Wiley

Mammalian interstitial collagenases (E.C.3.4.24.7) are considered as key initiators of collagen degradation in periodontal diseases. However, the cellular sources of collagenases present in gingival crevicular fluid have not been completely clarified. Resident fibroblasts and epithelial cells as well as infiltrating neutrophils and monocyte/macrophages are potential sources of the enzymes. We have recently found significant differences in tetracycline inhibition between human neulrophil and fibroblast interstitial collagenases. To address the cellular source of collagenase present in gingival crevicular fluid in 2 distinct periodontal diseases, we studied the tetracycline inhibition of collagenase in gingival crevicular fluid of patients with localized juvenile periodontitis and adult periodontitis. Gingival crevicular fluid samples were collected from deep (<5 mm) periodontal pockets and assayed for collagenase in the presence of 0‐1000 μM doxycycline as well as a chemically modified tetracycline devoid of antimicrobial activity (4‐de‐dimethylaminotelracycline). The drug concentration required to inhibit 50% of collagenase activity (IC₅₀) in localized juvenile periodontitis gingival crevicular fluid was 280 μM for doxycycline and 470 μM for 4‐de‐dimethylaminotetracycline. Significantly lower values, 10–20 μM, were obtained for collagenase in gingival crevicular fluid of patients with adult periodontitis. We propose that systemic letracycline levels are efficient inhibitors of collagenase in gingival crevicular fluid in affected sites of patients with adult periodontitis but not of patients with localized juvenile periodontitis and that the fibroblast type interstitial collagenase is the predominant collagenase type in gingival crevicular fluid in affected sites of patients with localized juvenile periodontitis and the neutrophil collagenase in adult periodontilis gingival crevicular fluid. Furthermore, specific tetracycline inhibition can be used as a simple and practical “probe” to identify the cellular source of interstitial collagenases from in vivo samples.

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.

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.

A system was developed by which 2′,7′‐bis(carboxyethyl)‐4 or 5‐carboxyfluorescein could be used to monitor intracellular pH at the same time that proton excretion was being measured. Streptococcal cells were loaded with the dye, and after the addition of glucose protons were excreted and the intracellular pH increased quickly and remained higher than the extracellular pH of 7.0. The excretion of protons stopped and the intracellular pH returned to the original level when glucose was depleted. The intracellular level of ATP remained high during glucose metabolism and decreased with the depletion of glucose. At extracellular pH of 5.5, and 5.0, the intracellular pH of fasting cells was higher than the extracellular pH value. After addition of glucose there were initial lags of proton excretion and of increases in intracellular pH at the acidic extracellular pH values. In the presence of fluoride, a lag in proton excretion and a simultaneous decrease in intracellular pH were observed, indicating a partial and transient inhibition of proton‐ATPase activity.

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.

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.

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.

A replica‐plate assay was used to screen for the interaction of salivary molecules with dental plaque bacteria. Bacterial colonies cultured from supragingival plaque on sheep‐blood (SB) agar were replica‐plated onto nitrocellulose membranes overlaying SB or mitis‐salivarius agar. Membranes with attached colonies were removed and incubated with ¹²⁵I‐amylase or ¹²⁵I ‐proline‐rich glycoprotein (PRG). Positive interactions were detected by autoradiography. Only strains of Streptococcus gordonii and Actinomyces viscosus bound amylase, and strains of A. viscosus bound PRG. The results suggest that amylase and PRG bind to selected species of aerobic dental plaque bacteria.

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.

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.