Identification of Yeast Species Isolated from Dental Plaque of Periodontitis Patients and Healthy Subjects Using TYI-S-33 Medium

BACKGROUND:Periodontal disease is an inflammatory-destructive condition of the supporting tissues of the teeth. Microorganisms found in the dental plaque were considered to be the primary local etiologic factor responsible for the periodontal destruction. It is also evident that herpes simplex viruses may have an impact in the etiopathogenesis of periodontal disease.AIM:This study has been made with the aim to analyse the prevalence of herpes simplex virus type 1 (HSV-1) in the dental plaque (supra- and subgingival) of patients with the chronic periodontal disease.MATERIAL AND METHODS:The study comprised a total of 89 patients with chronic periodontal disease divided into two groups (patients with moderate and severe periodontitis). Supragingival dental plaque samples were taken with sterile cotton (supragingival), and subgingival dental plaque samples were taken with paper absorbents. Samples were subjected to extraction of DNA and further analysis with multiplex PCR for the presence of herpes viral DNA.RESULTS:HSV-1 virus was detected In 24.7% of all patients included in the study. HSV-1 was detected in 22.2% of patients with the moderate stage of the disease, of which in all (100%) in the supragingival plaque samples and only 16.7% in subgingival plaque samples. In two patients HSV-1 was concomitantly detected in supra and subgingival plaque samples. In patients with advanced stage of the disease, the HSV-1 virus was detected in 28.6% patients. In two of the patients, HSV-1 was concomitantly detected in supra and subgingival plaque samples. Statistically, a significant difference was found in HSV-1 positive patients with a moderate stage of disease, between the presence of the virus in subgingival (100%) and subgingival (16.7%) dental plaque samples, p < 0.05.CONCLUSION:Herpes simplex viruses type 1 are present in supragingival and subgingival dental plaque.

The aim of this study was to assess the periodontal condition and the presence of putative periodontal pathogens in 30 Brazilian mothers, aging 21-40 years (28.4 4.49 years), and in their children, aging 5-6 years, since mothers can be a source of pathogens and, thus, influence their children's bacteriological and clinical condition. Besides assessing the plaque index (PI), gingival index (GI) and pocket probing depth (PD), the survey analyzed four subgingival dental plaque samples from mothers and children, as well as a sample of stimulated saliva from mothers. Those samples were analyzed by means of the slot immunoblot (SIB) technique, in order to determine the presence of Actinobacillus actinomycetemcomitans (Aa), Prevotella nigrescens (Pn), Porphyromonas gingivalis (Pg) and Treponema denticola (Td). The mean values and standard deviations of the evaluated clinical variables for mothers and children were, respectively: 1.86 0.67 and 1.64 0.68 for PI, and 1.24 0.67 and 0.82 0.37, for GI. Only for mothers, the total PD was 1.81 0.69 mm, and the PD of four sites was 4.03 1.40 mm. The Wilcoxon test revealed significant difference (p < 0.05) between mothers and their children only as to GI. The most prevalent bacteria in mothers were, in decreasing order: Aa, Pn, Pg and Td. The children presented patterns of oral hygiene and bacterial profiles similar to those of their mothers, in spite of the fact that most of them did not present enough subgingival plaque for testing. The comparison between mothers' subgingival dental plaque and saliva samples revealed statistically significant differences (p < 0.05) for all bacteria, with greater positivity and scores in the saliva, which demonstrates that it is an indicator of oral colonization and can work as a vehicle for the transmission of periodontopathogens from mothers to their children.

The primary habitats of oral veillonellae are the tongue, dental plaque, and the buccal mucosa. Isolates were obtained from each habitat and tested for coaggregation with a battery of other oral bacterial strains. All 59 tongue isolates tested for coaggregation were Veillonella atypica or Veillonella dispar. All but one of them coaggregated with strains of Streptococcus salivarius, a predominant inhabitant of the tongue surface but not subgingival dental plaque. These tongue isolates were unable to coaggregate with most normal members of the subgingival flora such as Actinomyces viscosus, Actinomyces naeslundii, Actinomyces israelii, and Streptococcus sanguis. In contrast, 24 of 29 Veillonella isolates, of which 20 were Veillonella parvula from subgingival dental plaque samples, coaggregated strongly with the three species of Actinomyces, S. sanguis, and other bacteria usually present in subgingival plaque, but they did not coaggregate with S. salivarius. The majority of isolates from the buccal mucosa (42 of 55) has coaggregation properties like those from the tongue. These results indicate that the three human oral Veillonella species are distributed on oral surfaces that are also occupied by their coaggregation partners and thus provide strong evidence that coaggregation plays a critical role in the bacterial ecology of the oral cavity.

Background:Bacterial colonization of dentition in different age groups can impact prognosis in different dental diseases. Latest diagnostic technique such as matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) is increasingly being used for accurate identification of bacteria. This study was undertaken to evaluate the MALDI-TOF MS technique to identify bacterial pathogens from dental plaques in subjects with primary, mixed, and permanent dentition.Materials and Methods:The study included 150 subjects of different age groups and were divided into three groups - Group A: Subjects with primary dentition (n = 50), Group B: Subjects with mixed dentition (n = 50), and Group C: Subjects with permanent dentition (n = 50). Subgingival dental plaque samples were collected from buccal and lingual surfaces of premolar and molar teeth. Clinical parameters such as gingival index were recorded. Samples were cultured in routine aerobic and anaerobic medium. Bacterial growths were assessed by semiquantitative methods. Bacterial isolates were confirmed by MALDI-TOF MS technique.Results:MALDI-TOF MS detected all the culture-grown bacteria. In primary dentition group, purple and yellow complex bacteria predominated. Streptococcus spp. was the predominant bacteria (51%) followed by Escherichia coli (19%) and Veillonella spp. (19%). In mixed dentition and permanent group also, Streptococcus spp. was predominant (46%) followed by Veillonella spp. (24%) and E. coli(19%). However, in both groups, orange complex bacteria (bridge complex) such as Prevotella nigrescens and red complex bacteria (Porphyromonas gingivalis, 3%) were seen. For majority of bacteria, the load increased with age.Conclusions:The bacterial isolates showed a distinct age-specific colonization. The use of advanced technique such as MALDI-TOF MS is helpful in the detection of periodontal pathogens, and the effective oral health programs can be implemented to minimize the risk of periodontal diseases.

IntroductionWe evaluated the presence of Porphyromonas gingivalis (Pg) DNA in the synovial tissue through synovial biopsy and in other compartments of rheumatoid arthritis (RA) patients in comparison with patients affected by other arthritides. Possible links with clinical, immunologic and genetic features were assessed.MethodsPeripheral blood (PB), sub-gingival dental plaque, synovial fluid (SF) and synovial tissue samples were collected from 69 patients with active knee arthritis (32 with RA and 37 with other arthritides, of which 14 had undifferentiated peripheral inflammatory arthritis - UPIA). Demographic, clinical, laboratory and immunological data were recorded. The presence of Pg DNA was evaluated through PCR. The HLA-DR haplotype was assessed for 45 patients with RA and UPIA.ResultsNo differences arose in the positivity for Pg DNA in the sub-gingival plaque, PB and SF samples between RA and the cohort of other arthritides. Full PB samples showed a higher positivity for Pg DNA than plasma samples (11.8% vs. 1.5%, P = 0.04). Patients with RA showed a higher positivity for Pg DNA in the synovial tissue compared to controls (33.3% vs. 5.9%, P <0.01). UPIA and RA patients carrying the HLA DRB1*04 allele showed a higher positivity for Pg DNA in the synovial tissue compared to patients negative for the allele (57.1% vs. 16.7%, P = 0.04). RA patients positive for Pg DNA in the sub-gingival plaque had a lower disease duration and a higher peripheral blood leucocyte and neutrophil count. The presence of Pg DNA did not influence disease activity, disease disability or positivity for autoantibodies.ConclusionsThe presence of Pg DNA in the synovial tissue of RA patients suggests a pathogenic role of the bacterium. The higher positivity of Pg DNA in full peripheral blood and synovial tissue samples compared to plasma and synovial fluid suggests a possible intracellular localization of Pg, in particular in patients positive for HLA-DR4.

The aim of this study was to compare the detection frequencies of 25 bacterial species in subgingival and supragingival plaque of 18 untreated periodontitis subjects and 12 periodontally healthy subjects. Genomic DNA was extracted from subgingival and supragingival plaque samples, and bacterial detection was performed by polymerase chain reaction of the 16S rRNA genes. Fourteen bacteria showed no relationship with periodontitis, and 11 of these 14 species were frequently detected (> or =50%) in subgingival plaque in both periodontitis and healthy subjects. Nine bacteria such as Eubacterium saphenum, Prevotella intermedia, and Treponema denticola seemed to be related to periodontitis; their detection frequencies in subgingival plaque samples were higher in periodontitis than in healthy subjects, but these differences were not statistically significant by multiple comparisons (0.002< or =P<0.05). Two species (Mogibacterium timidum and Porphyromonas gingivalis) were detected significantly more frequently in subgingival plaque of periodontitis subjects than of healthy subjects (P<0.002), with P. gingivalis being detected only in periodontitis subjects, suggesting that these two species are closely related to periodontitis. There were no significant differences in the detection frequencies of the 25 bacteria between subgingival and supragingival plaque, suggesting that the bacterial flora of supragingival plaque reflects that of subgingival plaque.

Yeasts are essential contributors to the ripening and flavor development of white brined cheeses. This study aimed to investigate and compare the microbial load and yeast species composition in artisanal and industrial white brined cheeses. The influence of key physicochemical parameters (salt content, acidity, fat content, moisture, and ripening stage) on yeast count and species composition was analyzed. A total of 100 white brined cheese samples produced in Bulgaria were analyzed. Yeast species were identified using MALDI–TOF MS, and physicochemical properties were assessed according to ISO standards. The predominant yeast species identified were Torulaspora delbrueckii, Debaryomyces hansenii, Saccharomyces cerevisiae, and Candida sphaerica. D. hansenii was the dominant species in industrial samples, while S. cerevisiae was more frequently isolated from artisanal cheeses. Statistical analyses showed that the physicochemical parameters most influencing yeast species composition were salt content and acidity. A statistically significant correlation between yeast count and salt content was observed only in industrial cheeses, with D. hansenii showing greater salt tolerance. Yeast counts were higher in cheeses with higher salt content, particularly in industrial samples. This study highlights the distinct influence of production methods and physicochemical parameters on the yeast ecology of white brined cheeses.

The highly destructive pathogenic processes in patients with periodontitis are attributed to the presence of subgingival biofilms comprising key periodontal pathogens, such as Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, as well as other periodontopathogens including Fusobacterium nucleatum, Selenomonas spp., Centipeda spp., and Campylobacter spp. Considering the vast microbial diversity in periodontitis, we aimed to analyze the presence of various bacterial species in subgingival dental plaque, with a special focus on Selenomonas spp. We first developed a phylogenetic tree for Selenomonas–Veillonella clusters, using in silico analysis followed by fluorescence in situ hybridization (FISH) on subgingival dental plaque samples from 22 patients with a history of chronic periodontitis, by using specific 16S rRNA oligonucleotide probes. These oligonucleotide probes’ specificity and hybridization conditions were determined on previously characterized bacterial strains. Qualitative and quantitative analysis of FISH slides was carried out by using an epifluorescence microscope. The majority of the patient samples showed high fluorescence signals with the oligonucleotide probes SEL1150, Sspu439, and SEL1469 (specific for Selenomonas spp.), ACI623 (identifying Selenomonas, Veillonella, and Dialister spp.), Tfor127 (T. forsythia) and L-Pgin1006-23 (P. gingivalis). SEL1150 showed specificity for bacterial species in the subclusters A, B, and C, namely S. dianae, S. infelix, S. flueggei, C. periodontii, S. artemidis, S. noxia, and S. sputigena; Sspu439 for S. sputigena; SEL1469 for subclusters A and B, and for S. sputigena; ACI623 for bacterial species in subclusters C and F, namely the S. sputigena and Veillonella species. The experimentally observed specificities of the oligonucleotide probes corresponded with our in silico analysis. Selenomonas spp. may play a role in the subgingival microbiome of periodontitis and contribute to the disease process. Targeting Selenomonas spp. with specific therapeutic strategies could offer new insights into the management of periodontitis. However, further studies are needed to determine a definite functional significance.

The prevalence and distribution of the putative periodontal pathogen Eikenella corrodens in the human oral cavity was examined. A total of 508 oral bacterial samples were taken from 10 periodontally healthy adults (PH), 11 adult periodontitis patients (AP), and 6 localized juvenile periodontitis patients (LJP). From each subject, samples of supra- and subgingival plaque were obtained from six to eight teeth as well as samples from buccal mucosa, lateral and dorsal surfaces of tongue, tonsil, and saliva. E. corrodens was cultured from 70% of healthy subjects and 100% of periodontitis patients. Dental plaque appears to be the main oral ecological niche of E. corrodens in PH subjects since it was found in, respectively, 26% and 31% of supra- and subgingival plaque samples and rarely found in other oral sites in these subjects. It was found in 59% of both supra- and subgingival plaque samples from AP subjects, as well as 48% and 64% of supra- and subgingival plaque samples of LJP subjects. In contrast to healthy subjects, E. corrodens was found on the buccal mucosa, tongue, tonsil and in the saliva of patients with periodontitis. The microorganism constituted, on average, 1% to 2% of the total cultivable bacteria in supra- and subgingival plaque samples. The prevalence of E. corrodens in plaque samples was higher in AP and LJP subjects and was significantly different from PH subjects. Within the AP group, the prevalence of E. corrodens in subgingival plaque is significantly higher from sites with GI greater than or equal to 2. These data suggest that E. corrodens is an indigenous oral microorganism which may be an opportunistic pathogen associated with gingival inflammation.

The extracellular matrix is a critical component of microbial biofilms, such as dental plaque, maintaining the spatial arrangement of cells and coordinating cellular functions throughout the structure. The extracellular polymeric substances that comprise the matrix include carbohydrates, nucleic acids, proteins, and lipids, which are frequently organized into macromolecular complexes and/or are associated with the surfaces of microbial cells within the biofilm. Cariogenic dental plaque is rich in glucan and fructan polysaccharides derived from extracellular microbial metabolism of dietary sucrose. By contrast, the matrix of subgingival dental plaque is a complex mixture of macromolecules that is still not well understood. Components of the matrix escape from microbial cells during lysis by active secretion or through the shedding of vesicles and serve to anchor microbial cells to the tooth surface. By maintaining the biofilm in close association with host tissues, the matrix facilitates interactions between microorganisms and the host. The outcome of these interactions may be the maintenance of health or the development of dental disease, such as caries or periodontitis. The matrix affords microbial cells protection against chemical and physical insults and hinders the eradication of pathogenic dental plaque. Therefore, strategies to control the matrix are critical to maintain oral health. This review discusses recent advances in our understanding of the composition, origins, and function of the dental plaque matrix, with a focus on subgingival dental plaque. New strategies to control subgingival dental plaque based on targeting the biofilm matrix are also considered.

Exploring the prevalence of oral trichomonads in patients with periodontitis at a tertiary teaching hospital.

To discuss the relationship between uncultivated pathogenic bacteria and periodontitis. Specific polymerase chain reaction (PCR) primers were designed for phylotypes AU126 and X112; PCRs were applied to determine the prevalence of these phylotypes in 35 patients with chronic periodontitis, 26 patients with plaque-induced gingivitis and 20 healthy control subjects. The specificity of each primer is validated on the basis of the results from sequence analysis of PCR products. AU126 and X112 were detected in the subgingival plaque samples in all the three groups. The prevalence of AU126 in subgingival plaque in chronic periodontitis (77.1%) and plaque-induced gingivitis (61.5%) is relatively higher than that in the healthy subjects (10.0%), and the difference is statistically significant (P < 0.01). The prevalence of X112 in subgingival plaque in periodontitis patients (85.7%) is higher than that in healthy subjects (30.0%), the difference (P < 0.01) being equally statistically significant. The difference between the chronic periodontitis group and the plaque-induced gingivitis group (50.0%) is statistically significant (P < 0.05). It might be assumed that the novel uncultivated AU126 phylotype could possibly be related to chronic periodontitis and plaque-induced gingivitis, and that X112 might play a role in the progress of lesion from gingivitis to periodontitis.

Two unique forms of periodontal disease, HIV-gingivitis and HIV-periodontitis, have been described in patients with Acquired Immunodeficiency Syndrome (AIDS). In order to determine the bacterial species associated with periodontitis in AIDS patients, the predominant cultivable microflora was examined in 21 subgingival plaque samples from 11 AIDS patients with periodontitis. The presence of putative periodontal pathogens including Actinobacillus actinomycetemcomitans, Bacteroides intermedius, Porphyromonas gingivalis (formerly B. gingivalis), and Wolinella recta was examined by immunofluorescence in 128 subgingival dental plaque samples from 50 AIDS patients including 32 patients with periodontitis. Of 666 bacterial strains isolated from the 21 subgingival plaque samples, Streptococcus sanguis II was the most frequently recovered species comprising 18.5% of the total number of isolates followed by Lactobacillus acidophilus (12.2%), Porphyromonas gingivalis (12%), Fusobacterium nucleatum (11.4%), Staphylococcus epidermidis (8.7%), Actinomyces naeslundii (7.5%), and Actinomyces viscosus (4.7%). Fusobacterium nucleatum was the most prevalent species and was found in 76% of the sites and 91% of the patients. Enteric species including Enterococcus avium and Enterococcus faecalis, Clostridium clostridiiforme and Clostridium difficle as well as Klebsiella pneumoniae also were recovered. Immunofluorescence assays detected similar carriage rates of A. actinomycetemcomitans, B. intermedius, and P. gingivalis in both gingivitis patients and periodontitis patients, while four times more periodontitis patients demonstrated W. recta. Subgingival yeast was a frequent finding in these AIDS patients, present in 62% of the subjects and 55% of the sites. This study indicates that subgingival plaque in AIDS patients with periodontitis can harbor high proportions of the same periodontal pathogens as are associated with periodontitis in non-HIV infected subjects as well as high proportions of opportunistic pathogens.

Culture has always been recognized as the gold standard for detecting oral anaerobes in dental plaque samples. However, many of the bacterial morphotypes observed by microscopy are difficult to culture, thus necessitating the need for alternative methods of detection. The objective of this study was to evaluate the sensitivity and specificity of BANA (N-benzoyl-DL-arginine-B-naphthylamide) and PCR (Polymerase Chain Reaction) as reliable detection methods for detecting oral anaerobes such as Porphyromonas gingivalis,Tannerella forsythia and Treponema denticola (often referred to as the “red complex”) in subgingival dental plaque. Of the 372 samples analysed, 7.25% tested positive for the BANA test and 36.29% yielded a positive PCR test. This study showed that PCR was more sensitive than BANA in detecting members of the “red complex” in plaque samples. Key words: Polymerase chain reaction (PCR), N-benzoyl-DL-arginine-B-naphthylamide (BANA), red complex, subgingival plaque, periodontal disease.

Fixed orthodontic appliances cause plaque accumulation around bands and brackets. Since the microbiological composition of dental plaque is closely connected to periodontal tissue health, the aim of this study was to determine the effects of fixed orthodontic appliances on subgingival microflora and periodontal status. This prospective study was carried out on 32 adolescents scheduled for fixed orthodontic treatment. Subgingival dental plaque samples and periodontal records (pocket probing depth and clinical attachment level) were obtained in four recording times: before bonding of fixed appliances (T0), 1 (T1), 3 (T2) and 6 (T3) months after the beginning of orthodontic therapy, in order to detect the changes in periodontopathic anaerobe microbial flora and its effects on periodontal status. The values of pocket probing depth, total number of microorganisms and number of patients with positive findings of Prevotella intermedia and other periodontopathic anaerobes increased from T0 to the maximum obtained in T2 recording time. Both clinical and microbiological values decreased 6 months after the beginning of orthodontic therapy. The therapy with fixed appliances may transitionally increase the growth of periodontopathogenic bacteria and consequently result in gingival inflammatory response but without destructive effect on deep periodontal tissues.