Involvement of PorK, a component of the type IX secretion system, in Prevotella melaninogenica pathogenicity.

Prevotella melaninogenica is a gram-negative anaerobic commensal bacterium that resides in the human oral cavity and is isolated as a pathogen of suppurative diseases both inside and outside the mouth. However, little is known about the pathogenic factors of P. melaninogenica. The periodontal pathogens Porphyromonas gingivalis and Tanerella forsythia secrete virulence factors such as protease and bacterial cell surface proteins via a type IX secretion system (T9SS) that are involved in pathogenicity. P. melaninogenica also possesses all known orthologs of T9SS. In this study, a P. melaninogenica GAI 07411 mutant deficient in the orthologue of the T9SS-encoding gene, porK, was constructed. Hemagglutination and biofilm formation were decreased in the porK mutant. Furthermore, following growth on skim milk-containing medium, the diameters of the halos surrounding the porK mutant were smaller than those of the wild-type strain, suggesting a decrease in secretion of proteases outside the bacterium. To investigate this in detail, culture supernatants of wild-type and porK mutant strains were purified and compared by two-dimensional electrophoresis. In the mutant strain, fewer spots were detected, indicating fewer secreted proteins. In infection experiments, the mortality rate of mice inoculated with the porK mutant strain was significantly lower than in the wild-type strain. These results suggest that P. melaninogenica secretes potent virulence factors via the T9SS that contribute to its pathogenic ability.

Dysbiotic oral microbiota and infected salivary glands in Sjögren's syndrome.

Key events in the pathogenesis of SjÓ§gren syndrome (SS) include the change of salivary gland epithelial cells into antigen-presenting cell-like phenotypes and focal lymphocytic sialadenitis (FLS). However, what triggers these features in SS is unknown. Dysbiosis of the gut and oral microbiomes is a potential environmental factor in SS, but its connection to the etiopathogenesis of SS remains unclear. This study aimed to characterize the oral microbiota in SS and to investigate its potential role in the pathogenesis of SS. Oral bacterial communities were collected by whole mouthwash from control subjects (14 without oral dryness and 11 with dryness) and primary SS patients (8 without oral dryness and 17 with dryness) and were analyzed by pyrosequencing. The SS oral microbiota was characterized by an increased bacterial load and Shannon diversity. Through comparisons of control and SS in combined samples and then separately in non-dry and dry conditions, SS-associated taxa independent of dryness were identified. Three SS-associated species and 2 control species were selected and used to challenge human submandibular gland tumor (HSG) cells. Among the selected SS-associated bacterial species, Prevotella melaninogenica uniquely upregulated the expression of MHC molecules, CD80, and IFNλ in HSG cells. Concomitantly, P. melaninogenica efficiently invaded HSG cells. Sections of labial salivary gland (LSG) biopsies from 8 non-SS subjects and 15 SS patients were subjected to in situ hybridization using universal and P. melaninogenica-specific probes. Ductal cells and the areas of infiltration were heavily infected with bacteria in the LSGs with FLS. Collectively, dysbiotic oral microbiota may initiate the deregulation of SGECs and the IFN signature through bacterial invasion into ductal cells. These findings may provide new insights into the etiopathogenesis of SS.

Suppurative parotitis caused by anaerobic bacteria in newborns.

Staphylococci are the usual bacterial etiology of suppurative parotitis in newborns. This report describes for the first time recovery of anaerobic bacteria from aspirates of the infected gland in two infants with suppurative parotitis. Peptostreptococcus intermedius and Prevotella melaninogenica were isolated from one child and Prevotella intermedia from the other patient. Complete recovery occurred after 4 weeks of antimicrobial therapy.

Pathogenicity of capsulate and non-capsulate members of Bacteroides fragilis and B. melaninogenicus groups in mixed infection with Escherichia coli and Streptococcus pyogenes.

The relationships between capsulate and non-capsulate Bacteroides fragilis strains and Escherichia coli, and between capsulate and non-capsulate strains of the B. melaninogenicus group and Streptococcus pyogenes, were studied in a subcutaneous abscess model in mice. Selective antimicrobial agents directed against either aerobic or anaerobic bacteria were used alone or in combination to explore the effect of eradication of one component of the mixed infection. Single agent therapy effective against both aerobic and anaerobic flora was also employed. Single therapy of mixed infection directed at the elimination of only one organism (S. pyogenes, E. coli or Bacteroides sp.) caused significant reductions in the numbers of sensitive organisms and also smaller yet significant decreases in the numbers of insensitive organisms. However, the abscesses were not eliminated after such therapy. Combination therapy or use of a single agent (cefoxitin) directed against the aerobic and anaerobic components of the infection was more effective. Non-capsulate Bacteroides spp. became capsulate after passage in mice mixed with either S. pyogenes or E. coli. Therapy directed at the elimination of S. pyogenes and E. coli did not prevent the emergence of capsulate Bacteroides spp. These data demonstrate the synergy between all members of the B. fragilis group and E. coli and between the B. melaninogenicus group and S. pyogenes, and reiterate the need to direct antimicrobial therapy at the eradication of the aerobic and anaerobic components of mixed infections.

Pathogenicity of encapsulated Bacteroides melaninogenicus group, B. oralis and B. ruminicola subsp. brevis in abscesses in mice.

The pathogenicity of 27 clinical isolates of the Bacteroides melaninogenicus (BM) group and four clinical isolates of B. oralis and B. ruminicola subsp. brevis were investigated by inoculating them into mice and subsequently determining their ability to cause subcutaneous (SC) or intraperitoneal abscesses. Only 11 isolates of BM group and one B. ruminicola induced abscesses in mice, and all were found to be heavily encapsulated on recovery from the abscesses (more than 50 per cent of the organisms were encapsulated). When the other 23 isolates, however, were injected SC in combination with either Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus or Streptococcus pyogenes, abscesses were formed in 16 of the 23 combinations. The Bacteroides spp. recovered from the mixed infection were heavily encapsulated. Capsules also formed in Bacteroides if the organisms were injected together with capsular material or formalin killed cells of K. pneumoniae or encapsulated Bacteroides sp. Once non-encapsulated or only slightly encapsulated strains acquired a capsule, they could induce abscesses on reinoculation into mice.

Experimentally induced subcutaneous infection by black pigmented Bacteroides species in rats.

In a rat model of anaerobic subcutaneous abscess developed to study the pathogenicity of pure cultures of black pigmented Bacteroides sp. (Bacteroides melaninogenicus and Bacteroides intermedius) maximum lesions were seen between 7 and 15 days after inoculation. Abscesses were produced by one type strain each of B. melaninogenicus and B. intermedius and by 20 of 25 clinical isolates of black pigmented Bacteroides species.

Association of black-pigmented bacteria with endodontic infections.

Black-pigmented bacteria (BPB) have been associated with endodontic infections. The purpose of this study was to evaluate further the presence of BPB with the clinical signs and symptoms associated with endodontic infections. Microbial samples were collected from the root canals of 40 intact teeth with necrotic pulps and apical periodontitis. Conventional laboratory methods were used for identification of the strains of BPB isolated in pure culture. In addition, the polymerase chain reaction and specific primers for 16S r-RNA genes were used to differentiate Prevotella nigrescens from Prevotella intermedia. Twenty-two (55%) samples were positive for the growth of BPB. Of those, 11 of 22 (50%) were identified as P. nigrescens, 8 of 22 (36%) were P. intermedia, 2 of 22 (9%) were Porphyromonas gingivalis, and 1 of 22 (5%) was Prevotella melaninogenica. Sixteen of the 22 root canals positive for the growth of BPB were associated with purulent drainage either from the root canal or an associated sinus tract. Statistical analysis did not show a significant relationship for the presence of BPB with clinical signs and symptoms.

Characterization of volatile sulphur production by pathogenic and non-pathogenic strains of oral Bacteroides.

Marked differences were observed in intermediate sulphur metabolism between non-pathogenic strains of Bacteroides melaninogenicus var melaninogenicus (CP-) and pathogenic Bacteroides melaninogenicus asaccharolyticus (CP+). The CP+ strains, which produced collagenase and protease and caused formation of abscesses when injected subcutaneously into groins of guinea pigs, produced copious amounts of volatile sulphur compounds (VSC) which consisted predominantly of CH3SH and (CH3S)2. Hydrogen sulphide occurred in considerably lesser amounts. CP+ cultures yielded 8-fold more total volatile S, 15-fold more CH3SH and 260-fold more (CH3S)2 during 24 h of incubation in trypticase-yeast extract medium. Whereas H2S accounted for 60 per cent of the total volatile S content of the head-space of CP- cultures, it represented only 8 per cent of the volatile S in CP + systems. Although the CP-organisms did not grow as well as CP +, the differences in concentration of VSC may be only partly related to the disparity in growth rates. When the VSC concentrations were calculated on the basis of equivalent optical density of 1.0, the CP + strains still produced over 3-fold more total volatile S, 6-fold more CH3SH and 100-fold more (CH3S)2. A similar allowance for growth rate suggests that CP-strains may possess a greater potential to produce H2S. Both groups metabolized S-containing amino acids and serine, resulting in appreciable increases in H2S production by CP-. However, the two groups appeared to metabolize the carbon moiety of cystine an cysteine by different pathways. The addition of glucose to the medium depressed total volatile S production by both CP+ and CP-strains, attributable mostly to lower H2S levels. Whereas the omission of yeast extract and charcoal treatment of trypticase did not adversely effect the activity of CP+, it further markedly reduced the capacity of CP-cultures to produce VSC. These results suggest that VSC analysis offers a convenient means of assessing strain differences and pathogenic potential of B. melaninogenicus.

The role of Fusobacterium necrophorum and Bacteroides melaninogenicus in the aetiology of interdigital necrobacillosis in cattle.

When cultures of known pathogenic strains of Fusobacterium necrophorum, isolated either from cattle or sheep were injected through the interdigital skin of cattle typical lesions of interdigital necrobacillosis were produced. The inclusion of Bacteroides melaninogenicus in the inoculum did not appear to contribute to the development of lesions.

[Anaerobic bacteria and their role in recurrent tonsillitis]. / Bakterie beztlenowe i ich udzial w nawracajacym zapaleniu migdalków podniebiennych.

The presence of anaerobic bacteria in 100 palatal tonsils obtained during the tonsillectomy, was evaluated in quality and in quantity. 17 species of anaerobic bacteria were found. The most frequent were: Bacteroides melaninogenicus, Peptostreptococcus anaerobius, Peptostreptococcus intermedius, Propionibacterium, Veillonella parvula and Peptococcus asaccharolyticus. In most evaluations their density was 10(5)-10(6) in gram of tonsil tissue, sometimes 10(7). Its role in recurrent tonsils infections was discussed.

A hemagglutinating variant of Prevotella melaninogenica isolated from the oral cavity.

Strains resembling Prevotella melaninogenica were isolated from healthy subjects and patients with periodontal disease and were identified using: a 5-test phenotypic screen; commercial identification kits; and a 16S rRNA-based polymerase chain reaction (PCR) method. Eleven clinical isolates closely resembling P. melaninogenica, and all from patients with periodontitis, were able to agglutinate erythrocytes. In the electron microscope, hemagglutinating isolates showed fimbria-like structures, that were not seen on non-hemagglutinating isolates. Some strains were further classified with PCR-restriction fragment-length polymorphism (RFLP) of 16S rRNA genes. Amplified 16S rDNA was digested using five different endonucleases, separated with agarose gel electrophoresis, stained and photographed. Photographs were then scanned, digitized and a distance matrix calculated using Dice coefficient, where the presence or absence of a band was used as a character. The distance matrix was plotted as a phenogram. At 70% similarity six clusters were seen. Type strains of separate Prevotella species did not fall into any cluster. Hemagglutinating isolates fell into three clusters: four clustered with the type strains of P. melaninogenica and Prevotella veroralis; four with other P. melaninogenica isolates and two hemagglutinating isolates clustered together Prevotella loescheii. The PCR-RFLP results showed that the hemagglutinating strains did not form a homogenous group inside the Prevotella genus.

Characterization of Bacteroides asaccharolyticus and B. melaninogenicus oral isolates.

Experiments were designed to characterize a number of oral "pigmented" Bacteroides isolates with regard to their pathogenicity in an experimental model system and a number of other properties. T these include fatty acid determination, hemagglutination studies, collagenase and protease activities, and vitamin K dependency. Oral B, asaccharolyticus and B, melaninogenicus isolates differed from one another in phenylacetic acid production, hemagglutination, collagenase activity, and pathogenicity. All B. asaccharolyticus were found to be pathogenic in the vivo mixed infection model and this property could be correlated with biochemical enzymatic activities.

[Phlegmon of the fore arm succeeding to a dog-bite : respective role of the miscellaneous bacteria involved in this mixed infection (author's transl)]. / Phlegmon de l'avant-bras après morsure de chien : rôle respectif des diverses bactéries impliquées dans cette infection mixte.

The authors report a case of a severe mixed infection (phlegmon of the fore arm) succeeding to a dog bite. A complex flora has been isolated from this phlegmom : Pasteurella multocida, Pasteurella pneumotropica, Streptococcus mitis, Actinomyces sp., Bacteroides melaninogenicus, and a Gram- bacteria related to group II j. The pathogeny of such diseases is discussed, dwelling in one hand on the part of synergistic development of these various germs and in the other hand on the part played by saliva in the severity of animal or human bites. This kind of wound requires careful disinfection and radical debridment without omitting a preventive broad-spectrum antibiotherapy.

[Anaerobes in infected canals: a preliminary study].

Anaerobes of 17 infected canals with periapical periodontitis were studied. Most of root flora were not only anaerobes but also aerobes. Anaerobes were predominant in chronic periapical periodontitis. Major anaerobes isolated from canals were peptostreptococcus, B. melaninogenicus and B. oralis. The chi-square results indicated that the peptostreptococcus were significantly related to apical radiolucency and B. melaninogenicus were significantly related to percussion or foul smell. Animal experiment results showed that rats inoculated with mixed flora developed many abscesses. But the monoinfected rats had infiltration of PMNs, dilation and hypermia of vessels. B. melaninogenicus, B. oralis and mixed flora had liquefied gelatin and indicated these bacteria had collagenase which could dissolve collagen fibers and significantly related to destroy of collagen fibers and bone in periapical tissues.