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Frequency and distribution of Mutans Streptococci in dental plaque from

caries-free and caries-affected Venezuelan children

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 63

FREQUENCY AND DISTRIBUTION OF MUTANS STREPTOCOCCI

IN DENTAL PLAQUE FROM CARIES-FREE AND CARIES-AFFECTED
VENEZUELAN CHILDREN

Ana M. Acevedo, Maria V. Ray, Mairobys Socorro, Fátima Rojas-Sánchez

Institute of Dental Research, School of Dentistry, Central University of Venezuela,

Caracas, Venezuela

ABSTRACT tococci. The bacterial identification procedure was done using

Mutans Streptococci, in particular S. mutans and S. sobrinus, the API Rapid Strep System. The criteria used to determine dmft
are generally considered to be the prime etiological bacteria and DMFT was established by Klein and Palmer (1941).
of human dental caries. Mean dmft and DMFT were 6.4±3.2 and 4.4±2.9, respectively.
The aim of this study was to determine the frequency of mutans Ten (33%) out of 30 caries-affected children harbored mutans
streptococci in dental plaque in three groups of caries-free and streptococci. The species most frequently found were S. mutans
caries-affected Venezuelan children aged 2-6, 7-12, 13-19 (20%), S. sobrinus (10%) and S. rattus (3.3%). Meanwhile, in
years, and their possible association with dental caries. the caries–free group only 6 out of 18 children (33%) harbored
The frequency of mutans streptococci was determined in sam- mutans streptococci, specifically S. mutans and S. sobrinus, both
ples of pooled dental plaque collected from all detectable at 17%, with no significant difference between the two groups.
sources of 30 (62.5%) caries-affected and 18 (37.5%) caries- These results indicate that the percentage of children that harbored
free children. The samples were collected from all available mutans streptococci was similar in both groups, suggesting that other
tooth sites using a Hollenbak probe and immediately suspend- acidogenic species may be responsible for caries development.
ed in Ringer’s solution, serially diluted and cultured in Mitis
Salivarius (MS) agar for total streptococci determination and Key words: dental deposit, Mutans streptococci, dental caries,
Mitis Salivarius Bacitracin (MSB) for isolation of mutans strep- children.

PRESENCIA DE STREPTOCOCCUS MUTANS EN LA PLACA DENTAL PROVENIENTE

DE NIÑOS VENEZOLANOS CON CARIES Y LIBRES DE CARIES

RESUMEN El examen clínico para determinar el ceod y CPOD fue reali-

Los Streptococcus del “grupo” mutans en especial Streptococ- zado siguiendo los criterios de Klein y Palmer (1941).
cus mutans y Streptococcus sobrinus han sido considerados El ceod y CPOD promedio fueron 6,4 ±3,2 y 4,4 ± 2,9, respec-
como las principales bacterias responsables del desarrollo de tivamente. De los 30 niños con caries evaluados solo en 10
la caries dental. (33%) de estos se encontró Streptococcus del grupo mutans en
El propósito principal de este estudio fue determinar la frecuen- la placa dental. Las especies del grupo mutans que se identifi-
cia de estreptococos del grupo mutans presentes en la placa caron fueron S. mutans (20 %), S. sobrinus (10 %), y S. rattus
dental de niños venezolanos divididos en tres grupos etarios, 2- (3,3%). En el grupo libre de caries el porcentaje de niños con
6, 7-12 y 13-19 años, con caries y libres de caries y establecer Streptococcus del grupo mutans en su placa dental fue similar
una posible asociación con el desarrollo de caries dental. al grupo afectado por caries y las especies presentes fueron S.
Muestras de placa dental de 30 (62,5%) niños con caries y 18 mutans (17%) y S. Sobrinus (17%).
(37,5%) niños libres de caries fueron recolectadas de todas las Estos resultados indican que el porcentaje de niños que presen-
superficies disponibles utilizando una espátula Hollenbak. Las taron Streptococcus del grupo mutans en su placa dental fue
muestras fueron recolectadas e inmediatamente suspendidas similar en los niños afectados por caries como en los libres de
en solución Ringer, luego de lo cual se realizaron diluciones caries, lo que sugiere que otras especies acidogénicas de la cavi-
seriadas. Las muestras de placa dental fueron cultivadas en dad bucal diferentes a las del grupo mutans parecieran estar
Agar Mitis Salivarius para la identificación de estreptococos jugando un papel importante en el desarrollo de caries dental.
totales y en Agar Mitis Salivarius con Bacitracina para la
determinación de Streptococcus del “grupo” mutans y poste- Palabras claves: caries dental, niños, placa dental, Strepto-
riormente identificadas utilizando el sistema API Rapid Strep. coccus mutans.

INTRODUCTION of bacterial and host origin that plays a primary role

In Venezuela, two surveys of dental caries have been in the etiology of dental caries. Among the many
carried out, showing high caries prevalence1,2. Den- bacterial species that have been identified in dental
tal plaque is the biofilm associated to teeth, consist- plaque, only a few are considered cariogenic3-5.
ing of a microbial community in a matrix of polymer Mutans Streptococci (MS) including S. mutans and

Vol. 22 Nº 1 / 2009 / ??-?? ISSN 0326-4815 Acta Odontol. Latinoam. 2009

64 Ana M. Acevedo, Maria V. Ray, Mairobys Socorro, Fátima Rojas-Sánchez

S. sobrinus are well known as the group of oral 6 years, mean age 5.0±0.9 years; Group B of 17
microorganisms which have virulence factors relat- children aged 7 to 12 years, mean age 8.5±1.6 years,
ed to cariogenicity. They possess a wide range of and group C of 17 children aged 13 to 17 years,
cariogenic traits which include a high rate of acid mean age 16.7±1.6 years. Each child was examined
production, high acid tolerance, sucrose-mediated for caries status (dmft or DMFT) following the cri-
extracellular polysaccharide synthesis, and intracel- teria established by Klein and Palmer33. Dental
lular glycogen synthesis from a variety of dietary examination was carried out by the same calibrated
carbohydrates3,6,7 which are significant determinants examiner (MVR) using artificial light, probe # 5
of the cariogenicity of plaque. and dental mirror # 23. The parents of all the chil-
For several decades, investigators have agreed on the dren were asked to sign a consent form that allowed
fact that MS generally are implicated as the principal us to examine their children’s teeth and to take
infectious bacteria responsible for dental caries in microbiological samples.
humans8-12, and are classified into seven species13. Of
these, S. sobrinus and S. mutans are the most frequent- SAMPLING AND CULTURING
ly isolated from the human oral cavity3. S. sobrinus is Two-hour fasting dental plaque samples were collect-
more cariogenic than S mutans, nevertheless, it has ed from each subject after clinical examination. After
been reported that strains of S. mutans and S. sobri- removal of saliva by a short blast of air, plaque was
nus isolated from infants and adolescents with vary- removed from all available tooth sites using a Hol-
ing caries experience did not show significant lenbak probe. Dental plaque was scraped from the
differences in aciduric and acidogenic activities14. S. available surfaces of the dentition in the morning (9
mutans and S. sobrinus are associated more often with to 10 am) from each patient. The pool plaque sample
the more acidic response of caries-affected plaque and was immediately placed in a sterile capped container,
research has indicated that the coexistence of S. sobri- transported to the laboratory, weighed immediately
nus and S. mutans is an important factor in the devel- and the wet weight was calculated by difference. The
opment of dental caries15, 16. Different studies have plaque was suspended in Ringer’s solution and
reported a correlation between plaque counts of MS homogenized by hand in a glass homogenizer. The
and both caries prevalence and incidence3, 17-22. Hama- suspension was serially diluted in ice-cold Ringer’s
da and Slade23, have shown that large numbers of S. solution for microbial analysis to yield a final con-
sobrinus are found in saliva of individuals with high centration of 5 mg wet weight plaque per mL.
caries scores, as well as in plaque of caries-affected,
but not caries-free mouths. Furthermore, it is reason- BACTERIOLOGICAL MEDIA
able to identify caries-susceptible individuals from Aliquots of 100µL of each appropriately serially
the correlation between the presence of mutans strep- diluted sample were plated in duplicate on Brain-
tococci and caries development24, 25. In contrast, other Heart-Infusion (BHI) for total microorganism count,
studies have reported that caries lesions can develop as well as in ready-poured Mitis Salivarius Agar
in the absence of S. mutans and their numbers are media with tellurite (0.1%) (Difco Labs. Detroit, Ml)
often low even when caries are present6-7, 26-28. In addi- for total streptococci count and Mitis-Salivarius
tion, supporting this statement, other investigations Bacitracin (MSB) agar (0.2 units/ mL) for mutans
have found no correlation between the S. mutans streptococci. Acidogenic ratio determinations34 were
count and the presence of caries29-32. carried out on bromocresol purple agar. The media
Based on the controversial results presented in lit- contained glucose 12 g; K2HPO4 1.2 g; KH2PO4
erature, the aim of this study was to determine the 1.2 g; tryptone 5 g; yeast extract 5 g; agar 15 g;
frequency and distribution of Mutans Streptococci bromocresol purple 16 ml of a 0.2% sterile stock
in dental plaque from caries-free and caries-affect- solution. The pH was adjusted to 7.4 with KOH.
ed Venezuelan children of different age groups and After inoculation, all media were incubated anaero-
to associate their occurrence with the presence of bically in jars (Gas Pack System) at 37ºC for 48 h.
dental caries.
BACTERIAL COUNT AND ISOLATION
PATIENTS AND METHODS Each colony was counted for total Streptococci on
A total 48 children that attended the Department of MS agar and a representative colony of each dis-
Pediatric Dentistry of the Faculty of Dentistry, Cen- tinct colonial morphology from MSB was selected
tral University of Venezuela and lived in the capital and grown overnight in 5 ml of pre-sterilized Todd-
city (Caracas) were selected for the study. The Hewitt broth at 37ºC. Each colony isolated was
patients were subsequently divided into three then identified biochemically using the API Rapid
groups: Group A made up of 14 children aged 2 to Strep System (API SYSTEMS S.A.). The results

Acta Odontol. Latinoam. 2009 ISSN 0326-4815 Vol. 22 Nº 1 / 2009 / ??-??

 Mutans Streptococci in Venezuelan Children 65

were recorded after 24 h of Table 1: Sample distribution according to caries experience expressed
isolation and tests showing as a percentage
unclear or weak results were
re-checked using biochemical Age range Nº Percentage of Percentage of
caries-free children caries-affected children
tests (fermentation of mani-
tol, sorbitol, and enzymatic 2-6 14 43 (6)* 57 (8)
tests such as catalase activi- 7-12 17 24 (4) 76 (13)
ty). Acidogenic organisms 13-19 17 47 (8) 53 (9)
were recognized by the color Total 48 38 (18) 63 (30)
change from purple to yellow
and the acidogenic ratio was *Number in parenthesis corresponds to the number of children.
calculated.

RESULTS Table 2: Mean dmft and DMFT in caries-affected

Sample distribution according to caries experience children
expressed as a percentage is illustrated in Table 1.
Children under study were divided into three Age Group dmft DMFT
groups, according to age: Group A – children aged 2-6 6.4 ± 3.2* —
2 - 6 years, of whom 57% were caries-affected and 7- 12 —
⎫
4.8 ± 3.1 **
42% were caries-free; in group B, 76% and 24% of 13-19 — 3.8 ± 2.6 ⎬
the subjects were caries-affected and caries-free Total — 4.4 ± 2.9 ⎭
respectively; and group C was made up of children *Data show the mean dmft and DMFT indexes in each age group.
aged 13 to 17 years, of whom 53% were caries- A high dmft was found in the 2 to 6-year-old children. The children
affected and 47% caries-free. in the group of 7 to12-year-olds showed a higher but non-signifi-
cant DMFT index when compared to data for 13 to19-year-olds
Table 2 shows the mean dmft and DMFT indexes according to Student T test (p>0.05).
in each age group. A high dmft was found in the 2
to 6-year-old children. The children belonging to
the 7 to 12-year-old age group showed a higher but
non-significant DMFT index compared to the group analyze the distribution of mutant streptococci
of 13 to 19-year-olds. The total mean DMFT for the (MS) species in dental plaque samples of the chil-
caries-affected children was 4.4± 2.2. dren in the different age groups. The percentages of
The mean values for total streptococci count caries- affected and caries-free subjects that har-
(CFU) in dental plaque samples from children in bored mutant streptococci species are shown in
different age groups as well as from caries-free Table 4. The results from the children of all ages
and caries-affected children are shown in Table 3. showed that caries-free children harbored S.
The mean total values of streptococci counts pres- mutans, (17%) and S. sobrinus (17%) but not S. rat-
ent in the plaque from children aged 2-6, 7-12 tus. On the other hand, 10% of the caries-affected
and 13-19 years were not statistically different children showed the presence of S. sobrinus, 20%
(p>0.05). In caries-free and caries-affected chil- showed S. mutans and 3% showed S. rattus.
dren, the total streptococci CFU was similar for In the group 2 to 6-year-old children affected by
two age groups, except in the group of 7 to 12- caries, none of the MS species analyzed was
year-old caries-free children, which showed a detected, although S. sobrinus was found in the
higher number of bacterial
counts. However when these
values were analyzed, no sta- Table 3: Mean values of total streptococci count in dental plaque samples
of caries-free and caries-affected children expressed as
tistical difference was found CFUx105/mg wet weight¹.
(p>0.05). Similarly, when the
total CFU counts in plaque Age Group Total streptococci Caries-affected Caries-free
for the three age groups were 2-6 18.5 ± 7.1* 19.6 ± 6.1 17.3 ± 8.0
compared, no significant dif- 7 - 12 22.9 ± 16.5 16.2 ± 10.7 29.6 ± 22.3
ference was observed. 13 -19 17.2 ± 6.9 16.9 ± 6.7 17.5 ± 7.1
Since the total number of
streptococci in caries-affected *Data are expressed as a mean (±SD). No differences in total Streptococci in caries-free
and caries-affected children were observed when data were compared according to Stu-
and caries-free children was dent T Test (p>0.05).
not different, we decided to

Vol. 22 Nº 1 / 2009 / ??-?? ISSN 0326-4815 Acta Odontol. Latinoam. 2009

66 Ana M. Acevedo, Maria V. Ray, Mairobys Socorro, Fátima Rojas-Sánchez

Table 4: Distribution of Mutans streptococci (MS) species in dental plaque samples from caries-free and
caries-affected children.

Frequency of occurrence (%)

Age Group Caries-affected Caries-free

S. sobrinus S. mutans S. rattus S. sobrinus S. mutans S. rattus

2-6 0 0 0 17 0 0
7 - 12 8 31 8 25 25 0
13 -19 22 22 0 13 25 0
Total 10 20 3 17 17 0

caries-free subjects. A higher percentage with MS DISCUSSION

was found in the 7 to 12-year-old group (Table 4). Mutans streptococci (MS) have been considered the
It is noteworthy that the percentage of children main etiological agents in caries development3,13.
with S. mutans in the caries-affected group was Later on, van Houte35,36 suggested the importance
slightly higher than in the caries-free subjects, but of streptococci other than MS in the initiation of
the percentage of children with S. sobrinus in the dental caries; however, even today there are con-
caries-free group was significantly higher than in flicting results on this subject.
the caries-affected subjects (p<0.05). The presence In this investigation we evaluate the total streptococci
of S. rattus was detected in 8% of the caries-affect- count, the distribution of S. mutans, S. sobrinus and S.
ed subjects. It is noticeable that S. rattus was not rattus in dental plaque from caries-affected and caries-
present in any of the caries-free children. On the free children, since it has been shown that the coexis-
other hand, the results obtained from caries-free tence of S. sobrinus and S. mutans is an important
and caries-affected children grouped in the 13 to factor in the development of dental caries36 as well as
19-year-old age range showed that the percentage the total acidogenic and non-acidogenic count as a
of individuals with S. mutans count was similar parameter to be associated with caries occurrence.
(25 and 22% respectively), S. rattus was not found, The estimation of the prevalence of mutans strepto-
and the percentage of children that harbored S. cocci was based on the condition that each subject
sobrinus was higher but not significant in caries- was considered as the basic unit influencing the
affected subjects. prevalence of these bacteria from individual sites.
Table 5 shows the mean values of total, acidogenic All comparisons between mutans scores were there-
and non-acidogenic bacterial count in dental fore first made within each individual and then sta-
plaque expressed in CFU x105/mg wet weight-1. tistically analyzed for all subjects.
Caries-free and caries-affected children 2-6 years The present study has confirmed the patterns
old showed a similar number of acidogenic bacte- observed in other studies for the distribution of
ria (10.2±2.6 and 10.3±3.4 respectively). Howev- mutans streptococci in caries-affected and caries-free
er, non-acidogenic bacteria were slightly higher, children and that local variations exist in the preva-
though not significantly, in the caries-affected lence of cariogenic species within dental plaque. S
group as compared to the caries-free children mutans and S. sobrinus were nevertheless found on
(8.3±4.9 and 5.2±4.1 respectively). The total num- apparently healthy sites, which has been seen in other
ber of CFU was lower in the caries-free group than studies previously reported by Babaahmedy et al.16.
in the caries-affected. However the differences Several reports have demonstrated that MS are isolated
observed in all bacterial types were not statistical- more frequently as the child grows older37-39. Moreover,
ly significant (p>0.05). many authors have reported that children commonly
A similar pattern was found in the 7 to 12 and 13 to acquire mutans streptococci between the 1st and 3rd year
19-year-old children when the acidogenic, non-aci- of life10,40. In this investigation mutans streptococci
dogenic and total counts were analyzed. When the species were not detected in plaque from 2 to 6-year-
mean total number of acidogenic, non-acidogenic old, caries-free or caries-prone children, except for S.
and acidogenic plus non-acidogenic (total) bacteri- sobrinus, which was found in 1 child in the caries-free
al counts were compared in the three age groups, group. This result suggests a delay in the colonization
the dif.ferences were also not statistically signifi- of mutant streptococci in the dental plaque of this group
cant (p>0.05). of children. However, the high caries rate observed in

Acta Odontol. Latinoam. 2009 ISSN 0326-4815 Vol. 22 Nº 1 / 2009 / ??-??

 Mutans Streptococci in Venezuelan Children 67

the caries-affected children may Table 5: Mean total count values, acidogenic and non-acidogenic in
suggest that Streptococcus, in dental plaque samples from children in each group studied
addition to species such as lacto- (expressed in CFUx105/mg wet weight¹).
bacilli and bifidobacterium41, Group Acidogenic Non-acidogenic Total
could be involved in the devel- N N Count Count Count
opment of dental caries.
2-6
On the other hand, all the caries-
Caries-affected 8 10.3 ± 3.4 * 8.3 ± 4.9 18.6 ± 5.2
free and caries-prone subjects in
Caries-free 6 10.2 ± 2.6 5.2 ± 4.1 15.4 ± 4.6
the 7 to 12 and 13 to 19-year-old
groups harbored both S. mutans 7-12
and S. sobrinus in their dental Caries-affected 13 8.6 ± 5.5 11.6 ± 7.4 20.4 ± 9.2
plaque. Moreover, the percent- Caries-free 4 15.5 ±15.1 10.6 ± 6.9 26.0 ± 22.0
age of children with S. sobrinus
13-19
in the caries-free subjects was
Caries-affected 9 8.3 ± 4.0 9.5 ± 3.5 19.0± 5.3
significantly higher than in the
Caries-free 8 10.2 ± 6.5 7.9 ± 3.8 18.2± 5.2
caries-affected group (Table 4).
However, the percentage of * Data are expressed as a mean (±SD).
children with S. mutans in the
caries-free group was lower
than in the caries-affected group. This result suggests subjects. However, the results of this investigation
that a higher number of S. mutans associated to S. indicate that the proportions of acidogenic to non-
sobrinus may be needed to increase the risk of devel- acidogenic in dental plaque from caries-affected and
opment of dental caries. In addition, Lang et al.17 sug- caries-free children were similar (Table 5). This
gested that local factors such as differences in indicates that factors other than the microbiological
exposure of saliva or variations of fluoride levels42 are determinant in the initiation of the caries process.
could be implicated in the association of mutans strep- Controversial results have been reported in relation to
tococci with healthy sites. the association between the presence of mutans strep-
The role of acid-producing bacteria in the carious tococci and dental caries45,46. In this study, no associa-
process has been reviewed by different authors6, 17,43. tion was observed to support the idea that caries could
Stephan44 was the first to show that the plaque pH occur in the absence of mutans streptococci47.
can fall below 5.0 after a sugar rinse and it is rea- The results of this investigation indicate that the
sonable to predict from the acidogenic theory of microbiological analysis of dental plaque could be
caries that plaque samples from caries-affected indi- a useful tool to identify the caries risk at individual
viduals will have a larger proportion of acidogenic or community level, as well as to improve diagno-
organisms than similar samples from caries-free sis and treatment.

ACKNOWLEDGMENTS CORRESPONDENCE
We wish to thank the parents and children who took part in this Dr. Ana Maria Acevedo
investigation. This work was supported by the Faculty of Den- Instituto de Investigaciones Odontológicas “Raúl Vincentelli”
tistry, Central University of Venezuela. Facultad de Odontología
Universidad Central de Venezuela
Los Chaguaramos, Caracas, 1050.
Venezuela
e-mail: [email protected] / [email protected]

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