Periodontal diseases in Thai schoolchildren. Clinical and microbiological observations.

The prevalence of periodontitis among Thai schoolchildren is unknown. In a cross-sectional study, the prevalence and severity of periodontal diseases, in a group of Thai schoolchildren, along with the presence and numbers of bacterial species commonly associated with periodontitis were investigated. A consent form was sent out to 192 schoolchildren in one school (Chanachanupathom School) in Chana, Southern Thailand (in the age range of 12-18 years) and 119 attended for a clinical and microbiological examination. Clinical recordings included number of teeth present, DMFT, plaque index, bleeding index, clinical attachment loss (CAL), and probing pocket depth (PPD). Pooled plaque samples were analyzed with culture and qPCR against bacteria associated with periodontitis. The children had low caries experience (DMFT = 3.2 ± 2.3), poor oral hygiene, high bleeding scores, and 67 (56.3%) had at least one interproximal site with CAL ≥ 1 mm. Thirty-seven (31.1%) of the children were diagnosed with periodontitis stage I, and sixteen (13.4%) were classified as periodontitis Stage II. Aggregatibacter actinomycetemcomitans was sparsely found in all but the healthy clinical groups (gingivitis, periodontitis Stage I and II), while the groups showed a high prevalence of Fusobacterium spp., Prevotella intermedia/nigrescens, and Campylobacter species as well as of the periodontitis-associated species Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. Thai schoolchildren have poor oral hygiene with abundant amounts of plaque and high presence of bleeding. Early onset periodontitis is common but mostly in its mild form and is not associated with the presence of A. actinomycetemcomitans.

Microbial metabolites in the pathogenesis of periodontal diseases: a narrative review.

The purpose of this narrative review is to highlight the importance of microbial metabolites in the pathogenesis of periodontal diseases. These diseases, involving gingivitis and periodontitis are inflammatory conditions initiated and maintained by the polymicrobial dental plaque/biofilm. Gingivitis is a reversible inflammatory condition while periodontitis involves also irreversible destruction of the periodontal tissues including the alveolar bone. The inflammatory response of the host is a natural reaction to the formation of plaque and the continuous release of metabolic waste products. The microorganisms grow in a nutritious and shielded niche in the periodontal pocket, protected from natural cleaning forces such as saliva. It is a paradox that the consequences of the enhanced inflammatory reaction also enable more slow-growing, fastidious, anaerobic bacteria, with often complex metabolic pathways, to colonize and thrive. Based on complex food chains, nutrient networks and bacterial interactions, a diverse microbial community is formed and established in the gingival pocket. This microbiota is dominated by anaerobic, often motile, Gram-negatives with proteolytic metabolism. Although this alternation in bacterial composition often is considered pathologic, it is a natural development that is promoted by ecological factors and not necessarily a true "dysbiosis". Normal commensals are adapting to the gingival crevice when tooth cleaning procedures are absent. The proteolytic metabolism is highly complex and involves a number of metabolic pathways with production of a cascade of metabolites in an unspecific manner. The metabolites involve short chain fatty acids (SCFAs; formic, acetic, propionic, butyric, and valeric acid), amines (indole, scatole, cadaverine, putrescine, spermine, spermidine) and gases (NH3, CO, NO, H2S, H2). A homeostatic condition is often present between the colonizers and the host response, where continuous metabolic fluctuations are balanced by the inflammatory response. While it is well established that the effect of the dental biofilm on the host response and tissue repair is mediated by microbial metabolites, the mechanisms behind the tissue destruction (loss of clinical attachment and bone) are still poorly understood. Studies addressing the functions of the microbiota, the metabolites, and how they interplay with host tissues and cells, are therefore warranted.

The secretion of cytokines by peripheral blood mononuclear cells of patients with periodontitis and healthy controls when exposed to H2S.

Background: Hydrogen sulfide(H2S) is a bacterial metabolite produced as a result of bacterial growth in subgingival pockets, suggested to partake in the pathogenesis of periodontitis. H2S has previously been shown to induce the secretion of the pro-inflammatory cytokines IL-1ß and IL-18 via the NLRP3 inflammasome in monocytes. Objective: To investigate the non-NLRP3 inflammasome-dependent immunological response of human peripheral blood mononuclear cells (PBMCs) of periodontitis patients and healthy controls exposed to H2S in vitro. Methods: PBMCs of periodontitis patients(N = 31) and healthy controls(N = 32) were exposed to 1 mM sodium hydrosulfide (NaHS) at 37°C for 24 h and the secretion of cytokines was compared to resting cells. TNF-α, IFN-γ, IL-6, IL-8, IL-12p40, IL-12p70, IL-17, MCP-1, and IL-1Ra secretions were measured with Bio-Plex Pro™ Human Cytokine Assay. Results: H2S triggered the secretion of the pro-inflammatory IFN-γ, IL-6, IL-17, TNF-α, IL-12p40, and IL-12p70, while the reverse was seen for IL-1Ra. In addition, a higher basal secretion of IFN-γ, IL-6, IL-12p70, IL-17 and MCP-1 was seen from PBMCs of periodontitis patients compared to healthy controls. Conclusion: The bacterial metabolite H2S triggers the secretion of pro-inflammatory cytokines from PBMCs and may thus have a prominent role in the host-bacteria interplay in periodontitis.

Short chain fatty acids released by Fusobacterium nucleatum are neutrophil chemoattractants acting via free fatty acid receptor 2 (FFAR2).

Fusobacterium nucleatum is a gram-negative and anaerobic oral commensal that is implicated in inflammatory conditions of the tooth-supporting structures, that is, periodontal diseases. One of the main characteristics of these conditions is an accumulation of neutrophil granulocytes in the gingival pockets where bacteria reside. Neutrophils are recruited to tissue-residing microbes by gradients of bacteria derived chemoattractants, and the cellular migration over the pocket epithelium into the gingival pocket is likely governed by chemoattractants released by the amino acid fermenting anaerobes typically colonising this site. However, the chemoattractants released by F. nucleatum and other oral anaerobes have long been unidentified. In the present study, we show that the major chemoattractants released during the growth of F. nucleatum are short chain fatty acids (SCFAs), primarily acetate and butyrate. These SCFAs, that are released at high levels as end-products of the metabolism of F. nucleatum, trigger chemotaxis of human neutrophils, as well as cytosolic Ca2+ signals, via free fatty acid receptor 2 (FFAR2). This finding establishes the SCFA-FFAR2 interaction as an important mechanism in the recruitment of neutrophils to the periodontal pocket, but could also be of importance in the pathogenesis of other medical conditions involving colonisation/infection of F. nucleatum.

Explorative study on Lactobacillus species and their acid-producing capacity and anti-microbial activity in head and neck cancer patients.

OBJECTIVE: To determine acid-producing capacity and anti-microbial activity of Lactobacillus species collected pretreatment and post treatment in head and neck cancer patients. MATERIAL AND METHODS: Lactobacillus isolates from 21 patients pretreatment and post treatment were identified using molecular methods. The patients' stimulated salivary secretion was determined pretreatment, and 6 and 12 months post treatment and caries lesions/new filled surfaces registered at 24 months post treatment. The acid-producing capacity of the Lactobacillus isolate was analyzed using a colorimetric fermentation test in microtiter plates. The anti-microbial activity of the isolates against Streptococcus mutans associated with caries, and against the mucosal pathogens Staphylococcus aureus, Candida albicans, and Enterococcus faecalis was analyzed by determining inhibitory zones on agar plates. RESULTS: The most frequent species were L. paracasei (n = 21), L. casei/rhamnosus (n = 17) and L. fermentum (n = 10). Sixty-seven percent of the patients harbored L. paracasei either at 6 or 12 months post radiotherapy. The corresponding figures for L. casei/rhamnosus and L. fermentum were 62% and 33%. L. paracasei strains showed the best acid-producing capacity and L. fermentum strains the lowest. Strong acid-producing capacity was most common among isolates collected at 6 months post treatment. Seventy-two percent of the strains showed an anti-microbial activity against S. mutans, one strain against S. aureus and none against C. albicans or E. faecalis. CONCLUSION: The most frequent species isolated from head and neck cancer patients both pretreatment and post treatment were L. paracasei, L. casei/rhamnosus, and L. fermentum. L. paracasei showed the best acid-producing capacity and the highest proportion with anti-microbial activity against S. mutans.

Oral Lactobacillus strains reduce cytotoxicity and cytokine release from peripheral blood mononuclear cells exposed to Aggregatibacter actinomycetemcomitans subtypes in vitro.

BACKGROUND: This study evaluated the effect of oral lactobacilli on the cytotoxicity and cytokine release from peripheral blood mononuclear cells (PBMCs) when exposed to Aggregatibacter actinomycetemcomitans subtypes in vitro. The supernatants and cell wall extracts (CWEs) of eight A. actinomycetemcomitans strains, representing different subtypes, and three Lactobacillus strains were used. The PBMCs from six blood donors were exposed to supernatants and CWEs of A. actinomycetemcomitans or Lactobacillus strains alone or combinations and untreated cells as control. The cytotoxicity was determined by trypan blue exclusion method and IL-1ß secretion by ELISA. TNF-α, IL-6, and IL-8 secretions were measured using Bioplex Multiplex Immunoassay. RESULTS: Supernatants or CWEs from all bacterial strains showed cytotoxicity and IL-1ß secretion and the subtypes of A. actinomycetemcomitans showed generally a significantly higher effect on PBMCs than that of the Lactobacillus strains. Two highly toxic A. actinomycetemcomitans strains (JP2 and JP2-like) induced a higher response than all other strains. When combined, Lactobacillus significantly reduced the toxicity and the IL-1ß secretion induced by A. acinomycetemcomitans. The effect varied between the subtypes and the reduction was highest for the JP2 and JP2-like strains. The Lactobacillus paracasei strain SD1 had a higher reducing effect than the other Lactobacillus strains. This strain had a consistent reducing effect on all subtypes of A. actinomycetemcomitans cytotoxicity, and release of IL-1ß, IL-6, IL-8, and TNF-α from PBMCs of the blood donors. A strong and significant variation in cytokine release between the six blood donors was noticed. CONCLUSIONS: Lactobacillus spp. and L. paracasei SD1 in particular, showed a limited but statistically significant reducing interaction with A. actinomycetemcomitans toxicity and release of cytokines in vitro.

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.

H2S mediates increased interleukin (IL)-1ß 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)-1ß 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-1ß and IL-18. Since this response differs between individuals, it may also reflect the susceptibility of the host to develop periodontitis.

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.

Acid-producing capacity from sugars and sugar alcohols among Lactobacillus isolates collected in connection with radiation therapy.

OBJECTIVE: To investigate the acid-producing capacity from sugars and sugar alcohols of oral Lactobacillus collected in connection with radiation therapy (RT) to the head and neck region. DESIGN: Lactobacillus were collected from the tongue, buccal mucosa and supragingival plaque in 24 patients before, during, and after RT. The acid-producing capacity of Lactobacillus isolates (n=211) was analyzed using a colorimetric fermentation test in microtiter plates. Solutions containing 2% sugars (sucrose, glucose, fructose, lactose) or sugar-alcohols (sorbitol and xylitol) were used. After 24h of incubation, bacterial acid-producing capacity was determined as strong (pH<5), weak (pH ≥5-≤ 6) or low/absent (pH>6). Data regarding intake frequency of sugar-rich products and products with sugar-alcohols was collected. RESULTS: The highest acid-producing capacity using the sugars was seen for isolates collected during RT. Sorbitol was fermented to a higher extent during and post RT, especially among isolates from plaque. Lactobacillus fermenting xylitol showed the highest acid-producing capacity during RT (p<0.05). No statistically significant correlations between stimulated whole salivary secretion rate and acid-producing capacity, or between the intake frequency of sugar-rich products or sugar-alcohol containing products and Lactobacillus acid-producing capacity, were found. CONCLUSION: The results suggest that Lactobacillus isolates, collected from the tongue, buccal mucosa and supragingival plaque, have a higher acid-producing capacity using sugars and sugar-alcohols during RT than one year post RT.

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.

Hydrogen sulfide exposure induces NLRP3 inflammasome-dependent IL-1ß and IL-18 secretion in human mononuclear leukocytes in vitro.

The aim was to investigate if hydrogen sulfide (H2S) induces the formation of the NLRP3 inflammasome and subsequent IL-1ß and IL-18 secretion in human peripheral blood mononuclear cells (PBMCs) and in the human monocyte cell line THP1. Bacterial production of H2S has been suggested to participate in the inflammatory host response in periodontitis pathogenesis. H2S is a toxic gas with pro-inflammatory properties. It is produced by bacterial degradation of sulfur-containing amino acids, for example, cysteine. We hypothesize that H2S affects the inflammatory host response by inducing formation of the NLRP3 inflammasome and thereby causes the secretion of IL-1ß and IL-18. PBMCs from eight healthy blood donors, the human monocyte cell line THP1 Null, and two variants of the THP1 cell line unable to form the NLRP3 inflammasome were cultured in the presence or absence of 1 mM sodium hydrosulfide (NaHS) in 24-well plates at 37°C for 24 hr. Supernatants were collected and the IL-1ß and IL-18 concentrations were measured with DuoSet ELISA Development kit. PBMCs exposed to NaHS produced more IL-1ß and IL-18 than unexposed control cells (p = .023 and p = .008, respectively). An increase of extracellular potassium ions (K+) inhibited the secretion of IL-1ß and IL-18 (p = .008). Further, NaHS triggered the secretion of IL-1ß and IL-18 in human THP1-Null monocytes (p = .0006 and p = .002, respectively), while the NaHS-dependent secretion was reduced in the monocyte cell lines unable to form the NLRP3 inflammasome. Hence, the results suggest that NaHS induces the formation of the NLRP3 inflammasome and thus the secretion of IL-1ß and IL-18. Enhanced NLRP3 inflammasome-dependent secretion of IL-1ß and IL-18 in human mononuclear leukocytes exposed to NaHS in vitro is reported. This may be a mode for H2S to contribute to the inflammatory host response and pathogenesis of periodontal disease.

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.