764 2024


Salivary Caries-Related Microorganisms and Demographics of a Group of Children  
 

Vol 13  No 1  (2025)    DOI 10.5195/d3000.2025.764 

 h#p://den*stry3000.pi#.edu 

Salivary Caries-Related Microorganisms and Demographics 
of a Group of Children 
Alhan Ahmed Qasim, Muna Saleem Khalaf, Ghada Ibrahim Taha, Zainab Juma Jafar 

College of Dentistry, University of Baghdad, Baghdad, Iraq 

Abstract 

Objec&ve: The purpose of this study was to determine the associa4on between caries related 
microorganisms in children’s saliva, such as Streptococcus mutans and lactobacilli, and their demographic 
factors.  
Methods: This study involved a sample of 135, both sexes with an age range between 3 and 10 years. 
Uns4mulated saliva was obtained and diluted in normal saline. Saliva was then placed in selec4ve media. 
Salivaris agar was used for mutans streptococci while Rogosa agar for lactobacilli. ADer incuba4on, 
Streptococcus mutans coun4ng of CFU (colony forming units) with morphology characteriza4on and 
numbers of CFU per milliliter of saliva for lactobacilli. Demographic factors informa4on was collected 
using a ques4onnaire. 

Results: Both studied salivary microorganisms increased with age. Streptococcus mutans was higher in 
boys, and associated with breasNeeding, overweight, and not brushing their teeth. Lactobacilli were 
higher in girls, and associated with mothers who were government employee, mother occupa4on, 
children who during sleep were not nursed, and with normal weight. 

Conclusion: The present study found an 
associa4on between salivary Streptococcus 
mutans and lactobacilli and various 
demographic variables in a group of children. 

Keywords: Saliva, Streptococcus mutans, 
Lactobacilli, Health, Demographics.  

Cita;on: Qasim AA, et al. (2025) Salivary Caries-Related 
Microorganisms and Demographics of a Group of 
Children. Den;stry 3000. 2.a001 
doi:10.5195/d3000.2025.764 
Received: October 30, 2024  
Accepted: November 7, 2024 
Published: January 8. 2025 
Copyright: ©2025 Qasim AA, et al. This is an open access 
ar;cle licensed under a Crea;ve Commons AXribu;on 
Work 4.0 United States License. 
Email: dr.alhan_altaai@codental.uobaghdad.edu.iq

 

 

Introduction 

The surfaces of the oral cavity are 
covered by salivary fluids, which is 
produced via the salivary glands; 
this fluid has definitive essential 
roles for maintaining oral healthy 
(1]. Many bacteria are present in 
saliva [2]. In fact, saliva secreted 
into the oral cavity from salivary 
glands is sterile [3], but when 
washing with saliva, bacteria shed 
to saliva from different surfaces in 
the oral cavity including oral 
mucosa, gingival crevices, and 
dental plaque, but the main site of 
origins are throat, tongue and the 
tonsils [4]. The microbiota of saliva 
contains many species of bacteria 

that are temporally stable and 
have been shown to be 
individualized, playing a crucial 
role in disease as dental caries and 
health of oral cavity [5]. Public 
policies on oral health consistently 
address several factors associated 
with dental caries like oral hygiene 
rule, dietary habits, genetics, oral 
health knowledge, demographics 
and sociocultural environment. 
Many studies have revealed an 
influence of diet and lifestyle on 
microbiota of saliva [6,7]. It is a 
fact that dental developmental 
stages affected the microbiome of 
saliva. Key factors include teeth 

eruption, age, oral hygiene 
practices and dietary changes [8]. 

Dental caries is a chronic disease 
which is widespread and affect 
people of different ages and 
demographics [9]. It is caused by 
bacterial acid result from 
fermentation of free sugars and 
poor oral hygiene, leading to 
localized damage to the hard 
tooth tissue, associated with the 
structure of deciduous and 
permanent teeth [10,11]. Most 
types of cariogenic bacteria were 
found to differ according to host, 
age and race. Bacteria vary among 
individuals of the same race. 
Investigations have employed 



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saliva to determine the occurrence 
of caries by exploring levels of the 
bacteria. as many cariogenic 
bacteria are present in saliva. 
Mutans Streptococcus (MS) is 
categorized as primary 
contributors to dental caries. 
Another bacteria strain set linked 
to the advancing of dental caries is 
lactobacillus (LB). Salivary 
lactobacilli may have an indirect 
role with advancement of caries. 
Elevated concentration of S. 
mutans and Lactobacillus in saliva 
are significantly corelated to the 
early occurrence of dental caries 
[12-16]. Saliva is one of the most 
studied components of caries [15], 
and because of positive 
correlation between 
concentration of salivary SM and 
LB and caries, these bacteria may 
be potential indicators for dental 
caries. A significant increase of 
salivary SM and LB concentrations 
was found in a group of 
adolescents with nontreated 
caries [16]. This study was 
intended to investigate the levels 
of Streptococcus mutans and 
Lactobacillus species in saliva and 
their relationship with children’s 
demographic profiles and 
nutritional status. 

Material and Methods 

This research is structured as 
descriptive observational study 
with cross-sectional design. It 
includes children of both sexes 

and ages 3 to 10 years [17] being 
treated at the teaching hospital’s 
Pedodontic and Preventive 
Dentistry unit clinics, University of 
Baghdad’s College of Dentistry. 
This study enrolled children with 
active carious lesions. Official 
approval of study protocol was 
garneted by both Department of 
Pedodontics and Preventive 
Dentistry’s Scientific Committee 
and the College of Dentistry’s 
Institutional Research Ethics 
Committee at the University of 
Baghdad in Iraq (reference 
Number.:816, project Number. 
816323, Date;30-8-2023) 
(Chairman, Prof. Dr. Salwan Y. 
Bede). The parents of all children 
included in this study were asked 
to sign an informed consent 
document.  

Total sample of children 
participating in this study was 135. 
Each child received a 
questionnaire to be filled by their 
parents. Demographic variables 
included in this questionnaire 
were date of birth, age, sex, 
nursing pattern, nursing at sleep, 
added sweet to milk, tooth 
brushing, and occupation of 
mother and father. 

Following standard guidelines of 
Fejerskov and Thylstrup, in the 
morning between 10 to11 A.M. 
the samples of unstimulated saliva 
were collected [19]. Prior to this 
procedure, children rinsed their 
mouths with water and asked to 
spit the first mouthful of saliva 
into a small, pre-labeled plastic 
tube. One hour before collecting 
saliva, the child was asked not to 

eat or drink (except for water). A 
pre–sample period of 1 minute 
was followed. Then, unstimulated 
saliva was collected for 10 to 15 
minutes. Saliva was collected with 
the child sitting and relaxed on a 
common chair. Any samples 
containing blood were discarded. 
After completing saliva collection, 
normal saline was used for 
dilution of the sample in the 
laboratory. A micropipette was 
used for application of saliva on 
selective media surfaces - mitis 
salivaris agar targeting Mutans 
streptococci and Rogosa agar for 
Lactobacilli. Then plates 
anaerobically were incubated at 
37°C for 48 hours. For 
Streptococcus mutans, counting of 
Colony Forming Units (CFU) and 
morphology features were used, 
while numbers of CFU per ml of 
saliva were used for lactobacilli 
[20]. 

G power 3.1.9.7 (Program design 
by Franz-Faul at the University of 
Kiel, Germany) was used to 
calculate sample size of this study. 
Assuming 80% power, alpha error 
of 0.05, effect size of 0.25 
(medium) sample size of this study 
was 111. When adding 10% as an 
error rate, 122 subjects were 
suggested. Therefore, our sample 
of 135 was enough to detect 
differences in our study [21]. 

Daily entrance of data from the 
children’s case sheets was done 
into Microsoft office Excel 2013, 
and the Statistical Package for 
Social Science version 22 (SPSS) 
(Chicago, Illinois, USA) was 
implemented for statistical tests. 



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We determined frequency, 
percentages, means and standard 
errors (SE), unpaired T test, one 
way analysis of variance with 
Games-Howell and Pearson 
correlations. Statistically 
significant level was accepted at p-
value of less than 0.05. 

 

Results 

The overall sample were allocated 
based on the demographic 
variables as shown in Table 1. 
Comparison of Streptococcus 
mutans count in the saliva of 
children with their demographics, 
data revealed that the bacterial 
count is significantly increased 
with increased age. Additionally, 
the boys had significantly higher 
count than girls. Furthermore, 
count of salivary Streptococcus 
mutans showed a significant 
difference for type of nursing, 
status of nutrition and brushing 
time of teeth (Table 2).  

The count of bacterial 
Streptococcus mutans in saliva 
significantly increased in children 
who did not brush their teeth 
compared to those who brushed 
one time (Table 3). The count of 
lactobacilli in the saliva revealed 
that girls had significantly higher 
counts compared to boys, and a 
significant higher bacterial count 
were noted in children of 
government employee working 
mothers than in children of 

housewives (Table 4). Multiple 
comparisons for children’s 
nutritional status revealed that 
the differences were significant 
when comparing the obese group 
with the two other groups (Table 
5). 

 

Discussion 

Dental caries is multifactorial in 
nature, so salivary features, oral 
hygiene, nutritional status, and 
demographic factors should be 
registered to indicate their roles as 
risk factors for dental caries [22]. 
Saliva provides a complex mixture 
of microorganisms and collection 
of saliva is noninvasive and easy 
[5]. Streptococcus mutans detach 
to saliva from different oral 
surfaces and is considered as 
major cariogenic bacteria. 
Lactobacilli play a role in tooth 
decay development because it can 
produce extracellular 
polysaccharides and lactic acid 
[23]. Several studies have 
investigated the salivary bacterial 
levels of Streptococcus mutans 
and risk of dental caries [22,24]. 
For optimal health from childhood 
to adulthood, good oral hygiene 
practices throughout life are 
suggested [25,26]. Changes in the 
environment and lifestyle shifts 
such as high access to 
consumption of processed foods 
with an increased content of 

carbohydrates and urbanization 
have contributed to obesity and 
dental caries among children [27].  

The finding of this study showed 
an increase of salivary 
Streptococcus mutans and 
lactobacilli counts among children 
with increased age, in agreement 
with previous data [28,29].  

In general, girls grow faster than 
boys, which results in earlier teeth 
eruption and more exposure to 
cariogenic bacteria [30]. Many 
studies observed variations in the 
salivary microbiota between males 
and females, which might suggest 
that microorganisms have limited 
influence on determination of 
dental caries [31,32].  

Parents are responsible for 
providing a suitable environment 
for a healthy lifestyle and instilling 
good habits of oral hygiene [33]. In 
our study, children from mothers 
that were government employees 
had significantly higher counts of 
lactobacilli than children whom 
mothers were housewives, which 
agrees with previous work [34]. 
This could be since housewives 
put more effort in maintaining 
children’s oral health because they 
spend more time with them.  

Streptococcus mutans counts were 
higher in breastfeeding than other 
feeding patterns in our study. This 
is possibly because mothers 



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include foods and drinks other 
than breastmilk feeding, which 
increases susceptibility of the 
children to cariogenic 
microorganisms’ early childhood 
caries [35,36]. Also, our study 
showed that overweight children 
significantly have highest 
Streptococcus mutans counts. The 
same results were found in other 
studies [37-39], which linked an 
unhealthy body mass index or 
obesity with increased dental 
caries. Khalaf et al. and Raju et al. 
found inverse results, as their 
results showed that Streptococcus 
mutans counts in children with 
normal weight were higher than 
other BMI weights [40,41]. 
Children who did not brush their 
teeth had significantly higher 
Streptococcus mutans counts in 
their saliva in our study findings, 
which agrees with previous work 
[40]. Mechanical action of 
toothbrush bristles and active 
anticaries effects of toothpaste 
may provide this benefit of tooth 
brushing in decreasing the 
prevalence of dental caries. 
Brushing leads to not enough 
nutrients and time for growth of 
Streptococcus mutans and this led 
to a decrease in acid production 
[42]. 

This study has some potential 
limitations. The sample may not 
represent the broader population. 
Also, because this study is a cross-

sectional in design, it could not 
give the causal relation between 
salivary caries related 
microorganisms and development 
of caries, depending on different 
demographics. In addition, our 
study sample collection from a 
specific geographic and 
socioeconomic group may not 
apply to other populations or 
communities.  

  

Conclusion 

Microbial profile of both salivary 
Streptococcus mutans and 
lactobacilli correlate with 
demographic factors of children. 
Further investigations using 
longitudinal designs are needed to 
search for causative relations and 
the role of demographics.   

Ethical Approval 

This study was received ethical 
approval from the Research Ethics 
Committee of the College of 
Dentistry \ University of Baghdad 
Ref. number: 816, Date 30-8-2023. 

Conflicts of interest 

The authors advertise no 
competing interest. 

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Table 1. Descriptive statistics of the complete sample depending on the studied demographic variables. 

Demographic 
Variables Divisions No. % 

Child age 
3 to 6 years 80 59.26 
7 to10 years 55 40.74 

Child sex 
Males 69 51.11 
Females 66 48.89 

Father occupation 
Government 
employee 66 48.89 

Free jobs 69 51.11 

Mother occupation 
Government 
employee 109 80.74 

Housewife 26 19.26 

Adding sugar 
Yes 39 28.89 
No 96 71.11 

Type of nursing 
Breast milk feeding 93 68.89 
Bottle milk feeding 15 11.11 
Mixed 27 20.00 

Sleep nursing 
Yes 86 63.70 
No 49 36.30 

Nutritional status 

Normal weight 
percentile  78 57.78 

Over-weight 
percentile 31 22.96 

Obese percentile 26 19.26 

Tooth brushing 
No brushing 14 10.37 
One time 56 41.48 
Two time 65 48.15 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



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Table 2. The counts of S. mutans level in the saliva based on the demographic variables. 

Demographic Variables Mean SE Test P value 

Child age 
3-6 years .220 .031 

3.308 0.001 
7-10 years .462 .066 

Child sex 

Males .419 .054 

3.106 0.001 
Females .214 .038 
21-30 years .239 .041 
31-40 years .604 .083 
41-50 years .345 .016 

Father occupation 
Government employee .252 .042 

1.931 0.056 
Free jobs .382 .053 

Mother occupation 
Government employee .321 .041 

0.254 0.801 
Housewife .306 .044 

Adding sugar 
Yes .248 .066 

1.282 0.204 No .347 .040 
Positive history .671 .199 

Type of nursing  
Breast milk feeding .386 .044 

12.992 0.000 Bottle milk feeding .027 .018 
Mixed .247 .061 

Nutritional status 
Normal weight percentile .357 .039 

6.416 0.003 Over-weight percentile .424 .102 
Obese percentile .076 .024 

Sleep nursing 
Yes .348 .046 

1.237 0.219 
No .266 .047 

Teeth brushing 
No brushing .711 .154 

6.744 0.004 One time .205 .047 
Two time .332 .041 

 

 

 

 

 

 



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Table 3. Games-Howell test for multiple comparing of S. mutans in saliva.

Demographic 
Variables 

Comparisons MD P value 

Type of nursing 
Breast milk feeding 

Bottle feeding 0.360 0.000 

Mixed feeding 0.139 0.165 

Bottle milk feeding Mixed feeding -0.220 0.005 

Nutrition status 

Normal weight 
percentile 

Over-weight -0.067 0.813 

Obese percentile 0.281 0.000 

Over-weight 
percentile 

Obese percentile 0.348 0.006 

Tooth brushing 
No brushing 

One time 0.507 0.017 

Two time 0.380 0.076 

One time Two time -0.127 0.111 

 

 

 

 

 

 

 

 

 

 

 

 



Salivary Caries-Related Microorganisms and Demographics of a Group of Children  
 

Vol 13  No 1  (2025)    DOI 10.5195/d3000.2025.764 

 h#p://den*stry3000.pi#.edu 

Table 4. The counts of lactobacilli level in the saliva based on the demographic variables. 

Demographic Variables 
Saliva 
lactobacilli F P value 
Mean SE 

Child Age 
3 to 6 years .221 .036 

1.771 0.081 
7 to 10 years .372 .077 

Child Sex 
Boys .128 .033 

4.320 0.000 
Girls .444 .065 

Father occupation 
Government employee .281 .046 

0.042 0.967 
Free jobs .284 .061 

Mother occupation 
Government employee .316 .047 

3.086 0.003 
Housewife .143 .031 

Adding sugar 
Yes .302 .054 

0.367 0.714 
No .275 .050 

Type of nursing  

Breast milk feeding .275 .052 

0.449 0.639 Bottle milk feeding .383 .062 

Mixed feeding .253 .066 

Nutritional status 

Normal weight 
percentile 

.359 .061 

9.524 0.000 
Over-weight percentile .285 .053 

Obese percentile .052 .015 

Sleep nursing 
Yes .215 .029 

2.373 0.019 
No .402 .091 

Teeth brushing 

No brushing .154 .070 

0.954 0.388 One time .263 .052 

Two time .327 .065 

 

 

 

 

 

 

 



Salivary Caries-Related Microorganisms and Demographics of a Group of Children  
 

Vol 13  No 1  (2025)    DOI 10.5195/d3000.2025.764 

 h#p://den*stry3000.pi#.edu 

Table 5. Games-Howell test for multiple Comparing of lactobacilli in saliva. 

Variables Comparisons MD p value 

Nutritional 
status 

Normal weight 
percentile  

Over-weight 
percentile 

0.074 0.634 

Obese percentile 0.307 0.000 

Over-weight 
percentile 

Obese percentile 0.234 0.000