Bacterial strains were obtained from Japan Collection of Microorganisms (JCM; Japan), Center for Conservation of Microbial Genetic Resource, Gifu University (GTC; Japan), and American Type Culture Collection (ATCC; America). All bacterial strains used in the present study are listed in Table 1. Bacterial strains used in the present study were maintained by cultivating them on Bact^TM Brain Heart Infusion (BHI, Becton, Dickinson and Co., Sparks, MD, USA) and 1.5% agar (BHI agar). These organisms were cultured at 30˚C overnight under an aerobic condition.

Table 1. Recovey of A.baumannii and other bacteria on BHI agar and ABSM.

^aAve ± SD.

2.2.1. Evaluation of Base Medium

BHI agar supplemented with 1% yeast extract (BHI-Y), BHI-Y supplemented with 5% sheep blood (BHI-Y blood), Nutrient agar (NA), and CVT agar (Shimadzu Diagnostics Co., Tokyo, Japan) were examined as the base medium in the selective medium. Ten-fold dilutions of cultures were made in 0.9 ml of Tris-HCl buffer (0.05 M, pH 7.2) and aliquots of 0.1 ml were spread onto the test media. The plates inoculated with bacteria were cultured at 30˚C for 48 h under an aerobic condition. After cultivation, the number of colony-forming units (CFU)/ml was counted.

2.2.2. Susceptibility Tests

Preliminary studies of antibiotic selection were also performed using disk susceptibility tests (Sensi-Disk, Becton Dickinson Co., MD, USA). The microbroth dilution method was used for susceptibility testing [12].

The recoveries of the Acinetobacter reference strains and other representative bacteria were calculated as CFU/ml on selective medium and compared with those on BHI agar for total cultivable bacteria. All bacterial strains used in the present study are listed in Table 1.

All bacterial strains were pre-incubated in BHI broth at 30˚C overnight in an atmosphere of 5% CO₂ in a CO₂ incubator. Ten-fold dilutions of cultures were made in 0.9 ml of Tris-HCl buffer (0.05 M, pH 7.2) and aliquots of 0.1 ml were spread onto the test media. The plates inoculated with bacteria were cultured at 30˚C for 48 h under an aerobic condition. After cultivation, the number of CFU/ml was counted.

Thirty volunteers (15 men, 15 women; mean age 38 years, range 15 - 71 years) participated in the present study. They had no systemic disease and received no antibiotic therapy for at least 3 months. All participants were asked not to brush, rinse, or smoke immediately prior to the assessment and not to eat or drink for at least 2 h beforehand.

Paraffin-stimulated whole saliva samples were collected in a sterile microcentrifuge tube. Swab samples were collected from ten dental spittoon units in a dental hospital (Nihon University Hospital, School of Dentistry at Matsudo). All samples were dispersed by sonication for 30 s in an ice bath (50 W, 20 kHz, Astrason® System model XL 2020, NY, USA), and 0.1 ml of each was diluted and inoculated on BHI-Y and selective medium plates. BHI-Y plates for total cultivable bacteria were cultured at 37˚C for 2 days in an atmosphere of 5% CO₂ in a CO₂ incubator, and selective medium plates for A.baumanii were cultured at 30˚C for 2 days under an aerobic condition. After cultivation, CFU/ml in each sample was calculated. The present study was conducted in accordance with the principles of the Declaration of Helsinki, and was approved by the Ethics Committee of Nihon University School of Dentistry at Matsudo, Japan (EC23-012).

For each sample, 24 colonies were randomly selected from approximately 50 colonies that had grown on selective agar plates. Each selected colony was completely isolated and subcultured by streaking onto BHI-Y medium. Subsequently, bacterial species identification was performed using PCR analysis.

Design of species-specific primers for A.baumanii was performed as described previously [13]. Briefly, the 16S rRNA gene sequences of A.baumannii (accession no. AB594765), A.nosocomialis (HQ180192), A.pittii (MN307289), A. calcoaceticus (AB626122) and A. lwoffii (X81665) were obtained from the DNA Data Bank of Japan (DDBJ; https://www.ddbj.nig.ac.jp/services.html, Mishima, Japan), and a multiple sequence alignment analysis was performed with the CLUSTAL W program; i.e., the 16S rRNA gene sequences of five Acinetobacter species were aligned and analyzed, respectively. Homology among the primers selected for each Acinetobacter species and their respective 16S rRNA sequences was confirmed by a BLAST search.

Bacterial cells were cultured in BHI supplemented with 0.5% yeast extract for 24 h, and 1 ml of the samples were then collected in microcentrifuge tubes and resuspended at a density of 1.0 McFarland standard (approximately 10⁷ colony-forming units (CFU)/ml) in 1 ml of sterile distilled water. A total of 3.6 μl of the suspension was then used as a PCR template. The detection limit of PCR was assessed by serially diluting known numbers of bacterial cells in sterile distilled water and then subjecting each suspension to PCR. The PCR mixture contained 0.2 μM of each primer, 10 μl of 2× MightyAmp Buffer Ver.3 (Takara Bio Inc., Shiga, Japan), 0.4 μl of MightyAmp DNA Polymerase (Takara), and 5 μl of the template in a final volume of 20 μl. PCR reactions were performed in a DNA thermal cycler (Applied Biosystems 2720 Thermal Cycler; Applied Biosystems, CA, USA). PCR conditions included an initial denaturation step at 98˚C for 2 min, followed by 30 cycles consisting of 98˚C for 10 s, 62˚C for 15 s, and 68˚C for 1 min. PCR products were analyzed by 2.0% agarose gel electrophoresis before being visualized by electrophoresis in 1 × Tris-borate-EDTA on a 2% agarose gel stained with ethidium bromide. A 100-bp DNA ladder (Takara Biomed, Shiga, Japan) was used as a molecular size marker. All experiments were performed in triplicate.

The screening of antimicrobial susceptibility of isolated A.baumanii strains was performed by disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [14]. In total, the used antimicrobial agents included amikacin (30 µg), imipenem (10 µg), and levofloxacin (5 µg) (Sensi-Disk, Becton Dickinson Co., MD, USA). MDRA were defined as strains resistant to all agents in three antimicrobial categories. The susceptibility testing criteria for three antimicrobial agents were set as follows: For amikacin, an inhibition zone diameter of less than 14 mm; For imipenem, an inhibition zone diameter of less than 13 mm; For levofloxacin, an inhibition zone diameter of less than 13 mm.

3.1.1. Selection of Base Medium

The selection of a base medium for the growth of A.baumanii was performed. A.baumanii grew well on CVT agar as same as BHI-Y, BHI-Y blood, and NA (data not shown). To inhibit the growth of Gram-positive bacteria, CVT was ultimately selected as the base medium.

3.1.2. Susceptibility to Antibiotics

A.baumanii was more resistant to chloramphenicol than the genus Klebsiella. The minimal inhibitory concentration (MIC) of chloramphenicol for A.baumanii was more than 40 μg/ml. A.baumanii was more resistant to aztreonam than Escherichiacoli. The MIC of aztreonam for A.baumanii was 20 μg/ml. The genus Microbacterium was sensitive to 5 μg/ml of lincomycin. The MIC of lincomycin for A.baumanii was 20 μg/ml. A.baumanii was more resistant to sodium deoxycholate than Pseudomonas, Raoultella, Citrobacter, and Serratia species. The MIC of sodium deoxycholate for A.baumanii was 1000 μg/ml. The genus Pseudomonas were sensitive to 1 μg/ml of sodium deoxycholate. A.baumanii was more resistant to cefsulodin than the genus Staphylococcus. The MIC of cefsulodin for A.baumanii was 25 μg/ml. The genus Staphylococcus and Acinetobacter species excluding A.baumanii were sensitive to 6 μg/ml of cefsulodin.

3.1.3. Composition of New Selective Medium

The new selective medium, designated oral A.baumanii selective medium (ABSM), was composed of the following (per liter): 23.5 g of CVT agar, 15 mg of chloramphenicol, 3 mg of aztreonam, 5 mg of lincomycin, 250 mg of sodium deoxycholate, 6 mg of cefsulodin, and 15 mg of amphotericin B. Antibiotics, i.e., chloramphenicol, aztreonam, lincomycin, sodium deoxycholate cefsulodin, and amphotericin B were added after the base medium had been sterilized and cooled to 50˚C.

3.1.4. Recovery of A.baumanii and Inhibition of Other Representative Bacteria on Selective Medium

Table 1 shows the recovery of some A.baumannii reference strains on ABSM relative to BHI-Y. The growth recoveries of the A.baumannii reference strains on ABSM were between 97.5% and 99.3% (average 97.5%) that on BHI-Y.

Table 1 also shows the inhibition of other representative bacteria except A.baumannii on ABSM relative to BHI-Y. The growth of other representative bacteria was markedly inhibited on the selective medium.

3.2.1. Primer Design

The specific primer set covering the upstream region of the 16S rDNA sequence of A.baumannii was designed in the present study (Table 2). The amplicon size of A.baumanii was 562 bp.

Table 2. Locations and sequences of species-specific primers for the16S rDNA of A.baumannii.

3.2.2. PCR Condition

A PCR method for identifying A.baumannii successfully amplified DNA fragments of the expected size (Figure 1). The detection limit was assessed in the presence of titrated bacterial cells, and the sensitivity of the PCR assay was between 5 × 1 and 5 × 10 CFU per PCR template (5.0 μl) for the A.baumannii-specific primer set with strain JCM 6841 (data not shown).

3.2.3. Assay of A.baumannii and Representative Oral Bacteria

The PCR method used to identify A.baumannii produced positive bands from the A.baumannii reference strain JCM 6841 and GTC 03319 (Figure 1). No amplicons were produced from any of the representative oral bacteria and microorganisms colonizing the environment (data not shown).

Figure 1.Specificity of PCR assays for A.baumanii. The primer mixture contained ABF and ABR. Lanes: 1, A.baumannii JCM 6841; 2, A.baumannii GTC 03319; 3, A.calcoaceticus JCM 6842; 4, A.pittii GTC 00524; 5, A.nosocomialis GTC 03314; 6, A.lwoffii JCM 6840. M, molecular size marker (100-bp DNA ladder).

The detection frequencies of A.baumanni in the saliva samples from thirty healthy subjects and the swab samples from thirty dental spittoon units are shown in Table 3. A.baumannii was not detected at all in the saliva samples (0%). On the other hand, this microorganism was detected from nine swab samples. In positive samples, the mean number of this microorganism and its proportion relative to the total bacterial number were 1.10 × 10⁴ CFU/ml and 0.065%, respectively. MDRA was not detected in any of the samples.

Table 3. Detection frequency of A.baumannii in the human oral cavity and dental units.

In the first isolation, A.baumannii colonies on ABSM commonly had a smooth appearance resembling a fried egg. The colony’s color was reddish purple at the center, with white surrounding it. Therefore, ABSM could distinguish from other bacteria based on colony morphology. The average colony sizes of A.baumannii on ABSM were 2.1 mm in diameter (Figure 2).

Figure 2.Appearance of A.baumanii colonies on ABSM.