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Prevalence of Different Streptococci Species in the Oral Cavity of Children and Adolescents Patricia Amoroso 1 Fernando A

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Prevalence of Different Streptococci Species in the Oral Cavity of Children and Adolescents Patricia Amoroso 1 Fernando A Braz J Oral Sci. January/March 2003 - Vol. 2 - Number 4 Prevalence of different streptococci species in the oral cavity of children and adolescents Patricia Amoroso 1 Fernando A. de Ávila 1 Abstract Célia M. O. Gagliardi 1 Streptococci species were isolated, identified and counted in 262 sa- liva samples collected from 131 children and adolescents from a pub- 1 Agraries and Veterinary Sciences University lic school nursery at the city of Jaboticabal, SP. Four Streptococci Departament of Patology Veterinary - species harboring oral samples were identified biochemically, as Strep- Estadual Paulista University tococcus salivarius, Streptococcus mutans, Streptococcus rattus, Strep- Campus Jaboticabal – SP - Brazil [email protected] tococcus sobrinus, and streptococci of the mitis group. The mean Streptococcus counts obtained were 1.0 x 109 CFU/ml saliva for the 3 to 5 year age range, 1.5 x 109 CFU/mL saliva for the 6 to 8 year age range, and 2.0 x 109 CFU/mL saliva for the 12 to 14 year age range. The prevalence of Streptococci in saliva were: S. salivarius (89.31%), S. mutans (73.28%), S. salivarius + S. mutans (44.27%) by the stan- Received for publication: January 22, 2003 dard method. The tongue depressor method showed S. mutans Accepted: February 14, 2003 (62.59%), S. salivarius (77.86%), S. salivarius + S. mutans (33.58%). The analysis of both techniques showed significant agreement for Streptococcus isolation. Key Words Isolation, streptococci, children, saliva, collection techniques. Correspondence to: Patricia Amoroso Av. Eng. Necker C. Camargos, 1733 Cep: 14783-085 Barretos SP Brazil e-mail: [email protected] 164 Braz J Oral Sci. 2(4):164-168 Prevalence of Streptococcus of saliva of children and adolescents Introduction Carlsson11 studied the distribution of the Streptococci The study of microorganisms of the genus Streptococci is mutans in children from rural districts in Sudan aging 12- of great clinical interest due to their pathogenic potential. In year-old with low caries prevalence showing that 96% of Oral Microbiology, there is considerable concern about the this population harbored these microorganisms. Based on group of so-called “viridans streptococci”. The species most the international and national literature, the objectives of frequently isolated from the oral cavity are Streptococcus the present study are: 1) to isolate and identify biochemically salivarius, Streptococcus sanguis, Streptococus mitis, and Streptococcus sp strains from the saliva of children and mutans Streptococci 1,2. adolescents (3 to 14 years of age) attending public institutions According to the literature, these bacteria play an important in the town of Jaboticabal, SP, 2) to count the colony forming role in terms of its pathogenic potential and ecological units (CFU/mL) of Streptococci in the saliva of children and relationships that might become established. In the former adolescents, and 3) to observe the prevalence of oral case, particularly important is the role of Streptococcus Streptococci species by using standard methods and tongue sanguis and Streptococcus salivarius in the qualitative and depressor. quantitative regulation of the oral microbiota1. There are large numbers of cariogenic microorganisms that Material and Methods can be defined by their ability to colonize teeth, causing a A total of 262 saliva samples were obtained from 131 children marked reduction in pH in the presence of a sugar substrate and adolescents of both sexes aged 3 to 14 years from public and consequently inducing caries3,4. Streptococcus mutans, institutions in the town of Jaboticabal, SP. Two samples were Streptococcus sanguis, Lactobacillus casei and obtained from each child. One of them, of about 2.0 ml, was Actinomyces viscosus fulfill most of these criteria. However, collected without stimulation into a sterilized 10 ml flask, and Streptococcus mutans seems to be the most efficient the other was collected using a sterile tongue depressor cariogenic microorganism, rapidly inducing caries in germ- which was moved around 10 times in a circular manner in the free rodents3. The presence of Streptococcus mutans in a mouth of each child, with excess saliva being removed caries-free animal may be attributed to transient infection through closed lips. After this procedure, each depressor and caries activity in a mouse from whom this species cannot was placed in a sterilized test tube with 0.15M sterill saline be isolated suggests the possible existence of other caries- solution which was sealed and transported to the laboratory. inducing microorganisms3. The saliva samples collected with the tongue depressor were Species of mutans Streptococci comprise the major then seeded by pressing the two surfaces of the depressor microorganisms implicated in the etiology of dental caries on dishes containing MS (Mitis salivarius) agar (Difco, occurring on smooth surfaces5. Among the species of this Detroit, Michigan, USA) and incubated at 37oC for 48 hours groups, Streptococcus mutans is the major agent of this under conditions of microaerophilia using the carbon dioxide disease in man, followed by Streptococcus sobrinus, which system in polyethylene bags. The saliva samples were has also been implicated in this process5,6,7. Other species submitted to decimal dilution up to 10-6 in sterilized saline such as Streptococcus cricetus and Streptococcus rattus are and 0.05 ml aliquots of each sample were deposited on the less frequently isolated from humans since they are mainly center of dishes containing MS agar medium plus 20% related to dental caries in animals. Streptococcus ferus does sucrose and spread homogeneously with the aid of an angled not seem to be related to the etiology of dental caries5. sterilized glass rod. After seeding, the dishes were incubated The human mouth is usually sterile at birth but soon acquires at 37oC for 48 hours under conditions of microaerophilia using a predominantly streptococcal microbiota. Streptococcus the carbon dioxide system in polyethylene bags. After salivarius was isolated from infants 18 hours after birth and incubation, the bacteria were counted as CFU/mL. from 75% of children aged 1 to 5 years. With age, this microbiota Three colonies from each dish were used to prepare smears differentiates, although the species Streptococcus salivarius on slides, which were stained by the Gram method to continues to predominate on the tongue and oral mucosa8. confirm culture purity and microscopic morphology (Gram- Rogers9 in a study carried out in South Australia, isolated positive cocci in a chain-link conformation). After purity 82 streptococcal strains from the mouth of individuals was confirmed, three or more colonies were transferred to aged 13 to 25 years with active caries and classified them test tubes containing defibrinated horse blood and frozen into five biotypes using 20 biochemical tests9. Two of at –20oC. these biotypes were found to be related to the species Streptococci strains were primarily identified on the basis of Streptococcus sanguis and Streptococcus mutans. their characteristics colonial morphology colonies in blood Alaluusua and Renkonen10 studied Streptococcus mutans agar Gram-stained smears, and catalase test. Biochemical colonization in plaque and saliva from 39 children aged 2, tests were also performed by fermentation of adonitol, starch, 3 and 4 years from the suburbs of Helsinki and confirmed arabinose, cellobiose, dextrin, dextrose, dulcitol, galactose, its close relationship to dental caries. glucose, inulin, lactose, maltose, mannose, manitol, 165 Braz J Oral Sci. 2(4):164-168 Prevalence of Streptococcus of saliva of children and adolescents mellibiose, raffinose, sucrose, sorbitol, sorbose, xylose, and When the standard technique was used, the species arginine hydrolysis20. Streptococcus salivarius again was the most frequent, with 117 (89.31%) strains being isolated and identified, followed Results by Streptococcus mutans 96 (73.28%). Seventeen All strains submitted to Gram staining showed a typical Streptococcus sobrinus strains (13.74%), 30 (22.90%) chain-linked cocci morphology. All strains were negative to Streptococcus strains of the mitis group, 1 (0.76%) the catalase test, supporting the idea that the colonies in Streptococcus ratti strains, and 13 (9.92%) strains belonging question belong to the genus Streptococcus. to other species were also identified. Table 1 and 2 show the prevalence of oral Streptococci in Table 3 and 4 express the number and percentage of 131 children and adolescents at the city of Jaboticabal-SP. streptococci isolated from the samples of saliva children and A total of 393 Streptococcus strains were isolated from the adolescents in (3 to 14 year old) obtained with tongue samples collected with a tongue depressor. Of these, 107 depressors. (77.86%) were Streptococcus salivarius, 82 (62.59%) The highest mean Streptococcus counts observed as 1.0 x Streptococcus mutans, 30 (22.90%) Streptococcus sobrinus, 109 CFU/ml saliva for the 3 to 5 year age range, 1.5 x 109 CFU/ 22 (16.79%) Streptococcus of the mitis group, 2 (1.52%) ml saliva for the 6 to 8 year age range, and 2.0 x 109 CFU/ml Streptococcus rattus, and 34 (25.95%) belonged to other saliva for the 12 to 14 year age range. species. Table 3- Number and percentage of streptococci isolated by the Table 1. Number and percentage of streptococci isolated by the tongue depressor technique used, and biochemical identification standart collection technique used, and biochemical identification of the species in 262 saliva samples from children and adolescents of the species in 262 saliva samples from children and adolescents from public institutions in the town of Jaboticabal, SP, in 1999. from public institutions in the town of Jaboticabal, SP, in 1999. Table 4- Number and percentage of streptococci isolated by the Table 2- Number and percentage of streptococci isolated by the tongue depressor technique used, and biochemical identification of standart collection technique used, and biochemical identification the species in 262 saliva samples from children and adolescents of the species in 262 saliva samples from children and adolescents from public institutions in the town of Jaboticabal, SP, in 1999.
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