Phenotypic Adaptation to Antiseptics and Effects on Biofilm Formation Capacity and Antibiotic Resistance in Clinical Isolates of Early Colonizers in Dental Plaque.

Despite the wide-spread use of antiseptics in dental practice and oral care products, there is little public awareness of potential risks associated with antiseptic resistance and potentially concomitant cross-resistance. Therefore, the aim of this study was to investigate potential phenotypic adaptation in 177 clinical isolates of early colonizers of dental plaque (Streptococcus, Actinomyces, Rothia and Veillonella spp.) upon repeated exposure to subinhibitory concentrations of chlorhexidine digluconate (CHX) or cetylpyridinium chloride (CPC) over 10 passages using a modified microdilution method. Stability of phenotypic adaptation was re-evaluated after culture in antiseptic-free nutrient broth for 24 or 72 h. Strains showing 8-fold minimal inhibitory concentration (MIC)-increase were further examined regarding their biofilm formation capacity, phenotypic antibiotic resistance and presence of antibiotic resistance genes (ARGs). Eight-fold MIC-increases to CHX were detected in four Streptococcus isolates. These strains mostly exhibited significantly increased biofilm formation capacity compared to their respective wild-type strains. Phenotypic antibiotic resistance was detected to tetracycline and erythromycin, consistent with the detected ARGs. In conclusion, this study shows that clinical isolates of early colonizers of dental plaque can phenotypically adapt toward antiseptics such as CHX upon repeated exposure. The underlying mechanisms at genomic and transcriptomic levels need to be investigated in future studies.

The antimicrobial effect of Rosmarinus officinalis extracts on oral initial adhesion ex vivo.

OBJECTIVE: In the last few decades, there has been a growing worldwide interest in the use of plant extracts for the prevention of oral diseases. The main focus of this interest lies in the identification and isolation of substances that limit the formation of microbial biofilm which plays a major role in the development of caries, periodontitis, and peri-implantitis. In this clinical ex vivo study, we investigated the antimicrobial effects of Rosmarinus officinalis extract against oral microorganisms within in situ initial oral biofilms. MATERIALS AND METHODS: Initial in situ biofilm samples (2 h) from six healthy volunteers were treated ex vivo with R. officinalis extract at concentrations of 20 mg/ml and 30 mg/ml. The number of viable bacterial cells was determined by counting the colony-forming units. All surviving bacteria were isolated in pure cultures and identified using MALDI-TOF and biochemical testing procedures. Additionally, live/dead staining in combination with epifluorescence microscopy was used for visualizing the antimicrobial effects in the initial biofilms. RESULTS: The number of colony-forming units in the R. officinalis-treated biofilms was significantly lower than in the untreated controls (p < 0.001). The reduction range of log10 was 1.64-2.78 and 2.41-3.23 for aerobic and anaerobic bacteria, respectively. Regarding the bacterial composition, large intra- and interindividual variability were observed. Except for Campylobacter spp., the average amount of all bacterial taxa was lower after treatment with R. officinalis than in the untreated biofilms. A total of 49 different species were detected in the untreated biofilms, while only 11 bacterial species were detected in the R. officinalis-treated biofilms. Live/dead staining confirmed that the R. officinalis-treated biofilms had significantly lower numbers of surviving bacteria than the untreated biofilms. CONCLUSIONS: The treatment with R. officinalis extract has a significant potential to eliminate microbial oral initial biofilms. CLINICAL RELEVANCE: The results of this study encourage the use of R. officinalis extracts in biofilm control and thus in the treatment of caries and periodontitis as a herbal adjuvant to synthetic substances.

Long-Term Fluctuation of Oral Biofilm Microbiota following Different Dietary Phases.

Caries development is associated with shifts in the oral biofilm microbiota and primarily linked to frequent simple carbohydrate consumption. Different nutritional ingredients can either promote or prevent caries development. To investigate the effects of selected ingredients on the oral biofilm microbiota in situ, 11 study participants underwent 3-month-long dietary phases with intake of a regular diet (PI), additional frequent sucrose (PII), milk and yoghurt (PIII), and a diet rich in dietary fiber (PIV) and then returned to their regular diet (PV). Oral biofilm was sampled and analyzed applying 16S rRNA Illumina MiSeq sequencing. Additionally, the effect on the enamel was analyzed by measuring enamel surface roughness with laser scanning microscopy. The beta-diversity results showed that the microbiota in all the following phases differed significantly from PI and that the microbial community in PII was significantly different from all other phases. The abundance of the genus Streptococcus fluctuated over the course of the five phases, with a significant increase in PII (P = 0.01), decreasing in PIII and PIV (PIII and PIV versus PII: P < 0.00001) and increasing again toward PV. Other taxa showed various fluctuations of their abundances, with PV returning approximately to the levels of PI. In conclusion, while elevated sucrose consumption favored caries-promoting non-mutans streptococci, frequent milk and yoghurt intake caused a significant decrease in the abundance of these microbial taxa and in addition reduced enamel surface roughness. These results indicate that modulations of the oral biofilm microbiota can be attained even in adults through dietary changes and corresponding recommendations can be made for the prevention of caries development.IMPORTANCE Caries affects a large proportion of the population worldwide, resulting in high treatment costs. Its etiology can be ascribed to shifts of the microbiota in dental biofilms primarily driven by dietary factors. It is unclear how diet affects the microbial community of plaque biofilm in situ and whether it can be modulated to help prevent caries development. To address these issues, we analyzed changes of the in situ plaque microbiota following 3-month-long dietary changes involving elevated sucrose, dairy, and dietary fiber consumption over a period of 15 months. Applying high-throughput sequencing, we found non-mutans streptococci, a taxonomic group involved in the beginning stages toward microbial dysbiosis, in decreased abundance with elevated dairy and dietary fiber intake. Through analysis of the enamel surface roughness, these effects were confirmed. Therefore, correspondent dietary measures can be recommended for children as well as adults for caries prevention.

Combining culture and culture-independent methods reveals new microbial composition of halitosis patients' tongue biofilm.

BACKGROUND: Oral malodor is a very discomforting condition deriving from the presence of volatile sulfur compounds in the expired air. In halitosis of intraoral etiology, the volatile sulfur compounds are metabolic products of the oral microorganisms within the biofilm coating the tongue dorsum as well as other tissues in the oral cavity. The aim of this study was to characterize and compare the microbial composition of tongue biofilm in volunteers suffering from halitosis and healthy volunteers by means of both the culture method and culture-independent cloning technique. RESULTS: A high bacterial variety (more than 80 different species) was detected using the combination of both methods. A distinct bacterial composition was revealed in the halitosis-associated biofilms compared with the health-associated biofilms. Actinomyces graevenitzii was shown to be significantly associated with the halitosis condition. The culture method identified 47 species, included Veillonella rogosae, never isolated from the tongue biofilm of halitosis patients so far. In the healthy condition, the culture-dependent method showed that the most frequent species were Streptococcus parasanguinis among the aerobes and Veillonella spp. among the anaerobes. The culture-independent cloning method detected more than 50 species. Streptococci, in particular S. mitis/oralis, S. pseudopneumoniae, and S. infantis as well as Prevotella spp., were found most frequently in halitosis patients. Streptococcus salivarius and Rothia mucilaginosa were found more frequently in the healthy condition. CONCLUSIONS: The combination of the culture-dependent and culture-independent cloning techniques allowed for a widespread analysis of the tongue biofilm in halitosis patients. The results can support further pharmacological research for new antimicrobial agents and halitosis therapy strategies.

In-vivo shift of the microbiota in oral biofilm in response to frequent sucrose consumption.

Caries is associated with shifts of microbiota in dental biofilms and primarily driven by frequent sucrose consumption. Data on environmentally induced in vivo microbiota shifts are scarce therefore we investigated the influence of frequent sucrose consumption on the oral biofilm. Splint systems containing enamel slabs were worn for 3 × 7 days with 7-day intervals to obtain oral biofilm samples. After a three-month dietary change of sucking 10 g of sucrose per day in addition to the regular diet, biofilm was obtained again at the end of the second phase. The microbiota was analysed using Illumina MiSeq amplicon sequencing (v1-v2 region). In addition, roughness of the enamel surface was measured with laser scanning microscopy. The sucrose phase resulted in significant differences in beta-diversity and significantly decreased species richness. It was marked by a significant increase in abundance of streptococci, specifically Streptococcus gordonii, Streptococcus parasanguinis and Streptococcus sanguinis. Enamel surface roughness began to increase, reflecting initial impairment of dental enamel surface. The results showed that frequent sucrose consumption provoked compositional changes in the microbiota, leading to an increase of non-mutans streptococci, hence supporting the extended ecological plaque hypothesis and emphasizing the synergy of multiple bacterial species in the development of caries.

Antibiotic Resistance Genes and Antibiotic Susceptibility of Oral Enterococcus faecalis Isolates Compared to Isolates from Hospitalized Patients and Food.

Enterococcus faecalis, a commensal of the intestinal tract of humans and animals is of great significance as leading opportunistic pathogen, and also prevalent in oral diseases, such as endodontic infections, as well as the healthy oral cavity. To investigate the potential of oral E. faecalis to constitute a reservoir of antibiotic resistance, isolates from supragingival plaque/saliva and from endodontic infections were screened regarding their resistance to selected antibiotics in comparison to nosocomial and food isolates.70 E. faecalis isolates were analyzed with PCR regarding their equipment with the resistance genes tetM, tetO, ermB, ermC, vanA, vanB and blaTEM. Additionally, they were tested for their phenotypic resistance to doxycycline, azithromycin, rifampicin, amoxicillin and streptomycin using the Etest.High percentages of the plaque/saliva, nosocomial and food isolates were resistant to doxycycline and azithromycin, particularly plaque/saliva isolates (81%) and nosocomial isolates (73.3%) showed resistance to doxycycline, significantly more than among the food and endodontic isolates. Rifampicin resistance was widespread among isolates from plaque/saliva (52.4%), endodontic infections (50%) and nosocomial infections (40%); all isolates were susceptible to amoxicillin and all oral isolates to high-level streptomycin. TetM genes were detected in the majority of all isolates and ermB genes were present in many nosocomial and plaque/saliva isolates. Thirty percent of the endodontic isolates and 53% of the nosocomial isolates were equipped with blaTEM genes.The results suggest that the oral cavity can harbor E. faecalis strains with multiple resistances against different antibiotics and thus be regarded as a potential source of resistance traits.

Rapid species-level identification of vaginal and oral lactobacilli using MALDI-TOF MS analysis and 16S rDNA sequencing.

BACKGROUND: Lactobacillus represents a large genus with different implications for the human host. Specific lactobacilli are considered to maintain vaginal health and to protect from urogenital infection. The presence of Lactobacillus species in carious lesions on the other hand is associated with progressive caries. Despite their clinical significance, species-level identification of lactobacilli still poses difficulties and mostly involves a combination of different phenotypic and genotypic methods. This study evaluated rapid MALDI-TOF MS analysis of vaginal and oral Lactobacillus isolates in comparison to 16S rDNA analysis. RESULTS: Both methods were used to analyze 77 vaginal and 21 oral Lactobacillus isolates. The concordance of both methods was at 96% with five samples discordantly identified. Fifteen different Lactobacillus species were found in the vaginal samples, primarily L. iners, L. crispatus, L. jensenii and L. gasseri. In the oral samples 11 different species were identified, mostly L. salivarius, L. gasseri, L. rhamnosus and L. paracasei. Overall, the species found belonged to six different phylogenetic groups. For several samples, MALDI-TOF MS analysis only yielded scores indicating genus-level identification. However, in most cases the species found agreed with the 16S rDNA analysis result. CONCLUSION: MALDI-TOF MS analysis proved to be a reliable and fast tool to identify lactobacilli to the species level. Even though some results were ambiguous while 16S rDNA sequencing yielded confident species identification, accuracy can be improved by extending reference databases. Thus, mass spectra analysis provides a suitable method to facilitate monitoring clinically relevant Lactobacillus species.

Comparison of the bacterial composition and structure in symptomatic and asymptomatic endodontic infections associated with root-filled teeth using pyrosequencing.

Residual microorganisms and/or re-infections are a major cause for root canal therapy failure. Understanding of the bacterial content could improve treatment protocols. Fifty samples from 25 symptomatic and 25 asymptomatic previously root-filled teeth were collected from Sudanese patients with periradicular lesions. Amplified 16S rRNA gene (V1-V2) variable regions were subjected to pyrosequencing (FLX 454) to determine the bacterial profile. Obtained quality-controlled sequences from forty samples were classified into 741 operational taxonomic units (OTUs) at 3% dissimilarity, 525 at 5% dissimilarity and 297 at 10% dissimilarity, approximately corresponding to species-, genus- and class levels. The most abundant phyla were: Firmicutes (29.9%), Proteobacteria (26.1%), Actinobacteria (22.72%), Bacteroidetes (13.31%) and Fusobacteria (4.55%). Symptomatic patients had more Firmicutes and Fusobacteria than asymptomatic patients, while asymptomatic patients showed more Proteobacteria and Actinobacteria. Interaction of disease status and age was observed by two-way ANOSIM. Canonical correspondence analysis for age, tooth restoration and disease status showed a correlation of disease status with the composition and prevalence of different members of the microbial community. The pyrosequencing analysis revealed a distinctly higher diversity of the microbiota compared to earlier reports. The comparison of symptomatic and asymptomatic patients showed a clear association of the composition of the bacterial community with the presence and absence of symptoms in conjunction with the patients' age.

Comprehensive analysis of secondary dental root canal infections: a combination of culture and culture-independent approaches reveals new insights.

Persistence of microorganisms or reinfections are the main reasons for failure of root canal therapy. Very few studies to date have included culture-independent methods to assess the microbiota, including non-cultivable microorganisms. The aim of this study was to combine culture methods with culture-independent cloning methods to analyze the microbial flora of root-filled teeth with periradicular lesions. Twenty-one samples from previously root-filled teeth were collected from patients with periradicular lesions. Microorganisms were cultivated, isolated and biochemically identified. In addition, ribosomal DNA of bacteria, fungi and archaea derived from the same samples was amplified and the PCR products were used to construct clone libraries. DNA of selected clones was sequenced and microbial species were identified, comparing the sequences with public databases. Microorganisms were found in 12 samples with culture-dependent and -independent methods combined. The number of bacterial species ranged from 1 to 12 in one sample. The majority of the 26 taxa belonged to the phylum Firmicutes (14 taxa), followed by Actinobacteria, Proteobacteria and Bacteroidetes. One sample was positive for fungi, and archaea could not be detected. The results obtained with both methods differed. The cloning technique detected several as-yet-uncultivated taxa. Using a combination of both methods 13 taxa were detected that had not been found in root-filled teeth so far. Enterococcus faecalis was only detected in two samples using culture methods. Combining the culture-dependent and -independent approaches revealed new candidate endodontic pathogens and a high diversity of the microbial flora in root-filled teeth with periradicular lesions. Both methods yielded differing results, emphasizing the benefit of combined methods for the detection of the actual microbial diversity in apical periodontitis.