Detection of transient bacteraemia following dental extractions by 16S rDNA pyrosequencing: a pilot study

Abstract

Objective: The current manuscript aims to determine the prevalence, duration and bacterial diversity of bacteraemia following dental extractions using conventional culture-dependent methods and 16S rDNA pyrosequencing.

Methods: The study group included 8 patients undergoing dental extractions under general anaesthesia. Peripheral venous blood samples were collected at baseline, 30 seconds and 15 minutes after the dental extractions. Blood samples were analysed for bacteraemia applying conventional microbiological cultures under aerobic and anaerobic conditions as well as pyrosequencing using universal bacterial primers that target the 16S ribosomal DNA gene.

Results: Transient bacteremia was detected by culture-based methods in one sample at baseline time, in eight samples at 30 seconds, and in six samples at 15 minutes after surgical procedure; whereas bacteraemia was detected only in five blood samples at 30 seconds after dental extraction by using pyrosequencing. By applying conventional microbiological methods, a single microbial species was detected in six patients, and Streptococcus viridans was the most frequently cultured identified bacterium. By using pyrosequencing approaches however, the estimated blood microbial diversity after dental extractions was 13.4±1.7 bacterial families and 22.8±1.1 genera per sample.

Conclusion: The application of 16S rDNA pyrosequencing underestimated the prevalence and duration of bacteraemia following dental extractions, presumably due to not reaching the minimum DNA required for PCR amplification. However, this molecular technique, unlike conventional culture-dependent methods, revealed an extraordinarily high bacterial diversity of post-extraction bacteraemia. We propose that microorganisms recovered by culture may be only the tip of an iceberg of a really diverse microbiota whose viability and potential pathogenicity should be further studied.

Figure 1. Gel of electrophoresis of 16S rDNA amplified by nested-PCR.

A set of four samples from four different patients are shown according to time collection: blood sample before dental extraction, blood sample 30 sec after dental extraction, blood sample 15 min after dental extraction, and subgingival plaque sample of teeth to be extracted. The last three lanes on the right show the respective negative and positive controls. PCR product expected is ∼560bp in length.

Figure 2. Distribution and diversity of bacterial families.

Only families present in a percentage higher than 1% of the total bacterial population detected in subgingival and blood samples of each one of the 8 patients are shown.

Figure 3. Rarefaction curves which relate the sequencing effort to the number of putative species in the samples.

Blue curves represent diversity in subgingival samples whereas Grey curves show diversity in blood samples where transitory bacteraemia was detected. Rarefactions curves are uniformly presented from a sub-set of sequences randomly selected from each sample dataset. The number of species-level phylotypes was calculated by clustering sequences at 97% sequence identity, which has been determined as the threshold for species boundaries , .

Figure 4. Comparative bacterial diversity (at the genus taxonomic level) detected in blood samples in this study.

Genera at the top of iceberg (black labeled) are those detected by culture-based method. Genera at the submerged part of the iceberg (blue labeled) are those detected by pyrosequencing. Font size is correlated with the frequency of the respective bacteria determined by both methods.