Salivary levels of Bifidobacteria in caries-free and caries-active children

DOI: 10.1111/j.1365-263X.2011.01220.x

children
Salivary levels of Bifidobacteria in caries-free and caries-active
RAVINDER KAUR1, STEVEN C. GILBERT2, EVELYN C. SHEEHY1 & DAVID BEIGHTON2

1Department of Paediatric Dentistry, and 2Department of Microbiology, Dental Institute, King’s College London, London,

England

International Journal of Paediatric Dentistry 2012

Background Salivary levels of Bifidobacteria have

been shown to be significantly correlated with

caries experience in adults but not as yet in chil-

dren.

Hypothesis. Salivary levels of Bifidobacteria are

positively associated with caries experience in

children.

Aim. To compare the salivary concentrations of

Bifidobacteria of caries-free and caries-active chil-

dren.

Design. Saliva was collected using the tongue-loop

method from 38 caries-active children and from

22 clinically caries-free children, and the numbers

of Bifidobacteria, mutans streptococci, lactobacilli

and yeasts were determined. Additionally, the age

and gender of the children, a plaque index, sugar

Correspondence to:

D. Beighton, Department of Microbiology, KCL Dental

Institute Floor 17, Guys Wing, London Bridge, London SE1

9RT, UK. E-mail: [email protected]

� 2012 The Authors

International Journal of Paediatric Dentistry � 2012 BSPD, IAPD and Bla

amount in diet, sugar frequency in diet, hygiene

practice and fluoride toothpaste usage were

recorded.

Results. Bifidobacteria were isolated from 95% of

the caries-active children and from only 9% of

the caries-free children (P < 0.001). Salivary levels

of Bifidobacteria were significantly correlated with

amount of sugar in the diet, frequency of sugar

consumption and oral hygiene practice. The sig-

nificant variables that discriminated between the

caries-free and caries-active subjects were salivary

levels of Bifidobacteria, salivary levels of mutans

streptococci and oral hygiene practice (v2 = 72.57,

P < 0.001) and overall 90.0% of cases were cor-

rectly classified.

Conclusions. Salivary levels of Bifidobacteria are

significantly associated with caries experience in

children. The salivary levels of this genus may be

a useful marker of caries risk.

Introduction

Bifidobacteria and related genera, Scardovia

and Parascardovia, have been sporadically iso-

lated or detected in the oral cavity usually,

but not always, in relation to active caries1–12.

The method used to isolate or identify the

presence of these bacteria in oral samples has

a significant influence on the detection rates.

Thus, in a study in which 16S rRNA genes

were amplified and cloned from infected den-

tine or supra-gingival plaque associated with

root caries, none of 3544 clones were identi-

fied as being derived members of these three

genera13. Using a DNA–DNA checkerboard

approach in which DNA is extracted from oral

samples and used without any PCR amplifica-

tion, Bifidobacterium species were, however,

present in plaque isolated from cavitated

lesions in 16 of 30 subjects and in the dentin

of 21 of 30 subjects with early childhood car-

ies8. Using a semi-selective cultural approach,

Modesto et al.11 reported that Bifidobacteria

and related genera were present in 13 of 19

samples of infected dentine and in 11 of 15

supragingival plaque samples.

In order to try and unify these observa-

tions, we implemented the use of a selective

growth medium for the isolation of Bifidobac-

teria and related genera which had been used

successfully in the study of the caecal flora of

chickens14. The selective components of the

culture medium are mupirocin and a low pH

environment. Using this approach, we15 iso-

lated Bifidobacteria from the saliva of 94.2%

of 192 adults at concentrations not signifi-

cantly different to those of mutans strepto-

cocci but significantly greater than those

of lactobacilli with Bifidobacterium dentium

being the most frequently isolated species.

Subsequently, we identified significantly

ckwell Publishing Ltd 1

2 R. Kaur et al.

higher levels of Bifidobacteria in active root

carious lesions compared to inactive root car-

ies lesions in adults16 and higher levels in

occlusal carious lesions in both adults and

children than were found on non-carious

surfaces in caries-free individuals17.

The use of saliva as a diagnostic tool in car-

ies has been widely used with salivary levels

of mutans streptococci, lactobacilli or yeasts

being used individually or together to predict

caries risk or current caries activity. In a multi-

variate analysis, we found that the salivary

level of Bifidobacteria was more significantly

associated with caries experience in adults

than were the levels of mutans streptococci,

lactobacilli or yeasts18. No such information is

available for children. Therefore, we under-

took this study in which salivary levels of

these caries-associated bacteria, mutans strepto-

cocci, lactobacilli and Bifidobacteria, and

yeasts were determined in caries-free and

caries-active children.

Materials and methods

Subjects

Children, aged up to 11 years, attending

King’s College Dental Institute and Guy’s

Dental Hospital presenting with carious or

non-carious primary teeth were included. The

patients were medically healthy with a pri-

mary dentition or in the mixed dentition.

Patients not willing to participate, with no

primary teeth, with tooth structure defects,

undergoing orthodontic treatment or wearing

a removable appliance were excluded. Chil-

dren taking antibiotics or completed a course

of antibiotics within the last 4 weeks were

also excluded. Ethical approval for collection

of saliva samples was obtained by Bromley

Research Ethics Committee (RJ1 07 ⁄0231).

Based on previous studies, we expected that

the response within each subject group would

be normally distributed with a standard devi-

ation of 1 and expected a difference in the

mean salivary Bifidobacterial counts of the

two groups, expressed as log10[CFU ⁄ mL of

saliva], would be 1. We would need to study

a minimum of 22 experimental subjects and

22 control subjects to be able to reject

International Journal of Pa

the null hypothesis that the population

means of the experimental and control groups

are equal with probability (power) 0.9. The

Type I error probability associated with this

test of the null hypothesis is 0.05. Clinical

data and saliva samples were collected from

the dorsum of the tongue of caries-free chil-

dren (n = 22) and from caries-active children

(n = 38).

Saliva and clinical data collection

The following data, based upon previous

research on an interactive PC program for

caries risk evaluation19, were collected from

each subject: age, gender, ethnicity, amount

of sugar intake [0 = almost no sugar in diet,

1 = low sugar diet, 2 = moderate sugar diet,

3 = high sugar diet (including bottle feeding

or breast feeding on demand)], frequency

of sugar consumption [0 = max three times

per day, 1 = 4–5 times per day, 2 = 6–7 times

per day, 3 = >7 times per day], oral hygiene

practice [0 = helped with tooth brushing

twice or more times a day using an

electric ⁄ manual toothbrush, 1 = helped with

brushing once a day with using an

electric ⁄ manual toothbrush, 2 = no supervised

tooth brushing but brushes twice a day,

3 = no supervised brushing and brushes once

or less daily], fluoride toothpaste usage

[0 = maximum fluoride >1100 ppm fluori-

dated toothpaste, 1 = Children’s fluoridated

toothpaste, 1000–1100 ppm of fluoridated

toothpaste, 2 = 500 ppm of fluoridated tooth-

paste – milk toothpaste, 3 = too little fluoride,

avoids toothpaste with fluoride] and DMFT,

DMFS, dmft, dmfs according to WHO criteria

supplemented with radiographs. Briefly, a

modified version of the plaque index by Sil-

ness and Loe20 was used; the plaque was

scored on the distal, buccal, mesial and lin-

gual surfaces of 6 teeth; the most posterior

tooth in each posterior segment and the

upper right central incisor and lower left

central incisor and an average calculated.

Saliva sample processing

To collect a saliva sample, a 10-lL bacterio-

logical loop was drawn down the centre of


ediatric Dentistry � 2012 BSPD, IAPD and Blackwell Publishing Ltd

Salivary Bifidobacteria in children 3

the anterior two-thirds of the tongue of each

child until the loop was full of saliva equiva-

lent to 10 lL of saliva21. The saliva sample

was immediately dislodged into 1ml Fastidi-

ous Anaerobic Broth (FAB; Lab M Ltd, Bury,

Lancs, UK), effecting a dilution of 1 in 100 of

the saliva sample. The samples were placed

on ice and processed within 1–3 h. Duplicate

100 lL samples were spread onto the follow-

ing media: Sabouraud dextrose agar (Oxoid,

Basingstoke UK) for the isolation of yeasts,

mupirocin-containing selective medium15 for

the isolation of Bifidobacteria, Rogosa agar

(Oxoid) for the isolation of lactobacilli and

trypticase yeast-extract cysteine medium

(TYC; Lab M Ltd) supplemented with sucrose

and bacitracin (TYCSB) for the isolation of

mutans streptococci. The inoculated plates

were incubated and the number of each

taxon was determined as previously

described15. As the saliva sample (10 lL) was

dispersed in 1 mL of FAB and duplicate

100 lL aliquots were plated on to each selec-

tive medium, the detection limit was

500 cfu ⁄mL, and when none of a particular

genus was isolated from a sample, a value of

200 was included in the analysis.

Statistical Analysis

Microbial counts were expressed as log10

(CFU ⁄ mL of saliva) and mean and standard

deviations were calculated. Mean values of

Table 1. Comparison of clinical and microbiological variables of t(mean ± SE) in bold are significantly greater than the other value

Caries active (n = 3

Age (years) 5.17 ± 0.34dmft 7.68 ± 0.85dmfs 13.05 ± 1.15DMFT 0.11 ± 0.08DMFS 0.11 ± 0.08Plaque Index 0.83 ± 0.14Mutans streptococci* 4.41 ± 0.15 (95)† [2Bifidobacteria* 4.41 ± 0.13 (95) [2.Lactobacilli* 3.54 ± 0.17 (79) [2.Yeasts* 2.28 ± 0.09 (21) [2.

*as Log10[CFU ⁄ mL of saliva]. The detection limit was 500 CFU ⁄ mL anorganism was not isolated from the subject.†(per cent of subjects from whom organisms were isolated).‡(range of values as Log10[CFU ⁄ mL of saliva]).



microbial counts and caries scores were com-

pared using t-tests for independent samples.

Spearman correlation coefficients were calcu-

lated between caries-associated variables and

salivary levels of microbial counts. The distri-

butions of caries-associated variable scores

between the caries-free and caries-active

groups were compared using v2 tests, whereas

the variables discriminating between the two

groups were determined using discrimination

analysis with Wilk’s lambda to assess the sig-

nificance of the associations and the reliability

of the group prediction. All analyses were

performed using SPSSP software (17.0 SPSS

Inc., Chicago, IL, USA).

Results

The mean age of the caries-free group was

significantly greater than that of the caries-

active group (Table 1). The caries-active

group had a significantly higher mean plaque

index score than the caries-free group and

significantly higher levels of mutans strepto-

cocci, lactobacilli and Bifidobacteria. The fre-

quency of detection of mutans streptococci,

lactobacilli and Bifidobacteria, but not yeasts,

was significantly greater than in the caries-

free group (Table 1). Salivary Bifidobacterial

levels were significantly correlated (P < 0.001)

with salivary levels of mutans streptococci

(r = 0.732) and lactobacilli (r = 0.625) but

not with the salivary levels of yeasts

he caries-free and caries-active subjects. All valuesin the row.

8) Caries free (n = 22)

6.82 ± 0.4900000.11 ± 0.05

.0–5.70]‡ 2.39 ± 0.13 (22)† [2.0–4.35]0–5.82] 2.28 ± 0.15 (9) [2.0–4.45]0–5.74] 2.15 ± 0.09 (14) [2.0–4.20]0–4.06] 2.19 ± 0.15 (9) [2.0–5.18]

d a value of 2 [equivalent to 100 CFU ⁄ mL] was used when the

ckwell Publishing Ltd

Table 2. Associations as Spearman correlations between salivary levels of caries-associated microbiota and subjectcaries-associated behaviours (as Scale scores 0–3) in which Score 0 is most health associated and Score 3 is the least healthassociated (see text for explanation).

Variables

Salivary levels of

BifidobacteriaMutansstreptococci Lactobacilli Yeasts

Hygiene practice 0.340** 0.437*** 0.252 )0.022Fluoride toothpaste usage 0.215 0.105 0.265* 0.272*

Sugar: amount in diet 0.635*** 0.633*** 0.347** 0.283*

Sugar: frequency in diet 0.557*** 0.586*** 0.267* 0.263*

***P < 0.001; **P < 0.01; *P < 0.05

Table 3. Comparison of the distribution of the scale scores for health practices associated with caries of the subjects in thecaries-free and caries-active groups. Scale score 0 is the most health associated and 3 is the least health associated for eachof the variables (see text for explanation). All v2 values are P < 0.01.

Variable Group

Scale scores

v20 1 2 3

Hygiene practice Caries free 20 0 2 0 18.1Caries active 14 10 6 8

Fluoride toothpaste usage Caries free 17 1 3 1 13.53Caries active 15 18 5 0

Sugar: frequency in diet Caries free 18 2 1 1 19.4Caries active 9 19 7 3

Sugar: amount in diet Caries free 16 3 2 1 32.5Caries active 2 5 18 13

4 R. Kaur et al.

(r = 0.122). Salivary levels of Bifidobacteria

were significantly associated with the plaque

index (r = 0.522; P < 0.001) as were the sali-

vary levels of mutans streptococci (r = 0.376;

P < 0.01) and lactobacilli (r = 0.312; P < 0.05)

but at a lower level of significance. The sali-

vary levels of each of these organisms were

significantly positively correlated to the

amount and frequency of sugar consumption

(Table 2). Poor oral hygiene practice was

significantly associated with higher salivary

levels of only Bifidobacteria and mutans

streptococci, whereas reduced fluoride

exposure was associated only with increased

salivary levels of lactobacilli and yeasts

(Table 2). The caries-free children were signif-

icantly more likely than the caries-active

children to have overall better oral hygiene

practices, to use toothpaste with the highest

fluoride concentration, to have the lowest

frequency of sugar consumption and to con-

sume a diet with the lowest amount of sugar

(Table 3).


Discriminant analysis was used to deter-

mine which of the variable measured here

best enabled the correct classification of the

caries-free and the caries-active subjects into

their correct groups. Only the three variables,

salivary levels of Bifidobacteria, salivary levels

of mutans streptococci and oral hygiene prac-

tice were significant in the analysis (Wilks’

Lambda = 0.277; v2 = 72.57, P < 0.001). It

was found that the group status of 34

(89.5%) of the caries-active group was cor-

rectly predicted and that the group status of

20 (90.9%) of the caries-free group was cor-

rectly predicted; overall 90.0% of cases were

correctly classified.

Discussion

Bifidobacteria and related genera are aciduric

and acidogenic species being routinely iso-

lated on acidic culture media. Many different

species of Bifidobacteria are members of the

human gut flora22, whereas the oral species




appear to be limited to B. dentium, Parascardovia

denticolens, Scardovia inopinata and Scardovia

wiggsiae11,12 although others including B. sub-

tile, B. longum, B. scardovii, Alloscardovia omni-

colens, B. adolescentis and B. urinalis have been

isolated from oral samples but generally only

at a low prevalence. Here, we did not identify

the isolates, but based on previous extensive

analyses using these same analytical methods,

it is likely they will have been predominantly

B. dentium15.

The salivary levels of Bifidobacteria were

found not to be significantly different to those

of mutans streptococci with 94% of adults

harbouring detectable levels of Bifidobacte-

ria15. We subsequently demonstrated that the

proportions and frequency of isolation of Bifi-

dobacteria in samples of infected dentine

from root caries lesions were significantly

related to the clinical severity of the lesion16.

Similarly, the proportions of Bifidobacteria in

occlusal lesions in adults and children were

significantly greater than on sound tooth

surfaces17.

Many studies have demonstrated similar

relationships for lactobacilli and mutans

streptococci and that the salivary levels of

these taxa are related to caries experience in

most groups of individuals, irrespective of age

as the presence of these bacteria is due to

an oral environment conducive to the initiation

and progression of caries23. We have, how-

ever, previously only reported that the sali-

vary levels of Bifidobacteria are significantly

related to caries experience in elderly

adults18. This study has extended these obser-

vations as we have shown that the salivary

levels of Bifidobacteria are significantly

related to caries experience in children and

that the salivary levels of mutans streptococci,

lactobacilli and Bifidobacteria are significantly

correlated as was also demonstrated in

adults18.

In both the adult population we studied

and the children here, the salivary level of

Bifidobacteria was the variable, of those mea-

sured, most significantly related to caries

experience. In the children, the salivary levels

of Bifidobacteria and mutans streptococci

were both independent variables associated

with caries experience suggesting that differ-



ent mechanisms are responsible for their pres-

ence in the mouth. Mutans streptococci

colonise hard surfaces and their proliferation

is mediated by intake of fermentable carbo-

hydrates and the development of a low

intraoral, or intrasite, pH24. The predominant

Bifidobacterium is B. dentium, and we know

little of the factors that influence its colonisa-

tion of the dentition. Its numbers are very

low on caries-free tooth surfaces but high in

lesions16,17, and data from studies of denture

plaque provide further insight into its eco-

logy25. Unlike mutans streptococci, B. dentium

does not colonise hard surfaces per se since

denture plaque associated with denture

stomatitis harboured high levels of mutans

streptococci, lactobacilli and yeasts but not

B. dentium. This indicates that B. dentium does

not simply colonise hard surfaces and suggests

that surfaces or dentinal components exposed

in lesions facilitate the attachment and prolif-

eration of B. dentium. It may therefore be that

different factors mediate the establishment of

B. dentium and mutans streptococci in the

mouth and so each is represented in the dis-

criminant analysis as significant independent

factors related to caries experience.

The salivary levels of Bifidobacteria and

mutans streptococci were significantly corre-

lated with the amount of sugar consumed,

with the frequency of sugar consumption

and with oral hygiene practice. This is

the first time that such associations have

been demonstrated for Bifidobacteria but

such relationships should be expected given

the association between the salivary levels

and caries experience reported here and pre-

viously.

Bifidobacteria are used in probiotics to

improve general health and have often been

provided in yoghurt in attempts to modify

the oral environment and reduce the levels of

mutans streptococci. Bifidobacterium animalis

subsp. lactis DN-173010 is one Bifidobacteri-

um used in such studies, though other species

and strains may be used, and a typical

exposure is for the consumption of 200 g

fruit yogurt containing (2 · 108 CFU ⁄g) once

daily for periods of 2 weeks26. There have

been no reports of significant colonisation of

the oral cavity of individuals involved in


6 R. Kaur et al.

these studies, but such studies apparently

involve individuals with no active carious

lesions. It remains to be determined whether

Bifidobacteria, included in probiotic prepara-

tions, colonise active lesions and contribute to

the caries process.

Why this paper is important for paediatric dentists
d Paediatric dentists should be aware that caries is a bio-
film-mediated disease with the increased salivary levels

of Bifidobacteria, and other caries-associated organ-

isms, an indicator of an oral environment conducive to

the initiation or progression of dental caries.d Paediatric dentists should know that caries is not an

infectious disease but is caused by the interaction of

sugars in the diet and the normal oral microbiota resi-

dent on the dentition.d Paediatric dentists should be aware that poor oral

hygiene is associated with increased caries experience.

Acknowledgements

This study was supported in part by the

Dental Institute, King’s College London and

the Department of Health via the National

Institute for Health Research (NIHR) Compre-

hensive Biomedical Research Centre award

to Guy’s & St Thomas’ NHS Foundation Trust

in partnership with King’s College London

and King’s College Hospital NHS Foundation

Trust.

Conflict of Interest

The authors declare no conflict of interest.

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Salivary levels of Bifidobacteria in caries-free and caries-active children