Novel Selective Medium for the Isolation of Rothia aeria, Which Is an Inhabitant of the Human Oral Cavity

Roth aeria, Rothia dentocariosa, and Rothia mucilaginosa are isolated from the human oral cavity. Among them, R. aeria can cause severe systemic infection diseases (e.g., bronchitis, endocarditis, pneumonia, and sepsis). However, the veritable prevalence of this organism in the human oral cavity has not ever been known. Thus, the selective medium for the isolation and quantification of R. aeria is necessary to assess the prevalence of this organism, and to diagnose various R. aeria infection diseases. To investigate R. aeria distribution in oral cavities, a novel selective medium (RAESM) was developed for isolating and quantifying R. aeria. RAESM consists of sodium gluconate, tryptone, meat extract, sodium fluoride, acriflavin neutral, fosfomycin, lincomycin, colistin, aztreonam, and agar. Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S rDNA genes of R. aeria. The percentage of R. aeria in saliva samples collected from 20 subjects was examined. Moreover, we examined the antibiotic susceptibility of thirty isolates from six subjects. The average growth recovery of R. aeria on RAESM was 96.6% compared with that on Brain Heart Infusion supplemented with Yeast Extract (BHI-Y) agar. Growth of other representative oral bacteria, including other Rothia species, was remarkably inhibited on the selective medium. The PCR primers reacted to R. aeria and did not react to other Rothia species or representative oral bacteria. R. aeria was detected as 1.0% of the total bacteria, 5.9×107CFU/ml, on BHI-Y agar in the oral cavities of all subjects. R. aeria isolates obtained in this study were susceptible to most antibiotics; however R. aeria isolates from one subject were highly resistant to erythromycin, lincomycin, and clindamycin. R. aeria may be a part of the normal flora in the human oral cavities. A novel selective medium, RAESM, was useful for isolating R. aeria. Moreover, it was indicated that RAESM was useful for diagnosing R. aeria infections.

To investigate R. aeria distribution in oral cavities, a novel selective medium (RAESM) was developed for isolating and quantifying R. aeria. RAESM consists of sodium gluconate, tryptone, meat extract, sodium fluoride, acriflavin neutral, fosfomycin, lincomycin, colistin, aztreonam, and agar. Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S rDNA genes of R. aeria. The percentage of R. aeria in saliva samples collected from 20 subjects was examined. Moreover, we examined the antibiotic susceptibility of thirty isolates from six subjects.
The average growth recovery of R. aeria on RAESM was 96.6% compared with that on Brain Heart Infusion supplemented with Yeast Extract (BHI-Y) agar. Growth of other representative oral bacteria, including other Rothia species, was remarkably inhibited on the selective medium. The PCR primers reacted to R. aeria and did not react to other Rothia species or representative oral bacteria. R. aeria was detected as 1.0% of the total bacteria, 5.9×10 7 CFU/ml, on BHI-Y agar in the oral cavities of all subjects. R. aeria isolates obtained in this study were susceptible to most antibiotics; however R. aeria isolates from one subject were highly resistant to erythromycin, lincomycin, and clindamycin. R. aeria may be a part of the normal flora in the human oral cavities. A novel selective medium, RAESM, was useful for isolating R. aeria. Moreover, it was indicated that RAESM was useful for diagnosing R. aeria infections. [9,10]. Moreover, we have also previously reported selective media for the simultaneous isolation of three oral Rothia species (R. dentocariosa, R. mucilaginosa, and S. aeria) [11]. However, there has never been reported the selective medium for the isolation of R. aeria only. Therefore, a suitable selective medium for the isolation of R. aeria is necessary to assess the accurate prevalence of this organism in the human oral cavity.
The aim of the present study was to develop a selective medium for the isolation of R. aeria and reveal its distribution in the human oral cavity.

Materials and Methods
Bacterial strains and culture conditions Table 1 shows all bacterial strains used in this study. All strains were subcultured on brain heart Infusion (BHI, CM1135, Oxoid, UK) supplemented with 0.5% yeast extract (Oxoid, UK) and 1.5% agar (BHI-Y agar, Difco agar, BD, USA). Genus Rothia were cultivated at 37°C in the air for up to 24 hours. Representative oral bacteria strains except genus Rothia were cultivated at 37°C in 5% CO 2 for up to 24 hours. ORSM [11] was chosen as a base medium for the selective medium. Disk susceptibility tests were used for antibiotic selection. (KB-Disk, EIKEN CHEMICAL CO., LTD., Tokyo, Japan). After choosing appropriate antibiotics, the microbroth dilution method was performed [20,21].

Recovery of representative Rothia species
The recovery of Rothia species reference strains and isolates from the human oral cavity in our previous studies [11] were calculated as colony-forming units (CFU) /mL on the selective medium compared with those on BHI-Y agar for total cultivable bacteria.
Rothia species were cultivated at 37°C in the air for up to 24 hours. Rothia reference strains and isolates were suspended at three different concentrations (10 5 , 10 6 , and 10 7 CFU/mL), and then 0.1 mL Tris-HCl buffer (0.05 M, pH 7.2) of each suspension was inoculated in triplicates onto BHI and the selective medium. After the culture, CFU/ ml was calculated.

Clinical samples
Clinical samples were obtained from twenty volunteers (age 22-58, male 9, female 11). Slaiva samples stimulated with paraffin from each volunteer were collected in sterile vials containing 0.5 mL of 0.05 M Tris-HCl buffer (pH 7.2). This study was approved by the Ethics Committee of Nihon University School of Dentistry at Matsudo, Japan (EC 15-025). Samples were processed as described previously [11].

Identification of R. aeria isolated from clinical samples
Ten colonies (which appeared to be R. Erie based on colony morphology), per subject were subcultured to confirm the presence of R. aeria. Pure cultures of each isolate were identified by: (i) gram staining; and (ii) polymerase chain reaction (PCR) analyses.

Design of species-specific primers for representative Rothia species and PCR method procedure
The 16S rRNA sequences of R. aeria (accession no. AB071952) were obtained from the DNA Data Bank of Japan (DDBJ; Mishima, Japan). Design of species-specific primers and the procedure of PCR method were performed as described previously [11].

Antibiotics susceptibility tests of R. aeria isolates
Antimicrobial susceptibility testing of R. aeria isolates were evaluated using the microdilution method. Table 4 shows antibiotics used in this study. They are widely used in the treatment of Grampositive infections. Because there is not Clinical and Laboratory Standards Institute (CLSI) protocols for R. aeria, the organism's drug susceptibility utilizing the 2016 CLSI criteria (M100-S27) for staphylococci was substituted in this study.

Development of the selective medium
R. Dentocariosa, R. mucilaginosa, and R. aeria grew well and at similar ratios on a base medium, i.e. ORSM [11]. The minimal inhibitory concentration (MIC) of aztreonam for R. mucilaginosa and R. aeria were 100 μg/mL. R. dentocariosa was sensitive to 30 μg/ml of aztreonam. Moreover, R. aeria was more resistant to fosfomycin than R. mucilaginosa. The MIC of fosfomycin for R. aeria was 80 μg/mL. R. Mucilaginous was sensitive to 3 μg/ml of fosfomycin.
The selective medium for the isolation of R. aeria (RAESM) was composed of the following (per liter): 5 g meat extract (Sigma-Aldrich Co. LLC., Tokyo, Japan), 1 g tryptone (Sigma-Aldrich), 10 g sodium gluconate (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), 125 mg sodium fluoride (Sigma-Aldrich), 15 g agar (Difco agar, BD, USA), 3 mg acriflavine neutral (Sigma-Aldrich), 10 mg colistin sulfate salt (Sigma-Aldrich), 0.2 mg lincomycin hydrochloride (Tokyo Chemical Industry), 3 mg fosfomycin disodium salt (Tokyo Chemical Industry), and 30 mg aztreonam (Tokyo Chemical Industry). When the mixture except of the antibiotics was cooled to 50ºC, colistin sulfate salt, lincomycin hydrochloride, fosfomycin disodium salt, and aztreonam were added aseptically. Table 2 shows specific primer sets covering the upstream regions of the 16S rDNA sequences of R. aeria, and the amplicon size of this organism was 918 bp. The PCR method used to identify R. aeria produced positive bands from R. aeria ( Figure 1) and did not produce any amplicons from other Rothia species or any of the representative oral bacteria, i.e. some Streptococcus, Actinomyces, Neisseria, and Corynebacterium species. Table 1 shows the recovery of R. aeria and isolates on RAESM relative to BHI-Y agar. The growth recoveries of R. aeria reference strains and the isolates ranged from 95.1% to 97.7% (average 96.6%) on RAESM relative to that on BHI-Y agar. The growth of R. dentocariosa and R. mucilaginosa was markedly inhibited on the selective medium.

Clinical examination
The percentage of R. aeria in saliva from the twenty subjects on BHI-Y and RAESM is shown in Table 3. The mean number of total cultivable bacteria was 5.9 × 10 7 CFU/ml (range: 2.1 × 107 -9.8 × 10 7 ). The mean number of R. aeria was 5.6 × 10 5 CFU/ml (range: 0.3 × 10 5 -27 × 10 5 ). R. aeria accounted for 1.0% of the total cultivable bacteria number on the BHI-Y medium and was detected in all twenty subjects.

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adhered to the agar medium such that they were not easily scraped off. The average colony size of R. aeria on RAESM was 1.8 mm in diameter.

Antibiotics susceptibility tests of R. aeria isolates
R. aeria reference strains and isolates from subject A, C, D, E, and F were susceptible to most antibiotics (Table 4). On the other hand, R. aeria isolates from subject B were highly resistant to erythromycin, lincomycin and clindamycin.

Discussion
R. dentocariosa, R. mucilaginosa, and R. aeria are part of the normal flora in the human oral cavity and pharynx [8][9][10][11]. R. aeria was first isolated from air and condensation water samples from the Russian space station Mir [1]. R. aeria was originally classified as R. dentocariosa genomovar II before the report of Li et al. [1]. R. aeria is capable of causing serious systemic infections, such as sepsis, bronchitis, pneumonia, and endocarditis [12][13][14][15][16][17]. However, there has never been reported the selective medium for the isolation of R. aeria only. Therefore, a suitable selective medium for the isolation of R. aeria is necessary to assess the veritable prevalence of this organism in the human oral cavity and to diagnose R. aeria infections rapidly. To examine the bacterium population in the oral cavity, a novel selective medium, designated RAESM, was developed for the isolation of aeria in this study. RAESM was highly selective for R. aeria.
On clinical microbiological examination, Rothia species can be mistaken for bacteria such as Dermabacter hominis, Actinomyces viscosus, Propionibacterium avidum, Corynebacterium matruchotii, and Nocardia species because many laboratories are unfamiliar with these organisms, which may be difficult to culture due to having the same gram positive rods and to their variable aero-tolerance [22][23][24]. Moreover, the colonies of Nocardia species are similar to those of R. aeria [18]. R. aeria is capable of causing serious systemic infections [15][16][17][18][19][20]. Therefore, RAESM may contribute to the correct and rapid diagnosis of the infectious diseases caused by R. aeria.
In this study, R. aeria was detected in all subjects and accounted for 1.0% of total bacteria in saliva. These results indicated that R. aeria is a part of the normal flora in the oral cavity of humans and is not a microorganism peculiar to a specific environment, such as the space station. In our previous studies, R. dentocariosa and R. mucilaginosa accounted for 2.6% and 3.4% of total bacteria in saliva, respectively [9,10]. Consequently, it was indicated that R. aeria is a part of the normal flora in the human oral cavity, and the genus Rothia includes three species that inhabit the human oral cavity. aeria can be mistaken for Nocardia spp due to the morphological similarities, and discrimination between R. aeria and Nocardia spp needs further analyses, such as 16S rRNA sequencing [18]. Microorganisms of the genus Nocardia are branching and partially acid-alcohol-fast grampositive bacilli, and they belong to the order of Actinomycetales. Numerous species have been described and are being reclassified continuously thanks to the use of molecular biology techniques. Nocardiosis is caused by various species of the genus Nocardia. It can cause lung disease, skin disease, or systemic disorders by involving the central nervous system, but it can also colonize the airways asymptomatically. Saraya et al. [25]