Osamu Tsuzukibashi

Characterization of a new serotype g isolate of Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is usually isolated from the oral cavity where it is associated with active periodontitis. The species can be divided into six serotypes (a-f) according to their surface carbohydrate antigens. However, some clinical isolates cannot be grouped within these six serotypes. Gram-negative, facultative anaerobic, catalase-positive coccobacilli were isolated from a patient with periodontitis and identified by employing genetic, biochemical and serological analyses. Phenotypic data identified the isolate as A. actinomycetemcomitans. Serotype-specific polysaccharide antigen from the isolate was untypeable by immunodiffusion testing in comparison with reference A. actinomycetemcomitans serotype a to f strains. Biofilm formation by the isolate was strong but cytotoxic activity was low. Gas chromatography/mass spectroscopy analysis of partially methylated alditol acetates from surface polysaccharide showed the presence of 2,4-di-O-methyl-rhamnose and 2,3,6-tri-O-methyl-glucose, with a 1 : 1 m ratio. The (1)H- and (13)C-nuclear magnetic resonance spectra of the antigen showed that both constituent glycoses had alpha-anomeric configuration. It is proposed that the untyped strain is a new A. actinomycetemcomitans serotype, designated serotype g.

A novel selective medium for isolation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

BACKGROUND AND OBJECTIVE Conventional selective media have been used for the selection of Aggregatibacter (Actinobacillus) actinomycetemcomitans in clinical samples. The proportion of A. actinomycetemcomitans grown on the selective media in vitro may not reflect the true counts in vivo because of the low selectivity. A novel selective medium, designated AASM, was developed for the isolation of A. actinomycetemcomitans. MATERIAL AND METHODS AASM was prepared by adding of 200 microg/mL of vancomycin and 10 U/mL of bacitracin to AAGM, which contains dextrose, sodium bicarbonate, trypticase soy, yeast extract and agar. Clinical efficacy was evaluated by the recovery, on AASM, of A. actinomycetemcomitans from subgingival samples of 44 periodontally healthy subjects and 76 patients with chronic periodontitis. RESULTS All serotypes (a-f) of A. actinomycetemcomitans strains grew well, and the average growth recovery of A. actinomycetemcomitans on AASM medium was 94.4% (80.0-109.7%) of that on AAGM. The exclusive rate of other bacteria was 99.9% in clinical samples cultured on AASM. A. actinomycetemcomitans was not detected in periodontally healthy persons but was detected in 25 (32.9%) patients with chronic periodontitis. The predominant serotype was c, detected in 11 subjects. CONCLUSION The new selective medium, AASM, was highly selective for A. actinomycetemcomitans, eliminated possible false-positive results and was useful for the isolation of A. actinomycetemcomitans from clinical samples.

A selective medium for Rothia mucilaginosa and its distribution in oral cavities

A selective medium for Rothia mucilaginosa (RMSM) was developed to examine the population of R. mucilaginosa in oral cavities. The growth recovery of R. mucilaginosa on RMSM was 85.1% relative to HI medium. R. mucilaginosa was detected at 3.4% of total bacteria from stimulated saliva of 8 subjects.

A selective medium for the isolation of Corynebacterium species in oral cavities

Corynebacterium matruchotii is a microbial inhabitant in the oral cavity of humans and is associated with the formation of dental calculi. C. matruchotii forms highly specific morphological units, which are referred to as corn-cobs. Although other Corynebacterium species have frequently been isolated from the oral cavity of humans, their distribution has not been reported as extensively. The aim of the present study was to develop a selective medium to isolate the genus Corynebacterium and examine the distribution Corynebacterium species in the oral cavity of humans. The growth recoveries of representative Corynebacterium species on the selective medium were sufficient. Moreover, the growth of other representative oral bacteria was markedly inhibited on the selective medium. The proportion of Corynebacterium species in saliva samples collected from 20 subjects was examined. PCR primers were designed for the oral Corynebacterium species. C. matruchotii and Corynebacterium durum accounted for 0.3% and 1.5% of the total cultivable bacteria number on the BHI medium from saliva samples, respectively. The selective medium could distinguish C. matruchotii from C. durum by each colony color using differences in acid production from galactose. The selective medium, designated OCM, was useful for isolating oral Corynebacterium species.

Isolation and identification methods of Rothia species in oral cavities

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.

Isolation and Identification Methods for Slackia Exigua and Investigation of the Relationship between this Organism and Peri-Implantitis

In the genus Slackia, Slackia exigua (formerly Eubacterium exiguum) is isolated from the human oral cavity. A relationship was recently reported between S. exigua and periodontal disease, including chronic periodontitis and peri-implantitis. A suitable selective medium for the isolation of S. exigua is needed in order to assess the veritable prevalence of this organism in various lesions of the oral cavity. The purpose of the present study was to develop selective media for the isolation of S. exigua and investigate whether the monitoring of S. exigua levels is useful as a clinical indicator for the diagnosis of peri-implantitis. In order to examine the bacterium population in the oral cavity, a novel selective medium (SEXSM) was herein developed for the isolation of S. exigua. SEXSM consists of tryptic soy agar, yeast extract, hemin, Vitamin K1, L-cysteine, sheep blood, 2-phenylethanol, trimethoprim, nalidixic acid, and polymyxin B. The average growth recovery of S. exigua on SEXSM was 98.3% that on CDC blood agar. The growth of other representative oral bacteria, i.e., the genera Streptococcus, Actinomyces, Neisseria, Corynebacterium, Veillonella, Fusobacterium, and Rothia, was strongly inhibited on the selective medium. PCR primers were designed based on partial sequences of the 16S rDNA genes of S. exigua. These primers reacted with S. exigua, but not with other representative oral bacteria. These results indicate that these primers are useful for identifying S. exigua. The proportion of S. exigua in Gingival Crevicular Fluids (GCF) collected from periodontally Healthy with Implants (HI) and Peri-Implantitis (PI) groups was examined. Colonies on SEXSM were subcultured for confirmation by a PCR analysis using the primers designed in the present study. The growth recoveries of S. exigua strains on SEXSM were very satisfactory. S. exigua in GCFs of the HI and PI groups were detected at 0.002%, and 1.7% to the total bacteria number, respectively. The selective medium, designated SEXSM, and a PCR method using the primers designed in the present study were useful for the isolation and identification of S. exigua. Moreover, the monitoring of S. exigua levels was useful as a clinical indicator for the diagnosis of periimplantitis.

Primer Design for the Identification of Ten Oral Actinomyces Species Using Multiplex PCR

Background: Actinomyces is one of the predominant genera in the oral cavity. Oral Actinomyces species play a central role in the initial stages of biofilm formation on teeth; however, limited information is currently available on the distribution of individual species in different sites or clinical conditions. Moreover, a suitable method has yet to be developed to identify oral Actinomyces species because of the phenotypic and genetic similarities between these microorganisms. Objective: Actinomyces naeslundii, A. odontolyticus, A. oris, A. georgiae, A. gerencseriae, A. graevenitzii, A. dentalis, A. johnsonii, A. israelii, and A. meyeri among the genus Actinomyces are regarded as normal human oral Actinomyces species. The purpose of the present study was to design primers to identify oral Actinomyces species using multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S rDNA genes of the representative oral Actinomyces species. The 16S rDNA genes of the representative oral Actinomyces species were obtained from the DNA Data Bank of Japan, and a multiple sequence alignment analysis was performed with the CLUSTAL W program. Homology among the primers selected for oral Actinomyces species and their respective 16S rRNA sequences was confirmed by a BLAST search. Results: These primers were able to distinguish each oral Actinomyces species and did not display cross-reactivity with representative oral bacteria or other Actinomyces species. Moreover, we developed a multiplex PCR method with the ability to identify and differentiate oral Actinomyces species (i.e., A. naeslundii, A. johnsonii, A. oris, A. odontolyticus, A. israelii, A. georgiae, A. dentalis, A. graevenitzii, A. gerencseriae, and A. meyeri) using only two PCR tubes per sample. Conclusion: The present results indicate that our multiplex PCR method with these primers is useful for identifying the representative oral Actinomyces species. This method is easy because the use of Mighty Amp DNA Polymerase Ver.2 (Takara) means that DNA extraction may be avoided, and species identification using this method only takes approximately 2 hours. Thus, the method described herein will allow the prevalence of oral Actinomyces species and their involvement in oral infections to be fully clarified in future studies.

Complete Genome Sequence of Rothia aeria Type Strain JCM 11412, Isolated from Air in the Russian Space Laboratory Mir

ABSTRACT Here, we present the complete genome sequence ofRothia aeria type strain JCM 11412, isolated from air in the Russian space laboratory Mir. Recently, there has been an increasing number of reports on infections caused by R. aeria. The genomic information will enable researchers to identify the pathogenicity of this organism.

A selective medium for the isolation of Microbacterium species in oral cavities.

The genus Microbacterium has been isolated from the environment, dairy goods, and human clinical specimens. Although, in our previous studies, some Microbacterium species were infrequently detected in oral samples collected from humans, there is currently no report that these organisms, which are capable of causing serious systemic infections, were isolated from the human oral cavity. The aim of the present study was to develop a selective medium to isolate the representative Microbacterium species most frequently detected in human clinical specimens, and reveal the distribution of individual Microbacterium species in the oral cavity. The growth recoveries of representative Microbacterium species on the selective medium, designated as MSM, were sufficient. Moreover, the growth of other representative oral bacteria was markedly inhibited on the selective medium. The proportion of Microbacterium species in the saliva samples of 60 subjects, 20 of whom were removable denture wearers, was then examined. The proportion of these organisms was also examined in environmental samples obtained by swabbing 20 washstands. PCR primers were designed for representative Microbacterium species. The genus Microbacterium was detected in 45% of the saliva and denture plaque samples collected from the twenty removable denture wearers, but was absent in the saliva of the forty non-denture wearers. On the other hand, these organisms were detected in all environmental samples. The genus Microbacterium accounted for 0.00003%, 0.0001%, and 12.6% of the total cultivable bacteria number on the BHI medium in the saliva and denture plaque samples of removable denture wearers and in the environmental samples, respectively. The most predominant Microbacterium species in all positive samples was Microbacterium oxydans. These results indicated that the genus Microbacterium was not a part of the normal flora in the human oral cavity, except for subjects wearing dentures that were contaminated by the environment, and the selective medium, designated as MSM, was useful for isolating Microbacterium species, which are frequently encountered in human clinical specimens, from the various samples.

Complete Genome Sequence of Rothia mucilaginosa Strain NUM-Rm6536, Isolated from a Human Oral Cavity

ABSTRACT Here, we present the complete genome sequence of Rothia mucilaginosa NUM-Rm6536, a strain isolated from the tongue plaque of a healthy human adult. This strain is amenable to genetic manipulation by transformation and so provides a useful foundation for more detailed investigation of this species.