Antimicrobial susceptibility and RNA purification protocol for gene expression analysis of clinical isolates of Fusobacterium nucleatum

Author(s)

Raquel Zanin Midena

• Document type: Doctoral Thesis
• Press: Bauru.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia de Bauru (FOB/SDB)
• Defense date: 2015-09-02

Examining board members

Flaviana Bombarda de Andrade; Marco Antonio Hungaro Duarte; Flavio Augusto Cardoso de Faria; Giulio Gavini; Rafael Nobrega Stipp

• Advisor: Flaviana Bombarda de Andrade

Abstract

Fusobacterium nucleatum is a Gram-negative bacterial species, strict anaerobic and one of the species often found in primary infection of the root canal system. This species has great importance in biofilm formation to be a union bridge between species which are not able to act alone. The constituent microorganisms of the biofilm exchange each tother genetic material, increasing the strength of them. It is almost impossible for a clinical isolate have genes totally equal to a standard strain, such as strains of culture collections like ATCC (American Type Culture Collection). The present study investigated anaerobic bacterial species Fusobacterium nucleatum isolated from root canals, comparing them to the ATCC strain. The microbial in vitro susceptibility of biofilm and planktonic growth of the strains was compared by means of microbiological culture and the E-test method, with the antibiotics Amoxicillin, Amoxicillin + clavulanic acid, Ampicillin, Azithromycin, Clindamycin, Eritromycin and Metronidazole.. Also, a RNA Purification protocol for the strains under the same growth conditions was defined. Clinical isolates were obtained by microbiological samples of patients with teeth with pulp necrosis and apical periodontitis visible on radiographs. The species isolation and identification were performed using commercial biochemical tests (Sistema Api, Bio-Meriéux, France) and conventional PCR, obtaining four clinical isolates. The protocol for RNA purification was done with zirconia beads, bead beater device and commercial kit RNeasy (Qiagen) and transcribed into complementary DNA (cDNA). The quality of purification was tested for its ability of amplification by real time polymerase chain reaction (qPCR) using primer for the gene 16s RNA specific for F. nucleatum. All tested trains were 100% susceptible to amoxicillin + clavulanic acid, ampicillin, azithromycin, clindamycin and metronidazole. Both types of bacterial growth showed resistance to Erythromycin. Bacteria in biofilm showed a decrease in susceptibility to all antibiotics, but without statistical difference. The protocol proposed for the purification of RNA of F. nucleatum was effective, in the planktonic and the biofilm growth. The average yield of RNA samples for bacteria in planktonic growth was 514.2 ng /μL (SD ± 397.7) and the samples in biofilm was 377.1 ng / μL (SD ± 144.1). These found values suggest a good quality of RNA, free of protein contamination. The established protocol for the purification of the RNA of the Fusobacterium nucleatum strains, grown in biofilm and planktonic phase, had successfully amplified by qPCR. (AU)

• FAPESP's process: 12/07364-5 - Gene expression of virulence factors of clinical isolates of Fusobacterium nucletaum
• Grantee: Raquel Zanin Midena
• Support Opportunities: Scholarships in Brazil - Doctorate