Health Indicator Bacteria That Is Useful for Risk Assessment of Peri-Implantitis

We established a novel identification method for oral Rothia species using one-step multiplex PCR analysis to investigate whether the monitoring of oral Rothia species levels is useful for peri-implantitis risk assessment, and to examine the oxygen concentration that these organisms need for growth in vitro. The mean number and proportion of Rothia aeria in peri-implant sulcus fluid (PISF) samples was significantly higher in the healthy implant group than in the peri-implantitis group (P < 0.05). Moreover, R. aeria under aerobic conditions vigorously grew compared with those under anaerobic conditions, and this organism grew only at the upper layer where high oxygen concentrations existed in a semi-liquid nutrient medium. Therefore, the monitoring of R. aeria levels may be suitable as an indicator of healthy peri-implant tissue conditions for the prevention of peri-implantitis.


Introduction
The successful use of jawbone-anchored (osseointegrated) titanium dental implants for the rehabilitation of edentulous and partially dentate patients has been well documented [1]- [7]. The results of implant treatment have mostly been satisfactory with survival rates of 85% to 99%. However, infections such as implant mucositis and peri-implantitis occur around dental implants [8] [9] [10] [11] [12].
The prevalence of peri-implant infections, defined as bone loss ≥ 3.1 mm compared with one-year radiographic data after placement of the supra-structure, and bleeding on probing (BOP) approaches 20% of cases within a 15-year follow-up odontopathic bacteria such as red complex bacteria, including Porphyromonas gingivalis, and detect them qualitatively or quantitatively. However, those examinations do not reflect the peri-implant tissue conditions precisely, because periodontopathic bacteria may be detected from healthy peri-implant sites [23].
Consequently, we concluded that it is suboptimal to use periodontopathic bacteria as an unhealthy indicator, and have instead explored bacteria that indicate healthy peri-implant tissue conditions. We chose oral Rothia species, which are part of normal oral flora, to be examined as potential health indicators. These species include Rothia mucilaginosa, Rothia dentocariosa, and Rothia aeria [24].
In addition to periodontitis, peri-implantitis is primarily caused by bacterial infection and presents symptoms such as soft tissue inflammation and bone resorption, but often progresses asymptomatically. However, peri-implantitis rapidly progresses compared with periodontitis, and therapeutics for periodontitis have limited effectiveness against peri-implantitis [25] [26] [27]. The detachment of the implant body in severe peri-implantitis cases occurs by resorption of the supporting bone, thereby reducing the quality of life of patients. In order to prevent the onset of peri-implantitis, it is necessary to establish a useful bacteriological examination system.
In the present study, we established a high-precision novel identification method for oral Rothia species using one-step multiplex PCR analysis to investigate whether oral Rothia species levels are useful for peri-implantitis risk assessment.
We also examined the oxygen concentrations that this organism needs for growth in vitro. Furthermore, the relationship between red complex bacteria most involved in periodontal disease and peri-implantitis was also investigated using PCR analysis.

Bacterial Strains and Culture Conditions
The following bacterial strains were used in the present study: R. mucilaginosa

Design of Species-Specific Primers for Oral Rothia Species
The design of species-specific primers for oral Rothia species was performed as described previously [24]. Briefly, the 16S rDNA sequences of R. dentocariosa (accession no. M59055), R. mucilaginosa (accession no. X87758), and R. aeria (accession no. AB071952) were obtained from the DNA Data Bank of Japan (DDBJ; https://www.ddbj.nig.ac.jp/services.html, Mishima, Japan), and a multiplex sequence alignment analysis was performed using the CLUSTALW program; i.e., the 16S rDNA sequences of seven Rothia species were aligned and analyzed. Homologies among the primers selected for R. dentocariosa, R. mucilaginosa, and R. aeria were confirmed by a BLAST search.

Development of a Novel One-Step Multiplex PCR Method Using Designed Primers
Bacterial cells were cultured in BHI supplemented with 0.5% yeast extract for 24 h, and 1 ml samples were then collected in microcentrifuge tubes and resuspended at a density of 1.

Clinical Samples
Sixty patients attending Nihon University Hospital, School of Dentistry at Matsudo, participated in the present study. They were divided into two subject groups: healthy implant (HI) and peri-implantitis (PI) groups. Thirty HI and thirty PI subjects were selected by inclusion criteria for peri-implantitis as fol- tamin K 1 , hemin, L-cysteine, yeast extract, and sheep blood, and on selective medium plates that were developed for the isolation of oral Rothia species in our previous report [24]. Selective medium plates for oral Rothia species were cultured at 37˚C for three days in an atmosphere of 5% CO 2 in a CO 2 incubator.
CDC plates for total cultivable bacteria were cultured at 37˚C for five days under anaerobic conditions with a gas pack system. After the cultivation, the number of CFU was calculated. Also, the detection frequencies of red complex bacteria, i.e., P. gingivalis, Treponema denticola, and Tannerella forsythia in the remainder of each PISF sample were determined using PCR as previously described [28].
This study was approved by the Ethics Committee of Nihon University School of Dentistry at Matsudo, Japan (EC 19-033).

Comparison of Growth in a Semi-Liquid Nutrient Medium between R. aeria and Anaerobic Bacteria
A semi-liquid nutrient medium that consisted of BHI and 0.8% agar was prepared in a glass test tube. Bacterial suspensions of R. aeria JCM 11412 and P. gingivalis ATCC 33277 that were preincubated were inoculated into each medium with an inoculating needle and were cultured at 37˚C for two days under aerobic conditions. After cultivation, the growth of R. aeria and P. gingivalis in semi-liquid nutrient medium was macroscopically compared.

2.6.Statistical Analysis
The numbers of oral Rothia species and total bacteria in the PISF samples from the HI and PI groups were compared using the Mann-Whitney U test. Values of P < 0.05 were considered significant. The detection frequencies of red complex bacteria in both groups were compared using Fisher's exact test. Values of P < 0.05 were considered significant.

Primer Design
Six specific primers covering the upstream regions of the 16S rDNA sequences of three oral Rothia species were designed in the present study ( Figure 1). The specific forward primers were designated as RMFF for R. mucilaginosa, RDFF for R. dentocariosa, and RAFF for R. aeria, whereas the specific reverse primers were designated as RMFR for R. mucilaginosa, RDFR for R. dentocariosa, and RAFR for R. aeria. The amplicon sizes of R. mucilaginosa, R. dentocariosa, and R. aeria were 356 bp, 541 bp, and 924 bp, respectively.

Detection Limit
Our one-step multiplex PCR method for identifying three oral Rothia species, R. mucilaginosa, R. dentocariosa, and R. aeria, successfully amplified DNA fragments of the expected sizes for each species (Figure 2). 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 R. mucilaginosa-specific primer set with strain JCM 10910, the R. dentocariosaspecific primer set with strain JCM 306, and the R. aeria-specific primer set with strain JCM 11412 (data not shown).

Assay of Representative Rothia Species and Representative Oral Bacteria
The one-step multiplex PCR method used to identify R. mucilaginosa, R. dentocariosa, and R. aeria produced positive bands from each respective strain ( Figure 1) and did not produce any amplicons from other Rothia species or with Streptococci, Open Journal of Stomatology   Actinomyces, Neisseria, or Corynebacterium species used as representative oral bacteria with the designed primer sets. Moreover, three bands equivalent to R. mucilaginosa, R. dentocariosa, and R. aeria were produced from a mixed sample of the three oral Rothia species.

Clinical Examination
The clinical parameters of HI and PI groups are shown in ( CFU. The mean numbers of R. dentocariosa, R. mucilaginosa, and R. aeria in the HI group were 1.05 × 10 3 CFU, 4.97 × 10 2 CFU, and 6.66 × 10 3 CFU, respectively. The mean number of total bacteria in the PI group was 7.16 × 10 6 CFU. The mean numbers of R. dentocariosa, R. mucilaginosa, and R. aeria in the PI group were 4.23 × 10 3 CFU, 6.40 × 10 2 CFU, and 6.25 × 10 2 CFU, respectively. The mean number of R. aeria in PISF samples was significantly higher in the HI group than in the PI group (P < 0.05).
A comparison of the proportions of oral Rothia species between the two groups is shown in (Figure 3). R. aeria in the PISF samples of the HI and PI groups was detected at 0.998% and 0.008%, respectively, of total bacteria. R. mucilaginosa in the HI and PI groups was detected at 0.030% and 0.010%, respectively, of total bacteria. R. dentocariosa in the HI and PI groups was detected at 0.176% and 0.079%, respectively, of total bacteria. The proportion of R. aeria in PISF samples was significantly higher in the HI group than in the PI group (P < 0.05).
The detection frequencies of red complex bacteria in PISF samples obtained from the two groups are shown in (Table 3). While the detection frequencies of all red complex bacteria, P. gingivalis, T. forsythia, and T. denticola, in PISF samples were significantly higher in the PI group than in the HI group (P < 0.01), these organisms were also detected from some of the HI group.

Comparison of the Growth of R. aeria between Two Culture Conditions
A comparison of the growth of R. aeria between two culture conditions is shown  in (Figure 4). R. aeria under aerobic conditions vigorously grew compared with under anaerobic conditions.

Comparison of Growth in Semi-Liquid Nutrient Medium between R. aeria and Anaerobic Bacteria
A comparison of growth in semi-liquid nutrient medium between R. aeria and anaerobic bacteria (P. gingivalis) is shown in (Figure 5). R. aeria grew only at

Discussion
The genus Rothia is Gram-positive and includes seven species, R. aeria, Rothia amarae, R. dentocariosa, Rothia endophytica, R. mucilaginosa, Rothia nasimurium, and Rothia terrae [24]. Among the Rothia, R. aeria, R. dentocariosa, and R. mucilaginosa inhabit the human oral cavity and pharynx. All three have been identified as opportunistic pathogens that can cause septicemia, endocarditis, and other serious infections [29] [30] [31]. R. aeria, which was originally classified as R. dentocariosa genomovar II, was first isolated from air and condensation water samples in the Russian space station Mir [32]. There was only one report in which R. aeria was detected in the mouths of healthy individuals [33] until our previous study demonstrated that R. aeria is part of the normal flora in the oral cavity [24]. Upon clinical microbiological examination, Rothia species can be mistaken for bacteria such as Dermabacter hominis, Actinomyces viscosus, Propionibacterium avidum, Corynebacterium matruchotii, and Nocardia spp., 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 varying aero-tolerance [34] [35] [36]. Moreover, some studies have previously reported that it is difficult to identify isolates, and that routine biochemical tests might misidentify R. aeria as R. dentocariosa [37] [38]. In addition, R. aeria can be mistaken for Nocardia spp. due to morphological similarities, and discrimination between R. aeria and Nocardia spp. needs further analyses, such as 16S rRNA sequencing [39]. Sequence analysis of several target genes is the most reliable method. However, it is expensive, laborious, and time-consuming. Thus, a simple and more reliable assay for identifying oral Rothia species is required.
In the present study, species-specific primers to identify oral Rothia species were designed using a one-step multiplex PCR method. These primers were able to distinguish R. mucilaginosa, R. dentocariosa, and R. aeria and did not react with representative oral bacteria or other Rothia species. Moreover, the novel Open Journal of Stomatology Rothia species, which are part of the normal oral flora, as possible indicator species in the present study. As a result, the mean number and proportions of R. aeria in PISF samples were significantly higher in the HI group than in the PI group (P < 0.05); however, those of R. dentocariosa and R. mucilaginosa did not demonstrate significant differences between the groups. Moreover, the detection frequencies of all red complex bacteria were significantly higher in the PI group than in the HI group (P < 0.01); however, these organisms were also detected in some samples of the HI group. Renvert et al. also reported that the prevalence of red complex bacteria, considered as key pathogens in periodontitis, is low and does not seem to differ by implant status [23]. In addition, R. aeria under aerobic conditions vigorously grew compared with anaerobic conditions and grew only at the upper layer where high oxygen concentrations existed in a semi-liquid nutrient medium. These results indicated that a shallow healthy peri-implant sulcus, where high oxygen concentration exists, is preferable for R. aeria growth.
We developed a one-step multiplex PCR method for the identification of oral Rothia species. The method described herein will be useful for determining the distribution and role of these organisms in various locations in humans. Moreover, the monitoring of R. aeria levels may be suitable as an indicator reflecting healthy peri-implant tissue conditions to aid in the prevention of peri-implantitis.

Authors' Contributions
Suzuki H, Tsuzukibashi O, Fukatsu A, corrected the data. Suzuki H wrote the manuscript. The concept of this manuscript was devised by Suzuki H. All authors read and approved the final manuscript.

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.