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BALKAN JOURNAL OF STOMATOLOGY ISSN 1107 - 1141

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Effects of Cetylpyridinium Chloride in Overall and

Caryogenic Salivary Micro Flora Reduction

SUMMARY Aleksandar Dimkov1, Nikola Panovski2,

As an active ingredient of oral antiseptics, cetylpyridinium chloride Marija Stevanovic1, Elizabeta Gjorgievska1
(CPC) has a wide antimicrobial spectrum with a strong bactericidal effect on 1Department of Pediatric Dentistry, Faculty of
Gram positive microorganisms and a fungicidal effect on yeasts. It’s efficiency Dental Medicine
2Institute of Microbiology and Parasitology,
against Gram negative pathogenic microorganisms and mycobacteriums is Medical Faculty
questionable. Concentration of 0.05% of CPC in mouth-rinses results with Skopje, FYROM
immediate reduction of bacterial counts. The aim of this study was to esti-
mate the salivary levels of Streptococcus mutans (MS) and Lactobacillus
species (LB) in the saliva before and after mouth rinsing with an antiseptic
mouth-rinse containing CPC, and to compare the number of whole salivary
flora by saliva analysis before and after mouth rinsing. In order to accom-
plish this aim we used Aqua fresh mouthwashes (Smith Kline Beecham,
Great Britain). The group consisted of 12 healthy schoolchildren aged 9 -
13 of both sexes. The participants were with a good oral health and similar
DMF indices. For better precision and accuracy, the same group was a con-
trol group, too. The saliva samples were taken before, and 20 minutes after
tooth brushing, early in the morning, after at least 12 hours without oral
hygiene. The counts of MS and LB were determined with commercial CRT
bacteria strips produced by Ivoclar-Vivadent, Liechtenstein. The total count
of the saliva microorganisms was determined by standard microbiological
methods. A significant reduction in salivary MS and LB levels was observed
in all samples as well as a decrease in the total count of aerobe and anaero-
be bacteria and yeast.
Keywords: Cetylpyridinium Chloride; Antimicrobial Agents; Streptococcus mutans; ORIGINAL PAPER (OP)
Lactobacillus species; Dental Decay; Salivary Flora Balk J Stom, 2006; 10:

Introduction
The fact that microorganisms are one of the most
essential factors in dental disease aetiology arouses the
question of how to eliminate or reduce them to minimal
values. Because of that the major prerequisite is typologi-
cal and numerical verification of the microorganisms. On
the other hand, the key factor of the "struggle" against
them is proper maintenance of oral hygiene.
New achievements of science and technology in
the field of prevention and contemporary mechanical
and chemotherapeutical approaches to oral hygiene are
aimed at changing the oral micro flora and contributing to
healthy dental and periodontal tissue. There is a plethora
of agents and methods for oral health preservation. Some
of these are meant to be used at one's home; the others are
designated for professional use.
Various classifications of anti-microbial agents can
be found in the literature. One of these is the classification
into groups according to chemical characteristics, and it is
this classification that this paper shall follow. According to
this classification, anti-microbial agents are divided into:
● Cations - positively charged ions with the potential to
interfere with bacterial membrane functions, bacterial
adhesion and glucose uptake;
● Anions - negatively charged ions with the potential to
interfere with bacterial membrane functions, glycolytic
metabolism and glucose uptake;
2 Aleksandar Dimkov et al. Balk J Stom, Vol 10, 2006

● Non-Ionic Agents - non-charged ions are believed to bactericidal effect on Gram positive pathogens and a
inhibit membrane enzymes, which leads to reduced fungicide effect on yeasts in particular. There are gaps
glucose uptake; in its effectiveness against Gram negative pathogens and
● Enzymes - some enzymes are suggested to interfere mycobacteria. Application of CPC at a concentration
with bacterial adhesion and others to enhance the of 0.05% as a mouth-rinse results in an immediate
lysozyme activity; reduction in bacterial counts. These data indicate that
● Sugar Alcohols - polyols are believed to interfere with the use of CPC-containing mouthwashes does not alter
bacterial glycolysis. the composition of the normal oral flora or results in the
The cation-group of antimicrobial agents consists, establishment of non-oral and potentially pathogenic
among others, of: bacteria in the mouth. The balance of intra-oral bacterial
1. Chlorhexidine; flora is not disturbed even in the case of relatively long-
2. Metal ions (Zn2+, Cu2+, Sn2+); term use of CPC (up to 6 weeks). Taking these properties
3. Quaternary ammonium compounds; into consideration, CPC may be considered as an
4. Sanguinaria extracts (a natural herbal extract). alternative active ingredient in the case of chlorhexidine
Cations are attracted to the bacterial cell walls intolerance in the treatment and prevention of bacterial
because of the substance’s positive charge and the negative or fungal disorders of the oropharyngeal area. It is also of
charge of the bacterial cell wall. Gram positive bacteria are interest as an element for combination in the development
more sensitive to cations since they are more negatively of new types of oropharyngeal antiseptics14,15. The
charged. Mutans streptococci (MS) are gram positive formulation of CPC, chlorhexidine and zinc lactate mouth-
bacteria and are therefore very sensitive to cations. rinses results in a significant reduction of the anaerobic
The durability of the effect depends also on the
and aerobic salivary microbial which prevents from bad-
concentration and the substantivity of the antimicrobial
breath16.
agent, and on the selectivity of the solvent. “Substantivity”
3 general ways of action of quaternary ammonium
is a term from pharmacology that connotes the ability of
compounds, including CPC, on the bacterial cell are
a drug or antimicrobial to stay in contact with the target
known3 (Fig. 1):
organisms long enough to kill or debilitate them1,2,13.
Substantivity can be assessed by measuring the magnitude
and duration of the fall in salivary bacteria following
single rinses with antimicrobials12.
The quaternary ammonium compound group consists
of 2 antimicrobial agents, which are perhaps the most
frequently used ones, since they are the main antimicrobial
ingredients of a considerable number of products for mass
consumption, i.e. dentifrices and mouthwashes. These
are: Cetylpyridinium Chloride (CPC) and Benzalkonium
Chloride.
Although there are many stages in the historical
development of quaternary ammonium germicides,
there is general agreement on at least 2 truly historical
milestones. The first is the work of Jacobs et al., which
examined structure, preparation, and antimicrobial activi-
ty. Browning (1926) described the bacterial activity of
quaternary derivates of pyridine, quinoline, and other ring
structures. Hartmann and Kagi, in 1928, reported on the
antibacterial activity of quaternary ammonium compounds
of acylated alkylene diamines. In 1935, Domagk disclosed
the antimicrobial activity of the long-chain quaternary
ammonium salts, and it was the second and most important GTF – Glycosyltransferase 3 PG – 3-Phosphoglycerate
PMF – Protonmotiveforce 2 PG – 2-Phosphoglycerate
milestone in the development of antimicrobial quaternary PTS – Phosphotransferase system PEP – Phosphoenolpyruvate
ammonium compounds. After Domagk’s discovery of G-6-P – Glucose-6-Phosphate PYR – Pyruvate
the biocidal properties of cationic surface active agents, F-6-P – Fructose-6-Phosphate E1 – Enzyme 1
several generations of structurally variable quaternary F-1,6P2 – Fructose 1, 6-bisphosphate HPr – Histidine protein heat resistant
3-PGA – 3-Phosphoglyceraldehyde E2 – Enzyme 2
ammonium antimicrobials of commercial importance were DHAP – Dihydroxyacetonephosphate
developed.4
CPC, as active ingredient of antiseptic oral mouth-
Figure 1. Ways of action of quaternary ammonium compounds
rinses, has a broad antimicrobial spectrum with a rapid
Balk J Stom, Vol 10, 2006 Antimicrobial Effect of Cetylpyridinium Chloride 3

● Disturbing the normal membrane functions of the
bacteria and especially in the case of Streptococcus
mutans;
● Interfering with the bacterial adhesion on the tooth or
in the pellicle by affecting a surface enzyme;
● Interfering with a glycolytic enzyme, leading to a
reduced acid production by the bacteria.
The aim of this study was:
1. To estimate the salivary levels of Streptococcus
mutans (MS) and Lactobacillus species (LB) before and
after rinsing with mouthwashes contains CPC;
2. To compare the number of whole salivary flora by
saliva analyses before and after rinsing with mouthwashes
contains CPC.
Material and Methods
with CPC mouthwashes, into special sterile containers
made particularly for this purpose (Fig. 2).
The specimens used to determine the counts of MS
and LB in the saliva were taken with CRT bacteria -
commercially available strips (Ivoclar-Vivadent, Schaan,
Liechtenstein). These strips have selective culture media
for the determination of the MS count in the saliva or
plaque on the blue agar surface and for the determination
of the LB count in the saliva on the bright agar surface.
The first step of the procedure was to remove the agar
carrier from the test vial (Fig. 3). After that, a NaHCO3-
tablet was placed at the bottom of the vial. The protective
foils were carefully removed from the 2 agar surfaces,
taking care not to touch the agar. Both agar surfaces were
thoroughly moistened with saliva, using a pipette, allowing
the excess saliva to drip off. Finally, the agar carrier was
slid back into the vial, the vial was closed tightly and it
was then sent to the Institute of Microbiology.

The group consisted of 24 healthy schoolchildren

aged 9-13 of both sexes. The participants had good oral
health, similar hygiene, normal dietary regimen, and
similar DMF indices. In order to obtain higher precision
and accuracy, the same group was used as a control group,
too. Saliva samples were taken before and 20 minutes
after mouth rinsing, early in the morning, after at least
12 hours being without oral hygiene. The study subjects
were selected at the Faculty of Stomatology, Department
of Pediatric Dentistry in Skopje. The microbiological
analyses were carried out at the Institute of Microbiology
and Parasitology, Medical Faculty, Skopje.
In our study we used the Aqua fresh mouthwashes
(SmithKline Beecham, Great Britain). Besides CPC as
an active antimicrobial agent, its ingredients are: water,
Sodiumfluoride, Cocamidopropyl Betaine, Sodiumsaccharin,
Figure 2. Special sterile containers for collecting saliva
Sodiumcarbonate.

Sequence of Procedures for Collecting Saliva

Samples
1. Patients refrain from oral hygiene for at least 12
hours before the treatment;
2. First saliva sample is taken without any prior
food consumption, mouth rinsing or saliva stimulation;
3. Rinsing with Aqua fresh mouthwashes for
approximately 30 sec;
4. After rinsing, the mouth is rinsed with 200 ml of
water for about 15 sec;
5. The second saliva sample is taken following a 20
min. intermission.

Methods of Quantitative Evaluation of MS and LB

and of the Total Number of Salivary Microbials
In order to determine the total number of salivary
microorganisms, saliva samples were taken by spitting Figure 3. Procedure for determining Streptococcus mutans and
approximately 3-5 ml of saliva before and after rinsing Lactobacillus species
4 Aleksandar Dimkov et al. Balk J Stom, Vol 10, 2006

Determining the quantitative estimate of MS and
LB species counts in saliva samples
Planting was performed simultaneously with
sampling. After an incubation period of 48 hours at 35-
370C, the grown colonies (colony forming units - CFU)
were counted, provided their number is small, or are
compared with the chart supplied by the manufacturer
when their number is excessive, and are then interpreted
as 10,000; 10,000-100,000; 100,000-1,000,000 and
>1,000,000 CFUs (Fig. 4). The MS colonies are
translucent on the blue agar surface, while LB species
colonies are grey-white on the green agar surface. By
counting the colonies only the approximate number of
bacteria can be determined, because of the notion that 1
bacterial cell causes the growth of 1 colony, and is thus
being designated as “a colony forming unit (CFU)”. After
drying up, strips can be stored in a refrigerator at 2-80C,
where, being protected from light and from temperature
fluctuations, they can last for years, and be used for
comparison purposes at any time.
A, B and C. The capital A was used to mark the growth
density in the first sector, B was used for the second and C
for the third sector. The sector marked with A presented a
sector with such a high density of growth of colonies that
they could not be counted (A > 100 colonies within the
sector), B - a growth of 20 to 100 colonies, C - 5 to 20, and
0 - 0 to 5 colonies. With the aim to present the results in
a more convenient way, we have split the growth density
sectors in accordance with the number of colonies into high
growth density sectors (AAA, AAB, and AAC), medium
growth density sectors (ABB, ABC, ABO, and ACO), and
into sectors of low or no growth density (BBO, BCO, and
OOO). The sectors of the first group were labelled with
two pluses (++), those of the second group with one plus
(+), and the sectors of the third group with plus minus (+-).

Figure 4. Colony forming units - CFU for Mutans Streptococci and for Figure 5. Growth density sectors
Lactobacilli (standard according to manufacturer’s instructions)

Results
Semi quantitative determination of whole microbial
salivary counts Results are shown in tables 1-4 and figures 6 and 7.
Semi quantitative determination of the whole salivary
microbial counts was performed with a 4 mm diameter Table 1. Growth density of salivary aerobic and anaerobic
calibrated eza. On each of the 3 bases, 50μl of saliva bacteria before and after use of CPC Aqua fresh mouthwashes
were spread in the usual manner (routine processing). In Aerobic Anaerobic
Growth density
order to obtain isolated colonies by dilution, the material Before After Before After
was transplanted up to the half of the Petry-dish (sector 1) AAA / / / /
++ AAV / / / /
on 3 sectors of the Petry-dish (Fig. 5). Then the eza was AAS / / / /
sterilized by heating, and the materials from the 2 lines of AVV 2 2 1 /
the sector 1 were transplanted onto the quarter in the lower +
AVS 6 / 5 2
AVO 5 1 4 1
side of the Petry-dish (sector 2). At the end, the eza was ASO 11 3 11 3
again sterilized by heating and the material from the last 2 VVO / 2 1 /
lines of the sector 2 was transplanted onto the last quarter +- VSO / 6 2 8
OOO / 10 / 10
of the Petry-dish (sector 3). 2 χ2 – p < 0,05
The results were read out in a semi quantitative way, χ – p < 0,05
p < 0,05 (p =
(p = 0,0000004)
i.e. the density of growth was marked with the capitals 0,00004)
Balk J Stom, Vol 10, 2006 Antimicrobial Effect of Cetylpyridinium Chloride 5

Table 2. CFU in subjects with isolated Candida albicans before

and after use of CPC Aqua fresh mouthwashes

Colony number
Subject Before use of After use of
Aqua fresh Aqua fresh
1 3 0
2 5 0
3 1 0
4 1 0
N 10 0
Figure 6. Growth density of salivary aerobic and anaerobic bacteria
before and after use of CPC Aqua fresh mouthwashes

Table 3. Number of subjects with CFU (colony forming units) of

Streptococcus mutans and Lactobacillus species in 1 ml saliva
Before
before and after use of CPC Aqua fresh mouthwashes

After
Streptococcus Lactobacillus species
mutans CFU/ml CFU/ml
Before After Before After
Without growth / 16 (1) 16
102-3 / 3 1 2 Figure 7. CFU in subjects with isolated Candida albicans before and
after use of CPC Aqua fresh mouthwashes
103-4 2 2 3 1
104-5 3 1 3 2
105-6 5 1 4 /
106-7 14 1 12 2 Discussion
N 24 24 23 23
In this study we analyses the CPC, a cationic
antimicrobial compound, and its effects against whole oral
microbial flora, and especially against cariogenic microbial
flora. Taking into consideration that CPC has a positive
Table 4. Effects of CPC Aqua fresh mouthwashes on reduction of charge it is attached to the bacterial cell walls, which are
cariogenic salivary micro-flora counts (number of subjects with charged negatively. Gram positive bacteria, including
logarithmic reduction factor - log RF) MS, are more sensitive to cations, since they are more
negatively charged. Like other agents from this group, first
Streptococcus Lactobacillus
of all banzalkonium chloride and chlorhexidine, CPC has
log RF a broad antimicrobial spectrum with a rapid bactericidal
mutans species
effect on Gram positive pathogens, and a fungicide
0 1 2 effect on yeasts in particular. According to literature,
1 2 / its effectiveness against Gram negative pathogens and
mycobacteria is questionable. Compared to chlorhexidine,
2 / 3
CPC has a lesser number of side-effects, but it has a
3 4 4 weaker effect against plaque and gingivitis.
≥4 17 14 In order to observe the effects of CPC in the
reduction of salivary cariogenic microflora and whole
N 24 23 salivary flora, we used the Aqua fresh mouthwash, whose
active compound is precisely the CPC.
6 Aleksandar Dimkov et al.
Despite the fact that in recent years the market and
the pharmacies offer different mouthwashes of various
compositions, produced by numerous manufacturers, their
use in our country has been almost entirely incidental.
This became apparent from the results of a poll, according
to which none of the participants had used mouthwashes.
This was an interesting finding, for which several possible
explanations can be offered. On the one hand, it could
probably have been a result of the already obsolete
perception regarding the negative effects caused by the
application of chlorhexidine - the cationic antimicrobial
agent with the longest “season” and, until now, the most
used and most researched mouth-washing agent. On the
other hand, another reason could be found in the small
number of conducted studies regarding the antimicrobial
effects of the mouthwashes and their benefit in the
prevention of dental caries. One of the goals of this study
was to provoke massive usage of antiseptic mouthwashes
as preventive agents through familiarization with their
positive effects.
The positive effects of mouthwashes on cariogenic
salivary flora, first of all, but also on plaque and gingivitis,
are described in a considerable number of studies, most
of them longitudinal and prospective12-16. A part of this
research refers also to the effects of mouthwashes on
prevention of bad-breath. The usage of mouthwashes in
western societies is habitual.
Using the semi quantitative determination method for
the total microbial salivary counts6, aerobic and anaerobic
microorganisms were separately processed. There has
been a redistribution of the counts of colonies aerobic
and anaerobic microorganisms from sectors with high
and medium growth density (++ and +) before rinsing, to
sectors with low or without any growth density (sectors
+ and + –) after rinsing. However, the statistical analyses
performed through χ2 test have shown very high statistical
significance in the case of aerobic microorganisms
after rinsing with CPC mouthwashes - p<0.05. Similar
results were obtained through the analyses in the case
of anaerobic microorganisms. The statistical analyses
conducted for anaerobic microorganisms have shown high
significant reduction after rinsing with CPC mouthwashes
- p<0.05. However, when one considers the duration of
rinsing, which was limited to 1-2 minutes, it becomes
apparent that the decrease in growth density after rinsing
with CPC mouthwashes depends also on the mechanical
function of the mouthwashes during rinsing.
CPC mouthwashes performed very effectively
in the reduction of Candida albicans yeasts colonies.
Namely, none of the 4 participants, in which a total of 10
colonies had been isolated before rinsing with Aqua fresh
mouthwashes, exhibited any growth density after rinsing.
The total number of colonies was reduced by 10 times the
average. These analyses confirm the fungicide effect of
CPC described in the literature.
Balk J Stom, Vol 10, 2006
By reducing the principal amount of cariogenic
micro-flora, mainly the salivary MS, the incorporation of
microbials into the plaque is avoided, and their fermentable
properties at low pH values are suppressed as well, which
prevents the start of the demineralisation process. In our
investigation we obtained a significant difference in the
number of cariogenic microorganisms’ colonies before
and after rinsing with CPC mouthwashes. The reduction
was higher in MS than in LB species counts, expressed by
the numbers of subjects with logarithmic reduction factor
of cariogenic micro-flora. The number of subjects with log
RF = 0 for MS was 1, while it was 2 for LB species. 17
subjects had a logarithmic reduction factor of 4 (log RF
≥ 4) for MS, and 14 subjects had this reduction factor for
LB species.
At the end of the discussion, we have to emphasize
that until now no research of the effects of CPC
mouthwashes on the bacterial cell have been carried out in
our environment, so that the results discussed in this paper
can be considered as the results of a pioneering experiment
in this direction. Moreover, considering the action of
quaternary ammonium compounds reported in literature,
we can conclude that our results are in accordance with the
research of Jenkins et al12, Mandel13, Pitten and Kramer14,
Radford et al15, and Van Steenberghe et al16.
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Correspondence and request for offprints to:
Aleksandar Dimkov
bul. Partizanski odredi 8/37
1000 Skopje, Republic of Macedonia
E-mail: adimkov@mail.net.mk