Extra-oral halitosis: an overview

Halitosis can be subdivided into intra-oral and extra-oral halitosis, depending on the place where it originates. Most reports now agree that the most frequent sources of halitosis exist within the oral cavity and include bacterial reservoirs such as the dorsum of the tongue, saliva and periodontal pockets, where anaerobic bacteria degrade sulfur-containing amino acids to produce the foul smelling volatile sulfur compounds (VSCs), especially hydrogen sulfide (H(2)S) and methyl mercaptan (CH(3)SH). Tongue coating is considered to be the most important source of VSCs. Oral malodor can now be treated effectively. Special attention in this overview is given to extra-oral halitosis. Extra-oral halitosis can be subdivided into non-blood-borne halitosis, such as halitosis from the upper respiratory tract including the nose and from the lower respiratory tract, and blood-borne halitosis. The majority of patients with extra-oral halitosis have blood-borne halitosis. Blood-borne halitosis is also frequently caused by odorous VSCs, in particular dimethyl sulfide (CH(3)SCH(3)). Extra-oral halitosis, covering about 5-10% of all cases of halitosis, might be a manifestation of a serious disease for which treatment is much more complicated than for intra-oral halitosis. It is therefore of utmost importance to differentiate between intra-oral and extra-oral halitosis. Differences between intra-oral and extra-oral halitosis are discussed extensively. The importance of applying odor characterization of various odorants in halitosis research is also highlighted in this article. The use of the odor index, odor threshold values and simulation of bad breath samples is explained.     

Comparison of breath and in-mouth collection for the measurement of oral malodorous compounds by gas chromatography using sulfur chemiluminescence detection

Volatile sulfur compounds (VSCs), specifically hydrogen sulfide, methyl mercaptan and dimethyl sulfide, are generally considered to be the primary volatiles responsible for 'morning' malodors in breath. To date, the 'gold standard' for detecting VSC concentrations in breath is the use of gas chromatography coupled with sulfur chemiluminescence detection. Breath gas is often collected in a polypropylene syringe and then aliquots are injected into a gas chromatograph for analysis. The objective of this work was to compare the Twister? bar in-mouth extraction methodology for measurement of VSCs with the gas syringe breath-sampling collection technique. The Twister bar technology captures malodorous compounds in the mouth as opposed to breath gas. Using these techniques, comparable results were obtained in studies demonstrating the efficacy of a proprietary oral malodor counteraction system.     

Autogenous regulation of the inducible tryptophan synthase of Pseudomonas putida.

Mutants blocked before indole-3-glycerol phosphate formation in the tryptophan biosynthetic pathway of P. putida ("early-blocked" mutants) are unable to use indole as a source of tryptophan for growth on minimal medium. The uninduced level of tryptophan synthase [EC 4.2.1.20; L-serine hydro-lyase (adding indole)] in such mutants was thought to be responsible for this property. We have shown that levels of indole higher than those previously tested will support growth of these mutants. In addition, the growth rate of these mutants on a given indole concentration was shown to be proportional to the synthase level induced under the same conditions. This apparent induction of tryptophan synthase by indole in "early-blocked" mutants was shown to be caused by formation of the normal effector molecule, indole-3-glycerol-P, from indole. Secondary mutations occur in "early-blocked" trp strains, which enable them to grow on low concentrations of indole. One type of "indole-utilization" mutation occurs in the trpA gene, inactivating its product. Tryptophan synthase is readily induced by low concentrations of indole in these mutants, even though they are unable to convert indole to indole-3-glycerol-P. We propose that the alpha-chain of the synthase has an autogenous regulatory function, serving as the repressor or the indole-3-glycerol-P recognition component of the repressor of the trpAB operon (synthase alpha-and beta-chains). Our hypothesis holds that the trpA type of "indole-utilization" mutation alters the repressor (synthase alpha-chain) so that indole as well as indole-3-glycerol-P serves as an effector molecule for tryptophan synthase induction.     

Reduced Diversity and Imbalance of Fecal Microbiota in Patients with Ulcerative Colitis

Background

Clinical observations and experimental colitis models have indicated the importance of intestinal bacteria in the etiology of ulcerative colitis (UC), but a causative bacterial agent has not been identified.

Aim

To determine how intestinal bacteria are associated with UC, fecal microbiota and other components were compared for UC patients and healthy adults.

Methods

Fresh feces were collected from 48 UC patients. Fecal microbiota were analyzed by use of terminal-restriction fragment length polymorphism (T-RFLP), real-time PCR, and culture. The concentrations of organic acids, indole, and ammonia, and pH and moisture, which are indicators of the intestinal environment, were measured and compared with healthy control data.

Results

T-RFLP data divided the UC patients into four clusters; one cluster was obtained for healthy subjects. The diversity of fecal microbiota was significantly lower in UC patients. There were significantly fewer Bacteroides and Clostridium subcluster XIVab, and the amount of Enterococcus was higher in UC patients than in healthy subjects. The fecal concentration of organic acids was significantly lower in UC patients who were in remission.

Conclusion

UC patients have imbalances in the intestinal environment—less diversity of fecal microbiota, lower levels of major anaerobic bacteria (Bacteroides and Clostridium subcluster XIVab), and a lower concentration of organic acids.     

Direct evidence for in vivo hydroxyl-radical generation in experimental iron overload: an ESR spin-trapping investigation.

Although the hydroxyl radical is often implicated as the species responsible for the initiation of oxidative damage in iron-overload conditions, no ESR evidence for the formation of the radical in vivo has been reported. We have employed a secondary radical-trapping technique in which the hydroxyl radical reacts with dimethyl sulfoxide to form the methyl radical, which is then detected as its adduct of the spin trap N-t-butyl-alpha-phenylnitrone in the bile of animals given an intragastric dose of ferrous sulfate. The identity of this adduct was verified by isotope-substitution techniques. We show that unless measures are taken to inactivate the iron excreted in the bile of treated animals, reactions between iron, oxygen, dimethyl sulfoxide, N-t-butyl-alpha-phenylnitrone, and bile components lead to the formation of artifacts during sample collection.     

Appropriate sample bags and syringes for preserving breath samples in breath odor research: a technical note

It is now generally accepted that the volatile sulfur compounds (VSCs) hydrogen sulfide, methyl mercaptan and dimethyl sulfide are the main contributors to halitosis when of oropharyngeal origin. The VSCs hydrogen sulfide and methyl mercaptan are the major causes of bad breath in oral malodour whereas dimethyl sulfide is generally the major cause of bad breath in extra-oral halitosis. To facilitate research in the field of halitosis, it is highly advantageous to be able to preserve breath samples for longer periods of time before measurement of the VSCs, e.g. for sampling patients at home or when studying a large cohort of patients where an immediate measurement of the VSCs is not possible. After testing numerous sample bags, ultimately the foil balloons, coated inside with the synthetic polymer polyethylene, were the preferred ones. All the VSCs in breath remained quite stable for at least 3 days in these balloons. Besides the sampling bags, the use of an appropriate syringe for sampling mouth air and for injecting samples in e.g. a gas chromatograph is also of great importance. Usually, syringes with a rubber barrel seal are used. However, some rubbers quickly adsorb the VSCs in breath. When preserving breath samples for longer periods, the rubber also releases VSCs, especially methyl mercaptan. It was also found that these syringes release a compound which interferes with dimethyl sulfide, when using gas chromatographic measurements with the OralChroma. We now use all-plastic syringes (B/Braun Injekt), made of polypropylene and polyethylene, in which the VSCs in breath remain quite stable for at least 9 h.     

Evaluating gut microbiota profiles from archived fecal samples

Background

Associations between colorectal cancer and microbiota have been identified. Archived fecal samples might be valuable sample sources for investigating causality in carcinogenesis and biomarkers discovery due to the potential of performing longitudinal studies. However, the quality, quantity and stability of the gut microbiota in these fecal samples must be assessed prior to such studies. We evaluated i) cross-contamination during analysis for fecal blood and ii) evaporation in stored perforated fecal immunochemical tests (iFOBT) samples, iii) temperature stability as well as iv) comparison of the gut microbiota diversity and composition in archived, iFOBT and fresh fecal samples in order to assess feasibility of large scale microbiota studies.

Methods

The microbiota profiles were obtained by sequencing the V3-V4 region of 16S rDNA gene.

Results

The iFOBT does not introduce any cross-sample contamination detectable by qPCR. Neither could we detect evaporation during freeze-thaw cycle of perforated iFOBT samples. Our results confirm room temperature stability of the gut microbiome. Diverse microbial profiles were achieved in 100% of fresh, 81% of long-term archived and 96% of iFOBT samples. Microbial diversity and composition were comparable between fresh and iFOBT samples, however, diversity differed significantly between long-term archived, fresh and iFOBT samples.

Conclusion

Our data showed that it is feasible to exploit archived fecal sample sets originally collected for testing of fecal blood. The advantages of using these sample sets for microbial biomarker discovery and longitudinal observational studies are the availability of high-quality diagnostic and follow-up data. However, care must be taken when microbiota are profiled in long-term archived fecal samples.

     

"Electronic nose" detects major histocompatibility complex-dependent prerenal and postrenal odor components.

Mice prefer to mate with individuals expressing different MHC genes from their own. Volatile components presenting MHC-dependent odor types are present in urine and can be detected by mice, as shown by extensive behavioral studies. Similar odor types are suspected to influence human behavior as well. Although a recent report indicates that MHC expression influences the ratio of volatile compounds such as phenylacetic acid, so far no other means than studying the behavior of mice or rats has been available to assess odor types. Here, we report the ability of a gas sensor array (referred to as "electronic nose") to detect MHC-dependent odor types. The electronic nose consists of an array of chemophysical detectors, in our case quartz crystal microbalances and semiconducting metal-oxide sensors that change frequency or conductivity upon binding of very small numbers of individual molecules present in the gas phase of odorous fluids. The pattern of changes is characteristic for a particular smell. Our electronic nose distinguishes the urine odor types of MHC congenic mouse strains, MHC class I mutant mice, and HLA-A2 transgenic mice. In addition, MHC-dependent odor types can be detected in serum. The device also clearly differentiates between individual odor types of human sera from HLA homozygous individuals; however, HLA expression seems to have only a secondary influence. Thus, odor-type research can now be carried out with an objective and fast through-put system independent of behavioral studies.     

Attraction of the oriental fruit fly, Dacus dorsalis, to methyl eugenol and related olfactory stimulants.

The attraction of male oriental fruit flies to methyl eugenol and 34 analogues was investigated quantitatively using the characteristic feeding response. Methyl eugenol was the most active compound studied, with a feeding response to 0.01 mug, but saturation of the allyl side chain or replacement of allyl by allyloxy produced compounds almost as effective. Replacement of the methoxy groups by methylenedioxy, methyl, or chloro groups abolished all response. The ring geometry of the methoxy groups was critical, with orthodimethoxy most active and meta-dimethoxy inactive. Replacement of methoxy with hydroxy, methylthio, or amino groups did not abolish the response. The failure of the oriental fruit fly to respond to the methyl and chloro isosteres of methyl eugenol was contrasted with the response of a human odor panel which perceived these compounds as having weak floral odors.     

Molybdopterin guanine dinucleotide: a modified form of molybdopterin identified in the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides forma specialis denitrificans.

The nature of molybdenum cofactor in the bacterial enzyme dimethyl sulfoxide reductase has been investigated by application of alkylation conditions that convert the molybdenum cofactor in chicken liver sulfite oxidase and milk xanthine oxidase to the stable, well-characterized derivative [di(carboxamidomethyl)]molybdopterin. The alkylated pterin obtained from dimethyl sulfoxide reductase was shown to be a modified form of alkylated molybdopterin with increased absorption in the 250-nm region of the spectrum and altered chromatographic behavior. The complex alkylated pterin was resolved into two components by treatment with nucleotide pyrophosphatase. These were identified as di(carboxamidomethyl)molybdopterin and GMP by their absorption spectra, coelution with standard compounds, and by further degradation by alkaline phosphatase to dephospho [di(carboxamidomethyl)]molybdopterin and guanosine. The GMP moiety was sensitive to periodate, identifying it as the 5' isomer. Chemical analysis of the intact alkylated pterin showed the presence of two phosphate residues per pterin. These results established that the pterin isolated from dimethyl sulfoxide reductase contains the phosphoric anhydride of molybdopterin and 5'-GMP, which is designated molybdopterin guanine dinucleotide.     

Evolution of diabroticite rootworm beetle (Chrysomelidae) receptors for Cucurbita blossom volatiles

The diabroticite rootworm beetles coevolved with plants of the family Cucurbitaceae as demonstrated by their feeding dependence on the tetracyclic triterpenoid cucurbitacins. These beetles also exhibit strong attraction to phenylpropanoid volatile components of Cucurbita blossoms. A mixture of 1,2,4-trimethoxybenzene, indole, and (E)-cinnamaldehyde, all blossom components, is highly attractive to the several species of diabroticite cucumber beetles and corn rootworms and is considered a simplified Cucurbita blossom kairomone odor. The evolutionary divergence in antennal receptor complementarity is best understood by comparing the species-specific responses of several Diabrotica to structural analogues of (E)-cinnamaldehyde, the major attractant for Diabrotica undecimpunctata howardi. Cinnamyl alcohol is a strong attractant for Diabrotica barberi, and 4-methoxycinnamaldehyde is an exceptional attractant for Diabrotica virgifera. The very closely related species D. barberi and Diabrotica cristata are most strongly attracted to 4-methoxyphenethanol, which is unattractive to the other species studied.     

Molecular mechanisms underlying differential odor responses of a mouse olfactory receptor

The prevailing paradigm for G protein-coupled receptors is that each receptor is narrowly tuned to its ligand and closely related agonists. An outstanding problem is whether this paradigm applies to olfactory receptor (ORs), which is the largest gene family in the genome, in which each of 1,000 different G protein-coupled receptors is believed to interact with a range of different odor molecules from the many thousands that comprise "odor space." Insights into how these interactions occur are essential for understanding the sense of smell. Key questions are: (i) Is there a binding pocket? (ii) Which amino acid residues in the binding pocket contribute to peak affinities? (iii) How do affinities change with changes in agonist structure? To approach these questions, we have combined single-cell PCR results [Malnic, B., Hirono, J., Sato, T. & Buck, L. B. (1999) Cell 96, 713-723] and well-established molecular dynamics methods to model the structure of a specific OR (OR S25) and its interactions with 24 odor compounds. This receptor structure not only points to a likely odor-binding site but also independently predicts the two compounds that experimentally best activate OR S25. The results provide a mechanistic model for olfactory transduction at the molecular level and show how the basic G protein-coupled receptor template is adapted for encoding the enormous odor space. This combined approach can significantly enhance the identification of ligands for the many members of the OR family and also may shed light on other protein families that exhibit broad specificities, such as chemokine receptors and P450 oxidases.     

Molecular parameters and olfaction in the oriental fruit fly Dacus dorsalis

The methyl eugenol receptor of the male oriental fruit fly (Dacus dorsalis) has been further characterized by evaluating the role of the linear free energy parameters and σ in the depolarization of the receptor by 37 substituted 3,4-dimethoxybenzenes. There was a strong positive correlation between the hydrophobic character of the primary substituent and intense odor and a positive correlation between the electron donating property of the primary substituent and intense odor. Maximum odor intensity was also associated with substituents of 3 atomic diameters and was improved by a center of unsaturation. Preference tests suggest that this simple and versatile odor receptor can serve as a model for investigation of molecular interactions between receptors and odorant molecules.     

Bacteriophage-encoded shiga toxin gene in atypical bacterial host

Background

Contamination from fecal bacteria in recreational waters is a major health concern since bacteria capable of causing human disease can be found in animal feces. The Dog Beach area of Ocean Beach in San Diego, California is a beach prone to closures due to high levels of fecal indicator bacteria (FIB). A potential source of these FIB could be the canine feces left behind by owners who do not clean up after their pets. We tested this hypothesis by screening the DNA isolated from canine feces for the bacteriophage-encoded stx gene normally found in the virulent strains of the fecal bacterium Escherichia coli.

Results

Twenty canine fecal samples were collected, processed for total and bacterial fraction DNA, and screened by PCR for the stx gene. The stx gene was detected in the total and bacterial fraction DNA of one fecal sample. Bacterial isolates were then cultivated from the stx-positive fecal sample. Eighty nine of these canine fecal bacterial isolates were screened by PCR for the stx gene. The stx gene was detected in five of these isolates. Sequencing and phylogenetic analyses of 16S rRNA gene PCR products from the canine fecal bacterial isolates indicated that they were Enterococcus and not E. coli.

Conclusions

The bacteriophage-encoded stx gene was found in multiple species of bacteria cultivated from canine fecal samples gathered at the shoreline of the Dog Beach area of Ocean Beach in San Diego, California. The canine fecal bacteria carrying the stx gene were not the typical E. coli host and were instead identified through phylogenetic analyses as Enterococcus. This suggests a large degree of horizontal gene transfer of exotoxin genes in recreational waters.     

A novel, noninvasive method for the measurement of intestinal fat absorption

BACKGROUND & AIMS: The goal of the study was to facilitate fat balance measurements with an appropriate intestinal marker for the transit of dietary fat and thereby eliminate the need for complete diet and fecal collections. METHODS: Dietary fat containing 5% sucrose polybehenate was fed in a semisynthetic diet to rats and mice. Fat absorption was calculated from the ratios of behenic acid to other fatty acids in diet and feces as analyzed by gas chromatography of fatty acid methyl esters. The method was validated by measuring absorption of well-absorbed (safflower oil) and poorly absorbed (olestra; calcium soaps) dietary fats. The animals were fed meals containing test fats for 2 or 3 days, and fecal samples were collected. Fecal samples of approximately 10 mg (single fecal pellet from mice) were assayed. RESULTS: The method yielded values that were consistent with complete absorption of safflower oil and the nonabsorbability of olestra and calcium soaps. The results were reproducible and consistent among individual fecal aliquots. The method was compared with traditional fat-balance methods in animals fed both high- and low-fat diets. CONCLUSIONS: Sucrose polybehenate is an appropriate marker that allows the rapid measurement of fat absorption by analyzing aliquots of <1% of total feces. The method is noninvasive, does not require isotope analyses, and can be carried out as part of an animal's normal feeding regimen. The method may be a facile technique to assess fat absorption measurements in humans.     

Identification of Human Bocavirus type 4 in a child asymptomatic for respiratory tract infection and acute gastroenteritis – Goiânia,Goiás,Brazil

Human Bocavirus (HBoV) has been identified from feces and respiratory samples from cases of both acute gastroenteritis and respiratory illness as well as in asymptomatic individuals.The aim of this study was to detect and characterize HBoV from fecal samples collected from hospitalized children aged less than five years old with no symptoms of respiratory tract infection (RTI) or acute gastroenteritis (AGE). The study involved 119 children and one fecal sample was collected from each participant between 2014 and 2015. HBoV was detected using Nested-PCR, and the viral type identified by genomic sequencing. HBoV-4 was identified from one sample obtained from a hospitalized child with soft tissue tumor of the submandibular region. This is the first report of HBoV-4 identification in Brazil, but we consider that this type may be circulating in the country similar to the other types and new investigations are necessary.     

Can pancreatic steatorrhea be diagnosed without chemical analysis?

Summary Conclusion Visual observation of feces, considering fecal output, is considered to be an excellent method of detection of steatorrhea when judged by experienced doctors. Methods Feces from 192 patients with untreated chronic pancreatitis or under pancreatic enzyme therapy were investigated. Feces were collected for three consecutive days and homogenized with water. Fecal samples were freeze-dried and analyzed for fatty acids by gas chromatography (GLC). The quantity of fat was calculated from the amount of fatty acid to obtain daily fecal fat excretion. Comparison of GLC method with van de Kamer method gave a significant (p<0.01) positive correlation with correlation coefficient of 0.916 (n=38). Steatorrhea was defined as fecal fat excretion exceeding 5 g/d. Mild steatorrhea was defined as 5–10 g/d, and severe steatorrhea as more than 10 g/d. Results Three visual identification items were used to consider fecal output exceeding 200 g/d: fecal fat concentration exceeding 4%, appearance, and odor. The results were compared with the results from GLC method. Detection of steatorrhea by means of visual properties was the most accurate, and correlation coefficient was 0.843 (p<0.01) by Spearman’s rank correlation test. This detection method was also significantly effective for differentiation of normal stool from mild and severe steatorrhea. The sensitivity and specificity were 89.3 and 91.1%, respectively, indicating a favorable result.     

Generation of indole/skatole during malodor formation in the salivary sediment model system and initial examination of the oral bacteria involved

Gram negative anaerobic microbial degradation of proteins, peptides and amino acids in saliva leads to production of oral malodor. Volatile sulfur compounds (VSCs) from cysteine and indole/skatole (I/S) from tryptophan are two major components of breath odor. In this study, salivary mixed bacteria and oral pure cultures were compared for their ability to produce odor from cysteine, tryptophan and salivary supernatant. The VSC malodor inhibitor, zinc, was used to help identify the malodor processes involved. Salivary sediment and salivary supernatant were both obtained from wax-stimulated whole saliva. Gram positive (13) and Gram negative (12) oral pure cultures of common salivary bacteria were each grown in appropriate media and harvested. Incubation mixtures were prepared with salivary sediment at 16.7% (v/v) or bacterial pure cultures at 8.3% (v/v). Cysteine or tryptophan at 6 mM or salivary supernatant at 33.3% (v/v) was added. When included, zinc as ZnCl(2) was added at 6 mM. All incubations were carried out for 24 h at 37 °C. Odor generation was assessed organoleptically (0-4 severity scale); VSC was determined with a Halimeter (Interscan Corp., Chatsworth, CA) and I/S was determined using Kovac's colorimetric method. Organoleptic odor levels produced by salivary sediment mixed bacteria with the three substrates tested were comparable, though tryptophan and cysteine generated distinct and characteristic odors. With cysteine and supernatant, rapid and substantial VSC was generated; with tryptophan no such VSC formation was observed. Zinc inhibited VSC generation from cysteine and from salivary supernatant but had no effect on I/S from tryptophan. Surprisingly though, zinc which had no effect on I/S production from free tryptophan, reduced I/S production from supernatant. Of the 12 Gram negative bacteria, 75% produced I/S (mean 3.1 ± 1.5 SEM mg ml(-1)); most was generated by Porphyromonas intermedia, Porphyromonas gingivalis and Fusobacterium nucleatum. Gram positive pure cultures produced none. Comparable levels of organoleptic odor were produced by Porphyromonas gingivalis from tryptophan and cysteine. Malodor produced orally is complex because multiple microbial types, substrates, metabolic pathways and hence end products are involved. Zinc clearly inhibited I/S production in salivary supernatant, possibly by interfering with the release of tryptophan from peptides/proteins.     

Enzyme-linked immunosorbent assay using monoclonal antibodies for direct serotyping of enteric adenoviruses in feces

We were prepared three monoclonal antibodies in which the monoclone 12D was type specific for Adenovirus 40 (Ad40), 1F was type specific for Ad41 and 15D was group specific for Ads. For identification of enteric adenoviruses (EAd) in stool specimens, enzyme-linked immunosorbent assay (ELISA) test using monoclonal antibodies was developed. Results of identification by the ELISA tests using monoclonal antibodies to EAd on 15 fecal samples in which Ad particles were found by electron microscopy showed complete coincidence to those of Sma 1 restriction endonuclease cleavage. From these results, the ELISA tests employing EAds type specific monoclonal antibodies proved to be specific and this was a rapid technique for laboratory diagnosis of EAd in fecal specimens of viral gastroenteritis. Fifty-eight fecal samples with Ad particles positive by EM were serotyped by the ELISA using monoclonal antibodies. Eleven fecal samples were identified as Ad40, 25 as Ad41, 1 as double infection with Ad40 and Ad41, and 4 as non-EAd. These results indicated that Ad41 was more dominant than Ad40 during April, 1986 to January, 1989 in Matsuyama city.