The purpose of this study was to identify the oral bacterial species in sequestra from patients with bisphosphonate-related osteonecrosis of the jaw (BRONJ). Fifteen patients with BRONJ (2 males and 13 females) were evaluated. Clinical features, radiographic findings, and bisphosphonate intake history were investigated. All patients were treated with surgical methods (curettage or sequestrectomy). Infected bone samples were collected from the affected BRONJ site. Ten bacterial species were selected for polymerase chain reaction (PCR) detection. Two to nine bacterial species were detected by PCR. Gram-negative species were predominant and all identified bacteria were anaerobes. Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola were detected at high levels. These are major pathogenic species in periodontal disease. Orthopantomographic radiographs showed generalized alveolar bone loss in most patients. These radiographic findings may provide evidence of chronic periodontitis as a pre-existing inflammatory disease. Most patients had experienced a predisposing dental procedure, such as tooth extraction. Sequestra (necrotic bone) infected with oral bacterial species may be an important risk factor for BRONJ. As such, prevention and management of BRONJ may rely on effective control of bacteria in the oral cavity.

The purpose of this study is to develop the quantitative PCR(qPCR) assay that would enable the rapid identification and simultaneous detection of six different endodontic pathogenic bacteria in a single reaction. In this study, six pairs of primers for Treponema denticola, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Streptococcus mutans, and Staphylococcus aureus and two pairs of housekeeping genes were designed for a multiplex qPCR based on the SYBR Green method. The genomic DNA was extracted from reference strains and submitted to the qPCR reaction. The specificity of the amplified products was analyzed by melting curves. As a result, six distinct melting peaks were identified by the melting curve analysis and all of the target species were simultaneously discriminated. Therefore, the multiplex qPCR assay developed in this study can be used for rapid identification and detection of T. denticola, P. gingivalis, F. nucleatum, P. intermedia, S. mutans, and S. aureus at the same time. In combination with the melting curve analysis, the level of the target species and total bacterial load can be obtained.

Many previous ecological studies on three major bacterial symbionts and a newly discovered symbiont PAXS (pea aphid X-type symbiont) in the pea aphid Acrythosiphon pisum have shown that these symbionts are associated with the expression of a variety of host phenotypes, including resistance to parasitoid Aphidius ervi and tolerance to heat stress. The principal role of all four symbionts “Candidatus Serratia symbiotica”, “Candidatus Hamiltonella defensa”, “Candidatus Regiella insecticola”, and PAXS is to protect aphids against abiotic stress by preserving the cells in which most of symbionts dwell and by reducing the rate of parasitism. In this experiment, we detected endosymbionts from four aphid clones by means of genomic DNA extraction, PCR with gene specific primer, and restriction enzyme cutting. The patterns of PCR and restriction enzyme cutting were all identical in the four aphid clones. In order to specifically identify the endosymbiont, we searched the sequences using BLAST. The BLAST search revealed that nucleotide sequences of the symbiont were 98% identical to Serratia entomophil. S. entomophil is also known to provide tolerance to heat stress, resistance to parasitoid wasps, and restoration of reproduction in aphids, suggesting its role in host protection.