Wiley

Apical periodontitis is initiated primarily by the mixed microflora of infected root canals. Continuous flow of bacteria and their products through the apical foramen induces influx, activation and coordinated interaction of immune‐inflammatory cells within the periapical area. Successful mobilization of host defense mechanisms prevents abundant extraradicular bacterial invasion. However, anti‐infective effector mechanisms are not restricted to killing the invading microorganisms but also destroy normal tissue components and induce bone absorption, resulting ultimately in the loss of the affected teeth. Moreover, autocrine and paracrine loops of stimulation may lead to the perpetuation of the local inflammatory lesion and may also alter the function of remote tissues and organs. This review attempts to summarize current knowledge about the pathogenic mechanism of apical periodontitis, focusing on the formation of a special granulation tissue that effectively fights bacteria originated from the infected pulp chamber and, by exerting this protective function, also contributes to harmful local and distant events. The dynamic equilibrium between defensive and destructive mechanisms may provide a pathobiological basis for better understanding of clinical signs and symptoms of various forms of apical periodontitis lesions and influence treatment strategy and practice.

Bacteria that persist after endodontic disinfection procedures may lead to treatment failure. Over 50% of the bacteria found in endodontic infections are as‐yet‐uncultivated so investigations of bacteria that endure treatment procedures should include techniques that side‐step cultivation. This culture‐independent study evaluated the bacterial reduction promoted by intracanal disinfection procedures and identified the taxa persisting after treatment. Samples taken from the infected canals of teeth with apical periodontitis before treatment (S1), after instrumentation using NaOCl as irrigant (S2) and after interappointment medication with a calcium hydroxide paste (S3) were subjected to 16S rRNA gene clone library and real‐time polymerase chain reaction analyses. The S2 and S3 samples from five of the 15 canals showed negative results. In the other cases, instrumentation and instrumentation/medication promoted a significant reduction (99.67% and 99.85%, respectively) in the number of bacteria when compared to S1 samples. Forty‐three distinct bacterial taxa were identified, of which 24 (56%) were as‐yet‐uncultivated phylotypes. Nineteen of these 43 taxa (including eight as‐yet‐uncultivated phylotypes) were disclosed in post‐treatment samples, with streptococci being the most prevalent taxa. Findings demonstrated that culture‐independent methods provide a detailed insight into the effects of intracanal disinfection protocols, helping to define more effective strategies to deal with endodontic bacteria, including as‐yet‐uncultivated phylotypes.

Introduction:  The use of probiotic bacteria is increasing worldwide and at least some of them can transiently colonize the oral cavity. Several studies have shown that probiotic bacteria, which are often thought of in relation only to intestinal health, can also affect the oral ecology, but the mechanisms for this are largely unknown. The aim of this study was to investigate in vitro if the probiotic bacteria used in commercial products affect the protein composition of the salivary pellicle and the adherence of other oral bacteria.

Methods:  Salivary pellicle on hydroxyapatite and the adhesion of two oral streptococci, Streptococcus mutans and Streptococcus gordonii, were used as a model.

Results:  Probiotic bacteria that bound to saliva‐coated hydroxyapatite reduced the adhesion of S. mutans but the inhibitory effect on the adherence of S. gordonii was weaker. Salivary pellicle protein composition was modified by all the strains tested. The modifications in the pellicle affected the adherence of S. mutans but not of S. gordonii. Two of the proteins missing from the pellicles made of saliva‐treated with the probiotic bacteria were identified as salivary agglutinin gp340 and salivary peroxidase. All bacterial strains bound salivary agglutinin gp340. The ability of the probiotic bacteria to degrade peroxidase was demonstrated with purified bovine lactoperoxidase and two of the probiotic strains.

Conclusion:  This in vitro study showed that probiotic strains used in commercial products may affect the oral ecology by specifically preventing the adherence of other bacteria and by modifying the protein composition of the salivary pellicle.

In vivo dental plaque biofilms consist of complex communities of oral bacteria that are a challenge to replicate in vitro. The aim of this investigation was to establish human dental plaque microcosms in microplates to reflect conditions that are relevant to dental caries. Microcosm plaque biofilms were initiated from the saliva of two different donors, grown for up to 10 days in 24‐welled microplates on Thermanox^TM coverslips in various types of artificial saliva with and without sucrose, which were replaced daily. Microbiota composition of 40 species associated with oral health and dental caries was monitored in the plaques using Checkerboard DNA–DNA hybridization analysis. pH was measured as an indicator of cariogenic potential. The composition of the saliva inocula was different, and yielded plaque microcosms with different composition and growth responses to sucrose. Artificial saliva type and presence of sucrose, and the resulting growth and pH conditions, modified the growth of individual species and hence the ecological profile of the microplate plaques during development. Complex population shifts were observed during development, and older plaques comprised predominantly facultative anaerobic species. Sucrose supplementation limited the decline of Streptococci over time but did not increase the abundance of mutans Streptococci. Sucrose at 0.15% increased levels of caries‐associated species including Lactobacillus fermentum, Lactobacillus acidophilus and Actinomyces gerensceriae; these were further increased with sucrose at 0.5%, in addition to Actinomyces israelii, Rothia dentocariosa and Capnocytophaga gingivalis. The microplate plaques demonstrated complex community dynamics that appeared to reflect the maturation of natural plaques, and sucrose induced a cariogenic plaque composition and pH.

Antimicrobial peptides, like human β‐defensins, play an important role in the epithelial innate defense response. The aim of the present study was to investigate the quantitative expression of human β‐defensin‐1, ‐2, and ‐3 in inflammatory gingival diseases. Gingival biopsies were obtained from patients with healthy gingiva (n = 10), patients with gingivitis (n = 10), and patients with periodontitis (n = 10). The clinical diagnosis was verified by histology. Gingival tissues were used for RNA extraction followed by reverse transcription. Gene expression was quantified by real‐time polymerase chain reaction (normalization with GAP‐DH). Comparing the tissues with different clinical stages of health and disease, no significant differences in mRNA expression were found for any of the β‐defensins studied. Similar levels of expression were found in healthy gingiva, whereas in gingivitis samples there was a significantly higher expression of hBD‐2 compared to hBD‐1 (P = 0.004) and hBD‐3 (P = 0.016). Likewise, in periodontitis samples, hBD‐2 expression was significantly higher than hBD‐1 (P = 0.016); however, hBD‐2 expression was comparable to hBD‐3. In conclusion, the results of the present study showed a differential expression of human β‐defensins (hBD‐1, ‐2, ‐3) in tissues with inflammatory gingival disease.

Colonization with mutans streptococci was studied in 1095 1‐year‐old children living in suburban Stockholm. During a scheduled vaccination appointment at a child health centre, a bacterial sample was obtained from the child's tongue and a structured questionnaire was completed by the accompanying parent. Six percent of the subjects were colonized with mutans streptococci. The variables most strongly correlated with presence of mutans streptococci were: non‐Swedish background, consumption of sugar‐containing beverages at night and total consumption of sugar‐containing beverages. The results indicate that, by the age of 1 year, maternally influenced behaviour patterns such as dietary habits that may predispose to early colonization of mutans streptococci are already established. Such early colonization with mutans streptococci may predict high caries risk in the primary dentition.

Background/aims:  The genus Lactobacillus has been associated with dental caries in humans, although it is seldom speciated due to lack of simple and nonlaborious identification methods. A considerable heterogeneity among Lactobacillus species has been demonstrated. The purpose of this study was to develop simple methods combining restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)‐amplified 16S rRNA (16S rRNA gene PCR‐RFLP) and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) for the identification of 13 reference strains of Lactobacillus.

Methods:  The 16S rRNA gene sequences were amplified by PCR using universal primers and digestion of PCR products with the restriction endonucleases, HpaII and HaeIII. The 16S rRNA gene PCR‐RFLP is reproducible and has been proved to be useful for differentiating Lactobacillus strains to species level. Seventy‐seven Lactobacillus isolates from a Thai population were used to show the applicability of the identification test.

Results:  PCR‐RFLP alone had limitations, because the RFLP patterns of Lactobacillus casei and Lactobacillus rhamnosus and of Lactobacillus acidophilus and Lactobacillus crispatus showed similar patterns; however, these could be differentiated by SDS‐PAGE. Of the 77 isolates, 38 were identified as Lactobacillus fermentum, 25 as L. rhamnosus, 5 as Lactobacillus salivarius, 5 as L. casei, 3 as L. acidophilus and 1 as Lactobacillus plantarum.

Conclusion:  16S rRNA gene PCR‐RFLP, using HpaII and HaeIII, together with SDS‐PAGE protein profiles could be an alternative method for the identification of oral Lactobacillus strains to species level, and may be applicable for large‐scale studies on the association of Lactobacillus to dental caries.

In this study, we evaluated the current effectiveness of 11 β‐lactam antibiotics for treatment of orofacial odontogenic infections by determining the antimicrobial susceptibility of the major pathogens. The antimicrobial susceptibilities of viridans streptococci (n = 47), Peptostreptococcus (n = 67), Porphyromonas (n = 18), Fusobacterium (n = 57), black‐pigmented Prevotella (n = 59) and non‐pigmented Prevotella (n = 47) isolated from pus specimens of 93 orofacial odontogenic infections to penicillin G, cefmetazole, flomoxef, cefoperazone, cefoperazone/sulbactam, ceftazidime, cefpirome, cefepime, cefoselis, imipenem and faropenem were determined using the agar dilution method. Penicillin G, most cephalosporins, imipenem and faropenem worked well against viridans streptococci, Peptostreptococcus, Porphyromonas and Fusobacterium. Penicillin G and most cephalosporins, including fourth‐generation agents, were not effective against β‐lactamase‐positive Prevotella, though they were effective against β‐lactamase‐negative strains. Cefmetazole, cefoperazone/sulbactam, imipenem and faropenem expressed powerful antimicrobial activity against β‐lactamase‐positive Prevotella. In conclusion, penicillins have the potential to be first‐line agents in the treatment of orofacial odontogenic infections. Most of the other β‐lactam antibiotics, including fourth‐generation cephalosporins, were not found to have greater effectiveness than penicillins. In contrast, cefmetazole, cefoperazone/sulbactam, imipenem and faropenem were found to have greater effectiveness than penicillins.

Background:  Terminal restriction fragment length polymorphism (T‐RFLP) analysis is commonly used to analyze microbial communities, including oral microflora. However, accurate identification of terminal restriction fragment (T‐RF) origins is prevented by unpredictable errors in sizing, thus necessitating the clone library analysis. To minimize sizing errors, we proposed optimizing the size definition of internal standards.

Methods:  GeneScan‐1000 ROX was regenerated as an internal standard by redefining the fragment sizes in terms of molecular weight (MW) based on their mobility relative to 6‐carboxyfluorescein (FAM) ‐labeled restriction fragments derived from the 16S recombinant RNA gene of Porphyromonas gingivalis. Using the new size definition, the average sizing error among eight oral bacteria from six phyla was estimated and compared with that of the conventional method. Microbial communities isolated from saliva were analyzed using the new MW size definition. Bacterial species were assigned to peaks using TRFMA, a Web‐based tool for T‐RFLP analysis, and compared with those identified in a clone library analysis.

Results:  Using the new size definition, the average sizing error for 40 T‐RFs was drastically reduced from 2.42 to 0.62 bases, and large sizing errors (more than two bases) were eliminated. More than 90% of the total bacterial clones detected by the clone library analysis were assigned by T‐RFLP.

Conclusion:  The size definition of the newly constructed internal standards reduced fragment sizing errors and allowed for accurate assignment of bacteria to peaks by the T‐RFLP analysis. This provided a more effective means for studying microbial communities, including the oral microflora.

Oral spirochetes were co‐incubated with monolayers of endothelial cells seeded into multiwell plates or onto filters mounted in plastic chambers. Attachment was assessed using an enzyme‐linked immunosorbent assay and scanning electron microscopy. Invasiveness was determined by monitoring media beneath filters within chambers for spirochetes using darkfield microscopy. Transmission electron microscopy was used to estimate intercellular and intracellular passage of spirochetes through monolayers. All tested treponemes attached to monolayers in a dose‐ and time‐ dependent manner, except Treponema phagedenis. A few treponemes were observed within host cell cytoplasm. Unidentified spirochetes obtained from dental plaque were also invasive. Results indicate that oral spirochetes possess virulence‐associated characteristics shared with pathogenic spirochetes. Further studies should examine the possibility that invasive spirochetes could disseminate from within affected gingiva.