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Journal of Clinical Microbiology, May 2002, p. 1698-1704, Vol. 40, No. 5
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.5.1698-1704.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

F. Elizabeth Martin,* Mangala A. Nadkarni, Nicholas A. Jacques, and Neil Hunter

Institute of Dental Research, Westmead Centre for Oral Health, Westmead Hospital, Westmead, NSW, Australia

Received 14 December 2001/ Returned for modification 25 January 2002/ Accepted 12 February 2002

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.

* Corresponding author. Mailing address: Institute of Dental Research, Westmead Centre for Oral Health, Westmead Hospital, P.O. Box 533, Wentworthville NSW 2145, Australia. Phone: 61-2-9845-8763. Fax: 61-2-9845-7599. E-mail: efjelda{at}mail.usyd.edu.au.

Journal of Clinical Microbiology, May 2002, p. 1698-1704, Vol. 40, No. 5
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.5.1698-1704.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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