The mechanism on Prevotella melaninogenica promoting the inflammatory progression of oral lichen planus.

Oral lichen planus (OLP) is a common chronic inflammatory disease occurring in the oral mucosa. Bacteria are a key driver of mucosal immune responses and can induce changes in gene expression and function of epithelial keratinocytes. IL-36γ can induce the expression of antimicrobial peptides, cytokines, and chemokines, and is widely involved in many chronic inflammatory diseases. Our aim is to explore the role of IL-36γ in the pathological process of OLP when Prevotella melaninogenica (P. melaninogenica) invades the oral mucosa. The expression of IL-36γ in OLP lesions and mice was detected by immunohistochemistry. Recombinant human IL-36Gamma (rhIL-36γ) was used to treat oral keratinocytes and the expression levels of inflammatory cytokines were detected by qRT-PCR and ELISA. The expression of IL-36γ and TRPV1 was detected by western blotting following co-culturing P. melaninogenica with oral keratinocytes. The mRNA expression of IL-36γ was detected by qRT-PCR. From our results, IL-36γ was upregulated in OLP lesions. Exogenous rhIL-36γ promoted the expression of pro-inflammatory cytokines and antibacterial peptides in oral keratinocytes. The expression of IL-36γ was significantly increased following the stimulation of P. melaninogenica in oral keratinocytes and mice. TRPV1 activation was induced by P. melaninogenica and its activation enhanced the expression of IL-36γ. IL-36Ra could reduce the inflammation in OLP in vitro. In summary, overexpression of IL-36γ in OLP lesions could promote its pathogenesis by inducing inflammation. P. melaninogenica invasion of oral keratinocytes could induce the expression of IL-36γ by the activation of TRPV1, thereby regulating the interaction between bacteria and oral epithelial cells.

The presence of Prevotella melaninogenica within tissue and preliminary study on its role in the pathogenesis of oral lichen planus.

OBJECTIVE: Oral lichen planus (OLP) is a chronic inflammatory disease that occurs in the oral mucosa with characteristic white striations lesions, recurrent erosions, and pains. The etiology and pathogenesis of OLP are still unclear. MATERIALS AND METHODS: We analyzed the bacterial community structure of buccal mucosa in patients with OLP and normal controls by high-throughput sequencing. Fluorescence in situ hybridization (FISH) was used to detect Prevotella melaninogenica (P. melaninogenica) in 13 OLP samples and 10 controls. The amounts of P. melaninogenica in OLP buccal mucosa and the expression of inflammatory cytokines in co-culture of mouse-derived macrophages with P. melaninogenica were detected by RT-qPCR. RESULTS: The P. melaninogenica was more abundant in OLP than in healthy controls, and the differences were significant at the level of the phylum, family, genus, and species (p < .05). FISH showed that P. melaninogenica can invade the epithelium and even the lamina propria of OLP, while no invasion was found in the normal mucosa. Prevotella melaninogenica can adhere to and invade macrophages and then activate the transcription of IL-1ß, IL-6, and TNF-α in NF-κB signaling pathway. CONCLUSION: Prevotella melaninogenica may be involved in the pathogenic process of OLP, and its specific mechanism deserves further study.

Oxygen induces mutation in a strict anaerobe, Prevotella melaninogenica.

Strict anaerobes are highly sensitive to oxygen, but the mutagenicity of oxygen in strict anaerobes has not been well understood. Prevotella melaninogenica, a strict anaerobe, is susceptible to oxygen and shows an increase in oxidative DNA damage upon exposure to oxygen. In this study, we have investigated the mutagenicity of oxygen and the types of mutations induced by oxygen. Exposure to oxygen decreased cell survival and increased the levels of 8-oxo-deoxyguanosine (8-oxodG). The frequency of rifampicin-resistant mutants was markedly increased after exposure to oxygen. After sequencing a 254-bp fragment of the rpoB gene, which encodes the beta subunit of bacterial RNA polymerase, a target molecule of rifampicin, we found that most mutants induced by oxygen had GC to TA transversions, a signature of 8-oxodG. In addition, all detected single-nucleotide changes would lead to amino acid changes that confer rifampicin resistance. These results indicate that oxygen is mutagenic in a strict anaerobe, P. melaninogenica, and its mutagenic characteristics could be analyzed with this experimental system.

Induction of oxidative DNA damage in anaerobes.

We compared oxidative DNA damage in strictly anaerobic Prevotella melaninogenica, aerotolerant anaerobic Bacteroides fragilis, and facultative anaerobic Salmonella typhimurium after exposure to O2 or H2O2. Using HPLC with electrochemical detection, we measured 8-hydroxydeoxyguanosine (8OHdG) as a damage marker. O2 induced 8OHdG in P. melaninogenica but not in B. fragilis, which shows catalase activity, or in S. typhimurium. In P. melaninogenica, with catalase, O2 induced less 8OHdG; superoxide dismutase had no effect; with glucose and glucose oxidase, O2 induced more 8OHdG. H2O2 also markedly increased 8OHdG. O2 was suggested to induce 8OHdG through H2O2. O2 or H2O2 decreased survival only in P. melaninogenica. Highly sensitive to oxidative stress, P. melaninogenica could prove useful for investigating oxidative DNA damage.

Sphingolipid biosynthesis and vitamin K metabolism in Bacteroides melaninogenicus.

B. melaninogenicus provides a unique system for the study of the biosynthesis of an important group of lipids, the phosphosphingolipids. Sphingolipid biosynthesis can be repressed and induced by depletion and restoration of vitamin K. At least one enzyme involved in sphingolipid biosynthesis from the microorganism can be solubilized and so purified by conventional methods. Pathways involved in biosynthesis may differ from hitherto postulated pathways, for example, the incorporation of NH4+ into ethanolamine residue of ceramide phosphorylethanolamine. Moreover, the derivation of mutants defective in steps in sphingolipid biosynthesis would be of great value in these studies.

Rapid diagnosis of anaerobic infections by gas-liquid chromatography.

It was postulated that the short chain fatty acids (SCFA) produced by anaerobic bacteria might serve as microbial markers in purulent material. Eighteen pus specimens from various sources were analysed by gas-liquid chromatography (GLC), and the SCFA detected were compared with the microorganisms isolated by conventional methods. It was found that the detection of propionic, isobutyric, butyric, or isovaleric acids by direct GLC of pus specimens is strong evidence for anaerobic infection but not specific for Bacteroides fragilis. It was also shown that the presence of succinic acid in pus specimens does not necessarily indicate infection by anaerobes. It can be concluded that direct GLC of purulent material provides a rapid and reliable presumptive method for the differentiation between anaerobic and aerobic infections.

Cytotoxic activity of Bacteroides gingivalis and Bacteroides asaccharolyticus.

Culture filtrates of all eight strains of Bacteroides gingivalis and all five strains of B. asaccharolyticus were toxic for Vero cells. Cytotoxicity was in general greater with material from cultures of B. gingivalis than from B. asaccharolyticus but none of the culture filtrates from eight strains of B. melaninogenicus showed activity in this test. The toxic material was released during prolonged incubation and more detailed study of preparations from one strain indicated that it had a molecular weight of less than 3500 and was heat stable.

The isolation and identification of Bacteroides spp. from the normal human faecal flora.

Gram-negative anaerobic bacilli of the Bacteroides group were isolated on an enriched selective medium from specimens of faeces from 20 normal healthy adults and identified by conventional bacteriological methods. A heavy growth of Bacteroides spp. was obtained from all specimens and 10 representative colonies from each subject were identified. Most isolates (84%) belonged to the B. fragilis group. The commonest species identified in this group were B vulgatus and B. thetaiotaomicron (22% each), B. distasonis (18%) and the B. eggerthii/variabilis complex (14%), and only 9% were B. fragilis. B. vulgatus was isolated from 80% of subjects, B. thetaiotaomicron and B. distasonis from 70% each and the B. eggerthii/variabilis complex from 65%, but B. fragilis was detected in only 45% of specimens. Asaccharolytic species were isolated in smaller numbers from 14 (70%) subjects, but only five strains of the B. melaninogenicus/oralis group were isolated and fusobacteria were not detected.

Presence of diaminopimelic acid in propionate-negative Bacteroides species and in some butyric acid-producing strains.

AThe presence of diaminopimelic acid (m-DAP) in strains of Bacteroides melaninogenicus, B. bivius and other species as well as in unidentified strains of Bacteroides was investigated by thin-layer chromatography. Strains of B. bivius and B. disiens all contained m-DAP as did the subspecies intermedius and melaninogenicus of B. melaninogenicus. Strains of B. asaccharolyticus and similar black pigment-producing butyrate-positive isolates showed heterogeneity. Asaccharolytic strains were DAP negative, whereas two strains fermenting glucose were positive. Some of the non-pigmented propionate-negative and butyrate-negative unidentified strains also contained DAP. The consistent finding of m-DAP in strains of B. bivius, B. disiens, and B. melaninogenicus indicates that DAP detection might be of value in the identification of these species.

Inhibitory effect of lactoperoxidase-generated hypothiocyanite upon black pigmented anaerobe growth.

This study aimed to evaluate the in vitro effect of lactoperoxidase with or without its substrates (hydrogen peroxide, thiocyanate) on the growth of 4 different black pigmented anaerobe (BPA) strains associated with the development and progress of periodontal diseases: Porphyromonas gingivalis ATCC 33277, Prevotella intermedia NCTC 9336, Prevotella loescheii ATCC 15930, and Prevotella melaninogenica NCTC 9338. A 5-min lactoperoxidase-generated OSCN--HOSCN incubation at pH 6.0, 7.0 or 8.0 resulted in a decrease of the growth rate (tested by turbidimetry in liquid cultures) of the 4 BPA strains, whilst lactoperoxidase alone actually promoted bacterial growth.

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.

Infectious oral necrosis (cancrum oris) in Nigerian children: a review.

The devastating orofacial gangrenous disease known as cancrum oris (noma) is still commonly seen in underprivileged Nigerian children. These children are usually victims of such stressors as chronic malnutrition, numerous endemic communicable diseases and severe adverse physical conditions which may lead to depletion of their adaptive resources or produce physiological maladaptation to additional stressors. Measles is the most common infection preceding the development of noma in Nigerian children. Acquired immunodeficiency as well as the impaired endocrine balance of the chronically malnourished permits, for example, widespread infection with the measles virus. Anergy resulting from the combination of malnutrition and measles virus infection promotes selective overgrowth and invasion by an infective consortium consisting of anaerobic organisms and other species capable of elaborating necessary growth factors for the former. Because of the pre-existing depletion of adaptive physiologic resources in the malnourished child, the infection is not readily contained locally as necrotizing ulcerative gingivitis but instead spreads rapidly to the next naturally occurring anatomical barriers. This is then followed by continuing necrosis and possible sequestration as exemplified by noma.

[Detection of anaerobes by direct immunofluorescence on clinical samples : evaluation of fluoretec F and M (author's transl)]. / Détection des anaérobies par immunofluorescence directe sur les prélèvements cliniques : évaluation des fluoretec F et M.

Immunofluorescence is a method allowing rapid identification of bacteria. The authors study two reagents. Fluoretec F and M, respectively detecting bacteroidis fragilis and bacteroidis melaninogenicus. These reactions were carried out directly on the clinical specimens. After a study carried out with 132 clinical specimens, by comparison with the usual techniques for isolation and identification, the following results were seen : Fluoretec F showed 89 p. cent concordance, 0.8 p. cent false negatives and 3 p. cent false positives. In 7.2 p. cent of cases, the type of discordance could not be determined. Fluoretec M showed 87.8 p, cent concordance, 6.1 p. cent false negatives, 0.8 p. cent false positives and in 5.3. p. cent of cases, the discordance could not be specified. Application of direct immunofluorescence for these two specific tests allowed rapid orientation of diagnosis and allowed evaluation of reliability of culture techniques.

The porphyrin pigmentation of subspecies of Bacteroides melaninogenicus.

Various subspecies of Bacteroides melaninogenicus differ in their pigmentation. Subsp. asaccharolyticus produces protohaem almost exclusively, subsp. intermedicus both protohaem and a smaller proportion of protoporphyrin, and subsp. melaninogenicus mainly protoporphyrin with a trace of protohaem. As a consequence young colonies can be differentiated by their red fluorescence in u.v. light (365nm): subsp. asaccharolyticus does not fluoresce, subsp. intermedicus shows a limited fluorescence, and subsp. melaninogenicus shows a bright fluorescence. The pigments were isolated as the dimethyl esters of protohaemin and of protoporphyrin and identified by electronic spectroscopy, mass spectrometry and comparisons by t.l.c. Incorporation of delta-aminolaevulinate into these pigments was not detected, nor was porphobilinogen formation observed. Subsp. melaninogenicus grown in the presence of [14C]protohaemin formed [14C]protoporphyrin. This appears to represent a novel biological demetallation.

Production of phenylacetic acid by Bacteroides melaninogenicus ssp. intermedius serogroup C-1.

The methyl derivatives of broth cultures of black-pigmented Bacteroides were examined by gas chromatography for production of phenylacetic acid. Two serogroups of B. melaninogenicus ssp. intermedius described by Lambe differed in the ability to produce phenylacetate. Serogroup C failed to produce phenylacetic acid while serogroup C-1 produced small amounts of phenylacetate, which contributed 2.2-5.7% to the total nonvolatile acid profile. Holdeman's newly proposed species "B. intermedius" and "B. corporis" correspond to serogroups C and C-1, respectively. These data support the elevation of the two serogroups of B. melaninogenicus ssp. intermedius to species status. Bacteroides gingivalis produced phenylacetate in significantly larger quantities than B. corporis. Bacteroides melaninogenicus ssp. melaninogenicus, "B. melaninogenicus ssp. levii," and B. asaccharolyticus did not produce phenylacetic acid. These results indicate that phenylacetic acid production may be useful in distinguishing "B. corporis" and B. gingivalis from the other black-pigmented Bacteroides.

Fermentation of L-aspartate by a saccharolytic strain of Bacteroides melaninogenicus.

Resting cells of Bacteroides melaninogenicus fermented L-[14C]aspartate as a single substrate. The 14C-labeled products included succinate, acetate, CO2, oxaloacetate, formate, malate, glycine, alanine, and fumarate in the relative percentages 68, 15, 9.9, 2.7, 1.8, 1.0, 0.7, 0.5, and 0.06, respectively, based on the total counts per minute of the L-[14C]aspartate fermented. Ammonia was produced in high amounts, indicating that 96% of the L-aspartate fermented was deaminated. These data suggest that L-aspartate is mainly being reduced through a number of intermediate reactions involving enzymes of the tricarboxylic acid cycle to succinate. L-[14C]asparagine was also fermented by resting cells of B. melaninogenicus to form L-aspartate, which was subsequently, but less actively, fermented.

Fructose 6-phosphate phosphorylation in Bacteroides species.

6-Phosphofructokinase (6-PFK) activities have been measured in cell extracts from a number of Bacteroides species. Two main types of 6-PFK were found: an ATP-linked 6-PFK and a PPi-linked 6-PFK. In most strains both of these activities were found, although in two strains only ATP-linked 6-PFK was present. The PPi-linked 6-PFK activity was always higher, when both activities were present, and showed Michaelis-Menten kinetics with respect to fructose 6-phosphate. In contrast, the ATP-linked 6-PFK activity usually gave sigmoid kinetics with respect to fructose 6-phosphate, although several strains were found to have activities with Michaelis-Menten kinetics. Conditions for measuring maximum activities of ATP-linked 6-PFK were not identical for different strains, and the activities were rather unstable in extracts. The possible consequences of the observation that most Bacteroides strains possess both an ATP-linked and a PPi-linked 6-PFK are discussed.

Production of phenylacetic acid by strains of Bacteroides asaccharolyticus and Bacteroides gingivalis (sp. nov.).

Strains of Bacteroides asaccharolyticus and Bacteroides melaninogenicus subspecies isolated from human and animal sources were examined for the production of phenylacetic acid. B. asaccharolyticus strains isolated from sites in humans and monkeys always produced phenylacetic acid. B. asaccharolyticus strains isolated from human nonoral sites consistently failed to produce this product. This metabolic difference correlates with the genetic dichotomy recently found to exist between oral and nonoral B. asaccharolyticus strains.

Description of a polyvalent conjugate and a new serogroup of Bacteroides melaninogenicus by fluorescent antibody staining.

A polyvalent conjugate (fluorescein isothiocyanate-labeled antibody reagent) containing serogroups A, B, and C conjugates was prepared. This polyvalent conjugate gave a positive fluorescent antibody (FA) stain with 49 stains of Bacteroides melaninogenicus representing serogroups A, B, and C. When additional strains (92 strains) of the three subspecies of B. melaninogenicus were examined by the FA stain, with A, B, and C, and polyvalent conjugates, nine strains of B. melaninogenicus subsp. intermedius failed to give a positive stain with any conjugate. Therefore, an FA conjugate was prepared with the antiserum to one of these strains (532-70A); all nine strains stained positively with this conjugate. These nine strains were biochemically characteristic of B. melaninogenicus subsp. intermedius; thus, these strains were designated as a new serogroup, serogroup C-1. A new polyvalent conjugate containing serogroups A, B, C, and C-1 was prepared. This polyvalent conjugate stained positively with 23 representative strains from serogroups A, B, C, and C-1. The new conjugates failed to stain positively with other anaerobes and aerobes tested. The four individual conjugates, as well as the polyvalent conjugate, may be used for a more rapid identification of B. melaninogenicus than is possible by biochemical testing.