treated cells). Factors other than MPO or toxic oxygen products generated by the PMN respiratory burst are responsible for enhanced killing of penicillin-pretreated S. aureus 502A (enhancement preserved using MPO-deficient, azide-treated, or chronic granulomatous disease patient cells). These studies define methods to study the interaction of antimicrobial agents and PMNs in the killing of microorganisms. They also demonstrate that penicillin treatment can change the susceptibility of gram-positive cocci to the action of specific PMN microbicidal mechanisms. The mechanism of the enhancement appears to be bacterial strain-dependent and not predictable by bacterial autolysin or catalase activity.     

Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci

The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.     

The role of peptidoglycan in pathogenesis

Bacterial pathogens rely on a variety of virulence factors to establish the colonization of a new niche. Although peptidoglycan and its muropeptide derivatives have been known to possess potent biological properties, until recently the molecular bases were poorly understood. With the identification of the cytosolic surveillance mechanism mediated by the nucleotide-binding oligomerization domain (Nod)1 and Nod2 proteins, which detect unique peptidoglycan-derived muropeptides, these muropeptides should be considered as potential virulence factors. Recent research highlights the role of peptidoglycan in the pathogenesis of different human pathogens such as Streptococcus pneumoniae, Listeria monocytogenes or Helicobacter pylori.     

The role of anaerobic bacteria in bacteremia

Anaerobic bacteria remain an important cause of bloodstream infections and account for 1–17% of positive blood cultures. This review summarizes the epidemiology, microbiology, predisposing conditions, and treatment of anaerobic bacteremia (AB) in newborns, children, adults and in patients undergoing dental procedures. The majority of AB are due to Gram-negative bacilli, mostly Bacteroides fragilis group. The other species causing AB include Peptostreptococcus, Clostridium spp., and Fusobacterium spp. Many of these infections are polymicrobial. AB in newborns is associated with prolonged labor, premature rupture of membranes, maternal amnionitis, prematurity, fetal distress, and respiratory difficulty. The predisposing conditions in children include: chronic debilitating disorders such as malignant neoplasm, hematologic abnormalities, immunodeficiencies, chronic renal insufficiency, or decubitus ulcers and carried a poor prognosis. Predisposing factors to AB in adults include malignant neoplasms, hematologic disorders, transplantation of organs, recent gastrointestinal or obstetric gynecologic surgery, intestinal obstruction, diabetes mellitus, post-splenectomy, use of cytotoxic agents or corticosteroids, and an undrained abscess. Early recognition and appropriate treatment of these infections are of great clinical importance.     

The role of anaerobic bacteria in sinusitis

The normal oropharyngeal flora contained aerobic and anaerobic bacteria that can cause respiratory infections including sinusitis. Some of these bacteria can interfere with the growth of potential pathogens and may play a role in preventing infections. Anaerobic bacteria emerge as pathogens as the infection becomes chronic. This may be the result of the selective pressure of antimicrobial agents that enable resistant anaerobic organisms to survive, and from the development over time of conditions appropriate for anaerobic growth, which include the reduction in oxygen tension and an increase in acidity within the sinus cavity. Anaerobes were isolated in acute maxillary sinusitis of odontogenic origin and in over half of the patients with chronic sinusitis whenever proper techniques for their cultivation were employed. These organisms were also recovered in acute sinusitis that was associated with dental infections. The predominant isolates were pigmented Prevotella and Porphyromonas, Fusobacterium and Peptostreptococcus spp.     

Regulation of the tricarboxylic acid cycle in gram-positive, facultatively anaerobic bacilli.

The facultative anaerobes Bacillus polymyxa Hino G, B. polymyxa Hino J, and B.macerans were observed to have imcomplete tricarboxylic acid cycles. They were devoid of malate dehydrogenase and all had very low levels of alpha-ketoglutarate dehydrogenase. B. polymyxa Hino J was devoid of alpha-ketoglutarate dehydrogenase when grown aerobically and anerobically. Citrate synthase from B. polymyxa was inhibited by adenosine triphosphate but not reduced nicotinamide adenine dinucleotide and resembled enzymes from other gram-positive bacteria in this respect. Like the citrate synthases from gram-negative, facultative anaerobes and chemolithotrophs, the enzyme from B. polymyxa was inhibited by alpha-ketoglutarate. Inhibition by adenosine triphosphate was shown to be competitive with acetyl-coenzyme A and alpha-ketoglutarate inhibition was competitive with oxaloacetate.