On bacteria persisting root canal treatment. Identification and potential mechanisms of resistance to antimicrobial measures

Anti-microbial treatment of infected root canals using mechanical instrumentation and chemicals for disinfection does not always lead to complete bacterial elimination and treatment failure may therefore result. As certain bacteria appear more resilient to endodontic treatment than others, the present thesis addresses the species that can most frequently be isolated after treatment and explores mechanisms which could potentially afford these organisms resistance to anti-microbial treatment measures. In an initial assessment, consecutive root canal samples from a total of 200 clinical cases were analysed for cultivable microbes. Patients had presented with clinical and/or radiographic evidence of apical periodontitis. Root canal treatment had been initiated at one or more appointments prior to sampling. Results demonstrated a clear predominance of Gram-positive rods and cocci. These organisms outweighed other bacterial groups common in primary root canal infections including Gram-negative anaerobes. In a second study, a protocol for identification of Gram-positive rods at the species level was validated. Here, 86 cases presenting with Gram-positive rods in the first study as well as 53 new cases were included. Identification was based on a combination of selective media, gas-liquid chromatography and SDS-PAGE electrophoresis of whole-cell proteins. Of a total of 158 Gram-positive rods isolated, it was possible to identify 96% at the species level. The most common species were Olsenella uli, Lactobacillus paracasei and Propionibacterium propionicum. In a third study Streptococcus, being the second largest group of organisms isolated in the first study, were subject to identification. Extracellular proteins produced in culture media were also screened. A total of 100 cases (45 from study I, 32 from study II and 23 new cases) which showed growth of streptococcal species were included. Criteria for identification were based on physiological characteristics. The most frequently isolated species were Streptococcus gordonii, Streptococcus anginosus and Streptococcus oralis. The protein release pattern expressed by clinical isolates was different from that of corresponding laboratory strains. S. gordonii, S. anginosus, S. oralis and S. parasanguis produced the molecular chaperone DnaK extracellularly. The bacterial cell viability and protein response patterns to alkaline stress of a collection of seven organisms, isolated in the previous studies, were assessed in paper IV. When tested under planktonic growth conditions, cells were found to be less viable after an alkaline challenge than corresponding cells growing in biofilms. Excretion of proteins appeared to be a rapid response of these organisms prior to a switch into a stationary phase. When these strains were grown in mixed cultures (community assessments - unpublished data), viability of cells seemed to be less affected by alkaline challenge. In addition, the rate at which bacteria switched into an inactive phase appeared to increase.In conclusion, Gram-positive rods and cocci seem to be the most enduring organisms in infected root canals subjected to endodontic treatment. The formation of biofilms and expression of a variety of proteins, as well as growth in mixed communities, may provide these organisms with the capacity to resist root canal treatment