title: Isolation and identification methods of Rothia species in oral cavities.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, Nihon University, School of Dentistry at Matsudo, Chiba 271-8587, Japan. Electronic address: tsudukibashi.osamu@nihon-u.ac.jp.
authors.forename: Osamu
authors.initials: O
authors.lastname: Tsuzukibashi
authors.affiliation: Department of Crown Bridge Prosthodontics, Nihon University, School of Dentistry at Matsudo, Chiba 271-8587, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Uchibori
authors.affiliation: Department of Crown Bridge Prosthodontics, Nihon University, School of Dentistry at Matsudo, Chiba 271-8587, Japan.
authors.forename: Taira
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Department of Special Needs Dentistry, Nihon University, School of Dentistry at Matsudo, Chiba 271-8587, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Umezawa
authors.affiliation: Department of Bacteriology, Osaka Dental University, Osaka 573-1121, Japan.
authors.forename: Chiho
authors.initials: C
authors.lastname: Mashimo
authors.affiliation: Department of Bacteriology, Osaka Dental University, Osaka 573-1121, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nambu
authors.affiliation: Department of Microbiology and Immunology, Nihon University, School of Dentistry at Matsudo, Chiba 271-8587, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Saito
authors.affiliation: Department of Microbiology and Immunology, Nihon University, School of Dentistry at Matsudo, Chiba 271-8587, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Hashizume-Takizawa
authors.affiliation: Department of Microbiology and Immunology, Nihon University, School of Dentistry at Matsudo, Chiba 271-8587, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ochiai
mesh_terms: D000362:Agar; D003470:Culture Media / Q000737:chemistry*; D017931:DNA Primers; D005942:Gluconates; D006801:Humans; D008835:Micrococcaceae / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D009055:Mouth / Q000382:microbiology*; D010461:Peptones; D016133:Polymerase Chain Reaction; D012463:Saliva / Q000382:microbiology*
chemical_list: D003470:Culture Media; D017931:DNA Primers; D005942:Gluconates; D010461:Peptones; C001830:tryptones; D000362:Agar; C030691:gluconic acid
keywords: Genus Rothia; Oral cavity; Selective medium