Identification of bacteria enduring endodontic treatment procedures by a combined reverse transcriptase-polymerase chain reaction and reverse-capture checkerboard approach.
BACKGROUND
This study identified the bacterial taxa enduring endodontic treatment procedures by using a combined 16S ribosomal RNA-based reverse-transcriptase polymerase chain reaction (RT-PCR) and reverse-capture checkerboard hybridization approach.


METHODS
Samples were taken from infected canals of 15 teeth with apical periodontitis before treatment (S1), after chemomechanical preparation with NaOCl as the irrigant (S2), and after interappointment medication with a calcium hydroxide paste (S3). Bacterial presence was first screened by a DNA-based single PCR assay. RNA extracts were subjected to RT-PCR, and the resulting products were surveyed for the presence of 28 targeted taxa by using the checkerboard method.


RESULTS
Bacteria were found in all S1 samples. Detectable levels of bacterial ribosomal RNA, used as an indicator of viability, were observed in 60% of the cases after chemomechanical preparation and 53% after intracanal medication. The most prevalent taxa in S1 were Olsenella uli (67%), Pyramidobacter piscolens (60%), Streptococcus species (53%), and Bacteroidetes clone X083 (53%). Streptococcus species (47%), Fusobacterium nucleatum (40%), and O. uli (33%) prevailed in S2, whereas Streptococcus species (47%), Propionibacterium acnes (27%), and O. uli (27%) were the most frequent taxa in S3.


CONCLUSIONS
The present study with a combined molecular approach revealed that bacterial diversity was overall markedly reduced by treatment procedures. Although bacterial taxa more frequently identified in post-treatment samples emerge as potential risk factors for persistent disease, this remains to be determined by longitudinal studies.
Rôças
Isabela N
IN
Department of Endodontics and Molecular Microbiology Laboratory, Faculty of Dentistry, Estácio de Sá University, Rio de Janeiro, RJ, Brazil.
Siqueira
José F
JF
D001419:Bacteria / Q000187:drug effects; D002126:Calcium Hydroxide / Q000494:pharmacology; D002164:Camphor / Q000494:pharmacology; D002733:Chlorophenols / Q000494:pharmacology; D055113:Chronic Periodontitis / Q000188:drug therapy / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis; D003786:Dental Pulp Cavity / Q000382:microbiology*; D004338:Drug Combinations; D024881:Drug Resistance, Bacterial; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D010485:Periapical Periodontitis / Q000188:drug therapy / Q000382:microbiology*; D012329:RNA, Bacterial / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012387:Root Canal Filling Materials / Q000494:pharmacology*; D012388:Root Canal Irrigants / Q000494:pharmacology*; D018915:Root Canal Preparation; D012973:Sodium Hypochlorite / Q000494:pharmacology
D002733:Chlorophenols; D004269:DNA, Bacterial; D004338:Drug Combinations; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012387:Root Canal Filling Materials; D012388:Root Canal Irrigants; D002164:Camphor; C029804:camphorated parachlorophenol; D012973:Sodium Hypochlorite; D002126:Calcium Hydroxide