Isolation and chemical analysis of a lipopolysaccharide from the outer membrane of the oral anaerobic spirochete Treponema pectinovorum

Isolation of a putative lipopolysaccharide from the surface of the oral treponeme, Treponema pectinovorum, revealed it to contain larger amounts of 3-deoxy-D-manno-octulosonic acid compared with other oral Treponema species. This molecule was isolated from the outer membrane of T. pectinovorum and had chemical characteristics of a putative lipopolysaccharide. The yield of lipopolysaccharide was between 0.6% and to 1.1% of the bacterial dry weight. The purified molecule was resistant to the action of proteinases and consisted of both sugars and lipids. 3-Deoxy-D-manno-octulosonic acid and hexoses accounted for 6.1-8.7% and 17.6-20.2%, respectively of the dry weight. Carbohydrate compositional analysis revealed the presence of glucose, galactose, 2-acetamido-2-deoxy-glucose, rhamnose and 6-deoxy-talose in the molar ratio of 1.00:0.96:0.19:0.88:0.98, respectively. No heptose was detected. The fatty acid analysis determined the presence of straight chain, C13:00, C14:00, C15:00 and C17:00 acids, as well as branched chain, C13:00, C14:00 and two species of C15:00, acids. Electrophoretic analysis indicated that the lipopolysaccharide was present as two major species.     

Short- and medium-chain fatty acids exhibit antimicrobial activity for oral microorganisms

Objectives

This study assessed the antibacterial activity of short-, medium-, and long-chain fatty acids against various oral microorganisms.

Methods

The short-chain fatty acids [formic acid (C1), acetic acid (C2), propionic acid (C3), butyric acid (C4), isobutyric acid (C4), isovaleric acid (C5), hexanoic acid (C6)], medium-chain fatty acids [octanoic acid (C8), capric acid (C10), lauric acid (12)], and long-chain fatty acids [myristic acid (C14), palmitic acid (C16)], were investigated for antimicrobial activity against Streptococcus mutans, Streptococcus gordonii, Streptococcus sanguis, Candida albicans, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis.

Results

The data demonstrated that the fatty acids exhibited patterns of inhibition against oral bacteria with some specificity that appeared related more to the bacterial species that the general structural characteristics of the microorganism. As a group the fatty acids were much less effective against C. albicans than the oral bacteria, with effectiveness limited to hexanoic, octanoic, and lauric acids. Formic acid, capric, and lauric acids were broadly inhibitory for the bacteria. Interestingly, fatty acids that are produced at metabolic end-products by a number of these bacteria, were specifically inactive against the producing species, whilst substantially inhibiting the growth of other oral microorganisms.

Conclusions

The results indicate that the antimicrobial activity of short-chain fatty acids (SCFAs), medium-chain fatty acids (MCFAs), long-chain fatty acids (LCFAs) could influence the microbial ecology in the oral cavity via at least 2 potential pathways. First, the agents delivered exogenously as therapeutic adjuncts could be packaged to enhance a microbial-regulatory environment in the subgingival sulcus. Second, it would be the intrinsic nature of these fatty acid inhibitors in contributing to the characteristics of the microbial biofilms, their evolution, and emergence of species within the biofilms. Further studies on these functions are required to better understand the nature of these potential microbial interactions in the biofilms.     

Effects of ammonia and volatile fatty acids produced by oral bacteria on tissue culture cells

Culture filtrates of several bacterial species isolated from the oral cavity were tested for their effects on two types of tissue culture cells: Vero cells, the continuous cell line of African green monkey kidney cells; and chondrocytes, isolated from 15-day-old chick embryo tibiae. Only a limited number of bacterial species--i.e., the asaccharolytic black-pigmented Bacteroides species and Fusobacterium species--affected the two cell types. The effect on Vero cells, detected by the rounding of the cells, correlated with the butyric acid concentration in the bacterial supernatant, which confirms previous findings. A small enhancement of this effect was found with propionic acid and ammonium ions. The same strains which affected Vero cells also affected chondrocytes, detected by a vacuolization of the cells. However, volatile fatty acids on their own had no visible effect on these cells. Instead, ammonium ion in the culture filtrate, when present in concentrations of 20 to 60 mmol/L, proved to be responsible for vacuolization of the chondrocytes. The volatile fatty acids (butyric and propionic) had a limited additive effect. No effects were visible with cell extracts of the bacteria.     

Purification of arginine-sensitive hemagglutinin from Fusobacterium nucleatum and its role in coaggregation

Hemagglutinin of Fusobacterium nucleatum was extracted from Triton X-100-pronase P-treated cell envelopes, and was purified by affinity chromatography on L-arginine agarose. The hemagglutinin was inactivated by heating at 70°C for 1 min. The activity was inhibited by L-arginine but was not affected by any sugars or by EDTA. The hemagglutinin aggreggated 14 out of 17 strains of oral streptococci tested, and the bacterial aggregating activity was also inhibited by L-arginine. The results indicate the dominant role of this hemagglutinin in the adherence of this bacterium both to host cells and to other bacteria.     

Fatty acid profiles in smokers with chronic periodontitis

We hypothesized that tobacco smoke induces alterations to the 3-OH fatty acids present in lipid A in a manner consistent with a microflora of reduced inflammatory potential. Whole saliva samples and full-mouth clinical periodontal recordings were obtained from persons with (22 smokers; 15 non-smokers) and without (14 smokers; 15 non-smokers) chronic periodontitis. Clear differences in the contributions of multiple saturated 3-OH fatty acid species were noted in the group with disease compared with healthy individuals. Increases in the long-chain fatty acids associated with anaerobic bacterial periodontopathogens, particularly 3-OH-C(i17.0) (146.7%, relative to controls), were apparent. Significant reductions in the 3-OH fatty acids associated with the consensus (high potency) enteric LPS structure (3-OH-C(12.0) and 3-OH-C(14.0); 33.3% and 15.8% reduction, respectively) were noted in smokers compared with non-smokers with chronic periodontitis. Thus, smoking is associated with specific structural alterations to the lipid-A-derived 3-OH fatty acid profile in saliva that are consistent with an oral microflora of reduced inflammatory potential. These findings provide much-needed mechanistic insight into the established clinical conundrum of increased infection with periodontal pathogens but reduced clinical inflammation in smokers.     

Intra-oral acid production associated with eating whole or pulped raw fruits

The hypotheses that raw fruits, whether whole or pulped, were cleared rapidly from the mouth and that the sugars in the whole and pulped fruits are fermented with equal efficiency to acids by the oral microflora were tested in this study. Groups of 7-9 adult subjects chewed 10 g of raw, whole or pulped fruit (apple, banana, orange, pear and pineapple) for 1 min and whole, unstimulated saliva samples were collected during the following 60-min interval. Each saliva sample was assayed for the concentrations of fruit-derived sugars (glucose, fructose and sucrose), fruit-derived acids (malic and citric) and acids which may be produced as a result of bacterial fermentation (acetic, lactic, formic and succinic). We found the fruit-derived sugars were rapidly cleared from the mouth (within 5 min). The major bacterially produced acids were lactic and succinic, which reached maximum concentrations in the 5-min sample. There was no significant difference, within a fruit, in the salivary levels of any of the sugars or acids between the raw whole or raw pulped forms. In light of these findings it seems unwise to assume that fruits may be consumed without consideration of their acidogenic potential.     

Fatty acids and sugars in lipopolysaccharides from Bacteroides intermedius, Bacteroides gingivalis and Bacteroides loescheii

Lipopolysaccharide (LPS, endotoxin) was isolated from Bacteroides intermedius, Bacteroides gingivalis and Bacteroides loescheii by phenol extraction, purified by ultracentrifugation and investigated chemically by gas chromatography and mass spectrometry. Twenty different fatty acids were found. The fatty acid composition differed between the bacterial species, and intraspecies variation was also recorded. All endotoxins contained 3-hydroxy-15-methylhexadecanoic acid and 3-hydroxy-hexadecanoic acid as major hydroxy fatty acids. Additional fatty acids were straight chain or iso- or anteiso-branched fatty acids, and straight chain or iso-branced hydroxylated fatty acids. The sugar composition showed great variability between different strains of the same species. Heptose and 2-keto-3-deoxyoctonate (KDO) were detected in LPS from strains of B. gingivalis and B. intermedius. A tentatively identified glucosamine disaccharide was detected in all three Bacteroides species. This is indicative of lack of phosphorylation in the lipid A backbone, which is in concordance with the low general toxicity of Bacteroides LPS.     

Bacterial diversity in aphthous ulcers

INTRODUCTION: Recurrent aphthous ulcers are common lesions of the oral mucosa of which the etiology is unknown. This study aimed to estimate the bacterial diversity in the lesions and in control mucosa in pooled samples using a culture-independent molecular approach. METHODS: Samples were collected from ten healthy individuals and ten individuals with a clinical history of recurrent aphthous ulcers. After DNA extraction, the 16S ribosomal RNA bacterial gene was amplified by polymerase chain reaction with universal primers; amplicons were cloned, sequenced and matched to the GenBank database. RESULTS: A total of 535 clones were analyzed, defining 95 bacterial species. We identified 62 putative novel phylotypes. In recurrent aphthous ulcer lesions 57 phylotypes were detected, of which 11 were known species. Control samples had 38 phylotypes, five of which were already known. Only three species or phylotypes were abundant and common to both groups (Gemella haemolysans, Streptococcus mitis strain 209 and Streptococcus pneumoniae R6). One genus was found only in recurrent aphthous ulcer samples (Prevotella) corresponding to 16% of all lesion-derived clones. CONCLUSION: The microbiota found in recurrent aphthous ulcers and in the control groups diverged markedly and the rich variety of genera found can provide a new starting point for individual qualitative and quantitative analyses of bacteria associated with this oral condition.     

Factors affecting the formation of spherical bodies in the spirochete Treponema denticola

The oral spirochete Treponema denticola typically is a helically shaped, motile bacterial cell. However, morphological variations of T. denticola cells in the form of "spherical bodies" are sometimes seen. Little is known about the environmental factors that cause their formation. The effects of oxygen, growth temperature, nutrient depletion and the addition of metabolic end-products were tested to determine their role in the morphogenesis of the spherical bodies. It was found that the age of the culture, the omission of individual components (yeast extract, rabbit serum, volatile fatty acids or thiamine pyrophosphate) from the medium and the addition of the metabolic end product lactic acid enhanced the formation of these bodies. However, their formation was decreased upon omission of the medium components asparagine and sodium bicarbonate.     

Diverse activity spectra of bacteriocin-like inhibitory substances having activity against mutans streptococci

Mutans streptococci (MS) are known to be causative agents of dental caries. It has been suggested that these cariogenic bacteria could be eliminated from dental plaque by application of bacteriocins or bacteriocin-like inhibitory substances (BLIS). In the present study 272 bacterial strains of the genera Streptococcus, Enterococcus and Staphylococcus were tested for their production of BLIS activity against MS by use of a deferred antagonism test on agar media supplemented with either whole blood or yeast extract. Although only 14.3% of the test strains displayed anti-MS activity, the inhibitory agents produced by these strains were characterised by considerable diversity in the range of their inhibitory action against both MS and other common oral streptococcal species. It is suggested that combinations of relatively specifically targeted anti-MS BLIS may have potential application to the prevention of dental caries.     

Coaggregation of Candida albicans oral Actinomyces species

Eight strains of Actinomyces were examined for their ability to coaggregate in vitro with four strains of Candida albicans. The Actinomyces coaggregated to various degrees with all of the Candida strains. Exposure of the Candida but not the Actinomyces to heat, trypsin, proteinase K, amphotericin B or trichodermin abolished Coaggregation. All sugars tested did not inhibit any of the reactions. All coaggregating pairs were disaggregated by the addition of SDS, but nonionic detergents had no effect. The addition of urea or EDTA completely reversed Coaggregation. Actinomyces strains were sensitive to periodate oxidation, whereas the Candida strains were unaffected. These data suggest that the coaggregations involve a protein on the Candida surface that may interact with carbohydrates or carbohydrate-containing molecules on the surface of the Actinomyces. These observations expand the known range of intergeneric coaggregations occurring between human oral microbes and indicate that Coaggregation of C. albicans and Actinomyces may be an important factor in oral colonization by this yeast.     

Immunological labeling of oral bacteria after demineralization

Demineralization of teeth with their adherent bacterial flora is a prerequisite for the localization of selected species in sections of undisturbed bacterial deposits by immunoelectron microscopy. The effect of EDTA dernineralization on the immunological labeling of Gram-positive and Gram-negative oral bacteria was tested on strains of Actinomycesviscosus, A. israelii, Eikertella corrodens, Bacieroides meloninogenicus. Capnocytophaga sputigena and Wolinella recta . After 3 months in EDTA, cells prefixed in paraformaldehyde-picric acid were washed and incubated with homologous rabbit antiscrum followed by goat anti-rabbit IgG to enhance the immunocoating reaction. Samples were then processed for transmission electron microscopy. Control specimens included (1) demineralized cells incubated with pre-immune rabbit sera instead of homologous antisera (2) cells stored for 3 months in PBS instead of EDTA (3) fresh cells processed for immunocoating immediately after prefixation and washing. No detectable immunocoating layer was found on the cells of the control group incubated with pre-immune rabbitserum, EDTA treated cells demonstrated an immunocoating layer of similar density and thickness to that of cells stored in PBS, or fresh cells. The results indicate that immunoelectron microscopic localization of bacterial cells may be feasible in ullrathin sections of demineralized dental specimens.     

The formation of hydrogen sulfide and methyl mercaptan by oral bacteria

The capacity to form volatile sulfur compounds was tested in bacteria isolated from subgingival microbiotas and in a representative number of reference strains. A majority of the 75 tested oral bacterial species and 7 unnamed bacterial taxa formed significant amounts of hydrogen sulfide from L-cysteine. The most active bacteria were found in the genera Peptostreptococcus, Eubacterium, Selenomonas, Centipeda, Bacteroides and Fusobacterium. Methyl mercaptan from L-methionine was formed by some members of the genera Fusobacterium, Bacteroides, Porphyromonas and Eubacterium. When incubated in serum for 7 d, the most potent producers of hydrogen sulfide were Treponema denticola and the black-pigmented species, Bacteroides intermedius, Bacteroides loescheii, Porphyromonas endodontalis and Porphyromonas gingivalis. P. endodontalis and P. gingivalis also produced significant amounts of methyl mercaptan in serum. No other volatile sulfur compound was detected in serum or in the presence of L-cysteine and L-methionine. These findings significantly increase the list of oral bacteria known to produce volatile sulfur compounds.     

Effect of oral bacteria on growth and survival of Candida albicans biofilms

The purpose of this study was to evaluate the effect of eight aerobic and anaerobic oral commensal bacterial species on in vitro Candida albicans biofilm development. A single isolate of C. albicans 2560 g, and eight different species of oral bacteria comprising, Actinomyces israelii, Lactobacillus acidophilus, Prevotella nigrescens, Porphyromonas gingivalis, Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutans, and Streptococcus intermedius were studied using an in vitro biofilm assay. Biofilm formation was quantified in terms of the ability of Candida to grow on polystyrene plastic surfaces co-cultured with the foregoing bacteria. A viable cell count was used to quantify the sessile yeast growth and scanning electron microscopy was employed to confirm and visualize biofilm formation. Co-culture with differing concentrations of bacteria had variable effects on Candida biofilm formation. Co-culture with the highest concentrations of each of the foregoing bacteria resulted in a consistent reduction in the yeast counts in the candidal biofilm (P<0.05), except for L. acidophilus, S. mutans, and, S. intermedius co-cultures. Further, on regression analysis a significant negative correlation between the co-culture concentration of either P. gingivalis or E. coli and viable yeast counts in the biofilm was noted (P<0.05) although this was not evident for the other bacterial species. Taken together, our data indicate that, quantitative and qualitative nature of the bacteria modulate C. albicans biofilm formation in mixed species environments such as the oral cavity.     

Oral mycology

Yeasts occur commonly in the oral cavity in healthy individuals. The prevalent species is Candida albicans (about 60-70% of all isolates). C. glabrata and C. tropicalis come next, followed by other Candida species and genera (Rhodotorula, Saccharomyces, etc.) which are all of rare occurrence and transient. The yeast flora increases in many patient groups, especially those who are immunocompromised. C. albicans is the most important species, being the cause of almost all cases of yeast infections in the region, often in association with other species. The number isolated from the oral cavity depends on testing site and methods used. C. albicans can be typed by means of serology (types A and B), by biotyping, by morphology, by means of sensitivity to killer factors, by electrophoretic karyotyping, DNA fragments, and immunoblotting. Such methods may be of value epidemiologically. Switching in Candida morphology is associated with changes in micromorphology and physiology. Several non-yeast fungi may affect the oral cavity, most frequently in association with lung or disseminated infections.     

Coaggregation of Streptococcus salivarius with periodontopathogens: evidence for involvement of fimbriae in the interaction with Prevotella intermedia

Streptococcus salivarius is divided into two serological subgroups that carry either fibrils or fimbriae. Although fimbriae have been observed on up to 50% of S. salivarius strains in the human oral cavity, no function has yet been assigned to them. To determine whether S. salivarius fimbriae have a role in adhesion, we examined the ability of S. salivarius to coaggregate with selected microorganisms involved in periodontal diseases. Our results show that S. salivarius coaggregated with Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia. However, only fimbriated S. salivarius cells were able to coaggregate with P. intermedia, suggesting a specific role for these structures in the interaction. Heat treatment, sensitivity to sugars, amino acids, and EDTA, as well as protease treatment were also used to further characterize coaggregation between S. salivarius and periodontopathogens.     

Antimicrobial activity of n-6, n-7 and n-9 fatty acids and their esters for oral microorganisms

Objective

This study is to assess the antibacterial activity of omega-6, -7, -9 (n-6, n-7, n-9) fatty acids against various oral microorganisms.

Methods

The n-6, n-7, n-9 fatty acids, such as γ-linoleic acid (GLA), linoleic acid (LA), arachidonic acid (ARA), palmitoleic acid (PA), and oleic acid (OA), their fatty acid ethyl esters, GLA-EE, LA-EE, ARA-EE, PA-EE, OA-EE, and their fatty acid methyl esters, GLA-ME, LA-ME, ARA-ME, PA-ME, OA-ME, were investigated for antimicrobial activity against oral pathogens Streptococcus mutans, Candida albicans, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis. Various concentrations of the fatty acids, their methyl and ethyl esters were tested against various oral pathogens in 96-well plates and blood-agar plate. The plates were incubated anaerobically or aerobically at 37 °C for 48 h, and the colony forming units (CFU) were determined.

Results

The data demonstrated that select n-6, n-7, n-9 fatty acids and their esters exhibited strong antimicrobial activity against these oral microorganisms, demonstrating some specificity for individual microbial species.

Conclusion

The potential use or the combinations of the n-6, n-7, n-9 fatty acids and/or their esters, provided in a local delivery vehicle to infected sites in the oral cavity, could be considered as an additional therapeutic approach to improving oral health.