Importance of bacterial hydrogen sulfide in the pathogenesis of periodontal diseases

Hydrogen sulfide (H2S) is one of many end-products of the proteolytic activities in the subgingival microbiota in patients with periodontal diseases, such as gingivitis and periodontitis. Although H2S is generally regarded as toxic, the mechanisms that underlie its production and its effects on human cells and tissues are poorly understood. Therefore, the role of H2S in the pathogenesis of periodontal diseases was investigated. Two colorimetric methods, the bismuth test (BT) and the methylene blue (MB) method, were used to estimate the amounts of H2S produced by the bacteria in vitro and ex vivo (Papers I, II and V). Oral bacteria, e.g., Fusobacterium spp., Porphyromonas gingivalis and Treponema denticola, were found to have strong capacities to degrade cysteine and produce H2S in vitro (Paper I). The Fusobacterium spp. were found to express several enzymes that are involved in the production of H2S. The expression patterns of the different enzymes varied among Fusobacterium subspecies and strains (Paper III). In an ex vivo experiment using BT, we showed that the subgingival plaques of subjects (N=43) with poor oral hygiene had the capacity to produce H2S (Paper II). High levels of periodontitis-associated bacteria were detected, and the BT values reflected the proteolytic activities of the bacteria and gingival inflammation rather than disease progression and periodontitis. A correlation between a positive BT and gingival inflammation was confirmed in Paper V, where H2S-producing bacteria were significantly more prevalent in the subgingival pockets of periodontitis patients (N=32) than of healthy controls (N=32), which indicates potent bacterial proteolytic activities in the untreated deep periodontal pockets. Paper IV described how the peripheral blood mononuclear cells (PBMCs) of blood donors and a monocytic cell line increased their secretion of the pro-inflammatory cytokines IL-1β and IL-18 in vitro when exposed to the H2S-donor sodium hydrosulfide (NaHS). This secretion was shown to be mediated by the NLRP3 inflammasome. These results were verified in Paper V, where the PBMCs of periodontitis patients and healthy controls secreted significantly higher levels of IL-1β and IL-18 when exposed to NaHS. In addition, both unexposed and exposed PBMCs of the periodontitis patients secreted higher levels of the two cytokines than the corresponding cells of healthy controls. These results suggest that the susceptibility of the host to develop disease can be attributed in part to enhanced secretion of pro-inflammatory cytokines following exposure to bacterial metabolites, such as H2S. In summary, toxic bacterial metabolites, such as H2S, may play an important role by affecting the cells of the host immune system, thereby inducing and sustaining gingival inflammation

Importance of Virulence Factors for the Persistence of Oral Bacteria in the Inflamed Gingival Crevice and in the Pathogenesis of Periodontal Disease

Periodontitis is a chronic inflammation that develops due to a destructive tissue response to prolonged inflammation and a disturbed homeostasis (dysbiosis) in the interplay between the microorganisms of the dental biofilm and the host. The infectious nature of the microbes associated with periodontitis is unclear, as is the role of specific bacterial species and virulence factors that interfere with the host defense and tissue repair. This review highlights the impact of classical virulence factors, such as exotoxins, endotoxins, fimbriae and capsule, but also aims to emphasize the often-neglected cascade of metabolic products (e.g., those generated by anaerobic and proteolytic metabolism) that are produced by the bacterial phenotypes that survive and thrive in deep, inflamed periodontal pockets. This metabolic activity of the microbes aggravates the inflammatory response from a low-grade physiologic (homeostatic) inflammation (i.e., gingivitis) into more destructive or tissue remodeling processes in periodontitis. That bacteria associated with periodontitis are linked with a number of systemic diseases of importance in clinical medicine is highlighted and exemplified with rheumatoid arthritis, The unclear significance of a number of potential “virulence factors” that contribute to the pathogenicity of specific bacterial species in the complex biofilm–host interaction clinically is discussed in this review

Estimation of bacterial hydrogen sulfide production in vitro

Oral bacterial hydrogen sulfide (H2S) production was estimated comparing two different colorimetric methods in microtiter plate format. High H2S production was seen for Fusobacterium spp., Treponema denticola, and Prevotella tannerae, associated with periodontal disease. The production differed between the methods indicating that H2S production may follow different pathways

H2S mediates increased interleukin (IL)-1β and IL-18 production in leukocytes from patients with periodontitis

Background: The mechanisms involved in the interplay between the bacteria and the host cells in periodontitis are not fully understood. Aim: To investigate the effect of the bacterial metabolite H2S on the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 from periodontitis patients and healthy controls, and to evaluate the composition of the subgingival microbiota with its capacity to produce H2S. Methods: Subgingival bacterial samples from patients with periodontitis (N=32) and healthy controls (N=32) were investigated for H2S production and bacterial composition. Peripheral blood mononuclear cells (PBMCs) were cultured in the presence/absence of 1mM H2S for 24h and cytokine concentrations were measured. Results: Subgingival plaque from periodontitis patients had more H2S producing bacteria and produced more H2S, than healthy controls. PBMCs exposed to H2S secreted significantly more IL-1ß and IL-18 (p<0.0001) than untreated control PBMCs from both groups. PBMCs from the periodontitis patients secreted higher levels of the cytokines, both spontaneously (IL-1ß p=0.0001; IL-18 p=0.09) and after exposure to H2S (IL-1ß p=0.03; IL-18 p=0.04), which is a new finding not previously reported. Conclusions: H2S, from the subgingival microbiota, can contribute to a host inflammatory response through secretion of the pro-inflammatory cytokines IL-1β and IL-18. Since this response differs between individuals, it may also reflect the susceptibility of the host to develop periodontitis

The proteins of Fusobacterium spp. involved in hydrogen sulfide production from L-cysteine

Background: Hydrogen sulfide (H2S) is a toxic foul-smelling gas produced by subgingival biofilms in patients with periodontal disease and is suggested to be part of the pathogenesis of the disease. We studied the H2S-producing protein expression of bacterial strains associated with periodontal disease. Further, we examined the effect of a cysteine-rich growth environment on the synthesis of intracellular enzymes in F. nucleatum polymorphum ATCC 10953. The proteins were subjected to one-dimensional (1DE) and two-dimensional (2DE) gel electrophoresis An in-gel activity assay was used to detect the H2S-producing enzymes; Sulfide from H2S, produced by the enzymes in the gel, reacted with bismuth forming bismuth sulfide, illustrated as brown bands (1D) or spots (2D) in the gel. The discovered proteins were identified with liquid chromatography - tandem mass spectrometry (LC-MS/MS). Results: Cysteine synthase and proteins involved in the production of the coenzyme pyridoxal 5'phosphate (that catalyzes the production of H2S) were frequently found among the discovered enzymes. Interestingly, a higher expression of H2S-producing enzymes was detected from bacteria incubated without cysteine prior to the experiment. Conclusions: Numerous enzymes, identified as cysteine synthase, were involved in the production of H2S from cysteine and the expression varied among Fusobacterium spp. and strains. No enzymes were detected with the in-gel activity assay among the other periodontitis-associated bacteria tested. The expression of the H2S-producing enzymes was dependent on environmental conditions such as cysteine concentration and pH but less dependent on the presence of serum and hemin

Presence of Helicobacter pylori and Campylobacter ureolyticus in the oral cavity of a Northern Thailand population that experiences stomach pain

Objective: To investigate oral diseases and microbiological conditions, such as the presence of ureolytic bacteria in dental plaque, in relation to experience of stomach pain in a remote adult Asian population. Methods: Ninety-three adults, 40–60-years old, from the Karen Hill tribe in Northern Thailand with no regular access to dental care were examined. Clinical registrations were performed and interproximal gingival plaque samples were collected and analyzed with the checkerboard (CKB) method for the presence of 14 oral bacterial species. Results: A number of 61 subjects reported daily stomach pain while 32 subjects had no symptoms from the stomach. The subjects with stomach pain had fewer remaining teeth (p < 0.05), higher caries experience (p < 0.05) and less BoP (p < 0.01). Most of the bacterial species were clustered statistically in three factors in a factor analysis, which together explained 65% of the microbiological variance. Factor 1, explaining 43.0% of the variance, was statistically associated with stomach pain (p < 0.001). Conclusions: The interproximal plaque/biofilm in adults of the study population showed a common presence of two gastrointestinal pathogens H. pylori and C. ureolyticus. The study also indicates for the first time a potential association between C. ureolyticus and stomach pain

Diagnostics of common microdeletion syndromes using fluorescence in situ hybridization: single center experience in a developing country

Microdeletion syndromes are caused by chromosomal deletions of less than 5 megabases which can be detected by fluorescence in situ hybridization (FISH). We evaluated the most commonly detected microdeletions for the period from June 01, 2008 to June 01, 2015 in the Federation of Bosnia and Herzegovina, including DiGeorge, Prader-Willi/Angelman, Wolf-Hirschhorn, and Williams syndromes. We report 4 patients with DiGeorge syndromes, 4 patients with Prader-Willi/Angelman, 4 patients with Wolf-Hirschhorn syndrome, and 3 patients with Williams syndrome in the analyzed 7 year period. Based on the positive FISH results for each syndrome, the incidence was calculated for the Federation of Bosnia and Herzegovina. These are the first reported frequencies of the microdeletion syndromes in the Federation of Bosnia and Herzegovina