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Azerbaycan Saytlari

 »  Home  »  Endodontic Articles 9  »  Combinations of bacterial species in endodontic infections
Combinations of bacterial species in endodontic infections
Introduction - Materials and methods.



Introduction.
Bacteria that are found in an infected root canal with apical periodontitis form a selected group of the total oral microflora. When the pulp becomes necrotic there is an increase of strict anaerobic Gram-negative and Gram-positive species such as Fusobacterium spp., Prevotella spp., Porphyromonas spp., Peptostreptococcus spp.,Eubacterium spp., and Actinomyces spp., which occurs at the expense of facultative anaerobic species (Fabricius et al. 1982). Amongst the important ecological factors in this dynamic process are nutrition, pH, reduction-oxidation potential, temperature, host resistance and microbial interactions.
In the apical part of the root canal (where interaction with the periapical tissues takes place), proteolytic bacteria constitute the main proportion of the microflora (Slots & Taubman 1992). The close contact with vital periapical tissues may be presumed also to give a higher oxygen tension inhibiting their growth. However, the small proportion of facultative anaerobic species present in the apical region may consume the available oxygen resulting in a low reduction-oxidation potential favoring colonization of strict anaerobic species (Slots & Taubman1992).
The interactions between different species can be beneficial to one or more microorganisms, whilst with other microbes it can be antagonistic. Positive interactions, e.g. co adherence and nutrient supply have been described to occur in plaque (Hillman et al.1985, Grenier & Mayrand 1986, Marsh 1989), the periodontal pocket (van Winkelhoff et al. 1987, Dzink et al. 1988, Socransky et al. 1988, Ashimoto et al. 1996) and in the root canal (Fabricius et al.1982, Sundqvist1992, Gomes et al.1994).
Combinations of bacteria may be related to specific signs and symptoms of oral diseases. Socransky et al. (1988) related periodontal attachment loss and deepest pockets to specific clusters of bacteria. Gomes et al. (1996b) related pain of endodontic origin with combinations of Peptostreptococcus spp. and Prevotella spp., whilst Haapasalo (1989) found Porphyromonas endodontalis and P. gingivalis to be related to acute symptoms. Fabricius et al. (1982) showed that specific combinations of bacteria had the greatest potential to induce apical periodontitis.
The first step in the investigation of microbial interactions in the root canal of teeth with apical periodontitis is to study the prevalence and the simultaneous isolation of bacterial species. The aim of this study was to investigate positive and negative associations of bacteria found in root-canal infections of teeth with periapical bone destruction without clinical signs and symptoms.

Materials and methods.

Patient material and microbiological sampling.
The content of 58 root canals in 50 patients was sampled for microbial analysis. All selected teeth were asymptomatic, did not respond to sensitivity testing, had not received previous endodontic treatment and showed radiographic evidence of periapical bone loss. All patients were systemically healthy and had not used antibiotics 6 months prior to the study.
After cleaning of the tooth with pumice and isolation with rubber dam, the crown and surrounding rubber dam were disinfected with 80% alcohol for 2 min. An access cavity was made with sterile high-speed diamond burs under irrigation with sterile saline. Before entering the pulp chamber, the access cavity was disinfected again for 2 min with 80% alcohol. A swab sample was taken from the surface and streaked on blood agar plates to check disinfection. The pulp chamber was accessed with sterile burs and rinsed with reduced transport Јuid (RTF, Syed & Loesche 1972) and suctioned with sterile tips. RTF was then introduced to the root canal by a syringe with a sterile 27-gauge needle. Care was taken not to overfi ll the canal.With sterileHedstro« mfi les the canal was enlarged to a size 20 fi le to the estimated working length as calculated from the preoperative radiograph. Five sterile paper points were consecutively placed in the canal and left for10 s and thenplaced in sterile tubes containing 1mL RTF and transferred to the laboratory within15 min for microbiological processing.

Microbiological procedures.
Ten-fold serial dilutions of the samples were prepared and100 mL of each dilution was inoculated on blood agar plates supplemented with 5% horse blood, 5 mg L  hemin and 1mg L   menadione. Plates were anaerobically (80% N2, 10% H2,10% CO2) incubated in an anaerobic box at 378C for 7 days. After incubation, the total colony forming units (CFU) and the different colony morphotypes were counted with the use of a stereomicroscope at 16x magnification (Zeiss, Oberkochen, Germany).
All colony morphotypes were streaked to purity, incubated in air and 5% CO2 (BBL Gaspak CO2 systems, Becton Dickinson and Company, Cockeysville, MD, USA) anaerobically to determine strict anaerobic and facultative anaerobic growth. Identification was made on the basis of Gramstain, catalase activity and a commercially available identification kit ATB rapid ID32A (Biomerieux SA, Lyon, France), for strict anaerobes and ATB rapid ID32Strep for facultative anaerobic cocci (Biomerieux SA).
The blood agar plates with the total samples were kept incubated under the anaerobic conditions for up to 14 days to allows low growing species to develop. Newly emerging colonies were also streaked to purity and identified.

Statistics.
Differences in prevalence of different species in the 58 root canal samples were tested with Cochran test for nominal nonparametric data (present vs. not present).
The Fisher’s Exact test was applied to test the null hypothesis that there was no relationship between any of the species (P > 0.05).When there was a significant difference, indicating that a relationship between species was present, the Odds ratio was calculated. Positive associations were those with an Odds ratio >2 (the odds of detecting one organism was twice as likely with another organism present as with it absent) and negative associations were those with an Odds ratio <0.5.