IADR Abstract Archives
Characterization of Porphyromonas endodontalis Clinical Isolates in Patients with Chronic Apical Periodontitis
Objectives: To characterize Porphyromonas endodontalis (Pe)`s endodontic clinical isolates and ATCC strain in patients with chronic apical periodontitis based on their morphology, antimicrobial susceptibility, lipopolysaccharide (LPS) profile and its endotoxic activity.
Methods: Samples of endodontic exudates from patients with chronic apical periodontitis were obtained (N=30) in the Dental Clinic, Faculty of Dentistry, Universidad de Chile. Samples were grown in blood agar supplemented with hemin-menadione in anaerobiosis and black-pigmented colonies were selected for subcultures and DNA extraction. Pe identification was confirmed through PCR with specific primers and 16S ribosomal RNA sequencing (V3-V6 regions). Clinical isolates and Pe reference strain (ATCC 35406) were characterized by scanning electron microscopy and their susceptibility to amoxicillin, polymyxin B and chlorhexidine. To characterize LPS profiles, LPS was purified by phenol extraction and visualized on silver stained polyacriylamide gels. The endotoxic activity of the extracted LPS was quantified by Limulus Amebocyte Lysate test. The results were analyzed with Stata V 12, with α= 0.05.
Results: One type of Pe clinical isolate with two distinct colonies (4E col 4 and 4E col 7) was identified by PCR and 16S rRNA gene sequencing. Morphologically, ATCC 35406 strain presented a smooth surface, while 4E showed regular protrusions of the surface. Clinical Isolates showed lower susceptibility to amoxicillin (6.26-2.0 mg/mL), polymyxin B (≥0.5 mg/mL) and chlorhexidine (2%) than the reference strain. Both, clinical isolate and ATCC LPS showed antigen O molecules compatible with high molecular weight chains. There were no significant differences in the
endotoxic activity between 35406 and 4E LPS (p>0.05).
Conclusions: P. endodontalis clinical isolates showed different morphology and antimicrobial susceptibility compared to the reference strain.
Methods: Samples of endodontic exudates from patients with chronic apical periodontitis were obtained (N=30) in the Dental Clinic, Faculty of Dentistry, Universidad de Chile. Samples were grown in blood agar supplemented with hemin-menadione in anaerobiosis and black-pigmented colonies were selected for subcultures and DNA extraction. Pe identification was confirmed through PCR with specific primers and 16S ribosomal RNA sequencing (V3-V6 regions). Clinical isolates and Pe reference strain (ATCC 35406) were characterized by scanning electron microscopy and their susceptibility to amoxicillin, polymyxin B and chlorhexidine. To characterize LPS profiles, LPS was purified by phenol extraction and visualized on silver stained polyacriylamide gels. The endotoxic activity of the extracted LPS was quantified by Limulus Amebocyte Lysate test. The results were analyzed with Stata V 12, with α= 0.05.
Results: One type of Pe clinical isolate with two distinct colonies (4E col 4 and 4E col 7) was identified by PCR and 16S rRNA gene sequencing. Morphologically, ATCC 35406 strain presented a smooth surface, while 4E showed regular protrusions of the surface. Clinical Isolates showed lower susceptibility to amoxicillin (6.26-2.0 mg/mL), polymyxin B (≥0.5 mg/mL) and chlorhexidine (2%) than the reference strain. Both, clinical isolate and ATCC LPS showed antigen O molecules compatible with high molecular weight chains. There were no significant differences in the
endotoxic activity between 35406 and 4E LPS (p>0.05).
Conclusions: P. endodontalis clinical isolates showed different morphology and antimicrobial susceptibility compared to the reference strain.