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Acta Odontológica Latinoamericana

versión On-line ISSN 1852-4834

Acta odontol. latinoam. vol.22 no.2 Buenos Aires set. 2009

 

ARTÍCULOS ORIGINALES

Antagonistic action of indigenous Streptococcus mutans strains

 

Fredy Gamboa1,2, Margarita Chaves2, Claudia Lamby2, Ana Fajardo3, Azucena Arévalo3

1 Department of Microbiology, Faculty of Sciences.
2 Dental Research Centre, Faculty of Dentistry.
3 Bacteriologist. Javeriana University, Bogotá, Colombia.

CORRESPONDENCE Dr. Fredy Gamboa Departamento de Microbiologia (Facultad de Ciencias) y Centro de Investigaciones Odontologicas (Facultad de Odontologia) Pontificia Universidad Javeriana Carrera 7 No. 40-62 Phone: 3208320 Ext. 2899 / Fax: 3208320 Ext. 2884 Bogota - Colombia E-mail: gamboa@javeriana.edu.co


ABSTRACT

Dental caries is an infectious process which ultimately destroys the tooth. Streptococcus mutans is considered to be the main agent causing this disease. If microorganisms with antagonistic action on S. mutans were found, they might provide a way of avoiding or controlling the disease. Within the framework of the Oral Microbial Ecology approach, the aim of this project was to identify S. mutans strains with antagonistic effect upon S. mutans. Saliva samples were taken from 66 children and cultured on Blood agar and Mitis Salivarius Bacitracin agar. They were incubated at 37oC in anaerobic atmosphere for 48 hours, after which bacteria were counted and biochemical tests performed on colonies compatible with S. mutans. Antagonistic effect was determined using the double layer in agar technique. In children without and with caries, the frequency of S. mutans was 91.7% and 96.7%, respectively. In the group of patients without caries, only two strains had no antagonistic action, and three strains had full antagonistic action (100%), while the rest showed different kinds of inhibitory action. In the group of patients with caries, only 5 strains had no antagonistic action, 32 strains had full antagonistic action (100%) and the rest had variable inhibitory action. To conclude, 112 S. mutans strains with high antagonistic potential were identified, which, after other requirements are fulfilled, could be used in caries prevention or control strategies.

Key words: Dental caries; S. mutans; Antagonism; Probiotics.

RESUMEN

Acción antagónica de cepas indígenas de Streptococcus mutans

La caries dental es un proceso infeccioso que termina en la destrucción del diente. Streptococcus mutans es considerado el principal agente causal de esta enfermedad. La búsqueda de microorganismos con acción antagónica sobre S. mutans puede ser una alternativa con la cual se pueda evitar o controlar esta enfermedad. Este proyecto enmarcado dentro de la línea de Ecología Microbiana Oral, tuvo como objetivo identificar cepas S. mutans con efecto antagónico. Se tomaron muestras de saliva en 66 niños y se cultivaron en Agar Sangre y Agar Mitis Salivarius Bacitracina. Después de la incubación a 37oC en anaerobiosis durante 48 horas, se hizo el recuento bacteriano y las colonias compatibles con S. mutans fueron sometidas a pruebas bioquímicas. La determinación del efecto antagónico se realizo utilizando la técnica de doble capa en agar. En los niños sin caries y con caries la frecuencia de S. mutans fue, respectivamente, 91.7% y 96.7%. En el grupo de pacientes sin caries solo dos cepas no tuvieron ninguna acción antagónica, tres cepas tuvieron acción antagónica completa (100%), y las restantes presentaron diferentes modalidades de inhibición. En el grupo de pacientes con caries solo 5 cepas no tuvieron ninguna acción antagónica, 32 cepas tuvieron acción antagónica completa (100%) y las demás cepas tuvieron actividad inhibitoria variable. En conclusión, se identificaron 112 cepas S. mutans con gran potencial antagónico, las cuales después de cumplir con otros requerimientos podrían ser utilizadas en estrategias de prevención o control de la caries dental.

Palabras clave: Caries dental; S. mutans; Antagonismo; Probioticos.


 

INTRODUCTION

Dental caries is an infectious, multifactorial, localized, post-eruptive, transmissible pathological process that leads to the destruction of hard dental tissue1,2. The main microorganisms asociated to caries production are, in order of frequency: (1) Streptococcus mutans (mainly serotype c), an acidogenic, aciduric microorganism that normally colonizes the oral cavity, and to a lesser extent S. sobrinus and S. gordonii; and (2) species of Lactobacillus and Actinomyces1-3. Different studies have shown a strong correlation between S. mutans counts in dental plaque and the prevalence and incidence of caries1,4,5. The recognition of S. mutans as the most important microorganism initiating caries has led to the design of preventive measures aimed at eliminating or reducing it in the oral cavity6. Different strategies have been proposed for preventing and controlling dental caries6. In Colombia, the actions carried out by institutions and health guidelines for caries prevention and control have not sufficed to achieve a significant reduction in the disease, which still persists in over 90% of the Colombian population7. This situation calls for further study and review of all the specific dental caries prevention techniques or measures8.
The search for microorganisms with antagonistic action on caries-causing S. mutans strains, as well as the application of these microorganisms, belong to an increasingly researched strategy, which might allow the disease to be prevented or controlled8-10. Microbiological control (replacement therapy), more recently known as “probiotic therapy”, may be an alternative for controlling the microbial species involved in dental caries, without any negative effect on the other species that make up the oral microbiota8-10. This study is part of the Oral Microbial Ecology line of research, and the aim was to identify indigenous S. mutans strains with antagonistic effect.

MATERIALS AND METHODS

1. S. mutans isolation, count and identification
Study population
After obtaining informed consent from parents or tutors, and in compliance with bioethical standards for sampling and handling, 66 children aged 3 to 5 years from a pre-school in Bogota were included in the study. Each child was examined clinically for caries experience by an examiner who determined the dmft index (decayed, missing and filled teeth) according to the World Health Organization criteria11. No X-rays were taken of any of the children. Of the 66 children, 36 had no dental caries, while 30 had caries with an average dmft index of 3.3 (range 2-5). The 66 children included in the study had no infectious systemic disease and had not been under antimicrobial treatment during at least 7 days prior to the sampling. A sample of unstimulated saliva was collected from each child using gentle aspiration with a plastic pipette.

Sample processing
The saliva samples were vortexed for 15 seconds and serially diluted (1/10, 1/100 y 1/1000) with 0.05 M phosphate buffer. 50 ul of each dilution was plated on Lamb’s Blood agar and Mitis Salivarius Bacitracin agar (MSB, Difco Laboratories; Detroit, MI). The MSB agar was used to count S. mutans and carry out its selective isolation. For its definitive use, MSB agar contains digested pancreatic casein, proteose peptone No 3, proteose peptone, dextrose, 20% sucrose, dipotassium phosphate, trypan blue, crystal blue, agar, Chapman tellurite and bacitracin 0.2 U/ml. The Petri dishes containing the agars (Lamb’s Blood Agar and MSB Agar) were incubated at 37oC for 48 hours in anaerobic atmosphere (H2:CO2:N2 10:10:80). After bacterial growth on MSB Agar, colonies with morphological characteristics of S. mutans were counted12 and 5 colonies per sample were collected for Gram stain, examination of catalasic activity and biochemical tests. The samples were plated on Lamb’s Blood Agar to observe total growth of bacteria present and correlate it with the growth on MSB agar. On Lamb’s Blood Agar, the characteristics of colonies compatible with S. mutans can be observed. They are small, translucid, creamy, shiny and with type á or ã hemolysis. The number of S. mutans colonies on MSB Agar was expressed in colony-forming units (CFU) per ml of unstimulated saliva. The following biochemical tests were run to identify S. mutans: fermentation of raffinose, mannitol, melibiose, trehalose and inulin; esculin hydrolysis in presence and absence of bile; urease; arginine hydrolysis and bacitracin resistance. S. mutans has the following biochemical profile: positive fermentation of raffinose, mannitol, melibiose, trehalose and inulin; negative esculin hydrolysis in presence of bile and positive esculin hydrolysis in absence of bile; negative urease; negative arginine hydrolysis, and resistance to 2 U of bacitracin. The commercial Api 20S system (bioMerieux, Marcy-letoile, France) was also used for identifying strains.

2. Strain biotyping
All isolated S. mutans were biotyped using the api- ZYM (bioMerieux, Marcy- letoile, France) system according to the manufacturer’s instructions. The api-Zym system is a semi-quantitative micro-method for research that enables 19 enzymatic activities to be detected rapidly and simultaneously from small amounts of bacterial inoculum. It consists of a strip with 20 microtubes or wells (1 control and 19 tests), the bottoms of which contain the substrates and buffer. Microtube 1 is the control for the test, and substrates 2 to 20 correspond respectively to 2- naphtyl phosphate, 2-naphtyl butyrate, 2 naphthyl caprylate, 2-naphthyl myristate, L-leucyl-2- naphthylamide, L-valyl-2-naphthylamide, L-cystyl-2- naphthylamide, N-benzoyl-DL-arginine- 2-naphthylamide, N-glutaryl-phenylalanine-2-naphthylamide, 2-naphthyl phosphate, Naphthol-AS-BI-phosphate, 6-Br-2 naphthyl-Alfa D-galactopyranoside, 2-naphthyl- Beta D-galactopyranoside, Naphthol-AS-BIBeta D-glucuronide, 2-naphthyl-Alfa D-glucopyranoside, 6-Br-2naphthyl-Beta D-glucopyranoside, 1-naphthyl-N-acetyl-BD-glucosaminide, 6-Br-2- naphthyl-Alfa D-mannopyranoside, 2-naphthyl-Alfa L-fucopyranoside. The base of the system allows contact between the microorganism’s enzyme and the usually insoluble substrate. Substrates are inoculated with a dense bacterial suspension (turbidity equivalent to a McFarland number 5 or 6), which rehydrates the substrates and produces enzymatic action on them. The end-products formed during a 4-hour incubation period are detected by colour reactions after adding reagents. The tests are read by comparing the colours they produce to a colour code provided by the manufacturer. Biotyping was duplicated and the biotypes were assigned according to the action of the S. mutans strains on the system’s 19 substrates.

3. Determination of antagonistic effect in the isolated S. mutans strains
The antagonistic effect was determined by means of the double layer test in BHI (Brain Heart Infusion) Agar, on which strains that act as effectors and strains that act as indicators were plated. Effector strains are those that will have antagonistic action on indicator strains. To this end, two or three colonies of each S. mutans strain from the BHI Agar were re-suspended in BHI broth and incubated at 37oC in anaerobic atmosphere (H2:CO2:N2 10: 10:80) for 48 hours. This suspension was plated on BHI Agar (1.5% agar and 2% yeast extract) using a micro-pipette (2 ul) and incubated at 37oC in anaerobic atmosphere (H2:CO2: N2 10:10:80) for 48 hours. The indicator strains were subsequently placed upon the effector strains. Indicator strains are those that will be acted upon by the effector strains, and they were selected according to the frequency of biotypes present. To this end, two or three colonies of each S. mutans strain from the BHI Agar were re-suspended in BHI broth and incubated at 37oC in anaerobic atmosphere (H2:CO2:N2 10: 10:80) for 48 hours. Subsequently, 0.5 ml of this suspension was mixed with 5 ml of BHI Agar (0.75% agar and 2% yeast extract) and immediately added to the BHI agar (1.5% agar and 2 % yeast extract) on which the strains prepared in the previous step had grown. These Petri dishes with double layer BHI Agar, in which both the effector strains and the indicator strains are plated, were incubated at 37oC in anaerobic atmosphere (H2:CO2:N2 10: 10:80) for 48 hours. After these final 48 hours, antagonistic action is reflected by the presence of an inhibition halo produced by the effector strain on the indicator strain. Inhibition halos larger than 4 mm are considered in order to determine antagonistic effect13.

RESULTS

S. mutans frequency and count
In children without caries and with caries, the frequency of S. mutans was, respectively, 91.7% (33/36) and 96.7% (29/30). Children with caries had a higher S. mutans count than children without caries and the differences in counts between the two populations were statistically significant (Two-sample Wilconson rank-sum (Mann-Whitney) test, Prob > / z / = 0.0000). Of the 62 children (33 in the group without caries and 29 in the group with caries) from whom S. mutans was isolated, 119 colonies were identified as S. mutans: 51 in children without caries and 68 in children with caries.

S. mutans biotypes
Tables 1 and 2 show enzymatic behaviour on the substrates in the api-ZYM system, of the 119 S. mutans strains isolated from children with and without caries. Biotypes were assigned according to a previous standardization. The wide range of activity of these microorganisms allowed 85 biotypes to be formed from the 119 S. mutans strains isolated: 33 biotypes in the strains isolated from children without caries and 52 biotypes in the strains isolated from children with caries. The two groups of strains had 4 biotypes in common (biotypes 5, 6, 9 and 12). The most frequent biotypes in children without caries were 6, 9, 5 and 3, with 5, 4, 3 and 3 strains respectively; and in patients with caries the most frequent biotypes were 37, 39, 6 and 9, with
5, 3, 3 and 3 strains respectively (Tables 1 y 2). A large number of biotypes represented by a single strain was found in both groups of patients.

Table 1: Biotypes in the 51 Streptococcus mutans strains isolated from children without dental caries.

Table 2: Biotypes in the 68 Streptococcus mutans strains isolated from children with dental caries.

Antagonistic effect in S. mutans strains
The antagonistic effect was determined in all 119 S. mutans strains isolated in this study (Tables 3 and 4). It is important to specify, as mentioned in materials and methods, that the strains that have antagonistic effect are taken as effectors, and those that suffer the antagonistic effect are taken as indicators. In each group of patients, 12 indicator isolates were selected, including representatives of the most frequent biotypes, in order to find out the antagonistic effect of the 119 strains isolated from patients with and without caries (Tables 3 and 4). In the group of patients without caries, only two strains (28FS1 and 35FS3) had no antagonistic action on the 12 indicator strains (Table 3). In the same group, only three strains (22FS4, 23FS1 and 24FS1) had full antagonistic action (100%) on the 12 indicator strains. The rest of the strains (n=46) had different kinds of antagonistic action ranging from inhibition of 2 to 11 indicator strains. In the group of patients with caries, only 5 strains (1FC2, 4FC1, 6FC1, 8FC3 and 9FC3) had no antagonistic action on the 12 indicator strains (Table 4). In the same group, 32 strains (47%) had full antagonistic action (100%) on the 12 indicator strains. The rest of the strains (n=31) inhibited 3 to 11 indicator strains.

Table 3: Antagonistic effect of the 51 Streptococcus mutans strains isolated from patients without dental caries.

Table 4: Antagonistic effect of the 68 Streptococcus mutans strains isolated from patients with dental caries.

Table 4: (Continued).

 

DISCUSSION

A microorganism may survive and multiply if it manages to eliminate or displace an organism from an ecological niche that has a range of microbial species14. Studies on antagonism in dental caries began in 1972 with experiments with S. mutans and Veillonella alcalescens3; which showed that the growth of V. alcalescens in dental plaque was influenced by the anaerobic atmosphere of plaque and the amount of lactic acid produced by the plaqueforming organisms. The search for S. mutans strains with antagonistic capacity and their application in microbiological control to displace virulent native S. mutans strains has been ongoing for many years8-10.
Different studies show that the antagonistic capacity of S. mutans is due to the bacteriocins it produces, which could provide great capacity for displacing indigenous (native) strains of the same species15-17. The double layer test is often used to show the action of bacteriocins or bacteriocinlike inhibitory substances (BLIS) produced by effector strains on the selected indicator strains, therefore the antagonistic or inhibitory capacity of the strains is due to the action of these substances15,18.
In this study, antagonistic effect was determined in 119 S. mutans strains isolated: 51 strains from children without caries and 68 strains from children with caries. Biotyping with the Api-ZYM system has been valuable in several studies for typing and relating S. mutans strains19,20. Due to the wide range of biotypes found in this study and in order to have a more realistic approach to the effect of antagonistic strains on the most commonly found strains, the most frequent biotypes in the study population were used as indicator strains. In order to assess the antagonistic action of the strains, two groups were formed: the group of strains from patients without caries and the group from patients with caries. In order to assess the strains from the group of patients without caries, 12 indicator strains were selected, representing the most frequent biotypes from the same group of strains; and to assess the strains from the group o patients with caries, 12 indicator strains were selected, representing the most frequent biotypes of the same group of strains. One hundred and twelve (94 %) of the 119 S. mutans strains isolated showed high antagonistic action on the strains used as indicators. In contrast, the study by Balakrishnan et al.9, in which strains of different species were used as indicators, reported finding 39 strains belonging to the genera Streptococcus, Enterococcus and Staphylococcus with antagonistic capacity, representing 14.3% of the 272 strains evaluated. Kamiya et al.13 evaluated bacteriocin production in 319 S. mutans strains isolated from 8 patients with caries and 8 patients without caries against only 12 S. mutans strains, and found antagonistic effect in 254 strains (79.62%). These differences in antagonistic effect were probably caused by different conditions in the tests and the use of different indicator strains.
There has been much debate regarding the association between S. mutans and the onset of dental caries1,5. Many studies have shown that there is a direct relationship between S. mutans count in the oral cavity and the incidence and prevalence of dental caries1,5,13. In this study, even when the frequency of S. mutans in children with and without caries was high and very similar (91.7% vs. 96.7%), there were statistically significant differences in the S. mutans count between the two populations studied. These findings show the high S. mutans colonization rate in children with dental caries. Ecologically, dental caries is considered to be a consequence of an imbalance in the oral ecosystem leading to the prevalence of flora, before normal and then transformed in pathogenic1. Any replacement therapy or microbiological control must take into account the ecological aspects in the oral cavity, since replacing one bacterium with another might lead to more imbalance than balance1,3,8. There are currently different effector strains that come from oral bacteria for use in replacement therapy for caries8,10,21. The main problem that arises when applying this strategy in the oral cavity, specifically in prevention of dental caries, is due to the fact that in addition to being a normal inhabitant of the human oral cavity, S. mutans is also a pathogen there. Studies on humans need to find an effector strain that can colonize effectively as well as displace indigenous S. mutans strains that live naturally in the oral cavity21. Another important fact is that the local ecological implications that the absence of the indigenous S. mutans strain and the presence of other(s) could have on the oral cavity cannot be clearly predicted. Moreover, it is not known which species are best able to replace it (if it becomes absent) in its ecological niche. However, replacing it with a strain of the same genus and species with known genetic and phenotypic characteristics, which maintains the balance of the oral ecosystem, is an option worth considering in the prevention of caries10,21.
As a result of this project, there are biotyped S. mutans strains with high antagonistic potential which can be used in further studies on replacement therapy strategies. Subsequent action should be aimed at learning the characteristics of the antagonistic strains in their interaction with other microorganisms that are important in forming the oral biofilm and in models that are closer to the real model22.
To conclude, (1) in this study, 119 S. mutans strains were isolated and grouped in 85 biotypes; (2) the most frequent biotypes in patients without and with caries were, respectively 6, 9, 5 and 3, and 37, 39, 6 and 9; and (3) 112 S. mutans strains were found with high antagonistic potential and inhibition range of 3 to 12 indicator strains.

ACKNOWLEDGMENTS

This study was funded by the Administrative Department of Science, Technology and Innovation (Colciencias) of the National Health Science and Technology Programme, with number 097-2003.

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