International Medical Journal Vol. 26, No. 6, pp. 487 - 490 , December 2019

MICROBIOLOGY

Isolation and Characterization of Microbial Flora from Dental Plague Patients

U. S. Mahadeva Rao1), J. Sivakumar2), C. Shanmuga Sundaram3)

ABSTRACTObjectives: The present research is to explore the supragingival bacterial plaques in kids by caries. Methods: Totally 100 plaque samples were collected by a sterile curette from the supra-gingival and the sub-gingival areas of

the tooth by scraping from patients of age between 5 - 72 years and were given serial nos. Four different types of commercially available mouth rinses were taken to test their action against dental plaque with different concentrations.

Results and Discussion: Once the primary isolation was performed, the organisms were identified. As a result, totally six bac-terial isolates viz Streptococcus sp, Staphylococcus sp, Micrococci sp, Neisseria sp, Haemophilus sp, and Enterobacter sp were identified. During the isolation of bacteria from the patient sample, it was found out that bacterial diversity was varied from one age group to another. In this present study, the action of four commercial mouth rinses was studied against plaque by an in vitro method. The most effective mouth rinse on the entire patient's was determined and their effectiveness in percentage was calcu-lated.

Conclusion: The plaque is said to contain almost 500 bacteria of which only 50 are isolated and identified.

KEY WORDSdental plaque, Streptococcus sp, Staphylococcus sp, Micrococci sp, Neisseria sp, Haemophilus sp, Enterobacter sp,

Chlorhexidine

Received on January 15, 2019 and accepted on May 17, 20191) Faculty of Medicine, Universiti Sultan Zainal Abidin Terengganu, Malaysia 2) PG & Research Department of Biotechnology, Hindustan College of Arts & Science Padur, Chennai 603 103, India3) PG & Research Department of Microbiology, Hindustan College of Arts & Science Padur, Chennai 603 103, IndiaCorrespondence to: U. S. Mahadeva Rao(e-mail: raousm@gmail.com)

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INTRODUCTION

Dental plaque can be different as the diverse communities of micro-organisms establish on the tooth surface as a biofilm, implanted in an extracellular template of polymers of congregation and microbial source. There is a high concentration of alarm in the properties of bio-films and microbial communities across all sectors of industrialized, eco-logical and medical microbiology1). Research over more than a few decades has provided a hard base for present studies of oral biofilms. Various literary studies have revealed the diversity of the inhabitant oral microflora, both at the genus and species rank in wellbeing and ailment2).

Dental plaque is a composite of microbes rising as a biofilm on enamel surfaces. The etiology of both dental caries (tooth decay) and diverse forms of periodontal disease has documented to be connected to bacterial accumulations and plaque composition. Although widespread investigation of plaque samples from well and ailing subjects as well as facts resulting from gnotobiotic and untainted animal experiments, no single microbe has been branded which satisfies Koch's postulates for a contagious mediator in moreover caries or periodontitis3-5).

Salam compared the endurance of individual dental plaque flora in different transfer media. Their data showed that, the organisms had high survival rate in reduced transport fluid. The major side effects of chlor-hexidine are a brown tint of teeth and tongue, pattern of supragingival calculus, flavor change and oral desquamation in kids. Moreover aller-gic reaction has been reported in some patients particularly Asians6).

Loesche7) recommended that the pre - requisite for the formation of plaque is the occurrence of the foremost bacterium Streptococcus mutans. Metee and Gold8) have found that the salivary Streptococcus sp level is hardly or not at all subjective by the time of the day the sample is taken or by oral hygiene measures. Lijemark9) revealed that the early colonizers of the tooth surface include members of the genera Streptococcus sp, Haemophilus sp., Neisseria sp. and Veillonelle sp. Hillman10) indicated that practical regimen for contamination by an effector strain might be achieved for the alternative therapy of dental plaque. Mouthwash or mouth rinse is a product used for oral hygiene. Antibacterial and anti-plaque mouth rinse claims to destroy the bacterial plaque causing caries, gingivitis and bad breath. Anti-cavity mouth rinse uses fluoride to defend aligned with tooth decay.

Walker11) planned that Chlorhexidine, as a microbial agent is effec-tive in vitro against gram negative and gram positive bacteria as well as aerobes and anaerobes. Gibbons12) said that the bacteria stick on to the acquired enamel pellicle by precise and non - specific molecular exchanges among adhesions on the cell and receptors on the surface. Beighton13) proposed that there are diverse factors concerned in the pat-tern of the dental plaque is frequently affected by changes in dietary behaviour, prescription, ailment and denture wearing.

Rams14) over and done with that about 500 bacterial species was iso-lated from the dental plaque, a very good microbial niche. It forms in a regimented way and has a various combination of microbes. Daniluk15) had done that in adults the mainly predominant tiny anaerobes resem-bling Streptococcus sp, whereas in supra gingival plaque Streptococcus

C 2019 Japan Health Sciences University & Japan International Cultural Exchange Foundation

Mahadeva Rao U. S. et al.488

sp and staphylococcus sp were more predominant.The aim of the present research is to decide the supra gingival bac-

terial plaques in kids by caries would differ from persons found in car-ies-free controls and also deals with isolation and characterization of bacterial flora from dental plaque in patients of different age groups and action of four commercially available mouth rinses against plaque.

MATERIALS AND METHODS

Collection of SampleTotally 100 plaque samples were collected by a sterile curette from

the supra-gingival and the sub-gingival areas of the tooth by scraping from patients of age between 5 - 72 years and were given serial nos. Samples were collected in a wide mouthed screw cap bottle containing Robertson's Cooked Meat Medium. Media were sterilized by autoclav-ing at 121℃ for fifteen min in clean glassware's. The sample were immediately transported to the lab within 3 hours after collection or refrigerated.

Isolation of MicrobesThe isolated colonies grown were sub cultured in agar slants and

conserved under refrigeration temperature. The identification of bacteria was performed on the basis of macroscopic and microscopic examina-tion as per Bergey's Manual of Determinative Bacteriology16). The sam-ple were directly streaked on to Nutrient Agar, MacConkey Agar and Blood Agar and incubated at 37℃. Distinct colonies from these medium

were isolated and maintained as pure cultures on nutrient agar slants. Each isolates were examined many times for its size, shape, margin, consistency, opacity, elevation, pigmentation, gram reaction, motility, endospore staining and cell morphology. The various biochemical anal-yses were done to identify the organisms. Once all the biochemical anal-ysis and the carbohydrate fermentation have been performed, the organ-isms are identified by the help of Bergey's Manual. Skim Milk Agar was prepared by autoclaving at 121℃ for 15 mins, cooled and poured onto sterile Petri plates and allowed to solidify. The colonies from various pure cultures viz, 1A, 2A, 3B, 1C, 3D were streaked onto the skim milk agar and incubated at 37℃ for 24 hours and observed.

Mannitol Salt Agar was prepared by autoclaving the medium at 121℃ for 15 minutes. The colony from 3B (b) was streaked onto the medium and incubated 37℃ for 24 hours and observed. Loopful of col-ony from 2C (q) was streaked onto the medium and incubated at 37℃ for 24 hours and observed. The medium was prepared by autoclaving at 121℃ for 15 minutes, cooled. It was poured onto sterile petri plates and allowed to solidify. Colonies from 3A (a), 3B (b) were streaked onto the medium and incubated at 37℃ for 24 hours and observed.

Antimicrobial Testing with Mouth RinsesFour different types of commercially available mouth rinses were

taken to test their action against dental plaque such as Tantum, S- flo, Senquel and Rexidin and these are used, with different concentrations.

Sterile MHA plates were prepared, by autoclaving the medium at 121℃ for 15 min. the media was distributed onto sterile Petri plates and allowed to solidify. Samples from different age groups were taken and were swabbed with a sterile cotton swab. 4 wells were cut on the plates and marked with the respective names of the mouth rinses. 50 μl of the four mouth rinses were added to the wells and plates were incubated at 37℃ for 24 hours and were observed.

The samples were collected from the patients with dental plaque with the help of a curette from the supra gingival region and transferred to Robert Son's Cooked Meat Medium and transported to the lab within 3 hours and incubated at 37℃ for 2 days to obtained a confluent growth. Immediately, after incubation the samples were processed by performing various tests like, Gram's Staining, Mortality, Primary Isolation- Nutrient Agar, MacConkey Agar and Blood Agar.

Once the primary isolation was performed the organisms were iden-tified based on the biochemical and carbohydrate fermentation tests, with the help of Berge's manual. From these results, the predominant bacteria among all patients were determined. To maintain one's oral hygiene, not only tooth paste and brushes is useful but also certain com-mercially available products like mouth rinses or mouth washes. There are many types of mouth rinses that are used by millions of people now days. From these 4 different commercial preparations were taken and their action against dental plaque among all the patients was treated. The most effective mouth rinse on the entire patients was determined and their effectiveness in percentage was calculated.

Table. 1 Isolation of Bacteria from various age group patients

S.NO Age Group Number of Number of Range Patients Isolates in %

1 5 - 19 25 56 (25.42%)

2 20 - 34 25 45 (20.53%)

3 35 - 49 25 49 (22.25%)

4 > 50 25 70 (31.08%)

Table.2 Biochemical Characteristics of Bacterial Isolates. S. Test Staphylococcus Streptococcus Micrococci Neisseria Haemophilus Enterobacter NO sp sp sp sp sp sp

1 Gram + + + _ _ _ staining

2 Motility _ _ _ _ _ _

3 Catalase + _ + + + +

4 Oxidase + + + + + _

5 Indole _ _ _ _ _ _

6 Methyl red + _ + + _ +

7 Voges + _ _ _ _ + Proskauer 8 Citrate V _ _ + _ +

9 Urease V _ _ + _ +

10 H2S _ _ _ _ _ _

11 TSI + _ + _ + +

Figure 1. Colony Morphology of Bacterial Isolates from Dental Plague Patients

Isolation and Characterization of Microbial Flora 489

RESULTS

Among 100 patients with dental plaque, 70 (31.8%) of bacterial iso-lates were isolated from the age group greater than 50 years (Table. 1). As a result, totally six bacterial isolates viz Streptococcus sp, Staphylococcus sp, Micrococci sp, Neisseria sp, Haemophilus sp, and Enterobacter sp were identified. White and red coloured; margined and elevated colonies were observed in all the samples. In the Gram's staining one of the puri-fied strains was positive and the other produced negative results (Table.2). Based on the Bergey's manual's reference; the screened organisms were confirmed as Streptococcus sp, Staphylococcus sp, Micrococci sp, Neisseria sp, Haemophilus sp, and Enterobacter sp (Fig. 1).

The study carried out in 100 patients aged between 5 to 72. Among four groups such as aged 5-19 (mean age 11.4 years); 20-34 (mean age 28.6 years); 35-49 (mean age 43.4 years); greater than 50 (mean age 56.2 years). Caries prevalence was diagnosed visually. In each group, 25 patients with caries were taken for analysis. The numbering was done for each patient sample in all the age group with the starting letter 'S' followed by numerical number.

During the isolation of bacteria from the patient sample, it was found out that bacterial diversity was varied from one age group to another. The sampling number would be same from all the age group, but number of bacterial isolates was not correlated. Among the four dif-ferent age groups, aged > 50-year group patients had more number of isolates in the percentage of 31.8 which was not followed by the group aged 5-19 (25.42) (Table 1 and Fig. 2).

Mouth rinse Tantum showed the highest zone of inhibition 30.33 mm against Enterobacter sp where as the lowest zone of inhibition 18.66 mm against Haemophilus sp. Rexidin showed the Zone of inhibi-tion against all the isolated bacteria. S-flo and Senquel didn't show any Zone against Staphylococcus sp, Streptococcus sp and Micrococci sp. S-flo mouth rinse showed Zone of 12 and 12.66 mm against Haemophilus sp and Enterobacter sp. Whereas Senquel showed Zone of 18.33 and 14 mm against Neisseria sp and Enterobacter sp.

DISCUSSION

The oral cavity is similar to other sections of the digestive tracts in having inhabitant micro flora that develops naturally, and which has a distinguishing composition. Due to difference in restricted ecological conditions, the plaque micro flora differs from other mucosal surfaces. The resident micro flora of a site is of benefit to the host by acting as part of host defences by preventing colonization by exogenous spe-cies14). The dental plaque is a structurally and functionally organized bio-film. Plaque forms in an ordered way and has a diverse microbial composition. Several bacterial species or cluster of species has been involved in the aetiology of the dental plaque16).

The combined cultural and molecular analyses have shown that a diverse community is found in dental plaque and that numerous novel taxa are present17). The exposed surfaces of the tooth are to be consid-ered as the open dentin ecosystems. These exposed surfaces are rapidly colonized by microorganisms and covered by dental plaque18). About 1g of dental plaque contains more than 1011 organisms19). These bacteria remain to the acquired enamel pellicle by specific and non-specific molecular interactions between adhesions on the cell and receptors on the surface12).

In the current research among the four different age groups, aged > 50 year group patients had more number of isolates in the percentage of 31.8 which was not followed by the group aged 5-19 (25.42) Table 1 and Fig. 2. The results are coincided with the previous reports20).

Also a similar theory was that the ability of microorganisms to settle the different oral surfaces mainly depends on the binding potential of the organisms4). Factors that include in formation of plaque are often affected changes in dietary habits, medication, disease and denture wearing13). This was also confirmed by Cao et al,21) revealing that diverse ecological factors and concerned in the harbouring of microor-ganisms and microbial composition on dental plaque.

Studies on microbes causing plaque from different geographic regions including developed and developing countries will show varia-tions in the numbers and species of cultivable flora22). The dental plaque

Figure 2. Bacterial Isolates and Antibacterial activity against various brands of Mouth rinses

Mahadeva Rao U. S. et al.490

is material which supports the growth of wide variety of bacteria. There are almost 500 species of bacteria14). This was in contrast with the find-ing of Thylstrup A et al23) that, 700 bacterial species were present in the oral cavity, of which only 50 were cultivated and detected.

As mentioned earlier, several bacterial species are isolates from the dental plaque and studied. The isolation and identification bacteria from dental plaque are based on the appearance of colony and pigment pro-duction, staining reactions and biochemical results5,24). In this current investigation, the isolation and identification is also carried, on the basis of these criteria.

The plaque is a very novel microbial niche, which supports the growth of many microbes. The most predominant organisms in the supra - gingival plaque were Staphylococcus sp and Streptococcus sp15). The cu l t ivab le microf lo ra o f den ta l p laque cons t i tu ted of Staphylococcus sp (0 - 46%), Streptococcus sp (8 - 86%), Gram nega-tive cocci (0 - 46%). this was similar to the present study's findings, where both Staphylococcus sp and Streptococcus sp amounted to 25% among all samples. 239 strains were isolated from kids with plaque. Among the isolates Gram positive bacteria were more than Gram nega-tive, which was very much similar to the study conducted here on dental plaque. Streptococcus sp. was more predominant from dental plaque25-27).

The rates for Pseudomonas sp were higher in patients with dental plaque from hospital. This study was exactly similar to the present topic of consideration were Pseudomonas sp was one of the predominant organisms among all patients. Antimicrobial and plaque inhibitory agents in mouth washes / rinses are used to inhibit further formation of plague and as a result prevent Chronic Gingivitis on the gums and the tooth. Many mouth rinses are used, with different formulations and ach has a distinct or different amount of inhibition on the dental plaque. Regular rinsing with 0.1% - 0.2% Chlorhexidine have been reportedly shown to result in a decrease in plaque mass and reduction in the gingivitis28).

Chlorhexidine as a microbial agent is effective in vitro against aer-obes, anaerobes both gram negative and gram positive bacteria11) this finding correlated with that of the present study where also, Chlorhexidine emerged as the most effective mouth rinse. Three mouth rinses with Benzydamine Hydrochloride, separately or in combination did not affect the secretion of saliva or microbiota and dental plaque for-mation29).

In the present study Chlorhexidine was more efficient in reducing mutants streptococci count in saliva as compared to other mouth rinses, this is coincided with reports of30). Chlorhexidine as a microbial agent is effective in vitro against both gram positive and gram negative bacteria including yeasts5). In conclusions, culturing of organisms plays a vital role to detect from the dental samples. Antibiotic resistance data required for cultured microorganisms to elucidate of virulence mechanism. However, cultured bacteria may rapidly adjust their phenotypic features in vitro, and 50% of oral microorganism have not till now been cul-tured3,19). It will also be significant to control the oral microflora for sys-temic reasons since strong associates are being recognized between focal infection of oral origin and a range of systemic diseases as well as coro-nary heart disease, gastrointestinal disorders and low birth weight, apart from severe overt systemic function. These developments are divided from an improved understanding of the ecological nature of the microbi-al biofilm that is dental plague, and of its interactions with human host2).

CONCLUSION

Dental plaque is a soft tissue that accumulates on the teeth. The plaque is said to contain almost 500 bacteria of which only 50 are isolat-ed and identified. Dental plaque is a mild problem, when unattended may lead to damage to the tooth and the gingiva. Dental plaque can be decreased by various methods like regular brushing, use of mouth rinses and also by good oral habits. The most effective mouth rinse on the entire patients was determined and their effectiveness in percentage was calculated. Dental plaque is a slender, persistent microbial coat that forms on the tooth surfaces. It supports the growth of very many species. And the accumulation of it causes serious damages to the tooth. There is a wide variety of bacterial species like Bacillus sp, Pseudomonas sp, Staphylococcus sp, Streptococcus sp. Of which Bacillus sp and Pseudomonas sp. were most predominant. Accumulation of plaque on tooth is unavoidable, but can be prevented and taken care. In this present study, the action of four commercial mouth rinses was studied against plaque by an in vitro method. Rexidin (Chlorhexidine) was the most effective mouth rinse among all the mouth rinses.

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