Cultivable Microbiota in Mitis Salivarius Agar from Dental Plaque of Dogs

Periodontal disease, which affects the support structures and protection of teeth, is the main disease found in dogs, reaching about 85% prevalence in animals over 4 years old. The most important bacteria in the initial colonization of human dental plaque are Streptococcus, which produce exopolysaccharides that facilitate fixation of the bacteria to the surfaces. Some studies have sought to identify the dental plaque microbiota in dogs using non-selective culture media, and have reported small amounts of Streptococcus, however they did not provide ideal conditions for the growth of this bacteria. The aim of this study was to isolate and identify the initial dental plaque microbiota of dogs cultivable in a selective medium for Streptococcus, and verify the presence of this genus. Genotypic identification of the isolates were carried out by partial 16S rDNA sequencing. A total of 179 strains were obtained from the plaque of 10 dogs without periodontal disease. The technique employed allowed 15 different microbial groups to be isolated, including the genus Streptococcus. This represented 33 (18.4%) of 179 isolates, thus illustrating the partially selective ability of Mitis Salivarius Agar for samples of dental plaque in dogs.


Introduction
Periodontal disease, which affects the support structures and protection of teeth, is a disease that is the mostly found in dogs, reaching about 85% prevalence in animals over the age of 4 years [1]. The etiologic agent of this disease is the bacterial plaque which develops on the tooth surface and the of immune system's reaction to these agents. Plaque builds up with the initial colonization of the tooth surface by indigenous bacteria, and promotes the adhesion of other, more virulent bacteria [2].
In humans, the microbial profile of plaque is been defined. It is formed predominantly by Gram-positive, aerobic, nonmotile and anaerobic bacteria in the early stages of infection [3,4], but by Gram negative and mobile bacteria in the later stages of infection and inferred that the same microbial profile occurs in canine plaque [5].
The most important bacteria in the initial colonization of human dental plaque are Streptococcus [3], which produce exopolysaccharides that facilitate fixation of the bacteria to the surfaces, especially in places where minor irregularities occur, such as fissures or roughness [5]. Some studies have sought to identify the dental plaque microbiota in dogs using non-selective culture media, and have reported small amounts of Streptococcus [6][7][8]. However, since special conditions are required to isolate this kind of bacteria from samples with a diverse microbiota, its possible that it growth was inhibited by other bacteria. Other work presented low presence of Streptococcus spp. in enamel surface of dogs, performing the identification without culture techniques, using pyrosequencing of 16S rDNA, and presented bacteria of Neisseria, Moraxella and Bergeyella species as the main bacterial groups in this environment [9]. However in human studies is presented the need to use a selective medium such as Mitis Salivarius Agar to obtain higher presence of Streptococcus spp., from dental plaque samples [10].
Thus, the importance of the genus Streptococcus in the composition of canine dental plaque remains unknown. Such knowledge is essential to determining therapeutic procedures for the treatment or prevention of periodontal disease in dogs. The aim of this study was, therefore, to isolate and identify the initial dental plaque microbiota of dogs cultivable in a selective medium for Streptococcus and to verify the presence of this genus in the samples.

Animals and Sampling
Bacterial isolates were obtained from the supragingival plaque of 10 mongrel dogs aged between 1 and 2 years, without clinical signs of periodontal disease, periodontal pockets, gingival swelling or redness. All the animals were from the experimental kennels of the Department of Veterinary Medicine or treated at the Veterinary Hospital of the Federal University of Viçosa. All procedures complied with the rules established by the Ethics Committee in the use of animals at the Federal University of Viçosa, and were approved by the same protocol under No. 18/2010.

Sample Processing
The collection of dental plaque was performed using a modified version of the method described by [8]. Plaque was obtained from the fourth premolars of 10 dogs by friction, with the aid of sterile swabs. Following collection, the swabs were transferred to tubes containing a solution of Tris-HCl, pH 7.2. Glass beads were introduced and the tubes were placed in a vortex model MS1 minishaker (IKA Laboratory Equipment, Artur Nogueira, Brazil) at 2200 rpm for 2 minutes.
Suspensions were serially diluted on a decimal scale to 1:10000. All were inoculated on the surface of plates containing 20 ml Mitis Salivarius Agar (Himedia, Mumbai, India) and potassium tellurite 1%, prior to incubation at 37°C for 72 hours in microaerophilic environmet, in order to provide optimal conditions for the isolation of Streptococcus sp. using BBL GasPak Plus Anaerobic System Envelopes with Palladium Catalyst kit (BD Biosciences, New Jersey, USA).
After incubation, colonies with different morphological characteristics were selected and streaked on Brain Heart Infusion agar (BHI) (Himedia, Mumbai, India), after which they were again incubated, this time at 37°C for 24 hours, in order to obtain pure cultures. Following purification, one colony from each culture was transferred to a tube containing BHI and once again incubated for 24 hours at 37°C, at which point glycerol (20%v/v) was added and the cultures were frozen in liquid nitrogen and maintained at -80°C.
To subsequently activate the cultures, a 10 µL aliquot of each stock was transferred to a BHI broth and incubated at 37°C for 24 hours.

Phenotypic Determinations
Upon reactivation in BHI broth, the isolates were subjected to Gram staining and tested for catalase production as described by American Society for Microbiology [11,12].

Polymerase Chain Reaction -PCR
DNA was extracted using a Wizard® Genomic DNA Purification Kit (Promega Corporation, Madison, USA) according to the manufacturer's protocol. DNA extraction was performed using 1.5 mL of the activated culture. The DNA was purified using a Genomic DNA Kit™ GeneJET Purification (Fermentas International Inc., Maryland, USA) according to the manufacturer's protocol, eluted with 70 µL of water and stored at -20°C. DNA from Streptococcus salivarius CDC 262 was used as a positive control in the PCR and Nuclease-Free Water (Promega Corporation, Madison, USA) was used as a negative control.

Sequencing and Data Analysis
Amplified fragments of expected size, together with the positive control, were sent for sequencing by the Macrogen Company (in Korea), which used the method previously described by Sanger et al. [14].
The sequences obtained were edited using Sequencher software (version 4.

Nucleotide Sequence Accession Numbers
The nucleotide sequences determined in this study have been deposited in GenBank [GenBank: HQ717175 to HQ717353].

Results
A total of 179 strains were obtained from the plaque of 10 dogs without periodontal disease ( Figure 1). As expected, Gram staining indicated 90% positive cocci and rods, representing 164 isolates. These were 66% catalase negative. The 179 isolates had fragments of 16S rDNA amplified and sequenced. The sequences obtained were deposited in GenBank ( Supplementary Material 1 and 2) and identified by comparison with other gene sequences from the same database. Of the 179 isolates sequenced, 46 were identified to species level, while the other 133 were not (data not shown). Despite the targeting of the isolation technique for the genus Streptococcus, the bacterial diversity found in this study indicated a total of 14 genera belonging to three phyla. The genera Aerococcus, Bacillus, Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Staphylococcus and Streptococcus represented the phylum Firmicutes (low G+C, Gram-positive), Actinomyces, Arthrobacter and Micrococcus represented the phylum Actinobacteria (high G+C, Gram-positive), and Neisseria, Haemophilus and Pasteurella represented the phylum Proteobacteria (Gramnegative). A total of six isolates of the bacteria known as Bisgaard taxon 16 [16] were also found in two dogs.

Discussion
The technique employed in this study not only enabled the isolation of bacteria of the genus Streptococcus, but also other bacteria (Figure 1), including rods and Gram-negative, demonstrating the partially selective capability of the Mitis Salivarius Agar. Among the main genera isolated, Streptococcus was the third most common, surpassed by both Staphylococcus and Enterococcus (Figure 1).
Elliot et al. [6] identified the microbiota in the subgingival plaque of 10 dogs, with Streptococcus sp. representing only 1% of the total. Those investigations, however, utilized plates of a different nature and different culture media from those employed in the present study. From a total of 32 isolates obtained from dogs without gum disease using non-selective culture media, Riggio et al. [8] identified only one isolate of Streptococcus minor, which was also identified in this study, as well as in other studies examining plaque from dogs and cats [6,17,18]. Takada et al. [10] were able to isolate Streptococcus using Mitis Salivarius Agar with supplements, with a recovery rate of 21.2% of all isolates. They also reported that the medium removed 89.7% of total bacteria grown in BHI from the same blood sample. The main species identified by Takada et al. [10] were S. mitis and S. sanguis, demonstrating the bias of this medium towards the growth of Streptococcus.
It is noteworthy that none of the above studies was able to isolate S. mutans, which is considered the most important bacteria in human plaque formation. This corroborates the observation of Dent and Marsh [19] that there is a correlation between the presence of S. mutans and diets rich in sucrose, which is not the case with dogs. Takada et al. [10] suggest that the different pH of a dog's oral cavity, compared to that of a human, may be a factor responsible for the absence of this type bacteria in these animals.
Studies on the microbiota of supragingival plaque from beagle dogs, assessed using DNA-DNA hybridization, identified several Streptococcus species, including some associated with dental plaque microbiota in humans, such as S. mitis, S. constelattus, S. oralis and S. sanguis [20]. These species were not isolated in the current study, since the sequences of Streptococcus sp. obtained showed no similarity with the examples of these species deposited in GenBank. Controversially, Holcombe et al. [9] did not presented significant presence of Streptococcus spp., in enamel dental plaques of 12 dogs with initial dental plaque formation between 24 and 48 hours after dental professional cleaning, which could suggest that the presence of this bacterial group could be increased just after later events in the development of dental plaque in animals.
The difficulty in identifying bacteria to the species level, especially when dealing with the genera Staphylococcus, Streptococcus and Enterococcus, can be because closely related species may have virtually identical 16S rDNA gene sequences. Some species of Streptococcus group D, for example, share a 99% similarity in their 16S rDNA sequence [21].
Bacteria of the genera Actinomyces, Porphyromonas, Fusobacterium, Neisseria, Staphylococcus, Streptococcus and Veillonella were isolated in most samples analyzed by Dent and Marsh [19], Kim et al. [22], Zambori et al. [23] and Jeussete et al. [24], suggesting that representatives of these genera can be considered as characteristic or indigenous members of the microbial community in dental plaque. This was confirmed in the present study, which verified the presence of the genera Staphylococcus, Streptococcus, Actinomyces and Neisseria (as shown in Figure 1). As expected, the genera Porphyromonas, Fusobacterium and Veillonella, found in other works [19,22,23,24] were not isolated as result of the methodology applied and the nutritional and strict anaerobic needs of these bacteria.
Study by Nowroozilarki et al. [25], presented Helicobacter spp. and Wolinella spp., in oral cavities of toy breed dogs, different of our findings. However, the dog population studied differ from that used in our work. In that work the aim was to study dogs older from six years with periodontal disease, meanwhile in the present study were used young dogs without oral diseases, suggesting that the inclusion of that genera [25] occur in a new environment provided in oral dental plaque of older dogs by the evolution of that specific oral disease.
It is suggested that Bisgaard taxon 16 [16], that has not yet formally referred in the bacterial taxonomy, it is part of oral microflora of dogs, since the few reports of them were obtained from this site [6,16] and in this study, it was obtained from two different dogs.
The sequence analyses and literature review indicated that 59/179 (33%) of the bacteria isolated from dogs in this study are not normally found in human oral cavities [26,27]. Only 5/15 (33.3%) of the identified groups (14 genera plus Bisgaard taxon 16), namely Actinomyces, Neisseria, Haemophilus, Streptococcus and Lactobacillus, are considered members of human dental supragingival plaque [22,23].
Recent works [23,[28][29][30] presented the sensitivity profile of some isolates presented in this work when challenged by natural compounds from vegetable and other bacteria, with important findings in the study of dental plaque control in dogs. These studies have to be conducted to confirm these alternative therapic drugs to replace chlorhexidine use, that presents a set of side effects on its long-term use [5].

Conclusions
It follows that the technique employed in this study allowed 15 different microbial groups to be isolated, including the genus Streptococcus, which represented 18.4% of 179 isolates. This demonstrates the partially selective ability of Mitis Salivarius Agar when analyzing samples of dental plaque from dogs. In addition the results indicated that the genus Streptococcus spp. bacteria can be present in higher levels in dogs dental plaque environment, particularly those included in group D streptococci.