Tsuzukibashi Osamu, Uchibori Satoshi, Kobayashi Taira, Umezawa Koji, Mashimo Chiho, Nambu Takayuki, Saito Masanori, Hashizume-Takizawa Tomomi, Ochiai Tomoko
Department of Microbiology and Immunology, Nihon University, School of Dentistry at Matsudo, Chiba 271-8587, Japan.
Department of Crown Bridge Prosthodontics, Nihon University, School of Dentistry at Matsudo, Chiba 271-8587, Japan.
J Microbiol Methods. 2017 Mar;134:21-26. doi: 10.1016/j.mimet.2017.01.005. Epub 2017 Jan 10.
Rothia dentocariosa and Rothia mucilaginosa which are Gram-positive bacteria are part of the normal flora in the human oral cavity and pharynx. Furthermore, Rothia aeria, which was first isolated from air samples in the Russian space station Mir, is predicted to be an oral inhabitant. Immunocompromised patients are often infected by these organisms, leading to various systemic diseases. The involvement of these organisms in oral infections has attracted little attention, and their distribution in the oral cavity has not been fully clarified because of difficulties in accurately identifying these organisms. A suitable selective medium for oral Rothia species, including R. aeria, is necessary to assess the veritable prevalence of these organisms in the oral cavity. To examine the bacterial population in the oral cavity, a novel selective medium (ORSM) was developed for isolating oral Rothia species in this study. ORSM consists of tryptone, sodium gluconate, Lab-Lemco powder, sodium fluoride, neutral acriflavin, lincomycin, colistin, and agar. The average growth recovery of oral Rothia species on ORSM was 96.7% compared with that on BHI-Y agar. Growth of other representative oral bacteria, i.e. genera Streptococcus, Actinomyces, Neisseria, and Corynebacterium, was remarkably inhibited on the selective medium. PCR primers were designed based on partial sequences of the 16S rDNA genes of oral Rothia species. These primers reacted to each organism and did not react to other non-oral Rothia species or representative oral bacteria. These results indicated that these primers are useful for identifying oral Rothia species. A simple multiplex PCR procedure using these primers was a reliable method of identifying oral Rothia species. The proportion of oral Rothia species in saliva samples collected from 20 subjects was examined by culture method using ORSM. Rothia dentocariosa, Rothia mucilaginosa, and R. aeria accounted for 1.3%, 5.9%, and 0.8% of the total cultivable bacteria number on BHI-Y agar in the oral cavities of all subjects, respectively. It was indicated that among oral Rothia species, R. mucilaginosa is most predominant in the oral cavity of humans. A novel selective medium, ORSM, was useful for isolating each oral Rothia species.
龋齿罗氏菌和黏液罗氏菌属于革兰氏阳性菌，是人类口腔和咽部正常菌群的一部分。此外，最早从俄罗斯和平号空间站的空气样本中分离出的空气罗氏菌预计也是口腔中的常住菌。免疫功能低下的患者常被这些微生物感染，从而引发各种全身性疾病。这些微生物在口腔感染中的作用很少受到关注，而且由于难以准确鉴定这些微生物，它们在口腔中的分布情况尚未完全阐明。为了评估这些微生物在口腔中的实际流行情况，需要一种适用于包括空气罗氏菌在内的口腔罗氏菌属的选择性培养基。在本研究中，为了检测口腔中的细菌群落，开发了一种新型选择性培养基（ORSM）用于分离口腔罗氏菌属。ORSM由胰蛋白胨、葡萄糖酸钠、牛肉浸膏粉、氟化钠、中性吖啶黄素、林可霉素、黏菌素和琼脂组成。与在脑心浸液酵母提取物（BHI-Y）琼脂上相比，口腔罗氏菌属在ORSM上的平均生长恢复率为96.7%。在这种选择性培养基上，其他代表性口腔细菌，即链球菌属、放线菌属、奈瑟菌属和棒状杆菌属的生长受到显著抑制。基于口腔罗氏菌属16S rDNA基因的部分序列设计了PCR引物。这些引物能与每种微生物发生反应，而不与其他非口腔罗氏菌属或代表性口腔细菌发生反应。这些结果表明这些引物可用于鉴定口腔罗氏菌属。使用这些引物的简单多重PCR程序是鉴定口腔罗氏菌属的可靠方法。采用ORSM培养法检测了从20名受试者采集的唾液样本中口腔罗氏菌属的比例。在所有受试者口腔的BHI-Y琼脂上，龋齿罗氏菌、黏液罗氏菌和空气罗氏菌分别占可培养细菌总数的1.3%、5.9%和0.8%。结果表明，在口腔罗氏菌属中，黏液罗氏菌在人类口腔中最为常见。一种新型选择性培养基ORSM可用于分离每种口腔罗氏菌属。
