Salivation pattern of Rhodnius prolixus (Reduviidae; Triatominae) in mouse skin

The objective of this work was to study the pattern of salivation of triatomines during feeding in mouse skin. Rhodnius prolixus was fed with a solution of the dye acridine orange or fluorescein. The saliva was efficiently labelled with acridine orange, probably due to the difference in pH between the salivary gland (6.0) and the hemolymph (6.5-7.0). This procedure was not effective at labelling the saliva of Triatoma infestans, however, fluorescent labelling of R. prolixus saliva allowed us to demonstrate that salivation occurs during entire feeding process. The saliva is released soon after the bite. In the probing phase, saliva is pumped continuously in the host skin, including around the blood vessels. During the engorgement phase, saliva is observed in a bolus within the blood vessel and some of it is sucked up by the insect, together with blood. The frequency of saliva emission inside the vessels was low (0.51+/-0.18 Hz). The saliva deposition in the microcirculation is continuous and modulated by the frequency of the cibarial pump because, when functioning at high frequency, cibarial pump sucks almost all saliva to the insect gut. This mechanism would determine the quantity of saliva deposited in the microcirculation as necessary, and consequently minimizing the host's immune response to salivary antigens.     

Scavenger receptor gp340 aggregates group A streptococci by binding pili

Group A streptococci (GAS) are the most frequent cause of bacterial pharyngitis. The first obstacle to GAS colonization of the pharynx is saliva. As well as forming a physical barrier, saliva contains components of innate and acquired immunity. Previous work has shown that saliva induces bacterial aggregation, which may serve as a clearance mechanism. As the aggregation of some oral streptococci in saliva is mediated by long proteinaceous appendages, we hypothesized that pili of GAS might behave similarly. Wild-type GAS M1 strain SF370 aggregated in saliva, while pilus-defective mutants did not. Similarly, heterologous expression of diverse GAS pili on the surface of Lactococcus lactis induced aggregation in saliva, while control strains were unaffected. Further studies revealed that aggregating bacteria bound salivary component gp340. Purified gp340 aggregated wild-type GAS and L. lactis expressing GAS pili, but not control strains. GAS pilus-defective mutants were abrogated in gp340 binding and aggregation. Furthermore, gp340-mediated aggregation reduced bacterial adhesion to human epithelial cells, suggesting a role in host defence.     

Production of nitric oxide-derived reactive nitrogen species in human oral cavity and their scavenging by salivary redox components

Nitrite is reduced to nitric oxide (NO) in the oral cavity. The NO generated can react with molecular oxygen producing reactive nitrogen species. In this study, reduction of nitrite to NO was observed in bacterial fractions of saliva and whole saliva. Formation of reactive nitrogen species from NO was detected by measuring the transformation of 4,5-diaminofluorescein (DAF-2) to triazolfluorescein (DAF-2T). The transformation was fast in bacterial fractions but slow in whole saliva. Salivary components such as ascorbate, glutathione, uric acid and thiocyanate inhibited the transformation of DAF-2 to DAF-2T in bacterial fractions without affecting nitrite-dependent NO production. The inhibition was deduced to be due to scavenging of reactive nitrogen species, which were formed from NO, by the above reagents. The transformation of DAF-2 to DAF-2T was faster in bacterial fractions and whole saliva which were prepared 1–4?h after tooth brushing than those prepared immediately after toothbrushing. Increase in the rate as a function of time after toothbrushing seemed to be due to the increase in population of bacteria which could reduce nitrite to NO. The results obtained in this study suggest that reactive nitrogen species derived from NO are continuously formed in the oral cavity and that the reactive nitrogen species are effectively scavenged by salivary redox components in saliva but the scavenging is not complete.     

Production of nitric oxide-derived reactive nitrogen species in human oral cavity and their scavenging by salivary redox components

Nitrite is reduced to nitric oxide (NO) in the oral cavity. The NO generated can react with molecular oxygen producing reactive nitrogen species. In this study, reduction of nitrite to NO was observed in bacterial fractions of saliva and whole saliva. Formation of reactive nitrogen species from NO was detected by measuring the transformation of 4,5-diaminofluorescein (DAF-2) to triazolfluorescein (DAF-2T). The transformation was fast in bacterial fractions but slow in whole saliva. Salivary components such as ascorbate, glutathione, uric acid and thiocyanate inhibited the transformation of DAF-2 to DAF-2T in bacterial fractions without affecting nitrite-dependent NO production. The inhibition was deduced to be due to scavenging of reactive nitrogen species, which were formed from NO, by the above reagents. The transformation of DAF-2 to DAF-2T was faster in bacterial fractions and whole saliva which were prepared 1-4 h after tooth brushing than those prepared immediately after toothbrushing. Increase in the rate as a function of time after toothbrushing seemed to be due to the increase in population of bacteria which could reduce nitrite to NO. The results obtained in this study suggest that reactive nitrogen species derived from NO are continuously formed in the oral cavity and that the reactive nitrogen species are effectively scavenged by salivary redox components in saliva but the scavenging is not complete.     

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.     

Functional anatomy of the hypopharynx and the salivary pump in the feeding apparatus of the assassin bug Rhodnius prolixus (Reduviidae, Heteroptera)

Focussing on the blood-feeding reduviid Rhodnius prolixus, we investigated the structure and function of the hypopharynx in (1) conducting the saliva towards the mouthparts and (2) bringing together the salivary pump and the stylets to ensure the difficult task of supplying the two closed antidromic streams of blood and saliva, while allowing the mouthparts to be moved forth and back during the feeding process. The distal apex of the hypopharynx forms a needle-like structure that is X-shaped in cross section. It arranges the interlocking of the maxillae in a manner resembling the fixed slider of a zip-lock. Further proximal, the hypopharynx extends into the maxillary food channel as a wide tongue. The salivary pump possesses two separate efferent ducts. The dorsal duct originates in the retrograde angle of the cupula (part of the salivarium) and conducts saliva directly into the maxillary salivary channel. The ventral duct originates at the distal opening of the cupula. It extends into a bag, the distal opening of which can be closed by a ventral bolster-like cuticle and opened by muscles. We show for the first time for heteropteran mouthparts that the saliva is not exclusively discharged into the maxillary salivary channel (via the dorsal efferent duct of the salivary pump), but that a large amount of saliva directly flows into the tube of the labium (via the ventral efferent duct of the salivary pump), which encloses the piercing stylets. However, within a short section, saliva may also pass from the ventral salivary duct into the maxillary salivary channel. Similar double salivary efferent ducts are present in the reduviids Triatoma dimidiata, T. infestans, Dipetalogaster maxima, Panstrongylus megistus, in the pyrrhocorid Pyrrhocoris apterus, and in the pentatomid Troilus luridus. It might thus be a more common feature of the Heteroptera.     

Influence of the blood group reactive substances in saliva on the aggregation of Streptococcus rattus

The interaction of blood group reactive substances in saliva with bacteria was investigated by testing saliva from persons with different blood groups in a bacterial aggregation assay with Streptococcus rattus HG 59, originally S. rattus BHT. For blood group A, saliva from 10 persons out of 11 aggregated S. rattus and for blood group O, saliva from 10 persons out of 16 aggregated S. rattus. For blood group B, saliva from 6 persons out of 8 aggregated S. rattus weakly and the average aggregation activity of blood group B was much lower than for blood group A or O. In addition, saliva from 3 non-secretors did not aggregate S. rattus. The role of blood group antigens in bacterial aggregation was confirmed by inhibition studies with blood group specific sugars and various other sugars. GalNAc, specific for blood group A, inhibited bacterial aggregation by saliva whereas D-galactose, specific for blood group B, and D-fucose, specific for blood group O, did not. In addition, sialic acid, a major terminal sugar residue in mucins, also inhibited the bacterial aggregation. This study shows that the blood group and secretor status of a person may influence the interaction of saliva with bacteria in the oral cavity.     

Adherence ofStreptococcus gordonii HG 222 in the presence of saliva

The influence of the presence of saliva from different salivary glands on the adherence ofStreptococcus gordonii strain HG 222 to saliva-coated polystyrene surfaces was tested. In the presence of undiluted parotid saliva or diluted whole, submandibular and sublingual saliva the adherence of HG 222 was enhanced by the formation of small aggregates on the attachment surface. In the presence of undiluted whole, submandibular and sublingual saliva large aggregates were formed and the adherence to saliva-coated polystyrene surfaces was inhibited.Adherence in the presence of whole saliva compared to adherence in buffer was decreased when lower densities of bacterial suspension were used, although in this case in the presence of whole saliva smaller bacterial aggregates were formed.In conclusion, these results suggest that the presence of saliva in solution may both enhance and decrease the adherence ofS. gordonii HG 222 to saliva-coated polystyrene surfaces, partly depending on the size of bacterial aggregates that are formed in the presence of saliva.Abbreviations CHW saliva clarified human whole saliva - PAR saliva parotid saliva - SM saliva submandibular saliva - SL saliva sublingual saliva     

Bacterial diversity in the human saliva from different ages

To obtain primary idea on oral bacterium species that are generally present in periodotally healthy Koreans, the oral bacterial flora in the saliva of four periodontally healthy Koreans at different ages (5, 32, 35, 65) was investigated in this study. For this investigation, 16SrRNA gene clone libraries were generated from the saliva of the four healthy Koreans, and 50 clones were randomly selected from each saliva clone library and sequenced. Totally, 37 different kinds of bacterial 16S rRNA gene sequences were identified based on sequence homology search through GenBank database. The 37 kinds of saliva clone sequences were classified to 14 genera and 2 uncultured and 1 unidentified bacteria. Among the 14 identified genera, Streptococcus, Prevotella, and Veillonella were common genera, and Streptococcus was dominant genus that accounted for 7 different species. Among the seven Streptococcus species, S. salivarius appeared as the most common species. More numbers of species belonging to the genera Streptococcus and Prevotella was present in saliva from ages 32 and 35. While saliva from ages 5 and 65 showed more numbers of species belonging to the genera Rothia, including potential pathogenic species. Overall, saliva of a young child and a senior showed higher bacterial diversity than that of young adults.     

DNA methylation analysis from saliva samples for epidemiological studies

Saliva is a non-invasive, easily accessible tissue, which is regularly collected in large epidemiological studies to examine genetic questions. Recently, it is becoming more common to use saliva to assess DNA methylation. However, DNA extracted from saliva is a mixture of both bacterial and human DNA derived from epithelial and immune cells in the mouth. Thus, there are unique challenges to using salivary DNA in methylation studies that can influence data quality. This study assesses: (1) quantification of human DNA after extraction; (2) delineation of human and bacterial DNA; (3) bisulfite conversion (BSC); (4) quantification of BSC DNA; (5) PCR amplification of BSC DNA from saliva and; (6) quantitation of DNA methylation with a targeted assay. The framework proposed will allow saliva samples to be more widely used in targeted epigenetic studies.     

Compositional Stability of a Salivary Bacterial Population against Supragingival Microbiota Shift following Periodontal Therapy

Supragingival plaque is permanently in contact with saliva. However, the extent to which the microbiota contributes to the salivary bacterial population remains unclear. We compared the compositional shift in the salivary bacterial population with that in supragingival plaque following periodontal therapy. Samples were collected from 19 patients with periodontitis before and after periodontal therapy (mean sample collection interval, 25.8±2.6 months), and their bacterial composition was investigated using barcoded pyrosequencing analysis of the 16S rRNA gene. Phylogenetic community analysis using the UniFrac distance metric revealed that the overall bacterial community composition of saliva is distinct from that of supragingival plaque, both pre- and post-therapy. Temporal variation following therapy in the salivary bacterial population was significantly smaller than in the plaque microbiota, and the post-therapy saliva sample was significantly more similar to that pre-therapy from the same individual than to those from other subjects. Following periodontal therapy, microbial richness and biodiversity were significantly decreased in the plaque microbiota, but not in the salivary bacterial population. The operational taxonomic units whose relative abundances changed significantly after therapy were not common to the two microbiotae. These results reveal the compositional stability of salivary bacterial populations against shifts in the supragingival microbiota, suggesting that the effect of the supragingival plaque microbiota on salivary bacterial population composition is limited.     

Novel antibody assessment method for microbial compositional alteration in the oral cavity

Recently, it has been demonstrated that dysbiosis, an alteration in commensal microflora composition, is intimately involved in the onset of a variety of diseases. It is becoming increasingly evident that the composition of commensal microflora in the oral cavity is closely connected to oral diseases, such as periodontal disease, and systemic diseases, such as inflammatory bowel disease. Next-generation sequencing techniques are used as a method to examine changes in bacterial flora, but additional analytical methods to assess bacterial flora are needed to understand bacterial activity in more detail. In addition, the oral environment is unique because of the role of secretory antibodies contained in saliva in the formation of bacterial flora. The present study aimed to develop a new method for evaluating the compositional change of microbiota using flow cytometry (FCM) with specific antibodies against the bacterial surface antigen, as well as salivary antibodies. Using specific antibodies against Streptococcus mutans, a causative agent of dental caries, and human IgA, bacterial samples from human saliva were analyzed via FCM. The results showed that different profiles could be obtained depending on the oral hygiene status of the subjects. These results suggest that changes in the amount and type of antibodies that bind to oral bacteria may be an indicator for evaluating abnormalities in the oral flora. Therefore, the protocol established in this report could be applied as an evaluation method for alterations in the oral microbiota.     

茶小绿叶蝉成虫唾液细菌蛋白的鉴定

[目的]通过鉴定茶小绿叶蝉Empoasca flavescens成虫水状唾液蛋白中的细菌蛋白,解析叶蝉唾液的细菌种类.[方法]利用自制的收集装置和Parafilm膜夹营养液法对叶蝉成虫唾液进行收集,经超滤浓缩和电泳分离后,使用膜辅助样品制备技术法(filter-aided sample preparation,FASP...     

RND家族外排泵及其与微生物群体感应系统的相互关系

抗生素耐药性一直是细菌病害防治的难题,药物外排泵过量表达是细菌耐药性形成的重要机制之一。在革兰氏阴性细菌中,RND(Resistance-nodulation-cell division)家族外排泵在耐药性中发挥着重要作用,近年来的研究表明,依赖于小分子信号物质进行调控的群体感应系统与RND外排泵家族之间存在紧密的相互作用关系。本文在介绍RND家族外排泵的结构、转运机理和群体感应系统的类型及调控方式的基础上,剖析了群体感应系统对RND外排泵的调控机理以及RND外排泵对群体感应系统信号分子转运的影响。深入研究RND家族外排泵与群体感应系统之间的相互依赖、相互制约关系有利于阐明RND家族外排泵的调控机理,并有可能为克服微生物耐药性问题提供新的思路。     

Effect of saliva processing on bacterial DNA extraction

More than 700 bacterial species inhabit oral cavity of humans. Various oral diseases are related to changes in the structure of this complex community. Their pathogenesis can, thus, be better understood by study of oral microbial flora. As many bacteria are refractory to cultivation, molecular approaches based on PCR followed by downstream analysis are more suitable for community analysis than culture dependent methods. Effective DNA extraction from the sample matrix is a fundamental part of the pre-analytical phase but it can be influenced by processing of the starting material. The aim of this study was to analyze the effects of saliva processing on DNA extraction using several non-commercial isolation procedures. Bacterial chromosomal DNA was extracted from three different sample matrices: fresh saliva, diluted saliva and pelleted saliva using four different extraction methods: phenol chloroform protocol, benzyl-chloride protocol, extraction with Chelex-100 and extraction with Triton X. Extraction from different saliva samples and the use of different extraction methods significantly affected the effectiveness of DNA extraction. The most suitable material for bacterial DNA extraction for molecular analysis is a fresh saliva sample. The most effective methods for isolating salivary DNA are the benzyl-chloride protocol and Chelex-100 extraction. Our results have implications for studies concentrating on salivary microbiome and its role in the pathogenesis of oral diseases.     

Anatomy of the oral valve in nymphalid butterflies and a functional model for fluid uptake in Lepidoptera

The food canal of the proboscis of Lepidoptera serves for the uptake of nutrient fluids and the discharge of saliva. A valve was discovered at the entrance to the sucking pump in the head that separates these countercurrent flows in nymphalid butterflies. Three species of Nymphalidae were examined by dissections and light microscopic serial semithin sections. The sucking pump is a unit composed of three structures: (1) the oral valve, which is a projection of the epipharynx extending into the anterior cibarial lumen, (2) the expandable lumen, and (3) the posterior sphincter valve which controls influx into the oesophagus. Based on the microanatomical results, a functional model is presented to account for the uptake and swallowing of fluids and for the control of the salivary flow into the food canal of the proboscis. Dilator muscles of the sucking pump expand the lumen by pulling on the muscular dorso-anterior side. This opens the oral valve and fluid can be drawn into the lumen from the food canal of the proboscis. Circular compressor muscles which attach to both sides of the sclerotized ventro-posterior wall of the sucking pump reduce the size of the lumen; passively they close the oral valve and press fluid through the relaxed posterior sphincter opening into the oesophagus. According to this model saliva can be discharged into the food canal during the swallowing phase. The oral valve and pumping unit are similar in all studied species despite the fact that saliva presumably plays a special role in the derived pollen-feeding behaviour of one of them, viz. Heliconius melpomene.     

Comparative information value of humoral immunity characteristics in some bacterial and viral infections

Serological examination of 144 patients with different bacterial and viral infections was carried out. Antibodies to Brucella were detected in blood serum in 42 patients (85.7%) with the average titer of 1:996 and in saliva in 41 patients (83.7%) with the average titer of 1:567 by passive hemagglutination test with brucella erythrocyte diagnosticum. Out of 26 dysentery patients, antibodies in blood serum were detected in the diagnostic titer in 17 patients (65.4%) with the average titer of 1:282 and in saliva in 21 patients (80.8%) in the titer of 1:100 and higher. Anti-HAV and anti-HBc IgM were detected in specimens of saliva from patients with serologically confirmed viral hepatitis A and B in 100% of cases. The presence of HBsAg in saliva from hepatitis B patients was established in 95.4% of cases. In blood serum and in specimens of saliva anti-HCV IgM were detected in 100% and 85.7% of cases respectively. Out of 25 women with aggravated obstetric history, IgG antibodies to CMV were detected in blood serum in 23 women (88.5%) and in saliva in 22 women (84.6%). The results of these investigations revealed that the detection rate of antibodies in blood serum and saliva in cases of infections, both bacterial (brucellosis, shigellosis) and viral (hepatitis A, B, C and CMV infection), was not essentially different. The simplicity of obtaining material for analysis make it possible to recommend the use of saliva for diagnosing bacterial and viral infections, especially in mass epidemiological surveys.     

Nitrate reductase activity of bacteria in saliva of term and preterm infants

The salivary glands of adults concentrate nitrate from plasma into saliva where it is converted to nitrite by bacterial nitrate reductases. Nitrite can play a beneficial role in adult gastrointestinal and cardiovascular physiology. When nitrite is swallowed, some of it is converted to nitric oxide (NO) in the stomach and may then exert protective effects in the gastrointestinal tract and throughout the body. It has yet to be determined either when newborn infants acquire oral nitrate reducing bacteria or what the effects of antimicrobial therapy or premature birth may be on the bacterial processing of nitrate to nitrite. We measured nitrate and nitrite levels in the saliva of adults and both preterm and term human infants in the early weeks of life. We also measured oral bacterial reductase activity in the saliva of both infants and adults, and characterized the species of nitrate reducing bacteria present. Oral bacterial conversion of nitrate to nitrite in infants was either undetectable or markedly lower than the conversion rates of adults. No measurable reductase activity was found in infants within the first two weeks of life, despite the presence of oral nitrate reducing bacteria such as Actinomyces odontolyticus, Veillonella atypica, and Rothia mucilaginosa. We conclude that relatively little nitrite reaches the infant gastrointestinal tract due to the lack of oral bacterial nitrate reductase activity. Given the importance of the nitrate-nitrite-NO axis in adults, the lack of oral nitrate-reducing bacteria in infants may be relevant to the vulnerability of newborns to hypoxic stress and gastrointestinal tract pathologies.     

The conditioning role of saliva in streptococcal attachment to hydroxyapatite surfaces

The solution properties of saliva and its role in conditioning both the substrate and the bacterial surface have been investigated with regard to the attachment of oral streptococci to hydroxyapatite surfaces. Saliva from eight subjects was used and the attachment of three organisms, Streptococcus mutans strains FA-1 (serotype b) and KPSK2 (serotype c) and S. sanguis T175-1, was studied. An adsorbed salivary layer on a hydroxyapatite surface substantially reduced the affinity of the organisms for the surface. Adsorbed saliva on the bacterial surfaces, however, tended to increase the organisms' affinity for saliva-coated apatite. The source of saliva was important in determining the extent of inhibition of attachment. The data indicated that the negatively charged and hydrophilic nature of salivary conditioning films was important in controlling bacterial adsorption to hydroxyapatite. The results also suggested that hydrophobic salivas could promote binding of the more hydrophobic bacteria known to be early colonizers of the teeth.