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Introduction

 The oral cavity harbors over 700 species of bacteria that contribute to the health and physiological status of oral cavity [1]. To understand the role of oral microbiota in the oral cavity, it is important to analyze its fundamental characteristics and dynamics. The oral microbiota in a healthy oral cavity versus a diseased one is distinctly different, which indicate that there may be a profile for oral microbiota [1]. Understanding the microbial differences between health and disease may give clinicians to recognize and diagnose diseases at an earlier and reversible stage [2].

In health, microbes may prevent disease progression in several ways. They can prevent the adherence of pathogens onto specific surfaces by occupying the niche preferred by pathogens. They can hinder pathogens’ abilities to multiply and degrade pathogens’ virulence factors [3]. In disease, the relationship between microbes are altered from mutualistic to parasitic and from commensal to opportunistic [4, 5]. As pathogenic bacteria flourish, host becomes infected or prones to infection [6]. 

Oral diseases such as periodontal disease and oral cancer are diseases worldwide affecting old ages [7, 8]. Periodontal disease results from subgingival plaque accumulation that causes shifts in the microbiota from healthy state to diseased state [9]. Microbes within biofilms begin to form pathogenic characteristics that aggravate and inflame the gingiva [10]. The predominant pathogens involved in periodontitis are Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Tannerella forsythia, Eikenella corredens, and Treponema denticola [9]. 

 Another oral disease that frequently affect the old ages is oral cancer. Oral cancer is the sixth most prevalent cancer, affecting over 300,000 people each year around the world [11]. Correlation between the structure and function of oral microbiota and oral cancer has been suggested [12]. Microbes can impact signal pathways that initiate and progress oral cancer [13]. Inflammation is usually the first symptom of compromised oral health and it gets worse as health regresses. Approximately 15–20% of human tumors contain pathogenic agents derived from inflammatory infections [12].

 In this study, we compared the detection rate of oral bacteria species between healthy volunteers and patients with periodontitis or oral cancer. The purpose of present study was to determine whether the salivary microbiota in healthy subjects would differ from those of the patients with periodontitis or oral cancer in Korean.

Materials and Methods

Patient and healthy donor selection

 Gargling samples used in this study were obtained from healthy volunteers and the patients with periodontitis or oral cancer. Since the definition of healthy state is quite controversial, samples from young volunteers were collected. Samples from healthy donor were obtained from School of Dentistry, Pusan National University. Samples from periodontitis patients were obtained from Pusan National University Dental Hospital. Samples from oral cancer patients were obtained from Dongnam Institution of Radiological and medical cancer. Criteria for the selection of periodontitis patient and healthy volunteers were: (1) absence of systematic diseases, (2) no antibiotics taken for at least 6 months before sampling, and (3) non-smokers. Sites with no overt signs of gingival inflammation and with a probing depth of < 3mm were defined as clinically healthy; sites with obvious alveolar bone loss detected radiographically and a probing depth > 3were defined as exhibiting signs of periodontitis. Informed consent was obtained from all donors and their rights were protected according to the protocol reviewed and approved by the institutional review board of Pusan National University Dental Hospital and Dongnam Institution of Radiological and medical cancer.

Sample collection

 To collect the samples, participants gargled 15 ml of distilled water for 30 sec and the water was carefully collected in 50 ml tube. The samples were centrifuged at 3000 rpm for 10 min and the pellets were immediately transferred and stored at - 20°C before extraction of genomic DNA.

DNA isolation and PCR detection

 DNA isolation was performed by using genomic DNA isolation kit (Qiagen, Valencia, CA, USA). The DNA concentrations in clinical samples and the concentrations of the reference DNA were determined by spectrophotometer measurement (Nanodrop, Thermo, Wilmington, DE, USA) of the absorbance at 260 nm. The PCR reaction used to assess the occurrence of all target taxa was performed with 20 μL of reaction mixture containing 1 pM of each specific primer and 2x master mix. PCR amplification was performed in a PCR Thermal Cycler (Eppendrof, Hambrug, Germany) programmed for 10 min at 95 °C for initial heat activation, followed by 35 cycles of 30 sec at 94 °C for denaturation, 30 sec at 60 °C for annealing, and 30 sec at 72 °C for extension, and 10 min at 72 °C for final extension. The predicted sizes of PCR products with species-specific primers are listed in Table 1. PCR products were separated on 1.5 % agarose gels, stained with ethidium bromide, and photographed under ultraviolet light. The sizes of PCR products were compared with a molecular size marker and confirmed to correspond to those listed in Table 1.

Table 1. Species-specific and ubiquitous polymerase chain reaction primers for 28 oral bacteria

Table 1. Species-specific and ubiquitous polymerase chain reaction primers for 28 oral bacteria

Data analysis

 Student t-test and Fisher’s exact probability test were used to analyze significance. P-values of < 0.05 were considered statistically significant.

Fig. 1. Representative results of electrophoresis of PCR products from saliva samples. Saliva samples were collected from healthy volunteers, periodontitis patients and oral cancer patients. Bacterial DNA was extracted from saliva samples and PCR was performed as described in Materials and Methods. The PCR products were separated by electrophoresis on 1.5% agarose gels.

Results

Patients and healthy volunteers

 Bacteria in the saliva isolated from 36 subjects were determined in this study. Healthy volunteers were free from any oral disease (n=16). Periodontitis patients were diagnosed at the department of periodontology, Pusan National University (n=11). Oral cancer patients include hypopharynx (n=2), tongue (n=2), tonsil (n=4) and maxillary sinus cancer (n=1). The mean ± SD age of the healthy volunteers, periodontitis patients and oral cancer patients group was 30 ± 2.70, 62.13 ± 12.52, 55.22 ± 7.60 years old, respectively. The age of healthy volunteers was significantly lower than other groups (p< 0.001). The age difference between the periodontitis patients and oral cancer patients was insignificant.

Detection rate of oral bacteria

 To characterize the oral bacteria frequently found in each study group, frequently isolated bacterial species were grouped together. Bacterial species more frequently detected in oral cancer patients are shown in Fig. 2A. Capnocytophaga ochracea, Gemella morbillorum, and Streptococcus salivarius were detected significantly more frequently in oral cancer patients. Streptococcus constellatus and Streptococcus gordonii were detected significantly more frequently in oral cancer patients compared to the healthy volunteers and periodontitis patients, respectively.

Fig. 2. Detection rates of oral bacteria in the saliva samples from healthy volunteers, periodontitis patients and oral cancer patients. (A) Bacterial species more frequently found in oral cancer patients. (B) Bacterial species less frequently found in oral cancer patients. (C) Bacterial species more frequently found in periodontitis patients. (D) Bacterial species more frequently found in healthy volunteers. * p < 0.05, ** p < 0.01, *** p < 0.01.

In Fig. 2B, bacterial species more frequently found in healthy volunteers and periodontitis patients are shown. F. nucleatum/polymorphym and Prevotella nigrescens were significantly lower in oral cancer patients compared to both the healthy volunteers and periodontitis patients. Actinomyces israelii was less frequently detected in oral cancer patients compared to the healthy volunteers. 

In Fig. 2C, bacterial species that were more frequently found in periodontitis patient are shown. P. gingivalis and T. denticola were more frequently found in periodontitis patients. Among fungus, C. albicans, frequently found in oral cavity, was more frequently found in periodontitis patients. 

 In Fig. 2D, bacterial species more frequently detected in healthy volunteers are shown. Peptostreptococcus anaerobius was more frequently found in healthy volunteers compared to the periodontitis patients and oral cancer patients.

Discussion

 Oral diseases such as dental caries and periodontal disease are the most prevalent diseases worldwide [14]. Most of periodontitis more frequently occur in the aged population and another oral disease which threatens the aged population is oral cancer. There are several studies which report oral bacterial species often found in periodontitis patients or oral cancer patients compared to healthy subjects. Since most of the studies were conducted in western nations, we surmised that different ethnic background and lifestyle may influence the microbiota. In this study, we compared the detection rate of several oral bacterial species between young healthy volunteers and periodontitis patients and oral cancer patients in Korean.

 Studies have reported that certain common oral bacteria are elevated in periodontitis patients. Most commonly accepted periodontopathogens are T. denticola, P. gingivalis, T. forsythus, P. intermedia, and E. corrodens [15, 16]. Consistent with other previous reports, high detection rate of P. gingivalis and T. denticola was observed. Also, P. intermedia and E. corrodens showed high rate of detection. Interestingly, C. albicans detection rate was also high in periodontitis patients. Our results showed similar pattern of bacteria species detection rate compared to other pervious reports studied in the western world [15, 16].

 Certain common oral bacteria have been reported to be elevated in oral and esophageal cancer lesions [17-19]. Facultative oral streptococci, Prevotella, Veillonella, Porphyromonas and Capnocytophaga species were reported to be elevated [20-22]. Among Streptococcus, the detection rate of S. constellatus was significantly higher in oral cancer patient compared to periodontitis patients. S. constellatus belongs to the anginosus group of streptococci. The anginosus groups are facultative anaerobic gram-positive cocci and can cause serious infections in humans [23]. They possess pathogenicity based on tolerance to polymorphonuclear leukocytes, cellular components such as capsules, and extracellular enzymes [24, 25]. S. constellatus preferentially colonize the oral soft tissues and saliva compared to the teeth [26]. Since we used gargling wash to collect bacterial DNA, bacteria preferentially colonized to oral soft tissue could have been detected more frequently. Mager et al. reported that Capnocytophaga gingivalis, Prevotella melaninogenica, and Streptococcus mitis counts were significantly increased among the oral cancer patients [27]. In our study, only C. ochracea was significantly increased among the oral cancer patients while C. gingivalis showed rather decreased incidence.

 Periodontitis and oral cancer develop mostly in aged population. Comparing the detection rate of oral bacterial species, several species were distinctively detected in periodontitis and oral cancer patients. Several similar results were observed between this study and other previous reports, suggesting that further study should improve our understanding on oral microbiota. However, small number of samples in this study limits its significance. Increasing the total number of clinical samples could strengthen the overall result and its significance.

Acknowledgements

 This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) (NRF-2011- 0013215).