This study was carried out to evaluate levels of infection of bacteria involved in caries and oral streptococci and their colonization after use of mouthrinses with NaF, chlorhexidine and chlorhexidine plus NaF. Combination of chlorhexidine and fluoride was the only agent that reduced levels of Streptococcus mutans (group) for 30 days

Beyruth, Janine B.

Lima, Kenio Costa

Magalhães, Fernando A. C.

Neves, Aline A.

Uzeda, Milton de

Lima, Kenio Costa de

Repositório Institucional da Universidade Federal do Rio Grande do Norte

Brazilian Journal of Microbiology (2001) 32:158-161
ISSN 1517-8382
158
LEVELS OF INFECTION AND COLONIZATION OF SOME ORAL BACTERIA AFTER USE OF
NAF, CHLORHEXIDINE AND A COMBINED CHLORHEXIDINE WITH NAF MOUTHRINSES
Kenio Costa Lima1,2; Aline A. Neves3; Janine B. Beyruth1; Fernando A.C. Magalhães1; Milton de Uzeda1*
1Instituto de Microbiologia “Prof. Paulo de Góes”, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
2Departamento de Odontologia, Universidade Federal do Rio Grande do Norte, Natal, RN, Brasil.
3Faculdade de Odontologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil
Submitted: November 28, 2000; Returned to authors for corrections: April 03, 2001; Approved: June 21, 2001
SHORT COMMUNICATION
ABSTRACT
This study was carried out to evaluate levels of infection of bacteria involved in caries and oral streptococci and
their colonization after use of mouthrinses with NaF, chlorhexidine and chlorhexidine plus NaF. Combination of
chlorhexidine and fluoride was the only agent that reduced levels of Streptococcus mutans (group) for 30 days.
Key words: chlorhexidine, sodium fluoride, cariogenic bacteria, oral streptococci
Mechanical methods are known for prevention and control
of oral diseases. Some authors consider that they consume
time for both professional and patients (14). It is important to
consider yet that they are laborious and difficult for many
patients (14,24) and impossible to others (24). Based upon these
considerations and aiming to make dental plaque control more
accessible to a significant proportion of population less
laborious methods are wished (32). Among these methods are
the chemical controls that have fascinated men for over 400
years (7). Chlorhexidine was originally introduced as commercial
products in 1957 in Europe as an antiseptic cream for skin
wounds (18). It has generated an increasing interest among
dental community since its introduction as a 0.2% mouthrinse
in experimental gingivitis study in 1970 (16). Apparently by
acting via a generalized perturbation of the bacterial membrane
rather than the antibiotic method, there is much less opportunity
for bacterial resistance development, which was an early concern
with it (18). Concerning fluoride, the first mouthrinse
recommended in the United States was in the fourties for
schoolchildren as caries prevention. Nowadays, FDA authorized
the sale under control with mouthrinses of 0.05% sodium fluoride
(NaF) (10). In spite of several antibacterial mechanisms
suggested for the action of fluoride, it seems that the effect
* Corresponding author. Mailing address: Laboratório de Microbiologia Oral, Instituto de Microbiologia “Prof. Paulo de Góes” - UFRJ, Caixa Postal
68040. Cidade Universitária, 21941-590, Rio de Janeiro, RJ, Brasil. Tel.: (+5521) 260-4139. Fax: (+5521) 560-8344. E-mail: immmmuz@microbio.ufrj.br
over plaque activity is low, reducing only its metabolic activity
(2,9) but not reducing bacterial concentration (17). Therefore,
the purpose of this study was to evaluate levels of infection of
major bacteria involved in caries process and the major group
of bacteria isolated in the mouth, the streptococci, and their
colonization after use of mouthrinses with NaF, chlorhexidine
and chlorexidine plus NaF.
The subject population was selected from a group of military
cadets from 18 through 25 years of age by means of previous
screening based on levels of infection of mutans streptococci
(>250.000 CFU / ml saliva) and therefore submitted to a high
microbiological risk of caries (30). All the 60 selected patients
signed an agreement term where they became aware of aims, risks
and benefits of the study, allowing their inclusion. The subject
population was divided into three groups according to products
that they would use. This study was a double-blind where each
group had 20 subjects. Subjects rinsed twice a day during 15
days with 10 ml of only one of the following solutions: a) NaF
(0.05%, aqueous commercial solution prepared by Herpo, Rio de
Janeiro, RJ) b) 0.12% of chlorhexidine digluconate
(Periogard®,Colgate-Palmolive, Osasco, SP), c) 0.05% of NaF plus
0.12% of chlorhexidine digluconate (Duplak®, Herpo Ltda, Rio
de Janeiro, RJ) according to recommendations for patients with
Use of mouthrinses
159
high risk of caries (15). Thirty minutes after toothbrushing, the
individuals rinsed with solutions containing chlorhexidine to avoid
interference of sodium lauryl sulfate from dentifrices (1). When
the period of 15 days was over, the patients remained under
observation for a month for disclosing concentration of bacterial
population studied. To verify levels of infection and colonization
by mutans streptococci, lactobacilli and total oral streptococci,
saliva stimulated with gum base was collected from all patients in
three periods: a) before treatment with each product (baseline), b)
immediately after finishing treatment (T0), c)15 days after (T15), d)
30 days after (T30). Saliva was collected and transported in a cooler,
and was immediately processed. Concerning total oral
streptococci, diluted saliva was inoculated on Mitis-Salivarius
Agar (Difco Laboratories, Detroit, Michigan) (3). For cultivation
of mutans streptococci diluted saliva was inoculated on Mitis-
Salivarius Agar added with 15% of sucrose, 0.1% potassium
tellurite solution and bacitracin (0.2 IU/ml) (6). These plates were
incubated in anaerobic atmosphere for 48 h and after this period
colonies were counted. Concerning oral lactobacilli, diluted saliva
was inoculated with pour-plate technique on Rogosa-SL Agar
(Difco Laboratories, Detroit, Michigan) (25) and incubated in
aerobic atmosphere at 37ºC for 72h. The same procedure used for
other bacteria was employed for counting these microorganisms.
Data obtained were statistically analyzed for differences within
and among groups and periods of experiment into each groups
by means of the ANOVA and Tukey’s tests. The significance
level was established at 5% (p < 0.05).
Considering all subject groups in relation to oral streptococci,
in Table 1 is observed that there was no significant difference
among them (p < 0.05) nor among period of experiment within
each group (p < 0.05). According Table 1, subjects that mouthrinsed
with NaF (group 1) showed no reduction of their levels of infection
by mutans streptococci alongside the study with no significant
difference among periods of experiment (p < 0.05). However,
concerning those subjects who mouthrinsed chlorhexidine
combined with NaF (group 2), there was a significant difference
among experimental period (p > 0.05). This difference was observed
among values of baseline and at different times (T0, T15 and T30,).
Concerning individuals from group 3, who mouthrinsed with
chorhexidine, levels of infection by mutans streptococci were
significantly reduced (p > 0.05) between baseline and T0.
Comparing to other periods with baseline we can observe that
there is no significant difference (p < 0.05). Comparing levels of
infection by mutans streptococci with periods among groups no
significant difference was observed (p < 0.05). This shows that
the reduction observed in group 2 (T0, T15 and T30) and, that
observed in group 3 (T0) was not sufficient to reduce levels below
those observed in group 1 at the same period. Considering all
groups, in Table 1 is observed with relation to lactobacilli there
was not a significant difference among them (p < 0.05) nor among
periods for each group (p < 0.05).
Recently, the association of chlorhexidine and fluoride was
proposed. Thus, this study was carried out to evaluate levels
of infection and colonization of the major cariogenic bacteria
and oral streptococci after use of fluoride, chlorhexidine and
fluoride plus chlorhexidine mouthrinses. After two weeks of
mouthrinses of fluoride no reduction was observed on the
levels of infection by oral streptococci, mutans streptococci
and oral lactobacilli nor any difference among baseline, at T0,
T15 and T30 of this treatment group regarding the other groups.
Concerning fluoride it was reported that minimal inhibitory
concentration to viridans streptococci was 0.125% (23),
therefore a concentration higher than our study (0.05%). Major
properties of fluoride in plaque control and as an antimicrobial
product are the abilities to change the enamel surface energy
(28) and inhibit the glycolysis and transport of nutrients based
on phosphoenolpyruvate (8,17) and in a system based on
ATPase linked to cell membrane by pH gradient (9). Some oral
lactobacilli are ten times more resistant to fluoride than
streptococci (8,17,22). In our study the reduction of lactobacilli
was not observed due to low concentration used in daily
mouthrinses. Combination of fluoride and chlorhexidine was
effective to reduce levels of mutans streptococci (around 1 log.)
and to keep it reduced for 30 days. However no reduction was
observed with oral streptococci and lactobacilli. Our results are
in accordance with most the authors where is observed a broad
effect of this association to reduce mutans streptococci
(12,20,29,30). This effect is formerly derived from bactericidal
Oral streptococci Streptococcus mutans (group) Lactobacilli
Periods NaF NaF+CHX CHX NaF NaF+CHX CHX NaF NaF+CHX CHX
Baseline 7.87 ± 0.66 7.86 ± 0.71 7.85 ± 0.50 5.86 ± 0.69 6.10 ± 0.65 5.86 ± 0,68 3.82 ± 1.10 4.36 ± 1,91 4.78 ± 1.54
T0 7.95 ± 0.45 7.63 ± 0.66 8.06 ± 0.62 5.60 ± 0.70 5.30 ± 0.72* 5.20 ± 0,55* 4.11 ± 1.38 3.88 ± 1,96 4.00 ± 1.57
T15 7.83 ± 0.52 7.78 ± 0.64 8.01 ± 0.48 5.43 ± 0.74 5.48 ± 0.66* 5.36 ± 0,99 4.26 ± 1.36 4.06 ± 1,69 4.23 ± 1.34
T30 7.97 ± 0.43 7.94 ± 0.55 7.99 ± 0.59 5.58 ± 0.72 5.45 ± 0.90* 5.34 ± 0,80 4.10 ± 1.77 3.56 ± 1,99 3.85 ± 1.59
Table 1. Means ± standard-deviations (in LOG) of number of CFUs of oral streptococci, Streptococcus mutans ( group ) and lactobacilli
/ ml of saliva in several periods (in days) and groups of patients treated with NaF, NaF + Chlorhexidine (CHX) and CHX.
* values different significantly (p > 0,05); baseline = before treatment; T0 = immediately after treatment; T15 = 15 days after treatment;
T30 = 30 days after treatment.
160
K.C. Lima et al.
action of chlorhexidine (19,20) followed its bacteriostatic action
that in association with fluoride would inhibit bacterial
metabolism in different sites. Bacterial recolonization is observed
after some days (26,29) or months based on adopted treatment,
infection levels, sucrose ingestion or dental restorations. In
this study the recolonization of mutans streptococci did not
occur 30 days after treatment, probably due to combined action
of both products and because infection level at baseline was
high. Despite reduction of mutans streptococci levels, oral
streptococci were not total, suggesting that the site and function
of mutans streptococci in oral ecosystem may be occupied by
other bacteria, probably some strain of streptococci not reduced
by this treatment. This hypothesis is supported by the fact that
some oral streptococci strains show variable susceptibility to
chlorhexidine and fluoride (17,19,21). Concerning oral
lactobacilli, levels were not reduced by fluoride and
chlorhexidine probably because these microorganisms were
hidden in habitats not affected by these products as observed
among irradiated patients (12). Daily mouthrinses with
chlorhexidine alone reduced levels of infection by mutans
streptococci immediately after treatment (T0), afterwards they
returned to baseline levels. Despite of some other studies had
observed that this reduction had been more prolonged (4,5,11),
in our study this fact was not observed probably due to low
concentration used. Toxic effects of chlorhexidine on mutans
streptococci is due to affect the hydrofobicity (13). Oral
streptococci and lactobacilli were not affected by treatment with
chlorhexidine mouthrinses. Oral streptococci shows varied
susceptibilities to chlorhexidine (19,21) from 4 to 32 µg / ml (27).
Oral lactobacilli frequently are not affected by chlorhexidine
because of difficulty to achieve niches of lactobacilli into mouth
(4,17). Regardless of reduction promoted by the use of
chlorhexidine with fluoride for 30 days after mouthrinses and
chlorhexidine immediately after treatment, these reductions must
be questioned. Indeed infection levels observed after those
rinses did not reduce as significantly as in fluoride group. In
addition those solutions probably causes shifts in oral microbial
ecosystem, thus, we can suggest that these compounds can
not be used without prescriptions and should be specifically
indicated for some patients. On the other hand, we observed
that the combination of chlorhexidine and fluoride solution is
able to reduce mutans streptococci by 30 days. Finally, we
concluded that the use of this solution, if not well controlled,
could be selective for other streptococci in oral environment.
ACKNOWLEDGMENTS
The authors are most grateful to the staff of Centro de
Estudos de Pessoal (Brazilian Army, Rio de Janeiro, RJ, Brazil)
where part of the research was carried out. It was supported by
grants and scholarships from the following Brazilian agencies:
CNPq, PRONEX-MCT, CAPES, FAPERJ and FINEP.
RESUMO
Níveis de infecção e colonização de algumas
bactérias orais após o uso de soluções de
fluoreto de sódio, clorexidina e de uma combinação
de fluoreto de sódio e clorexidina
O objetivo deste estudo foi avaliar os níveis de infecção de
bactérias cariogênicas e estreptococos orais e sua colonização
após uso de bochechos com NaF, clorexidina e clorexidina e
NaF associados. Associação de clorexidina e NaF foi o único
agente que reduziu os níveis de Streptococcus mutans (grupo)
por 30 dias.
Palavras-chave: clorexidina, fluoreto de sódio, bactérias
cariogênicas, estreptococos orais.
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Levels of infection and colonization of some oral bacteria after use of naf, chlorhexidine and a combined chlorhexidine with naf mouthrinses

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