JOURNAL OF DENTISTRY AND DENTAL MEDICINE (ISSN:2517-7389)

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, 5.9×10⁷ 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.

Rothia aeria; Genus Rothia; Selective medium; Oral cavity; PCR

The genus Rothia comprises 8 species (www.bacterio.net/rothia.html), Rothia aeria [1], Rothiaamarae [2], Rothia dentocariosa [3], Rothia mucilaginosa [4], Rothia nasimurium [4], Rothia terrae [5], Rothia endophytica [6], and Rothia saerolata [7], which were isolated from an air sample, sludge of a foul water sewer, a human oral cavity, a human pharynx, the nose of a healthy mouse, subtropical fields, plant tissues, and pig barn respectively. Among the genus Rothia, R. dentocariosa, R. mucilaginosa, and R. aeria are found in the oral cavity and pharynx of humans [8-11]. Concerning R. aeria, it was first isolated from air and condensation water samples from the Russian space station Mir. Initially, it was known as R. dentocariosa genomovar II [1]. R. aeria is capable of causing serious systemic infections, such as sepsis, bronchitis,pneumonia,and endocarditis [12-17]. R. aeria is difficult to identify because of its similar morphology and colony appearance to those of Nocardia species. Therefore, 16S rRNA sequencing is required to distinguish R. aeria from Nocardia species [18].

R. aeria was detected in the mouths of healthy individuals [11,19]. We have previously reported selective media for the isolation of R. dentocariosa and R. Mucilaginous, respectively [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.

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% CO₂ for up to 24 hours.

Development of the selective medium

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 (10⁵, 10⁶, and 10⁷CFU/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 TrisHCl 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 Gram-positive 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.

PCR analyses

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.

Recovery of R. aeria on the selective medium

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.

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 × 10⁷ CFU/ml (range: 2.1 × 107 - 9.8 × 10⁷ ). The mean number of R. aeria was 5.6 × 10⁵ CFU/ml (range: 0.3 × 10⁵ - 27 × 10⁵ ). R. aeria accounted for 1.0% of the total cultivable bacteria number on the BHI-Y medium and was detected in all twenty subjects. 

In the first isolation, R. aeria colonies on RAESM commonly exhibited a rough, dry, folded, and convex appearance (Figure 2) and 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.

R. dentocariosa, R. mucilaginosa, and R. aeria are part of the normal flora in the human oral cavity and pharynx [8-11]. 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 [12-17]. 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 [22-24]. Moreover, the colonies of Nocardia species are similar to those of R. aeria [18]. R. aeria is capable of causing serious systemic infections [15-20]. 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] has reported that R. aeria should be considered in the differential diagnosis of Nocardia spp, especially in immunocompromised patients who are vulnerable to Nocardia infections. Selective medium for the isolation of R. aeria, i.e. RAESM, and the PCR analysis developed in this study may help us rapidly diagnose the infectious diseases caused by R. aeria or Nocardia spp.

This work was supported by JSPS KAKENHI Grant Number JP17K17384.

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