Quantitative Microbiological Study of Human Carious Dentine by Culture and Real-Time PCR: Association of Anaerobes with Histopathological Changes in Chronic Pulpitis

Abstract

The bacteria found in carious dentine were correlated with the tissue response of the dental pulps of 65 teeth extracted from patients with advanced caries and pulpitis. Standardized homogenates of carious dentine were plated onto selective and nonselective media under anaerobic and microaerophilic conditions. In addition, real-time PCR was used to quantify the recovery of anaerobic bacteria. Primers and fluorogenic probes were designed to detect the total anaerobic microbial load, the genera Prevotella and Fusobacterium, and the species Prevotella melaninogenica, Porphyromonas endodontalis, Porphyromonas gingivalis, and Micromonas (formerly Peptostreptococcus) micros. The pulpal pathology was categorized according to the cellular response and degenerative changes. Analysis of cultured bacteria showed a predominance of gram-positive microorganisms, particularly lactobacilli. Gram-negative bacteria were also present in significant numbers with Prevotella spp., the most numerous anaerobic group cultured. Real-time PCR analysis indicated a greater microbial load than that determined by colony counting. The total number of anaerobes detected was 41-fold greater by real-time PCR than by colony counting, while the numbers of Prevotella and Fusobacterium spp. detected were 82-and 2.4-fold greater by real-time PCR than by colony counting, respectively. Real-time PCR also identified M. micros, P. endodontalis, and P. gingivalis in 71, 60, and 52% of carious samples, respectively. Correlation matrices of the real-time PCR data revealed significant positive associations between M. micros and P. endodontalis detection and inflammatory degeneration of pulpal tissues. These anaerobes have been strongly implicated in endodontic infections that occur as sequelae to carious pulpitis. Accordingly, the data suggest that the presence of high levels of these bacteria in carious lesions may be indicative of irreversible pulpal pathology.The microbial populations involved in dental caries are known to be highly complex and variable and have not yet been fully identified, although key organisms are generally recognized to be associated with disease progression. The bacteria involved in caries initiation and early caries development, particularly the mutans group streptococci and lactobacilli, have been well documented (34). As the lesion progresses, there is a transition from predominantly facultative gram-positive bacteria in early caries to anaerobic gram-positive rods and cocci and gram-negative rods in deep carious lesions (15). Previous research has associated the presence of anaerobic gram-negative rods, such as Fusobacterium, Prevotella, and Porphyromonas, with symptomatic teeth (11, 19), infected pulps (38), and periapical abscesses (29, 35), whereas anaerobic gram-positive cocci, such as peptostreptococci, have been associated with apical infections (10).While it is well recognized that bacteria and their products play a major role in dental caries and associated pulpal inflammation (5), attempts to correlate clinical signs an...

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