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ORIGINAL ARTICLE
Year : 2005  |  Volume : 23  |  Issue : 3  |  Page : 126-130
 

Estimation of salivary bacteria capable of inhibiting and stimulating Streptococcus mutans and its correlation to dental caries and untreated carious teeth


1 Department of Pedodontics and Preventive Dentistry, Yenepoya Dental College, Mangalore, India
2 Department of Pedodontics and Preventive Dentistry, MCODS, Mangalore, India
3 Department of Pedodontics and Preventive Dentistry, Yenepoya Dental College, Mangalore, Karnataka, India

Correspondence Address:
S K Hegde
Department of Pedodontics and Preventive Dentistry, Yenepoya Dental College, Mangalore
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-4388.16884

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   Abstract 

Bacteriocins are bacteriocidal proteinaceous molecules produced by the Gram-positive bacteria not active against the produced strain. Many investigations have revealed that certain bacteria using antibacterial or the inhibitory substance inhibit some other bacteria. A study was conducted in a group of 60 children to ascertain whether any correlation exists between the proportion of salivary bacteria inhibiting and stimulating Streptococcus mutans and the oral health indices (DMFT, deft and Community Periodontal Index of Treatment Needs). A definite inverse correlation was observed between the percentage of salivary inhibiting S. mutans and untreated carious teeth (UCT).


Keywords: Bacteriocins, Mutacins, Streptococcus mutans


How to cite this article:
Hegde S K, Kumar K B, Sudha P, Bhat S S. Estimation of salivary bacteria capable of inhibiting and stimulating Streptococcus mutans and its correlation to dental caries and untreated carious teeth. J Indian Soc Pedod Prev Dent 2005;23:126-30

How to cite this URL:
Hegde S K, Kumar K B, Sudha P, Bhat S S. Estimation of salivary bacteria capable of inhibiting and stimulating Streptococcus mutans and its correlation to dental caries and untreated carious teeth. J Indian Soc Pedod Prev Dent [serial online] 2005 [cited 2019 Dec 15];23:126-30. Available from: http://www.jisppd.com/text.asp?2005/23/3/126/16884


Dental infections such as dental caries and periodontal diseases are perhaps the most common bacterial infections affecting human beings.[1] These diseases are the resultant of alteration of the homeostasis among the indigenous oral bacterial population, which could be affected by various factors such as antimicrobial factors, or the inhibitory substances of human saliva.[2] The antimicrobial constituents of human saliva are divided into two main categories: nonantibody (innate) such as lysosymes, and antibody (acquired) factors such as IgG. Bacteriocins produced by the Gram-positive bacteria are bacteriocidal proteinaceous molecules not active against the producing strain. The role played by bacteriocins and other inhibitory substances in the ecology of the oral cavity remains controversial.[2] The bacterium Streptococcus mutans , which is indigenous to the mouth, is generally accepted to be the principal etiological agent of dental caries in human beings.[3] Among the different streptococcal species, S. mutans has shown to exhibit high cariogenic potential.[4]

The bacteriocins produced by the S. mutans are called mutacins. Some findings have failed to reveal an association between caries experience and mutacin activity.[5] However, it has been found by many in vivo investigations that mutacins reduce the number of viable salivary bacteria and strongly inhibit the cariogenicity of the cariogenic components like sucrose. It has been suggested that mutacins may be used as a preventive agent in the control of dental caries such as in mouth rinses.[3],[6],[7] Many investigations have revealed that certain bacteria using the antibacterial or inhibitory substances inhibit some other bacteria. These bacterial interactions maintain the homeostasis among the oral bacterial flora. [8],[9],[10] The existing literature in this aspect pertains to studies carried out in adults or in a population comprising of both children and adults.[2] Hence, this study was conducted entirely in a group of children to ascertain if any correlation exists between the proportion of salivary bacteria inhibiting and stimulating S. mutans and oral health indices.


   Aims and Objectives Top


1. To determine the correlation between the percentage of salivary bacteria capable of inhibiting or stimulating S. mutans and dental caries indices both in primary teeth and permanent teeth.

2. To determine whether there is any significant correlation between the percentage of salivary bacteria capable of inhibiting S. mutans and the percentage of untreated carious teeth (UCT).

3. To determine the correlation between the percentage of salivary bacteria capable of inhibiting or stimulating S. mutans and the Community Periodontal Index of Treatment Needs (CPITN).


   Materials and Methods Top


Sixty children (30 boys and 30 girls) aged between 7 and 14 years were randomly selected in the dental clinic. All children were healthy and were not taking any medication during the investigation period. Children with a history of consumption of antibiotics in the recent past were excluded from the study.

All the following procedures were evaluated by a single investigator, standardized by a pilot study and double-checked by the chief investigator. The dental caries status of each patient was evaluated using DMFT index (WHO 1986 criteria) for permanent dentition and deft index (as described by A.O. Gruebel in 1944) for primary dentition. Periodontal health status was evaluated using the WHO CPITN index (1982).

Paraffin-stimulated whole saliva samples were collected in a sterile bottle between 11:00 am and 12:00 pm, 1.5-2 hrs after eating. The child was asked to chew a piece of paraffin wax for 2 mins. After 2 mins, the child was asked to expectorate the accumulated saliva into the receiving sterile bottle. About 1-3 ml of saliva was collected from each patient. No transport medium was used as culturing was done within half an hour after collection of saliva samples. The sample was then subjected to microbiological analysis.

Saliva samples were cyclo-mixed with a cyclomixer for 1-2 min and subsequently diluted with phosphate buffered saline. The cyclomixer separates the bacteria from thick exudates and tissue. Saliva sample were plated on Todd-Hewitt agar enriched with 0.001% hemin and 0.0001% vitamin K using spiral plates [Figure - 1]. The total colony forming units were counted after incubating for 48 h, at 37C under anaerobic conditions.

Plates containing approximately 30 colonies were used for the deferred test. Plates containing less than 30 colonies were discarded. The plates were overlaid with 7 ml of soft Todd-Hewitt agar to which was added 400 ml of a cell suspension of a strain of S. mutans from an overnight culture in enriched Todd-Hewitt broth. The above mentioned cell suspension of S. mutans was isolated from 5 to 6 saliva samples by streaking it on mitis salivarius agar (MSA) and mitis bacteriocin agar (MBA) and then confirming the identification of S. mutans by catalase test (-ve) and mannitol motility medium. The plates were incubated at 37C for 48 h with S. mutans as an indicator strain. The colonies of salivary isolate producing a stimulatory zone (an overgrowth of overlayed bacteria around salivary isolate) or an inhibitory zone (nongrowth of overlaid bacteria around the salivary isolate) were counted and their proportion with respect to the total colony counts was determined for each saliva sample [Figure - 2]. For each patient one or two bacterial isolates showing an inhibitory or a stimulatory activity towards S. mutans were purified by restreaking twice on Todd-Hewitt agar and were then retested. The isolates inhibiting S. mutans were reassessed in a medium containing 1% arginine in order to reduce inhibition zone due to acid production. The bacterial isolates were all identified by the Vitek system.

Correlations between all parameters (CPITN, DMFT, deft, percentage of UCT, number of UCT, number of missing teeth, number of filled teeth, percentage of salivary isolates stimulating S. mutans , number of teeth present, and age) were calculated using the Spearman rank test. The significance threshold was fixed at P = 0.05. The significance of correlation was estimated by using the t -test.


   Results Top


Hundred and twelve salivary isolate inhibiting S. mutans were identified on first isolation. From this, nine did not survive the purification tests. From the 103 retested for inhibitory activity in the presence of arginine, only 23 were still truly positive. These 23 isolates were identified as follows:

1. Eight Streptococcus salivarius ;

2. Six Streptococcus sanguis ;

3. Six S. mutans ;

4. Three Streptococcus intermedius .

The percentage of isolates per salivary sample affecting S. mutans is presented in [Figure - 3]. The mean percentage of isolates inhibiting in UCT free and UCT bearing group is depicted in [Figure - 4]. The mean percentage of isolates inhibiting S. mutans was 59.91 with a SD of 14.23 for the total number of patients. The mean percentage of isolates stimulating S. mutans was 40.09 with a SD of 14.28. The range for the percentage of isolates stimulating S. mutans was 20-75.

No statistically significant ( P > 0.05) correlation was observed between the percentage of isolates inhibiting S. mutans and deft and CPITN [Figure - 5]. A statistically significant positive correlation was observed between the percentage of isolates inhibiting S. mutans and DMFT ( P < 0.05) [Table - 1]. A statistically significant negative correlation was observed between the percentage of isolates inhibiting S. mutans and number of UCT ( P < 0.001) [Figure - 6].

No statistically significant correlation ( P > 0.05) was observed between DMFT and deft index and CPITN index. No statistically significant correlation ( P > 0.05) was observed between the CPITN index and number of UCT and the percentage of UCT.

No statistically significant correlation ( P > 0.05) was observed between the percentage of isolates stimulating S. mutans and the DMFT and CPITN indices. A statistically significant positive correlation between percentage of isolates stimulating S. mutans and deft index ( P < 0.001) [Table - 1] percentage of UCT ( P < 0.001) number of UCT ( P < 0.001) and number of missing teeth ( P < 0.001), number of filled teeth ( P < 0.01) and age ( P < 0.001) was observed [Figure - 7].

When the total number of patients is considered, a significant negative correlation was found between the percentage of isolates inhibiting S. mutans and the percentage of UCT ( P < 0.001), and the number of UCT ( P < 0.001). No statistically significant correlation ( P > 0.05) was observed between percentages of isolates inhibiting S. mutans and CPITN index ( P > 0.05).

These results confirm that the salivary bacteria inhibiting and stimulating S. mutans plays a major role in dental caries in comparison to the periodontal diseases.


   Discussion Top


Dental caries and periodontal diseases may come into existence due to an alteration of the equilibrium of the oral bacterial population by many factors such as antimicrobial factors or the inhibitory substances of the human saliva.[2] Salivary peroxidase system, immunoglobulins, bacteriocins, and other inhibitory substances of saliva have been reported to exhibit a relation with caries prevalence[2],[3] Shiguki Hamada and Takashi Ooshima[10] have discussed the role of mutacin in the oral cavity and found that its role in the natural environment remains uncertain.

Interactions between the different inhibitory substances in the human saliva such as lysosymes, immunoglobulins, bacteriocins, etc., and S. mutans , its relation with dental caries and other dental diseases are a matter of controversy. Fukushima et al.,[6] Hirasawa et al.,[7] Ikeda et al.,[9] and others have reported that a strong relation exists between these substances and the dental diseases whereas Alaluusua et al.,[5] Erik Grahn et al.[11] and others did not find any correlation.

With so much of diversities in opinion, in the present study carried out on 60 healthy children in the age group of 7-14 years, an attempt was made to asses whether any correlation existed between the proportion of salivary bacteria capable of inhibiting and stimulating S. mutans and dental health indices.

The dental caries status was evaluated using the DMFT and deft indices, which are universally accepted criteria. The periodontal status was assessed by the CPITN index. Because Dasanayake et al.[12] has reported that stimulated saliva samples yielded higher number of S. mutans than unstimulated saliva, stimulated saliva was used for the study. Because stimulated saliva collected under standardized conditions was found to be appropriate for this study the collection of saliva was done as suggested by Higham and Edgar[13] and FDI Working Group 10, CORE.[14]

In this study there was a statistically significant positive correlation ( P < 0.05) between the percentage of salivary bacteria or isolates inhibiting S. mutans and the DMFT index ( P > 0.05). This may be because of more number of decayed teeth in the permanent dentition in the present population studied. There is no statistically significant correlation ( P > 0.05) between the percentage of isolates stimulating S. mutans and the DMFT index. This may be because the mean percentage of isolates stimulating S. mutans (40.09 with SD of 14.28) was less than the percentage of isolates inhibiting S. mutans (59.91 with SD of 14.23) observed in total population. This is in accordance with the study conducted by Goyette et al.[2]

In the current study, there is no statistically significant correlation between the percentage of isolates inhibiting S. mutans and the deft index ( P > 0.05). This agrees with the study done by Alaluusua et al.[5] However, there was statistically significant positive correlation ( P < 0.001) between the percentage of isolates stimulating S. mutans and deft index. This may be attributed to the extensive cavitations of the teeth in children, who usually present with excessive sucrose intake and elevated S. mutans and lactobacilli in the saliva. This was not reported by the study conducted by Alaluusua et al.[5] and Goyette et al.[2] A highly significant negative correlation was observed between the percentage of salivary bacteria inhibiting S. mutans and UCT. It was observed that there exists a proportional increase in percentage of isolates inhibiting S. mutans in a healthy mouth and its decrease in the mouth with dental caries. Therefore, this serves as evidence that certain amount of interaction is present between the percentage of isolates inhibiting S. mutans and the S. mutans in the arresting, susceptibility and activity of dental caries. This adds a possible therapeutic role of the isolates inhibiting S. mutans , tried successfully by Fukushima et al.,[6] Hirsawa et al.,[7] and Ikeda et al.[3] who used bacteriocin for the inhibition of dental caries. Because dental caries is a multifactorial disease, salivary bacteria inhibiting S. mutans contributed only slightly to the variability of these data. The studies carried out by Maries Demers et al.,[15] Herald M. Eriksen and Espen Bjertness,[16] and Jalil et al.[17] confirm this.

A statistically significant correlation was not observed between the percentage of isolates inhibiting and stimulating S. mutans and CPITN index ( P > 0.05). Because S. mutans participates in the plaque accumulation and plaque was not measured in this study, the results cannot be explained. There was statistically significant negative correlation ( P < 0.001) between the percentage of isolates inhibiting S. mutans and percentage of isolates stimulating S. mutans . However, it is very difficult to assess whether the above interactions actually exist in vivo because environmental conditions in vivo is entirely different. Because there is a highly significant negative correlation ( P < 0.001) between the percentage of isolates inhibiting and stimulating S. mutans and the percentage of UCT the importance of proportion of salivary isolates inhibiting and stimulating S. mutans in the dental caries is quite obvious. Future in vivo studies about the therapeutic use of the isolates inhibiting S. mutans in dental caries has to be carried out.

The following conclusions were drawn from the present study:

1. A positive relationship between the percentage of salivary bacteria or isolates inhibiting S. mutans and DMFT index was observed. On the other hand, DMFT index did not show any association with percentage of isolates stimulating S. mutans .

2. In this study, it was seen that the deft index showed a positive correlation between the percentage of isolates stimulating S. mutans in contrast to the no effect of isolates inhibiting S. mutans on the deft index.

3. There was a definite inverse correlation between the percentage of bacteria inhibiting S. mutans and UCT. On the contrary, a definite positive correlation observed showed an increase of UCT with an increase of percentage of isolates stimulating S. mutans .

4. There was no relationship between the percentage of salivary isolates affecting S. mutans and CPITN index.

The present study recommends use of salivary bacteria inhibiting S. mutans in the prevention of dental caries. Further in vivo studies are needed to confirm their efficacy.







 
   References Top

1.Walter J Loesche. Role of streptococcus mutans in human dental decay. Microbiological Reviews 1986;50:353-80.  Back to cited text no. 1    
2.Goyette N, Parrot M, Sutzescu D. Inverse correlation between the proportion of salivary bacteria inhibiting streptococcus mutans and the percentage of untreated carious teeth. J Oral Pathol Med 1995;24:462-7.  Back to cited text no. 2    
3.Ikeda T, Koulourides T, Kurita T. Anti dental caries effect in rats and man of a bacteriocin purified from the oral bacterium streptococcus mutans C3603. Archs Oral Biol 1985;30:381-4.  Back to cited text no. 3    
4.Chestnutt IG, Mac Farlane TW, Stephen KW. An in vitro investigation of the cariogenic potential of the oral streptococci. Archs Oral Biol 1994;39:589-93.  Back to cited text no. 4    
5.Alaluusua S, Takei T, Ooshima T, Hamada S. Mutacin activity of strains isolated from children with varying levels of mutans streptococci and caries. Archs Oral Biol 1991;36:251-5.  Back to cited text no. 5  [PUBMED]  
6.Fukushima H. Characterization and mode of action of a purified bacteriocin from the oral bacterium streptococcus mutans RM-10. Archs Oral Biol 1985;30:229-34.  Back to cited text no. 6    
7.Hirasawa M. Effect of mouthrinse containing a bacteriocin from streptococcus mutans on plaque microflora. J Dent Res 1984;63:1146.  Back to cited text no. 7    
8.Birgitta Lindquist. Claes-Goran Emilson: Interactions between and within streptococcus mutans and streptococcus sobrinus isolated from humans harbouring both species: Scand.J Dent Res 1991;99:498-504.  Back to cited text no. 8    
9.Ikeda T, Kurita T, Hirasawa M. Suppression of streptococcus sobrinus 6715(g) in the plaques by streptococcus mutans 32K(c). Journal of Oral Pathology 1988;17:471-4.  Back to cited text no. 9  [PUBMED]  
10.Shiguki Hamada. Takashi Ooshima:Inhibitory spectrum of a bacteriocin like substance (mutacin) produced by some strains of streptococcus mutans. J Dent Res 1975;54:140-5.  Back to cited text no. 10    
11.Erik Grahn. Antimicrobial systems of human whole saliva in relation to dental caries, cariogenic bacteria and gingival inflammation in young adults. Acta Odontol Scand 1988;46:67-74.  Back to cited text no. 11    
12.Dassanyake AP. Differences in detection and enumeration of mutans streptococci due to differences in methods. Archs Oral Biol 1995;40:345-51.  Back to cited text no. 12    
13.Higham SM, Edgar WM. Effects of paraffin and cheese chewing on human dental plaque pH and metabolism. Caries Research 1989;23:42-8.  Back to cited text no. 13  [PUBMED]  
14.FDI Working Group 10 CORE: Saliva:Its role in health and diseases. Inter Dental J 1992;42:291-304.  Back to cited text no. 14    
15.Maries Demers, Jean Marc Bourdeur, Paul L Simard. Caries predictors suitable for mass screenings in children: A literature review. Community Dental Health 1990;7:11-21.  Back to cited text no. 15    
16.Herald M Eriksen, Espen Bjertness. Concepts of health and diseases and caries prediction: A literature review: Scand. J Dent Res 1991;99:476-83.  Back to cited text no. 16    
17.Jalil RA, Ashley FP, Wilson RF, Wagaiyu EG. Concentrations of thiocynate, hypothiocynate, " free" and "total" lysosyme, lactoferrin and secretory IgA in resting and stimulated whole saliva of children aged 12-14 years and the relationship with plaque accumulation and gingivitis. J Periodont Res 1993;28:130-6.  Back to cited text no. 17  [PUBMED]  


Figures

[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6], [Figure - 7]

Tables

[Table - 1]


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[Pubmed]



 

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