Bacterial analysis of biofilms from tooth root surfaces presenting different caries activity / Análise bacteriológica de biofilmes de superfícies dentárias radiculares apresentando diferentes estágios de atividade de cárie

This study evaluated the numbers and determined the proportion of mutans streptococci and Lactobacillus spp., which are possible relevant cariogenic organisms, in biofilms recovered from lesions at root surfaces with active caries lesions (ARC), inactive caries lesions, and sound root surfaces (SRS). Samples were cultured in MSB agar for mutans streptococci counts, Rogosa agar for Lactobacillus spp. counts, and brain-heart infusion agar for total viable anaerobic counts. After incubation, the number of colony-forming units (CFUs) was determined and compared between groups by the Mann-Whitney U test with a significance level set at 95%. The proportion of counts of mutans streptococci and Lactobacillus spp. in the total viable microorganisms was also analyzed by Chi-square test. Ninety samples (30 from each surface) from 37 patients were cultured and analyzed. The CFU was similar between mutans streptococci and Lactobacillus spp.These species were present in at least half of the samples and no difference was found in the frequency of isolation of these species. Only 6 samples showed a proportion of more than 10% of mutans streptococci; 4 of the samples were from ARC. Most (93%) SRS samples did not contain viable Lactobacillus spp. The data indicate the low counts of mutans streptococci and Lactobacillus spp. in root sur-faces, regardless of the activity of caries lesions.

O estudo analisou contagens e proporções de mutans strep-tococci e Lactobacillus spp., que podem ser microorganismos importantes em lesões de cárie radicular com diferentes atividades. Biofilmes foram coletados em três locais: ARC ­ superfície radicular com lesão ativa de cárie; IRC ­ superfícies radiculares com lesão inativa de cárie; SRS ­ superfícies de raizes hígidas. As amostras foram cultivadas em agar MSB para contagens de mutans streptococci; agar Rogosa para Lactobacillus spp., e agar BHI para contagens de microrganis-mos viáveis anaeróbicos totais. Após a incubação, o número de unidades formadoras de colônias (UFCs) foi determinado e comparado entre os grupos pelo teste de Mann-Whitney U test. O nível de significância foi estabelecido em 95%. A pro-porção de contagem de mutans streptococci e Lactobacillusspp. no total de microrganismos viáveis também foi analisado através do teste de qui-quadrado. Um total de 90 amostras de 37 pacientes foram cultivadas e analisadas: 30 amostras de ARC, 30 de IRC e 30 de SRS. Números de UFC foram seme-lhantes entre os grupos para ambos, mutans streptococci e Lactobacillus spp. Estas espécies estavam presentes em pelo menos metade de todas as amostras e nenhuma diferença foi encontrada na frequência de isolamento dessas espécies dentro dos grupos. Apenas 6 amostras apresentaram mais de 10% de mutans streptococci e 4 foram de ARC. Em relação aos Lactobacillus spp., 93% das amostras não apresentaram proporção dessas bactérias nas SRS. Mutans streptococci e Lactobacillus spp. estão presentes em baixa proporção nas superfícies radiculares, independentemente da atividade das lesões de cárie.

The role of Candida albicans in root caries biofilms: an RNA-seq analysis

Abstract Objective This study sought to analyze the gene expression of Candida albicans in sound root surface and root caries lesions, exploring its role in root caries pathogenesis. Methodology The differential gene expression of C. albicans and the specific genes related to cariogenic traits were studied in association with samples of biofilm collected from exposed sound root surface (SRS, n=10) and from biofilm and carious dentin of active root carious lesions (RC, n=9). The total microbial RNA was extracted, and the cDNA libraries were prepared and sequenced on the Illumina Hi-Seq2500. Unique reads were mapped to 163 oral microbial reference genomes including two chromosomes of C. albicans SC5314 (14,217 genes). The putative presence of C. albicans was estimated (sum of reads/total number of genes≥1) in each sample. Count data were normalized (using the DESeq method package) to analyze differential gene expression (using the DESeq2R package) applying the Benjamini-Hochberg correction (FDR<0.05). Results Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) were up-regulated on SRS, and their functions are related to biofilm formation. Seven genes ( UTP20 , FDR=0.018; ITR1 , FDR=0.036; DHN6 , FDR=0.046; CaO19.7197 , FDR=0.046; CaO19.7838 , FDR=0.046; STT4 , FDR=0.046; GUT1 , FDR=0.046) were up-regulated on RC and their functions are related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation. The use of alternative carbon sources, including lactate, and the ability to form hypha may be a unique trait of C. albicans influencing biofilm virulence. Conclusions C. albicans is metabolically active in SRS and RC biofilm, with different roles in health and disease.

Gene expression profile of Scardovia spp. in the metatranscriptome of root caries

Abstract A few investigations of caries biofilms have identified Scardovia spp.; however, little is known about its involvement in caries pathogenesis. The purpose of this study was to assess the gene expression profile of Scardovia spp. in root caries, and compare it with other microorganisms. Clinical samples from active root caries lesions were collected. Microbial mRNA was isolated and cDNA sequenced. The function and composition of the Scardovia were investigated using two methods: a) de novo assembly of the read data and mapping to contigs, and b) reads mapping to reference genomes. Pearson correlation was performed (p < 0.05). Proportion of Scardovia inopinata and Scardovia wiggsiae sequences ranged from 0-6% in the root caries metatranscriptome. There was a positive correlation between the transcriptome of Lactobacillus spp. and Scardovia spp. (r = 0.70; p = 0.03), as well as with other Bifidobacteriaceae (r = 0.91; p = 0.0006). Genes that code for fructose 6-phosphate phosphoketolase (the key enzyme for "Bifid shunt"), as well as ABC transporters and glycosyl-hydrolases were highly expressed. In conclusion, "Bifid shunt" and starch metabolism are involved in carbohydrate metabolism of S. inopinata and S. wiggsiae in root caries. There is a positive correlation between the metabolism abundance of Lactobacillus spp., Bifidobacteriaceae members, and Scardovia in root caries.

The use of bur and laser for root caries treatment: a comparative study.

This research analyzed the influence of bur and erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser caries removal on cavity characteristics and marginal seal of composite resin restorations. One hundred and forty human dental root samples were used. After in vitro root caries induction using Streptococcus mutans, the carious lesions were removed either by a conventional technique using burs (G1=control) or by using an Er,Cr:YSGG laser (λ=2.78 µm, 20 Hz, pulse duration is approximately equal to 140 µs, noncontact mode using a 600-µm tip) with the following power outputs: G2: 1.0 W; G3: 1.25 W; G4: 1.5 W; G5: 1.75 W; G6: 2.0 W; G7: 2.25 W; G8: 2.5 W; G9: 2.75 W; G10: 3.0 W; G11: 3.25 W; G12: 3.5 W; G13: 3.75 W; and G14: 4.0 W. Samples in the 14 groups (n=10) were conditioned with Clearfil SE Bond and restored with a flowable composite. They were then thermocycled (1000 cycles) and immersed into a 2% methylene blue solution for microleakage analysis. The data were statistically compared (analysis of variance or Spearman correlation tests; p≤0.05). The lased groups showed significantly greater microleakage indexes, cavity depths, and presence of residual caries than did those of the control group. There was a strong positive correlation between residual caries and microleakage. The results indicate that Er,Cr:YSGG laser irradiation is not a good alternative to the use of burs for root caries removal since it may cause a significant loss of marginal sealing in composite resin restorations.

A preliminary study on the screening of emerging drug resistance among the caries pathogens isolated from carious dentine.

BACKGROUND: Dental caries being the commonest unmet public health problem indicates its need to urge the dentists to overcome this problem globally. Caries exhibit in different types and is found to be associated with co-aggregation property of microbial flora with other oral hygienic factors. In spite of the surgical removals, excavations and administration of antimicrobials for carious dentine, there seems to be repeated infection and chronic prevalence of caries. A complete understanding of microbial etiology and prevention of emerging drug-resistant strains will aid in the eradication of this chronic dentine problem condition from the oral cavity. AIM: This study is aimed to isolate the predominant bacterial pathogens associated with caries and to screen for the emergence of drug resistance among the isolated caries pathogens. MATERIALS AND METHODS: Carious dentine specimens were collected from 75 endodontic patients and the samples were processed microbiologically to isolate the caries pathogens. Identification of the strains was done by standard biochemical characterization studies. Statistical analysis of the isolates was done by Pearson Chi-square test and Fisher's exact test. The predominant isolates were subjected to antimicrobial sensitivity test using Kirby Bauer's method. The results were recorded and analyzed for drug resistance. RESULTS: Carious dentine samples yielded a high percentage of Lactobacillus sp., and Candida albicans from different type of caries. Among the study population, dentinal caries was the most predominant type affecting most males with other associated risk factors. Nearly 47.3% of the isolated Lactobacillus sp. and 55.5% of the yeast C. albicans were screened to show resistance against the antimicrobials used for the study. CONCLUSION: This study concludes by stating that Lactobacillus sp., and C. albicans are mostly involved in the caries etiology and show resistance to the commonest antimicrobial agent. This implicates the need for periodical antimicrobial susceptibility examination of the caries pathogens that will aid to prevent the emergence of resistance property among the dentinal pathogenic organisms.

Oral colonization by Streptococcus mutans and its association with the severity of periodontal disease in adults

Background: Streptococcus mutans (S. mutans) is associated with the onset of caries. Since root exposure in patients affected by periodontitis leads to higher caries rates, progressively more severe forms of periodontal disease might associate with elevated counts of S. mutans. Aim: To determine whether increasingly destructive forms of periodontal disease are associated with higher counts of S. mutans in untreated patients. Methods: 206 subjects aged 20-75 were classified into three groups according to the severity of periodontal disease: 1) gingivitis, 2) chronic slight periodontitis and 3) chronic moderate or chronic severe periodontitis. S. mutans counts (cfu/mL) were obtained by direct counting on selective agar plates from saliva samples. A cumulative proportional logistic regression model was adjusted for S. mutans counts. Results: The model failed to show differences by gender, but periodontal diagnosis had a significant effect on S. mutans counts depending on age. While in the group with moderate and severe periodontitis the probability of having high counts of S. mutans significantly increased with age, the probability remained unchanged in individuals with chronic slight periodontitis or gingivitis. Conclusion: High S. mutans levels appear directly co-associated with increased severity of periodontal disease at older ages in untreated patients.

Mutans streptococci in subgingival plaque of treated and untreated patients with periodontitis.

BACKGROUND: The etiology of root caries is thought to be identical to coronal caries, though root caries seem to be more complicated because of the higher susceptibility of exposed roots (dentin) by periodontal therapy to demineralization than intact enamel. This implies that mutans streptococci are the most likely pathogens in the development of root caries. Although it is known that both the numbers of mutans streptococci and the frequency of isolation in root caries lesions are negatively correlated with the distance from the gingival margin, the subgingival sulcus has not been considered a possible habitat for mutans streptococci. However, subgingival occurrence of mutans streptococci in both untreated and treated periodontal patients has not been documented well in the literature. OBJECTIVE: To investigate the presence and levels of mutans streptococci in the subgingival plaque of patients (n=154) in different stages of periodontal therapy. The subgingival sulcus may be a possible habitat for mutans streptococci. This localisation of mutans streptococci may be of importance in the development of root caries after periodontal surgery. MATERIALS AND METHODS: In this cross-sectional study, subgingival plaque samples from 154 consecutive adult periodontitis patients were tested for presence and levels of mutans streptococci and putative periodontal pathogens by anaerobic cultures. These patients were divided into 4 groups based on their stage of periodontal treatment: (1) untreated patients; (2) patients after initial periodontal therapy only; (3) patients in the maintenance phase who not underwent periodontal surgery; (4) patients after periodontal surgery. RESULTS: The prevalence of mutans streptococci in the 4 study groups varied from 82% in untreated patients to 94% in maintenance patients. The mean proportion of mutans streptococci was 6.65% in maintenance patients versus 1.86% in untreated patients (p=0.005) and 2.51% in patients after scaling and root planing (p=0.041). CONCLUSIONS: The subgingival area is a microbial habitat for mutans streptococci that may be of importance in the development of root caries in periodontitis patients.

Role of dentinal carious lesions in the pathogenesis of oral candidiasis in HIV infection.

The authors describe a clinicopathologic study that evaluated whether dentinal carious lesions are colonized by candidal organisms--and if so, whether there is a relationship between dentinal carious lesion colonization and clinical oral candidiasis, or OC, in HIV infection. Using light microscopy, the authors examined 30 extracted teeth with dentinal carious lesions from people in each of two groups: 30 consecutively treated HIV-positive patients and 30 consecutively treated HIV-negative patients. OC was diagnosed only in HIV-positive patients (40 percent). The dentinal carious lesion pattern in both groups was similar in occlusal, root and proximal caries. Candidal colonization of carious dentinal tubules was more frequent in HIV-positive subjects than it was in HIV-negative subjects. This research shows that it may be important to restore dentinal caries in HIV-infected patients to remove a protected niche for candidal organisms.

Root-surface caries in rats and humans: inhibition by a non-antimicrobial property of tetracyclines.

The incidence of root caries has been found to increase as the population ages and as edentulism becomes less prevalent due to improved dental awareness and care, and as exposure of roots due to gingival recession has also increased in the elderly. The mechanism of root caries is thought to be mediated by both bacterial and mammalian proteases produced by plaque and the periodontal tissues, respectively. In the current study, a rat model of periodontal disease was used in which gnotobiotic rats were infected intra-orally with a periodontal pathogen (P. gingivalis). Infecting the rats with P. gingivalis increased the collagenase activity in the gingival tissue in association with severe alveolar bone loss. Treating P. gingivalis-infected rats with doxycycline or CMT-1 prevented the destruction of the periodontium by MMPs, thus preventing exposure of roots to subgingival bacterial plaque and host tissue collagenases and the subsequent development of root caries. In addition, a low-dose doxycycline (LDD, 20 mg bid, non-antimicrobial dose) for 3 months was used in humans predisposed to increased root caries as the result of heavy use of smokeless (chewing) tobacco, causing gingival recession, subgingival plaque accumulation with Gram-negative bacteria, increased gingival crevicular fluid flow (GCF), and elevated GCF collagenase. Daily administration of LDD in smokeless tobacco patients reduced the GCF collagenase and prevented the further development of root caries.