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.

Phototargeting oral black-pigmented bacteria.

We have found that broadband light (380 to 520 nm) rapidly and selectively kills oral black-pigmented bacteria (BPB) in pure cultures and in dental plaque samples obtained from human subjects with chronic periodontitis. We hypothesize that this killing effect is a result of light excitation of their endogenous porphyrins. Cultures of Prevotella intermedia and P. nigrescens were killed by 4.2 J/cm2, whereas P. melaninogenica required 21 J/cm2. Exposure to light with a fluence of 42 J/cm2 produced 99% killing of P. gingivalis. High-performance liquid chromatography demonstrated the presence of various amounts of different porphyrin molecules in BPB. The amounts of endogenous porphyrin in BPB were 267 (P. intermedia), 47 (P. nigrescens), 41 (P. melaninogenica), and 2.2 (P. gingivalis) ng/mg. Analysis of bacteria in dental plaque samples by DNA-DNA hybridization for 40 taxa before and after phototherapy showed that the growth of the four BPB was decreased by 2 and 3 times after irradiation at energy fluences of 4.2 and 21 J/cm2, respectively, whereas the growth of the remaining 36 microorganisms was decreased by 1.5 times at both energy fluences. The present study suggests that intraoral light exposure may be used to control BPB growth and possibly benefit patients with periodontal disease.

Soft-tissue abscess involving Actinomyces odontolyticus and two Prevotella species in an intravenous drug abuser.

Skin and soft-tissue infections in intravenous users comprise a variety of microorganisms and anaerobic bacteria are frequently involved in these suppurative infections. A case of subcutaneous abscess into anterior femoral muscles involving Actinomyces odontolyticus and two Prevotella species (Prevotella buccae and Prevotella melaninogenica) in an intravenous drug abuser is presented. This combination of microorganisms has not previously been described in soft-tissue infections. The patient volunteering that he licked his hypodermic needle prior to cocaine injection supports that the implicating bacteria originated from the oral cavity. Eventually, the patient recovered and at a 6-month follow-up a gradual improvement of his subcutaneous infection was noticed.

Bacterial and fungal microleakage of AH26 and AH Plus root canal sealers.

AIM: To evaluate the penetration of Candida albicans alone and a combination of bacteria through root canals filled with gutta-percha and one or other root canal sealers, AH26 and AH Plus. METHODOLOGY: Eighty teeth were randomly divided into two groups of 40 teeth each and obturated with gutta-percha using either AH26 or AH Plus sealer. A further 10 teeth served as negative controls and 10 as positive controls. The external surface of each root, except the apical 2 mm, was covered with two layers of nail varnish. The teeth were inserted into Eppendorf plastic tubes and suspended in glass bottles containing sterile Schaedler broth. Streptococcus mutans, Streptococcus mitis, Prevotella melaninogenica and Lactobacillus acidophilus were placed in the access cavities of 20 teeth filled with AH26 and 20 with AH Plus. Candida albicans was placed in the access cavities of the other teeth. The culture medium with microorganisms was changed every 7 days. Every 72 h bacterial or fungal growth in the broth was tested up to a period of 90 days. RESULTS: Leakage in the experimental teeth occurred between 14 and 87 days. Leakage was present in 47% of all samples. From the samples with AH26, 45% leaked bacteria and 60% leaked fungi; whilst from the samples with AH Plus, 50% leaked bacteria and 55% fungi. There was no statistically significant difference in penetration of bacteria and fungi between the sealers. CONCLUSION: In this in vitro study, gutta-percha and the sealers AH26 and AH Plus allowed leakage of bacteria and fungi.

Salivary microbiota levels in relation to periodontal status, experience of caries and miswak use in Sudanese adults.

OBJECTIVES: The purpose of the present investigation was to assess the salivary levels of 25 oral bacteria in relation to periodontal status and experience of caries, and to compare the levels of these bacteria between habitual miswak and toothbrush users in adult Sudanese subjects. MATERIAL AND METHODS: The study subjects consisted of 56 individuals with age range 19-53 years (mean 35.2 years) and included 30 miswak and 26 toothbrush users. The periodontal status and presence of dental caries were assessed clinically. Whole saliva was collected from all subjects, and the levels of 25 selected bacterial species in saliva were assessed by the checkerboard DNA-DNA hybridization method using whole genomic DNA probes. RESULTS: A high percentage of the subjects had detectable levels (> or = 105 bacterial cells) of several bacterial species in saliva. Between 12% and 16% of the subjects showed high (> or = 106 cells) salivary levels of the periodontitis-associated bacteria A. actinomycetemcomitans, P. melaninogenica, P. intermedia, C. rectus and E. corrodens, whereas only two (3.6%) and four (7.1%) subjects had high levels of P. gingivalis and F. nucleatum, respectively. There were no significant differences in the levels of all or most bacterial species by age group, gender or periodontal status. Presence of > or = 105 L. acidophilus bacterial cells in saliva was associated with high caries scores (p = 0.02). There were significantly higher levels of A. actinomycetemcomitans, P. melaninogenica, C. rectus, P. micros, V. parvula, S. mutans, S. anginosus, A. israelii, C. sputigena, and C. gingivalis, and significantly lower levels of P. intermedia, F. nucleatum, S. sputigena, E. corrodens, L. acidophilus, S. sanguis, S. salivarius, S. oralis, and S. mitis in the miswak than in the toothbrush group. CONCLUSIONS: : The findings suggest that miswak may have a selective inhibitory effect on the level of certain bacteria in saliva, particularly several oral streptococci species. This is the first report that the checkerboard DNA-DNA hybridization method can be useful for assessing the levels of a wide range of bacterial taxa in saliva.

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.

Effect of Bacteroides melaninogenicus culture supernatant and deconjugated bile salt on lipid absorption.

Lipid malabsorption is a common clinical manifestation of small bowel bacterial overgrowth. Its pathogenesis, however, remains controversial. Bacteroides melaninogenicus ssp. intermedius, an anaerobic bacterium, is commonly isolated from the upper bowel of patients with small intestinal bacterial overgrowth. The effects of a culture supernate of this organism and deoxycholate, an unconjugated bile salt, on intestinal oleic acid absorption were examined using a rat closed-loop model. The supernatant reduced the in vitro uptake of oleic acid by 19% (P< 0.001). Deoxycholate did not significantly reduce the lipid absorption. Combined supernate and deoxycholate did not have an additive effect on absorption of oleic acid. We conclude that anaerobic bacterial products may contribute to the malabsorption of lipid in the setting of bacterial overgrowth of the small bowel.

An in-vitro study of the comparative viability on different swab types of simulated specimens of bacteria commonly present in oro-facial infections.

An in-vitro study was undertaken to investigate the survival on bacteriological swabs of three potentially pathogenic organisms found in oro-facial infections, by using simulated clinical specimens incubated in the presence or absence of Amies transport medium. Standard inocula of pure cultures of Streptococcus milleri, Prevotella melaninogenica and Peptostreptococcus anaerobius were soaked on to swabs and plated out on to solid media at various intervals up to 3 days, enabling an estimate of their viability to be made following further incubation. Differential survival of the three test organisms was observed, with Streptococcus milleri being generally the hardiest and Peptostreptococcus anaerobius the least hardy, whilst survival was consistently enhanced by the presence of a transport medium. Recovery of Prevotella melaninogenica was improved by incubation for 5 days. We conclude that transport swabs should be used in preference to plain swabs whenever immediate laboratory culture cannot be assured, to avoid loss of anaerobic bacteria.

Cross-inhibition between black-pigmented Bacteroides species.

Cross-inhibition within the group of black-pigmented Bacteroides, including both oral and non-oral strains, was studied by means of a membrane filter technique. It was found that B. gingivalis possessed the most extended inhibitory capacity among all species tested. B. gingivalis showed inhibitory activity against B. intermedius, B. endodontalis, B. loescheii, and B. melaninogenicus. B. endodontalis was active against some B. intermedius strains. Among the saccharolytic species, some B. melaninogenicus strains were inhibitory for some B. endodontalis strains, some B. gingivalis strains, and some B. intermedius strains. These inhibitory activities observed in vitro may play a role in the colonization of the periodontal pocket.

Nutritional relationships between oral bacteria.

Nutritional relationships were revealed during the coculturing of Bacteroides gingivalis with Wolinella recta and Bacteroides melaninogenicus with W. recta. W. recta produced a substance that stimulated the growth of B. gingivalis and B. melaninogenicus. Characterization by thin-layer chromatography and absorption spectrometry identified the compound as protoheme. Production of large amounts of formate by B. melaninogenicus stimulated the growth of W. recta. These nutritional relationships could represent examples of mechanisms favoring bacterial succession in periodontal sites.

A note on ultra-violet red fluorescence of anaerobic bacteria in vitro.

Anaerobes other than the Bacteroides melaninogenicus group isolated from clinical material produce an ultra-violet red fluorescence when grown under certain conditions in vitro. These organisms include other members of the genus Bacteroides as well as strains of some species of Clostridium, Bifidobacterium and Actinomyces. The major fluorescent pigment was identified as protoporphyrin IX. Factors necessary for the production of fluorescence are the presence of blood or haem and a fermentable carbohydrate during growth on a solid medium. Fluorescence intensity was related to the concentration of blood and fermentable carbohydrate present but was independent of inoculum size. Certain commercially available blood agar bases designed specifically for the isolation of fastidious anaerobes from clinical material which contain added carbohydrate were shown to induce fluorescence in certain organisms. This may lead to the misidentification of some anaerobes as B. melaninogenicus.

Use of the RapID-ANA System to screen for enzyme activities that differ among species of bile-inhibited Bacteroides.

The RapID-ANA System (Innovative Diagnostics Systems, Inc., Atlanta, Ga.) was used to test 102 strains of 14 species of phenotypically similar bile-inhibited Bacteroides from humans. Bacteroides oris, Bacteroides veroralis, Bacteroides buccalis, Bacteroides melaninogenicus, Bacteroides loescheii, and Bacteroides denticola had very similar enzyme activity profiles. Clear differentiation of these six species by the RapID-ANA System was not possible, but tests for arginine aminopeptidase and beta-glucosidase were helpful. Bacteroides oralis, Bacteroides intermedius, Bacteroides corporis, Bacteroides disiens, Bacteroides bivius, Bacteroides gingivalis, Bacteroides asaccharolyticus, and Bacteroides buccae each had unique enzyme activity profiles. No consistent differences in enzyme activities were found between the two DNA homology groups within Bacteroides melaninogenicus, Bacteroides loescheii, or Bacteroides intermedius. Tests for glycine aminopeptidase, alpha-galactosidase, arginine aminopeptidase, alpha-fucosidase, N-acetylglucosaminidase, reduction of triphenyltetrazolium, and production of indole were helpful in the differentiation of the species studied.

Effect of the growth of anaerobic bacteria on the surface pH of solid media.

Changes in surface pH occurring after varying periods of anaerobic incubation were measured for a total of 23 test solid media. There was little change in the surface pH of uninoculated plates, but plates inoculated with Bacteriodes fragilis showed a striking fall in pH, to pH 5 in the case of some of the test media. The problems of controlling the surface pH of solid media are discussed and possible methods of control are considered.

The influence of incubation time and medium composition on fatty acid production by some Bacteroides species.

Thirteen strains (eleven species) of Bacteroides have been examined for the production of short chain fatty acids (SCFAs). Two media, cooked meat glucose (CMG) and peptone yeast glucose (PYG) were examined quantitatively by gas liquid chromatography (glc) after anaerobic incubation at 37 degrees C for up to 7 days. Growth and medium pH were monitored in PYG for two species. The SCFA profile was established after 12 h and the amount of acids produced was greater in CMG than in PYG; both CMG and PYG contained SCFAs prior to inoculation. Maximum acid production and lowering of pH appear to coincide with the stationary phase of growth. Results are discussed in terms of a standardized approach to the identification of anaerobic bacteria.

Morphological changes in the bacterium Bacteroides melaninogenicus subspecies melaninogenicus isolated from the human mouth and grown in culture without added blood components.

Striking polymorphism in the cellular morphology could be induced by removal of blood components from the liquid growth medium, but the cells of Bacteroides gingivalis and B. melaninogenicus subspecies intermedius did not exhibit polymorphism when grown under these conditions. The major changes observed with light microscopy were an increase in cell size and extreme polymorphism. Electron microscopy of the polymorphic forms of B. melaninogenicus subspecies melaninogenicus strains showed that such cells lacked both the outer cell membrane and peptidoglycan layer. Serum promoted the growth of these strains, suggesting that some blood component is either conducive to the synthesis of the cell wall or masks an unknown inhibitor for cell-wall synthesis contained in the medium.

Effects of estradiol and progesterone on Bacteroides melaninogenicus and Bacteroides gingivalis.

Bacteroides melaninogenicus subsp. intermedius increases in the subgingival microflora during pregnancy. These studies evaluated direct interactions between hormonal steroids and oral Bacteroides species. Resting cell suspensions of pure cultures of plaque organisms were incubated anaerobically with [14C]estradiol and [14C]progesterone. Uptake of labeled compound per microgram of bacterial protein was determined by thin-layer chromatography and liquid scintillation counting. B. melaninogenicus subsp. intermedius and B. melaninogenicus subsp. melaninogenicus took up 2.6 x 10(-4) to 5.4 x 10(-4) mumol of estradiol or progesterone per microgram of cell protein. Minimal steroid uptake was observed with B. gingivalis and five other organisms. Uptake of steroids by B. melaninogenicus subsp. intermedius was temperature dependent and resulted in a labeled product as detected on thin-layer chromatography. Growth curves indicated that intermedius and melaninogenicus subspecies of B. melaninogenicus but not B. gingivalis could substitute progesterone or estradiol for vitamin K, an essential growth factor. Growth of B. melaninogenicus subsp. intermedius in steroids was concentration dependent. Addition of fumarate to resting cells of B. melaninogenicus subspecies as well as B. gingivalis increased steroid uptake by 70 to 500% and resulted in the gas-liquid chromatographic detection of succinate. Cultures given fumarate alone or steroids alone produced no succinate. Steroids appeared to directly interact with the fumarate reductase system and foster the growth of B. melaninogenicus subsp. intermedius. This interaction may be of ecological significance.