Waad M. Kheder, BDS, MSc (Conservative Dentistry), MSc /Dental specialtyProsthodontics. University of Toronto/ Toronto, Ontario/ Canada
In vitro evaluation of antimicrobial activity of chlorhexidine and
sodium hypochlorite irrigant solutions
Abstract:
The aim of this study is to investigate in vitro the antimicrobial activeness by the
agar diffusion test two different concentrations of two endodontic irrigants 0.1% or
0.2 % chlorhexidine and1% or 3% sodium hypochlorite to Staphylococcus aureus,
Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli and a mixer of
these species.
Under the condition of the agar diffusion test, all solutions has exhibited zones of
antimicrobial activity; however, chlorhexidine had the best antimicrobial effectiveness
for both Staphylococcus aureus and Enterococcus facealis in both concentrations,
while sodium hypochlorite has the best antimicrobial effectiveness against
Pseudomonas aeruginosa even with 1% concentration. On the other hand, low
concentration of chlorhexidine has been effective as the high concentration of sodium
hypochlorite for Escherichia Coli and the mixed culture of four types of the bacteria
are used in our study. Sodium hypochlorite and chlorhexidine gluconate at all tested
concentrations can be used as irrigating solutions owing to their antimicrobial
properties.
Key words: agar diffusion test, antimicrobial activity, chlohexidine, endodontic
irrigant, sodium hypochlorite
Introduction:
Chemo mechanical debridement of the root canal is an important phase of
endodontic treatment. Elimination of pulpal tissue, microbiote and their by-products,
organic and inorganic debris by mechanical instrumentation and chemical irrigation
are objectives of this essential part of endodontic procedure.
Despite all efforts to achieve a root canal system free of bacteria, it is evident that
bacteria can still survive in areas that are not accessible to current cleaning and
shaping procedures.1Irrigation is an essential part of root canal debridement because
it allows for cleaning beyond what might be achieved by root canal instrumentation
alone.
However; the endodontic success has been directly related to the presence of
negative bacterial culture before root canal filling.2
Although, various irrigant solutions have been indicated for the root canal
disinfection and many of them have shown varied degrees of antimicrobial
effectiveness, the choice of the ideal irrigant solution and its concentration is a
challenge. Byström and Sundqvist has showed that there is no antibacterial
difference between 0.5% and 5% sodium hypochlorite (NaOCl) solutions.3 Heling and
Chandler by analyzing the antimicrobial effect of irrigants, they concluded that 0.12%
chlorhexidine (CHX) and 1% NaOCl were similarly effective.4 While Ohara et al.
compared six irrigants on selected anaerobic bacteria and they concluded that 0.2%
CHX was the most effective, 5.25% NaOCl
was less effective while the saline
proved to be totally ineffective.5 Ayhan et al. reported the antimicrobial effects of
various endodontic irrigants on selected microorganisms and observed that 5.25%
NaOCl was superior and the reduced concentration of 0.5% resulted in significantly
decreased antimicrobial effectiveness.6
Facultative microorganisms such as Enterococcus faecalis (E. faecalis),
Staphylococcuc aureus (S. aureus), and even Candida albicans (C. albicans) are
considered to be the most resistant species in the oral cavity, and one possible
cause of root canal treatment failure.7
An endodontic irrigant should ideally exhibit powerful antimicrobial activity,
dissolve organic tissue remnants, disinfect the root canal space, flush out debris from
the instrumented root canals, provide lubrication, and have no cytotoxic effects on
the periradicular tissues, among other properties.8 NaOCl has many of these
properties but it has a cytotoxic effect when injected into the periapical tissues, a foul
smell and taste, a tendency to bleach clothes, and corrosive potential. It is also
noticed to produce allergic reactions.9 Therefore, an equally effective but safer
irrigant is desirable.
CHX is widely used as a mouth rinse in the prevention and treatment of
periodontal diseases and dental caries, and has been suggested as an irrigating
solution or intracanal dressing in endodontic therapy. The antimicrobial properties of
both irrigating solutions have been tested against E. faecalis.9
However, their antimicrobial activity against microorganisms such S. aureus and
C. albicans, which are also considered to be resistant to endodontic therapy, has not
been well investigated yet.
Therefore, the aim of this study is to investigate in vitro, the antimicrobial
activeness by the agar diffusion test, two different concentrations of two endodontic
irrigants solutions (0.1% or 0.2 % CHX and1% or 3% sodium hypochlorite) against
S. aureus , E. faecalis, Psedomonas aeruginosa (P. aeruginosa), Escherichia coli (E.
coli) and a mixed culture of these bacteria.
Materials and method
Four reference bacterial strains – S. aureus (ATCC 29213), E. faecalis (ATCC
29212), P. aeruginosa (ATCC 27853), and E. coli (ATCC 25922), has been obtained
from American Type Culture Collection, and one mixture of these is also included in
the study.
The strains were inoculated in 7 ml brain heart infusion (BHIA; Difco Laboratories,
Detroit, MI, USA) and incubated at 37oC for 24 h. The 5 biological indicators were
cultivated on the surface of brain heart infusion agar (BHIA; Difco Laboratories,
Detroit, MI, USA) following the same incubation conditions. Microbial cells were
resuspended in saline to give a final concentration of about 3 x 108 cells/ml, similar
to that of tube #1 of the Mac Farland scale. One ml of each of these pure
suspensions has been used to obtain a mixture of the test microorganisms.
The irrigant solutions tested in this experiment were 1%, %3 NaOCl, 0.2%, 0.1%
CHX, and sterile distilled water. Twenty five Petri plates with 20 ml of BHIA were
inoculated with 0.1 ml of the microbial suspensions, using sterile swabs that were
spread on the medium, obtaining growth in junction. Seventy five paper disks (9 mm
in diameter) were immersed in the experimental solutions for 1 min and then 3 paper
disks were placed over the BHIA surface in each agar plate. The plates were
maintained for 1 hour at room temperature, and then incubated at 37oC for 48 hours.
The diameter of microbial inhibition was measured around the paper disks containing
the substances. Positive and negative controls were done, maintaining the plates
inoculated and without inoculums, for the same time periods and under identical
incubation conditions. (Positive and negative controls consisted of plates incubated
for the same period under identical conditions with and without inocula) All assays
were carried out under aseptic conditions.
This procedure has been done in the microbiology lab of Gulf Medical college
(Dubai,UAE).
RESULTS:
The antimicrobial activities of 1%, 3% NaOCl, 0.2%, 0.1% CHX, and sterile
distilled water are shown in Table 1. The saline control groups did not inhibit growth
of any of the microorganisms tested. The magnitude of antimicrobial effect was
influenced by the concentration of irrigant solutions.
The positive control has showed bacterial growth, while the negative control has
showed no growth. CHX had the best antimicrobial effectiveness for both S. aureus
and E. facealis in both concentrations, while NaOCl had the best antimicrobial
effectiveness against P. aeruginosa even with 1% concentration. On the other hand,
low concentration of CHX was as effective as the high concentration of NaOCl for E.
Coli and the mixed culture of the four types of the bacteria used in our experiment.
Discussion:
NaOCl solution is, to date, the most commonly employed root canal irrigant, but no
general agreement exists regarding its optimal concentration, which ranges from
0.5% to 5.25%. According to our study, the antimicrobial effectiveness of NaOCl is
proportional to its concentration. In order to reduce the cytotoxic effects, 1% NaOCl is
recommended.
The high pH of NaOCl interferes in cytoplasmic membrane integrity with
irreversible enzymatic inhibition, biosynthetic alterations in cell metabolism and
phospholipid
destruction
observed
in
lipidicperoxidation.
The
amino
acid
chloramination reaction forming chloramines interferes in cell metabolism. Oxidation
promotes irreversible enzymatic inhibition of bacteria replacing hydrogen with
chlorine. Enzyme inactivation can be observed in the reaction of chlorine with amino
groups (NH2-) and an irreversible oxidation of sulphydryl groups (SH) of bacteria
enzymes (cystein). Thus, NaOCl presents antimicrobial activity with action on
bacterial essential enzymatic sites promoting irreversible inactivation originated by
hydroxyl ions and chloramination action. Dissolution of organic tissue can be verified
in the saponification reaction when NaOCl destroys fatty acids and lipids resulting in
soap and glycerol.10,11
CHX has been recommended as an alternative irrigating solution to NaOCl,
especially in cases of open apex owing to its biocompatibility or in cases of related
allergy to bleaching solutions.12 CHX is a cationic agent (biguanide group; 4chlorophenyl radical), which exhibits antibacterial activity. The cationic nature of the
compound promotes connection with anionic compound at the bacterial surface
(phosphate groups from teicoic acid at Gram-positive and lipopolysaccharide at
Gram-negative bacteria) capable of altering its integrity. The potassium ion, being a
small entity, is the first substance to appear when the cytoplasmic membrane is
damaged. The alteration of the cytoplasmic membrane permeability promotes
precipitation of cytoplasmic proteins, alters cellular osmotic balance, interferes with
metabolism, growth, cell division, inhibits the membrane ATPase and inhibits the
anaerobic process.13,14,15
Various results have been reported for irrigant antimicrobial effectiveness. Similar
results have been reported for NaOCl and CHX16,4,12 ; however, another research
has shown that NaOCl has better antimicrobial activity than CHX 6,17,18, or,
conversely, that CHX has better antimicrobial activity than NaOCl 5,19. Different
experimental methods, biological indicators, concentrations, exposure time or the
period of analysis may have caused these differences.
The irrigant of choice should be one that exerts its antimicrobial activity against the
majority of microorganisms found in the root canal and dentinal tubules. Other
properties beyond antimicrobial activity must also be investigated before the final
collection of an irrigant solution, such as minimum inhibitory concentration, tissue
dissolution
capacity,
detoxification
of
endotoxin
and
acceptable
biological
compatibility. For this reason, it is important to know both the type of an irrigant to kill
microorganisms and the irrigant’s minimum concentration that provide these
requirements.
In the present work, the highest antimicrobial effectiveness was for 0.2% CHX
against all tested microorganisms except P. aeruginosa; On the other hand, there is
no significant difference between the antimicrobial effectiveness of 1% & 3% NAOCl
against P. aeruginosa.
Although P. aeruginosa is not frequently isolated from root canals, it has been
considered, because it is a bacterium that is too difficult to eliminate from root
canals.20
The combination of NaOCl and chlorhexidine forms a precipitate. This NaOCl/CHX
precipitate tends to occlude the dentinal tubules. Therefore; caution should be
exercised when irrigating with NaOCl and CHX.21
We have concluded that NaOCl and chlorhexidine gluconate at all tested
concentrations can be used as irrigating solutions owing to their antimicrobial
properties. However using both types of irrigants as alternative steps during
treatment is advised to get the benefits of both and avoid NaOCl/CHX precipitate
formation on the same time. By using NaOCl we will overcome the shortcoming of
CHX as CHX does not dissolve organic tissues.
References:
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Table 1. Means of the diameters (in mm) of the inhibition zones by the agar
diffusion test.
S. aureus
1%
NaOCl
3%NaO
Cl
0.2%CH
X
0.1%CH
X
Distilled
water
E.faceali
s
P.
aeruginosa
E . Coli
Mixture
14.3mm
12.7mm
16mm
14.7mm
13.3mm
14.5+14+
12.6+12
16.1+16
15.1+14.6
13.4+13.2
14.3
.8+12.7
.3+16.5
+14.3
+13.3
19mm
14mm
17.7mm
17.3mm
15.7mm
19.3+18.7
13.8+14
17.6+18
17.2+17.3
15.5+15.6
+19
.1+14.2
+17.4
+17.4
+16.9
24mm
22mm
14.7mm
19.3mm
17.3mm
24.1+23.9
21.7+22
14.6+14
19.2+19.5
17.4+17.1
+24
.3+22
.5+14.9
+19.3
+17.4
21.7mm
17.3mm
12mm
17mm
15.7mm
21.7+22+
17.1+17
11.8+11
16.8+16.9
15.2+15.7
21.4
0
.4+17.5
0
.9+12.3
0
+17.3
0
+16.1
0
30
25
20
S. aureus
E.facealis
15
P. aeruginosa
E . Coli
10
Mixture
5
0
1% NaOCl
3%NaOCl
0.2%CHX
0.1%CHX
Distilled water
Graph 1: comparing the arithmetic mean of inhibition zones between experimental
solutions.