Development and pyrosequencing analysis of an in-vitro oral biofilm model.

BACKGROUND: Dental caries and periodontal disease are the commonest bacterial diseases of man and can result in tooth loss. The principal method of prevention is the mechanical removal of dental plaque augmented by active agents incorporated into toothpastes and mouthrinses. In-vitro assays that include complex oral bacterial biofilms are required to accurately predict the efficacy of novel active agents in vivo. The aim of this study was to develop an oral biofilm model using the Calgary biofilm device (CBD) seeded with a natural saliva inoculum and analysed by next generation sequencing. The specific objectives were to determine the reproducibility and stability of the model by comparing the composition of the biofilms over time derived from (i) the same volunteers at different time points, and (ii) different panels of volunteers. RESULTS: Pyrosequencing yielded 280,093 sequences with a mean length of 432 bases after filtering. A mean of 320 and 250 OTUs were detected in pooled saliva and biofilm samples, respectively. Principal coordinates analysis (PCoA) plots based on community membership and structure showed that replicate biofilm samples were highly similar and clustered together. In addition, there were no significant differences between biofilms derived from the same panel at different times using analysis of molecular variance (AMOVA). There were significant differences between biofilms from different panels (AMOVA, P < 0.002). PCoA revealed that there was a shift in biofilm composition between seven and 14 days (AMOVA, P < 0.001). Veillonella parvula, Veillonella atypica/dispar/parvula and Peptostreptococcus stomatis were the predominant OTUs detected in seven-day biofilms, whilst Prevotella oralis, V. parvula and Streptococcus constellatus were predominant in 14-day biofilms. CONCLUSIONS: Diverse oral biofilms were successfully grown and maintained using the CBD. Biofilms derived from the same panel of volunteers were highly reproducible. This model could be used to screen both antimicrobial-containing oral care products and also novel approaches aiming to modify plaque composition, such as pre- or probiotics.

Effects of Diluents, Saliva and Other Organics on the Microbicidal Activity of Cetylpyridinium Chloride and Povidone-iodine.

Povidone-iodine (PVP-I) is used for infection control and preoperative sterilization of the oral and pharyngeal regions. Marketed preparations containing cetylpyridinium chloride (CPC) are used to inhibit growth of oral bacteria. We conducted an in vitro study of the sterilizing effects of these microbicides on 10 oral bacterial strains and fungi related to pneumonia and periodontal disease, after dilution with phosphate-buffered saline (PBS), saliva, and components in saliva. The CPC solution was evaluated at 50 mg/100 mL, which is the concentration used in products. CPC sterilized all strains within 1 minute. Prolongation of the sterilization time associated with dilution was more gradual in comparison to PVP-I solution. CPC sterilized 7 of 10 microbial strains within 3 minutes at 3 mg/100 mL. At 500 mg/100 mL, which is near the upper limit of the concentration that is actually used, PVP-I solution sterilized 7 microbial strains within 3 minutes. However, PVP-I had no sterilization effect when diluted to 100 mg/100 mL or lower. With addition of saliva, PVP-I sterilized 2 microbial strains within 3 minutes at 500 mg/100 mL, whereas CPC solution sterilized 9 microbial strains within 1 minute at 50 mg/100 mL. Our results show that in use influenced by dilution with saliva, CPC is likely to maintain a strong sterilization effect, whereas PVP-I may have a reduced effect.

Efficacy of antibiotics against periodontopathogenic bacteria within epithelial cells: an in vitro study.

BACKGROUND: Periodontopathogenic bacteria can invade and survive within epithelial cells, but susceptibility of intracellular infection to antibiotics used in periodontitis treatment has not been studied to date. METHODS: KB cells were infected by Actinobacillus actinomycetemcomitans, strain NCTC 9710; Porphyromonas gingivalis, strains ATCC 33277 and JH16-1; or Streptococcus constellatus, strain J012b. After 2, 4, and 12 hours the bactericidal effect of antibiotics (clindamycin, doxycycline, metronidazole, and moxifloxacin) on intracellular microorganisms was tested at a concentration up to the 100-fold minimum inhibitory concentration (MIC) determined separately on planktonic bacteria. RESULTS: The P. gingivalis strains differed in their invasiveness and ATCC 33277 was 100-fold more invasive than JH16-1. Doxycycline and clindamycin at a concentration 10-fold MIC had no effect, but P. gingivalis intercellular infection was significantly reduced by metronidazole at 10-fold MIC after 2 and 4 hours. Moxifloxacin was effective, but a 100-fold MIC concentration was necessary to reduce P. gingivalis strains intracellular growth to 7% of the control. Other bacterial species grown inside the KB cells were more susceptible to antibiotics. Clindamycin at 10-fold MIC reduced the number of intracellular S. constellatus after 4 and 12 hours. This bacterium was eliminated by moxifloxacin at 50-fold MIC. Intracellular A. actinomycetemcomitans was killed by 10-fold MIC of doxycycline and moxifloxacin after 4 hours incubation. CONCLUSIONS: Moxifloxacin was the most efficient antibiotic to treat intracellular infection. However, taking into account the MIC values and the levels of antibiotics in gingival fluid, elimination of intracellular bacteria by antibiotics alone seems to be questionable.

Development of an ex vivo coculture system to model pulpal infection by Streptococcus anginosus group bacteria.

INTRODUCTION: Streptococcus anginosus group (SAG) bacteria are opportunistic pathogens and a major cause of pulpal infection and subsequent abscess formation. Understanding of the processes involved in SAG oral infections has been limited by the lack of an appropriate model system. METHODS: Cocultures of SAG bacteria and mammalian tooth slices were maintained using a combination of Dulbecco modified eagle medium and brain-heart infusion broth at 60 rpm, 37°C, 5% CO(2) for 4, 8, or 24 hours before histologic examination or staining with acridine orange/ethidium bromide. Tooth slices were also incubated as described with SAG bacteria stained with fluorescein diacetate. Pulps were extirpated from infected and sterile cultured tooth slices, messenger RNA was extracted and converted to complementary DNA, and polymerase chain reaction were performed for genes encoding tumor necrosis factor α, interleukin 1ß, and interleukin-6. RESULTS: SAG bacteria were able to adhere directly to the central region of the pulpal matrix in small foci that were associated with a localized matrix breakdown. Acridine orange-ethidium bromide staining and cell counts indicated a decrease in mammalian cell viability with increasing incubation times in the presence of SAG bacteria. The increased expression of tumor necrosis factor α and interleukin 1ß was detected in infected tooth slices. CONCLUSIONS: A novel ex vivo model system has been developed that allows coculture of SAG bacteria with a 3-dimensional organotypic tooth slice. The model allows observation of bacterial growth patterns and subsequent responses from host tissues. Therefore, it may be of future use in testing the efficacy of both antimicrobial and anti-inflammatory treatments for use in endodontic therapy.

Phototargeting oral black-pigmented bacteria.

We have found that broadband light (380 to 520 nm) rapidly and selectively kills oral black-pigmented bacteria (BPB) in pure cultures and in dental plaque samples obtained from human subjects with chronic periodontitis. We hypothesize that this killing effect is a result of light excitation of their endogenous porphyrins. Cultures of Prevotella intermedia and P. nigrescens were killed by 4.2 J/cm2, whereas P. melaninogenica required 21 J/cm2. Exposure to light with a fluence of 42 J/cm2 produced 99% killing of P. gingivalis. High-performance liquid chromatography demonstrated the presence of various amounts of different porphyrin molecules in BPB. The amounts of endogenous porphyrin in BPB were 267 (P. intermedia), 47 (P. nigrescens), 41 (P. melaninogenica), and 2.2 (P. gingivalis) ng/mg. Analysis of bacteria in dental plaque samples by DNA-DNA hybridization for 40 taxa before and after phototherapy showed that the growth of the four BPB was decreased by 2 and 3 times after irradiation at energy fluences of 4.2 and 21 J/cm2, respectively, whereas the growth of the remaining 36 microorganisms was decreased by 1.5 times at both energy fluences. The present study suggests that intraoral light exposure may be used to control BPB growth and possibly benefit patients with periodontal disease.

The acid-tolerant microbiota associated with plaque from initial caries and healthy tooth surfaces.

The intent of this study was to compare the inherent acid tolerance of bacteria in samples of dental plaque from tooth sites in subjects with and without initial caries. Plaque was collected from approximal surfaces showing early enamel caries and from healthy tooth surfaces in the same subjects, as well as from enamel surfaces of caries-free individuals. In addition to plating on blood agar, the plaque samples were plated directly on non-selective solid agar medium buffered to pH 7.0, 6.0, 5.5, 5.0, 4.5 and 4.0 to avoid any loss of adaptation to acid during primary isolation of plaque bacteria. The results showed that approximately 50% of the total cultivable plaque microbiota from caries, as well as healthy tooth sites, was able to grow at pH 5.5 and 1% at pH 5.0, pH values regarded as critical for the demineralization of tooth enamel. At pH 5.0, members of the genus Streptococcus were the dominant group, but mutans streptococci accounted for less than half of the streptococcal viable count. The other acid-tolerant streptococcal isolates included Streptococcus anginosus, Streptococcus constellatus, Streptococcus gordinii, Streptococcus intermedius, Streptococcus mitis, Streptococcus oralis, Streptococcus salivarius and SStreptococcus sanguis. Analysis of the results indicated that the mutans streptococci in dental plaque were highly variable with respect to acid tolerance, and that both caries and healthy sites harboured significant numbers of mutans streptococci that were not acid-tolerant.