INTODUCTION

Primary teeth are a child's most valuable assets since they are essential for mastication,
phonetics, aesthetics, and social acceptability. The issue of space loss is now resolved by pulp therapy
for primary teeth, which also preserves the integrity of jaw growth and assimilation (1). Pediatricians
find it extremely difficult to perform pulp therapy on primary teeth with necrotic pulp because of the
complicated structure of the canal system, which encourages microbial growth and invasion of the
lateral canals, apical deltas, and dentinal tubules (2).
Many bacteria, including Candida, Enterococcus faecalis, Staphylococcus aureus, Bacteroid
fragilis, E. coli, black pigmented bacilli, Mutans streptococci, and other anaerobic species, were
recovered from necrotic primary teeth. Getting rid of the bacteria that are the major cause of peri-
radicular lesions is one of the most important objectives of pulp treatment in deciduous teeth (3).
Although the amount of germs reduces when infected pulp tissue is removed, pulpectomy success
rates are improved by using an obturating substance with good antibacterial activity(4).
Primary teeth have been obturated using a number of different materials, although
traditionally, zinc oxide eugenol was utilised. Moreover, materials based on calcium hydroxide and
iodoform, such as Metapex, Vitpex, and Calpex, have become quite popular. Another product on the
market is called Endoflas, which is a combination of ZOE, calcium hydroxide, and iodoform (5).
Preserve main teeth with non-negotiable root canals by using antimicrobial agents alone, or in
conjunction with other non-antibiotic agents and/or vehicles (6). In addition to being recommended as
an intracanal medication for young, recalcitrant children, triple antibiotic paste has also been
recommended as an obturating substance (7,8). For this medication, a number of modifications have
been tested, including Augmentin, modified triple antibiotic paste (MTAP), and double antibiotic
paste (DAP), which contains metronidazole and ciprofloxacin(9,10).
Multi-antimicrobial preparations used intracanally decrease the likelihood of systemic harm
and virtually completely eliminate bacteria at extremely low doses. With varying degrees of efficacy,
topical multi-antimicrobial formulations have been employed against endopathogens (11). In order to
evaluate the antibacterial effectiveness of Calpex, endoflas, modified triple antibiotic and zinc
oxide/eugenol, as an obturating substance for primary teeth, the current vitro study was conducted.

MATERIALS AND METHODS

Study design
This study In-vitro study microbiological study, which study the Antimicrobial efficacy of
different obturation materials used for root canal treatment in primary teeth under investigation were
examined against Escherichia coli and Enterococcus faecalis bacteria and zone of inhibition were
measured for each group.
This study has been approved by the Research Ethics Committee (REC-PE-23-03), Faculty of
Dental Medicine for Girls, Al-Azhar University, Cairo, Egypt.
The four materials used in pulpectomy of primary teeth were examined at Al-Azha
University's Regional Institute for Mycology and Biotechnology in Cairo using an agar diffusion test
to determine their antibacterial activity.
The antibacterial efficacy of Cetapex, Endoflas, Modified Triple-Antibiotic Paste and ZOE
against Enterococcus faecalis and Escherichia coli was evaluated using the agar diffusion method by
measuring the inhibition zone surrounding each substance. The culture media was prepared agar
plates. Each agar plate's reverse was marked with a permanent marker bearing the group's name.

Preparation of culture medium:

After being made, Mueller Hinton agar was autoclaved to sanitize it. The substance was cooled until it
reached room temperature. Five petri dishes—for each kind of bacteria—were then filled with it.

Purification and incubation of bacterial culture:

A sterile swab was used to collect the common bacterial strains of E. faecalis and E. coli,
which were then transferred to fluid broth medium and cultured aerobically at 37°C. E. faecalis and E.
coli were inoculated onto the agar plates using a sterile swab and 0.1 mL of the primary broth vial in
one swift motion. The 1.5 ml mark on disposable droppers was clipped with sterile scissors.
To make an agar well for the obturating material pellet, agar was punched out of the agar
plates using a dropper. For each of the five agar plates, the wells (4 mm in depth by 6 mm in diameter)
were produced, totaling 20 wells for each group.

Preparation of the test materials and grouping:

The Regional Center for Mycology and Biotechnology prepared all of the obturating material
pellets at once, using the same clinically-recommended ratios, and placed them in the agar wells for
the agar diffusion test. For each species of bacterium, there are five agar dishes and a total of 20
mixes, one mix from each material category. For this work, a total of 40 obturating material pellets
were created.

Group I
A readymade intracanal calcium hydroxide and iodoform medication paste (Calpex, jK,
Egypt) was used. It was dissolved in normal saline, to achieve -consistency according to manufacturer
instructions using a stainless-steel spatula. Once a pellet was made it was directly placed in the labeled
agar well with a gloved finger.
Group II
Endoflas (Sanlor Laboratories, Cali, Colombia) powder and liquid were combined in a 4:1
ratio (0.28g: 0.07 ml by weight) as per the manufacturer's instructions, poured on a cold glass slab,
and stirred until consistency was achieved. After a pellet was created, it was immediately inserted with
a gloved finger into the designated agar well.
Group III
Modified triple antibiotic paste was created by mixing equal weights of the antibiotics
ciprofloxacin (Ciprobay, Hikma Pharma, Egypt), metronidazole (Flagyl, Sanofi-Aventis, Egypt), and
augmentin (GSK, Egypt) in a 1:1:1 ratio with a concentration of 1 mg/ml. A stainless-steel spatula was
used to combine the powder with normal saline in an exact ratio of 1:1 w/v to create a homogeneous
paste for application. When a pellet was ready, a gloved finger immediately placed it in the agar well
that was marked.
Group IV
Using a stainless steel spatula and a cold glass slab, zinc oxide powder and eugenol (4:1
(0.28g) 0.07 ml by weight) powder: liquid proportion) were combined to produce the desired
consistency. After a pellet was created, it was immediately inserted with a gloved finger into the
designated agar well.

Incubation of agar plates:

The agar plates were kept at room temperature for two hours to allow for material diffusion.
They were then incubated for 24 hours in anaerobic conditions at 37 °C in a Memmert incubator. The
inhibition zone around each tested obturating material was then measured to ascertain its antibacterial
potential.
Antimicrobial susceptibility Test (Screening test by agar diffusion method):
The agar plates were assessed after 24 hours. Using a vernier calliper, the inhibitory zone's
most consistent outside diameter was measured in millimetres. The gathered information was tallied
and forwarded for statistical evaluation. Zones were measured separately by two observers five times
for each substance mixture during the experiment. The data were then used to compute the mean zone
of inhibition for each filler material, which was then statistically analysed.
Data management and Stastiscal analysis:
Data management and statistical analysis were performed using the Statistical Package for
SPSS version 20 for social sciences. The mean, standard deviation, and confidence intervals were used
to summarise numerical data. By examining the data distribution, applying the Kolmogorov-Smirnov
and Shapiro-Wilk tests, and testing for normality, the data were examined.
One-way ANOVA and post hoc test were used to compare groups with regard to normally
distributed numerical variables. Every p-value is two-sided. P-values below 0.05 were deemed
significant.

RESULTS
I-Enterococcus Faecalis Inhibition zone
The highest mean value was recorded in Modified triple antibiotic paste (25.33 ± 0.58) Cfu/ml
forming unit, followed by Calcium hydroxide and iodoform (Calpex) (16.0 ± 1.0) Cfu/ml, then
Endoflas group (13.33 ± 0.58) Cfu/ml ; with the least mean value recorded in Zinc oxide/Eugenol
(10.33 ± 0.58) Cfu/ml. Post hoc test revealed a significant difference between groups. The different
studied groups according to Enterococcus Faecalis inhibition zone (mm). there was a statistically
significant difference between groups (p<0.001).
II- E. coli Inhibition zone
The highest mean value was recorded in Modified triple antibiotic paste (30.67 ± 1.53) Cfu/ml
forming unit, followed by then Endoflas group (20.67 ± 0.58) Cfu/ml, then Calcium hydroxide and
iodoform (Calpex) (17.33 ± 0.58) Cfu/ml; with the least mean value recorded in Zinc oxide/Eugenol
(15.67 ± 1.15) Cfu/ml. Post hoc test revealed a significant difference between groups. The different
studied groups according to E. coli inhibition zone (mm). there was a statistically significant
difference between groups (p<0.001).
 Table (1): mean value of Enterococcus Faecalis Inhibition zone (mm) and [Link] Inhibition zone (mm)
Calcium Endoflas Modified Zinc oxide/ F P
hydroxide and triple Eugenol
iodoform antibiotic
(Calpex) paste
E. Faecalis a b c d <0.001
16.0 ± 1.0 13.33 ± 0.58 25.33 ± 0.58 10.33 ± 0.58 252.167* *

E. Coli a b c a <0.001
17.33 ± 0.58 20.67 ± 0.58 30.67 ± 1.53 15.67 ± 1.15 125.00* *

Significance level p≤0.05, *significant

Post hoc test: means with different superscript letter are significantly different

Figure 1: Bar chart Representing Comparison of inhibition zone between the different studied
DISCUSSION
primary teeth should be preserved in order to maintain aesthetics, phonetics and mastication.
Moreover, primary teeth also act as natural space maintainers ensuring adequate room to the
success accessor teeth. When the pulp has become permanently diseased or necrotic, pulpectomy is
(12)
the best treatment to save the tooth . An efficient antimicrobial agent must be used for a certain
amount of time in the root canal space in order to guarantee the total eradication of root canal
bacteria (13).
Maybe a more efficient way to give an anti-microbial treatment is by local medication
(14)
administration within the root canal system . The goal of the current in vitro study was to
evaluate the effectiveness of MTAP, Metapex, Endoflas, and ZOE against E. faecalis and E. coli.
According to [Link] results of the current study revealed that all four obturating materials
have antibacterial effects when it came to E. coli development, however MTAP had the strongest
antimicrobial potential, followed by Endoflas, Metapex, and finally ZOE (p<0.001), Similar
findings were published in other studies(20,22).
The results of another studies (21,24) have caused a paradigm shift in the way primary teeth are
treated because 3Mix antibiotic paste showed a significantly more effective than Metapex and
traditional root canal therapy. It is also superior to calcium hydroxide paste as a pulp therapy agent
when used with the LSTR technique. Moreover, a prior study found that MTAP had significantly
higher antibacterial activity than ZOE and a mixture of calcium hydroxide and iodoform.
Nevertheless, the results of other study showed that ZOE was more effective than Endoflas.
Also, it was claimed that the most efficient obturating ingredients were pure Iodoform paste mixed
with calcium hydroxide. This variation in findings might be caused by a number of variables,
including the heterogeneity of the tested bacterial species and the use of several antibacterial effect
testing methods with various sensitivity ranges (25,26).
In the present study regarding the suppression of growth of E. faecalis, MTAP was the
superior this goes with other investigation(22,27) followed by Metapex, Endoflas and ZOE was least
effective and these results in accordance with another study(20,28,29).
The strongest antibacterial properties of MTAP with each type of selected bacteria might be
due to that 3 Mix paste produced effective destruction of all aerobic and anaerobic endodontic
pathogens. The efficacy of MTAP has been confirmed by in vitro studies conducted by several
authors(30,31).
In a prior in vitro study(32), comparing the antibacterial effects of the 3 Mix paste and the
calcium hydroxide paste on Enterococcus faecalis (E. faecalis), it was discovered that the 3 Mix
paste was extremely effective and could be considered a more potent root canal medication than
calcium hydroxide pastes.

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Conclusion: Within the limitations of this in vitro study, the following conclusion can be drawn
that all four obturating materials have antibacterial effects when it came to Enterococcus Faecalis
and E. coli development, however MTAP had the strongest antimicrobial potential, followed by
Endoflas, Calpex, and finally ZOE.

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