The presence of black-pigmented Gram-negative anaerobes in the oral cavity of edentolous subjects

Abstract The aim of this study was to investigate whether black-pigmented Gram-negative anaerobes are part of the indigenous oral flora of edentulous subjects with or without dentures. Group I consisted of 11 subjects with dentures (mean age 63.6 years, range 52–75) and Group II consisted of 39 subjects with complete dentures in both jaws (mean age 59.3 years, range 37–80). Two microbial samples for microbiological examination were taken from each subject. One of them was from the dorsum of the tongue and the other was from saliva. Black-pigmented Gram-negative anaerobes > 10⁷ cfu/ml were found in both samples. In 50 edentulous subjects, they were found more commonly from tongue (40%) than from saliva (26%). None of the subjects had Porphyromonas gingivalis . There was no statistically significant difference ( P > 0.05) between the presence of black-pigmented Gram-negative anaerobes on tongue and in saliva in the two groups. Our results suggested that high levels of black-pigmented Gram-negative anaerobes may belong to the indigenous oral flora in edentulous mouths with or without dentures.     

Cloning and expression of a pectate lyase from the oral spirochete Treponema pectinovorum ATCC 33768

The pelA gene, encoding a pectate lyase, from Treponema pectinovorum ATCC 33768 was isolated by heterologous expression of a cosmid library in Escherichia coli. In vitro transposon mutagenesis identified an open reading frame of 1293 bp capable of encoding a protein of 430 amino acids with a predicted amino-terminal signal sequence of 21 amino acids. Analysis of the amino acid sequence suggested that it is a member of the polysaccharide lyase family 10 of which all characterized members show pectate lyase activity. An amino-terminal His-tagged recombinant form of PelA was expressed and purified from E. coli. The recombinant enzyme has characteristics common to other bacterial pectate lyases such as an alkaline pH optimum, dependence on calcium ions for activity, and inhibition by zinc ions.     

Level of redox potential as a possible contributing influence in the pathogenicity of oral anaerobes

Dental plaque anaerobes may be associated with the etiology of periodontal disease. This has created an interest in the potential pathogenicity of oral anaerobes. We compared the metabolic activity of anaerobic corynebacteria (C. parvum, C. anaerobium) and corresponding aerobic species (C. diphtheriae, C. xerosis). The anaerobes exhibited lower levels of growth rate, m-RNA half-life and ribosomal efficiency but much higher levels of RNA synthesis, ranging from 5 to 10 fold over the aerobes. We further examined these anaerobes, plusActinomyces naeslundi N16 (isolated from the anaerobic region of periodontally-diseased tissues), for the influence of redox potential on RNA level and antigenic function. Notable increases in RNA were found at specific Eh levels; the extent and direction of the changes varied with the different organisms. This environmental feature appeared to effect corresponding changes in agglutinability and PCA reactivity with antisera against the anaerobes cultured at different redox potentials. For example, while antisera against certain organisms (C. parvum, A. naeslundi) cultured under the most reduced conditions showed an intense PCA reaction, other antisera against the same organism cultured under less reduced conditions were non-reactive. Hence, alterations in redox potential may lead to altered metabolism and to altered antigenicity. Our results imply such a microbial response to environmental stress.     

Toad egg-jelly as a source of divalent cations essential for fertilization

Dejellied uterine eggs of the toad Bufo bufo japonicus are not fertilizable in 1/20 De Boer's solution (1/20 DB), but are fertilized when inseminated in a uv-solubilized jelly (UVJ) or the dialyzate of UVJ (UVJD). The present study was carried out to define this fertilization-supporting activity of egg-jelly. Dejellied eggs were fertilized in a high frequency when inseminated in a medium containing the ashes obtained by heating UVJD at 600 degrees C for 16 hr. Similarly, a reconstituted salt solution (RSS), which mimics the ionic composition of UVJD, supported a high rate of fertilization. To be effective in fertilization, however, RSS had to be present at the time of insemination. Analyses of individual salts revealed that dejellied eggs are successfully fertilized in CaCl2 and/or MgCl2 at 1-5 mM, only slightly in KCl at 10 mM, but not at all in NaCl at any of the concentrations tested. The activity of UVJD was lost reversibly when divalent cations were chelated by EDTA. The fertilization of dejellied eggs is therefore possible in a medium without any organic components of egg-jelly, provided that 2-5 mM Ca2+ or Mg2+ is present. Sperm were motile in media containing cations below 20-25 mM, regardless of the ionic composition. The egg-jelly possessed cations in a concentration of about 130 mM, but most ions were lost from intact jelly on immersion of eggs in water for 2-3 min, accompanied by the acquisition of fertilizability by sperm. Examination of the behavior of salts on dialysis or gel-filtration of jelly molecules revealed that the jelly retains Ca2+ and Mg2+, and possibly K+ as well, but not Na+ and Cl-. We propose that toad egg-jelly plays a function in fertilization by retaining Ca2+ and/or Mg2+ around each egg at the level necessary for successful sperm entrance into the egg.     

Algal biomass as a source for novel oral nano-antimicrobial agent

In the present study, sulphated polysaccharide Ulvan from Ulva lactuca was used for the synthesis of biogenic Selenium Nanoparticles (SeNPs) conjugate and Mouth rinse was prepared using this conjugate. The synthesis of nanoparticles was confirmed by UV–Visible spectrophotometry and characterized using Fourier transform infrared spectroscopy (FTIR), transmission electron microscope (TEM) and X-ray diffraction (XRD). TEM showed that the average size of the nanoparticle was 85 nm and spherical in shape. Furthermore, nanoparticle conjugates were evaluated for cell viability using MTT assay 3T3-L1 cell line and at 30 µl/ml showed 34% cell viability. The antimicrobial activity of SeNPs mouth rinse was tested against oral pathogens such as Streptococcus mutans, Staphylococcus aureus, Lactobacillus, and Candida albicans and it was effective against all tested microorganism at the concentration of 100 µl/ml. The present study has shown that Ulvan from algal biomass can be a safe and effective source for the development of oral nano-antimicrobial agents.     

Investigation of archaeosomes as carriers for oral delivery of peptides

Oral administration of peptide and protein drugs faces a big challenge partly due to the hostile gastrointestinal (GI) environment. Lipid-based delivery systems are attractive because they offer some protection for peptides and proteins. In this context, we prepared a special lipid-based oral delivery system: archaeosomes, made of the polar lipid fraction E (PLFE) extracted from Sulfolobus acidocaldarius, and explored its potential as an oral drug delivery vehicle. Our study demonstrates that archaeosomes have superior stability in simulated GI fluids, and enable fluorescent labeled peptides to reside for longer periods in the GI tract after oral administration. Although archaeosomes have little effect on the transport of insulin across the Caco-2 cell monolayers, the in vivo experiments indicated that archaeosomes containing insulin induced lower levels of blood glucose than a conventional liposome formulation. These data indicate that archaeosomes could be a potential carrier for effective oral delivery of peptide drugs.     

Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein

Bacterial outer membrane proteins are supposed to span the membrane repeatedly, mostly in the form of amphipathic beta-sheets. The last ten C-terminal amino acid residues of PhoE protein are supposed to form such a membrane-spanning segment. Deletion of this segment completely prevents incorporation into the outer membrane. Comparison of the last ten amino acid residues of other outer membrane proteins from different Gram-negative bacteria revealed the presence of a potential amphipathic beta-sheet with hydrophobic residues at positions 1 (Phe), 3 (preferentially Tyr), 5, 7 and 9 from the C terminus, in the vast majority of these proteins. Since such sequences were not detected at the C termini of periplasmic proteins, it appears to be possible to discriminate between the majority of outer membrane proteins and periplasmic proteins on the basis of sequence data. The highly conserved phenylalanine at the C termini of outer membrane proteins suggests an important function for this amino acid in assembly into the outer membrane. Site-directed mutagenesis was applied to study the role of the C-terminal Phe in PhoE protein assembly. All mutant proteins were correctly incorporated into the outer membrane to some extent, but the efficiency of the process was severely affected. It appears that both the hydrophobicity and the aromatic nature of Phe are of importance.     

Isolation and identification of the oral bacteria and their characterization for bacteriocin production in the oral cavity

Oral cavity is a diverse ecosystem which harbors immense diversity of microorganisms like fungi, virus and bacteria. Some of these microorganisms are involved in causing multiple infections. Oral flora is continuously changing due to connection with the external environment and produce bacteriocin against each other to compete for nutrient in this mini ecosystem. Current study was aimed to explore and compare the bacterial fauna of both healthy and non-healthy dental samples, by isolation and identification with biochemical tests to characterize the bacteriocin production. During study 120 swabs were taken from both healthy and unhealthy subjects. Samples were collected from the dental clinics of Makkah City, in sterile eppendorfs containing 1 ml nutrient broth, and were incubated overnight using shaking incubator. Bacteria were isolated following identification through Gram staining, microscopy and biochemical test. Total 15 strains of bacteria were isolated during the study amongst which 8 strains were gram positive and 7 strains were gram negative. The most dominant species of the gram positive strains was Streptococcus pneumoniae (n = 26). On the other hand, Escherichia coli (n = 26) was the prominent specie amongst the gram negative strains. Overall, the dominated family was Enterobacteriaceae (19.36%) followed by Streptococcaceae with 13.83% abundance. One of the most cariogenic strain Klebsiella pneumoniae (n = 14) was also isolated. The bacterial strain diversity between these two type of ecosystem was approximately the same, with slight variation in Shannon (HS:2.627187, NHS:2.653594) and Simpson diversity (HS:0.923461, NHS: 0.92684) index. The current research revealed that bacteriocin production in the Enterobacter species was prominent against Escherichia coli and Klebsiella pneumoniae. Apart from this other strains like Klebsiella pneumoniae and Exiguobacterium spp were also able to produce bacteriocin against Enterobacter species and Bacillus cereus respectively.     

Environmental heavy metal as a potential risk factor for the progression of oral potentially malignant disorders in central Taiwan

ObjectiveOral cancer (OC) is a leading cause of death from cancer in men between the ages of 25 and 44 years in Taiwan. The overall 5-year survival rates for the four OC stages (I–IV) in Taiwan are approximately 70%, 30%, 20%, and 10%, respectively, indicating the importance of the early diagnosis of oral potentially malignant disorders (OPMDs). Previous studies indicated an association between the OC incidence and certain environmental heavy metal concentrations. If these associations do exist for OC, they may also be observed for OPMD. The purpose of this study is to explore the association between the development of OPMD to OC and environmental heavy metals. Oral submucous fibrosis (OSF) and oral leukoplakia (OL) are two major types of OPMD in Taiwan.Materials and methodsA retrospective cohort study was conducted by Changhua Christian Hospital, the sole medical center in Changhua County, where 2725 male adult patients diagnosed with either OSF or OL between 2000 and 2014 were recruited. Data were analyzed by Cox regression and adjusted for smoking and betel-quid chewing.Results and discussionOPMD patients who resided in areas with high nickel concentrations (polluted levels) exhibited hazard ratios of 1.8–2 for OC relative to those who lived in areas with low nickel levels (P < 0.01). Meanwhile, smokers with OPMDs had a hazard ratio of 2.8–2.9 relative to non-smokers. Betel-quid chewers had a 2.2–2.3 hazard ratio relative to non-chewers. Smoking, betel-quid chewing, and environmental nickel exposure are associated with an increased risk of OC development in OPMD patients. This study provides valuable findings on the environmental effects of heavy metals on human health. Enhanced surveillance of the condition of OPMD patients who have been exposed to high nickel concentrations may be crucial for OC prevention.     

Phytate as the sole phosphate source for growth of Tetrahymena

Phytate, the storage form of phosphate in seeds and grains, is a major form of environmental phosphate loading from fertilizer inputs and agricultural runoff. We have investigated the ability of Tetrahymena populations to grow on phytate as their sole phosphate source. Populations grew equally well in chemically defined medium with phosphate and medium in which the phosphate was replaced with phytate in comparable concentrations between 0.5 mM and 6 mM. Intracellular phytate concentrations of cells grown in phytate showed a 4-6-fold increase over those grown in phosphate when measured during the late stage of exponential growth. These results demonstrate that phytate can provide a source of adequate phosphate for sustained growth in phytate-rich environments.     

The mitochondrial dicarboxylate carrier is essential for the growth of Saccharomyces cerevisiae on ethanol or acetate as the sole carbon source

The dicarboxylate carrier (DIC) is an integral membrane protein that catalyses a dicarboxylate-phosphate exchange across the inner mitochondrial membrane. We generated a yeast mutant lacking the gene for the DIC. The deletion mutant failed to grow on acetate or ethanol as sole carbon source but was viable on glucose, galactose, pyruvate, lactate and glycerol. The growth on ethanol or acetate was largely restored by the addition of low concentrations of aspartate, glutamate, fumarate, citrate, oxoglutarate, oxaloacetate and glucose, but not of succinate, leucine and lysine. The expression of the DIC gene in wild-type yeast was repressed in media containing ethanol or acetate with or without glycerol. These results indicate that the primary function of DIC is to transport cytoplasmic dicarboxylates into the mitochondrial matrix rather than to direct carbon flux to gluconeogenesis by exporting malate from the mitochondria. The delta DIC mutant may serve as a convenient host for overexpression of DIC and for the demonstration of its correct targeting and assembly.     

Bacterial utilization of a hydrazine derivative as nitrogen source for growth.

The ability of microbes to metabolize the N--N bond seems rare. Pseudomonas sp. from soil can utilize 1,4,5,6-tetrahydro-6-oxo-3-pyridazinecarboxylic acid as C and N source. This appears to be the first report that a microbe can cleave a nitrogen--nitrogen bond in an organic compound and use the products for growth.     

Lippia gracilis Schauer essential oil as a growth promoter for Japanese quail

The use of antibiotics as performance enhancers in animal feeding is declining, so Lippia gracilis Schauer essential oil (LGSEO) could be used as a potential substitute for the conventionally used growth promoters. The LGSEO contains components such as carvacrol and thymol, which kill and/or control pathogenic bacteria, increase population of beneficial organisms, act against oxidative processes and onto nutrient digestibility and absorption. The aim of this study was to investigate the action and the effects of LGSEO as a growth promoter in the diet of Japanese quail by examining their productive performance, intestinal microbiology, blood biochemical parameters, hepatic thiobarbituric acid reactive substances (TBARS) content and intestinal gene expression. A total of 252 two-day-old quail (Coturnix coturnix japonica) were assigned to 3 treatments in 7 replicates, using 12 birds per experimental unit. The treatments consisted of a basal diet, basal diet + LGSEO at 400 mg/kg of diet and basal diet + chemical antimicrobial (bacitracin methylene disalicylate) at 500 mg/kg of diet. The experimental period was 34 days. The highest feed intake (P < 0.01) was found in the group receiving the conventional antimicrobial, whereas the best feed conversion (P < 0.01) was shown by the animals receiving LGSEO. Escherichia coli growth was restricted in the quail receiving the growth promoters. Salmonella spp. growth was controlled by the treatment containing the conventional antimicrobial. There was no difference between the treatments (P > 0.05) for the concentration of aspartate aminotransferase and alanine aminotransferase enzymes in the blood or hepatic TBARS content. Birds receiving negative-control treatment exhibited a higher expression of sodium-glucose cotransporter (SGLT1), while those receiving the treatment with essential oil showed lower catalase (CAT) and glutathione peroxidase (GPX7) expressions compared to the conventional antimicrobial and control groups, respectively. Lippia gracilis Schauer essential oil is a powerful performance enhancer for Japanese quail by virtue of its abilities to improve their intestinal environment, balance the microbial population and reduce energy expenditure for oxidative processes.     

Determination of the cell surface hydrophobicity of oral bacteria using a modified hydrocarbon adherence method

Abstract Hydrophobic interactions between bacterial cell surfaces and colonisable substrates have been implicated in the mechanisms of bacterial adherence. However, current methods of assessing bacterial hydrophobicity as a function of adherence to liquid hydrocarbons (especially hexadecane) do not always produce accurate or reproducible results. Therefore, the present technique was developed using xylene. The hydrophobic surface properties of fresh and type strains of Bacteriodes gingivalis, Bacteriodes intermedius, Capnocytophaga spp., Streptococcus salivarius and Streptococcus sanguis suspended either in saliva ions buffer (SIB) or in saliva diluted in SIB were measured. In SIB the test strains were predominantly hydrophobic. The addition of saliva caused a significant reduction ( P < 0.05) in hydrophobicity compared to SIB alone, with 80% of the strains tested. Since oral bacteria will be suspended in saliva in vivo, it is concluded that bacteria in the oral cavity may be less hydrophobic than previous studies have suggested.     

Evaluating the potential of a novel oral lesion exudate collection method coupled with mass spectrometry-based proteomics for oral cancer biomarker discovery

Introduction  

Early diagnosis of Oral Squamous Cell Carcinoma (OSCC) increases the survival rate of oral cancer. For early diagnosis, molecular biomarkers contained in samples collected non-invasively and directly from at-risk oral premalignant lesions (OPMLs) would be ideal.