Basic ResearchBiology

Effectiveness of Antibiotic Medicaments against Biofilm

Formation of Enterococcus faecalis and
Porphyromonas gingivalis
Alaa H.A. Sabrah, BDS, MSD,* Ghaeth H. Yassen, BDS, MSD, PhD, and Richard L. Gregory, PhD

Abstract
Introduction: In this study we compared the antibacte-
rial effect of triple antibiotic paste (TAP), double antibiotic
paste (DAP), and calcium hydroxide [Ca(OH)2] against
Enterococcus faecalis and Porphyromonas gin-
givalis biofilm. Methods: The minimum inhibitory
concentration (MIC), minimum bactericidal concentration
(MBC), minimum biofilm inhibitory concentration (MBIC),
and biofilm formation were measured by using microtiter
plate methods. The 2 bacteria were treated with different
dilutions of TAP, DAP, and Ca(OH)2 solutions. The turbid-
ities of the bacterial cultures in the microtiter plate were
measured by optical density at 490 nm by using a spectro-
photometer. Data were analyzed by 2-way analysis of
variance (a = 0.05). Results: For TAP, the MIC and
MBIC values were 0.003 mg/mL for E. faecalis and
0.006 mg/mL for P. gingivalis. The MBC values for
TAP were 0.3 mg/mL for both bacteria. The MIC and
MBIC values for DAP were 0.001 mg/mL for E. faecalis
and P. gingivalis. The MBC values for DAP were 0.14
mg/mL for both bacteria. Biofilm formation of the 2
bacteria was significantly decreased with TAP and DAP
at all tested dilutions (P < .0001) compared with control
groups; however, TAP and DAP biofilm formations were
not significantly different from each other. Ca(OH)2
significantly decreased bacterial biofilm formation
compared with the control, but it was significantly less
than TAP and DAP (P < .05). Conclusions: Both TAP
and DAP were more effective than Ca(OH)2 against E.
faecalis and P. gingivalis. DAP can be considered an
effective and comparable antibacterial substitute for
TAP. (J Endod 2013;39:13851389)
Key Words
Double antibiotic, calcium hydroxide, Enterococcus
faecalis, Porphyromonas gingivalis, triple antibiotic
T he endodontic regeneration procedure has received great attention in recent years
because it allows continuation of normal root development (13). The use
of antibacterial dressing to disinfect the root canal during endodontic regeneration
is an essential step during endodontic regeneration (1). Although calcium hydroxide
[Ca(OH)2] has been used in endodontic regeneration (4, 5), triple antibiotic paste
(TAP) is the most widely used medicament during endodontic regeneration (68). TAP
is a combination of metronidazole, ciprofloxacin, and minocycline, which was found to
be effective in root canal disinfection both in situ and in vivo (911). However, TAP
was associated with tooth discoloration caused by minocycline (7, 1214). Double
antibiotic paste (DAP) is another antibiotic mixture of metronidazole and ciprofloxacin
that has been used successfully in endodontic regeneration (15). The use of DAP was
suggested to overcome the discoloration problem associated with the minocycline in
TAP (16).
Biofilm is a slimy layer of polysaccharide, protein, and microbial cells forming
a matrix that provides bacteria protection from antibiotics or the host immune response
(17). To compare the effectiveness of antibacterial agents, biofilm inhibition is among
the most important criterion to examine. To the best of our knowledge, no previous
studies have compared the effectiveness of DAP, TAP, and Ca(OH)2 against bacterial
biofilm formation. Therefore, the purpose of this study was to compare the antibacterial
effectiveness and biofilm inhibition of TAP, DAP, and Ca(OH)2 against 2 bacterial
species; Enterococcus faecalis and Porphyromonas gingivalis.
Materials and Methods
Bacterial Strains and Media
E. faecalis and P. gingivalis were used in this study because they are the most
commonly isolated bacteria from root canal infections (18). Anaerobic blood agar
(CDC, BioMerieux, Durham, NC) plates were used to initially grow E. faecalis (ATCC
29212) and P. gingivalis (ATCC 33277) strains. Brain-heart infusion (BHI) broth sup-
plemented with 5 g yeast extract/L and 5% v/v vitamin K+ hemin (BHI-YE; Becton, Dick-
inson and Company, Franklin Lakes, NJ) was used to grow the bacteria. Bacterial strains
were grown at 37 C in an anaerobic environment by using gas-generating sachets (Gas-
Pak EZ; Becton, Dickinson and Company) to produce the required environment.
Saturated Solution Preparation
Typical working strength mixtures of each preparation were prepared. A saturated
solution of Ca(OH)2 paste (UltraCal XS; Ultradent, South Jordan, UT) was prepared by
mixing 16 mg Ca(OH)2 with 1 mL distilled water. For TAP (CHAMPS Medical, San

From the *Division of Dental Biomaterials, Department of Restorative Dentistry, Indiana University School of Dentistry, Indianapolis, Indiana; Department of Conser-
vative Dentistry, Faculty of Dentistry, University of Jordan, Amman, Jordan; Department of Pediatric, Orthodontic and Preventive Dentistry, Mosul University School of
Dentistry, Mosul, Iraq; and Department of Oral Biology, Indiana University School of Dentistry, Indianapolis, Indiana.
Address requests for reprints to Dr Alaa H. A. Sabrah, Division of Dental Biomaterials, Department of Restorative Dentistry, Indiana University School of Dentistry,
1121 West Michigan Street, Indianapolis, IN 46202. E-mail address: sabralolo@yahoo.com
0099-2399/$ - see front matter
Copyright 2013 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2013.05.003

JOE Volume 39, Number 11, November 2013 Effectiveness of Antibiotic Medicaments 1385
Basic ResearchBiology
Antonio, TX), 300 mg USP grade antibiotic powder compounded of
equal portions of metronidazole, ciprofloxacin, and minocycline was
dissolved in 3 mL distilled water (33 mg of each antibiotic/mL). For
DAP (CHAMPS Medical), 300 mg USP grade antibiotic powder com-
pounded of equal portions of metronidazole and ciprofloxacin was
dissolved in 3 mL distilled water (50 mg of each antibiotic/mL).
Ca(OH)2, TAP, and DAP mixtures were stirred for 4 hours at room
temperature. The mixtures were then centrifuged at 3000 rpm for 15
minutes to clarify the solutions, and the aqueous supernatant layers
were filter sterilized by using a sterile 25-mm syringe filter (Fisher
Scientific, Newark, DE). Furthermore, to report the concentrations of
the saturated solutions, the maximum amount of each tested material
dissolved in distilled water was calculated.
Determination of Minimum Inhibitory and
Bactericidal Concentrations
Minimum inhibitory concentration (MIC) is the lowest concentra-
tion of an agent that inhibits the visible growth of a microorganism, and
the minimum bactericidal concentration (MBC) is the lowest concen-
tration of an agent that kills the microorganism. MIC and MBC of
TAP, DAP, and Ca(OH)2 against E. faecalis and P. gingivalis were
determined by a 2-fold dilution method (19). Briefly, overnight, E.
faecalis and P. gingivalis cultures in BHI-YE were treated with 1:10,
1:20, 1:40, 1:80, 1:160, 1:320, 1:1000, 1:2000, 1:4000, 1:8000,
1:16,000, 1:32,000, and 1:64,000 dilutions of Ca(OH)2, TAP, or DAP
solutions for 24 hours in sterile 96-well flat-bottom microtiter plates
(Fisher Scientific). The turbidities of the bacterial cultures were
measured by optical density at 540 nm by using a spectrophotometer
(SpectraMax 190; Molecular Devices, Sunnyvale, CA). MIC was opera-
tionally defined as the lowest concentration of an agent that yielded
a turbidity change equal to or less than 0.050 (19, 20). To
determine MBC, bacterial cultures from the wells with Ca(OH)2, TAP,
and DAP concentrations equal to and higher than MIC were streaked
on blood agar plates and incubated for 48 hours. MBC was defined
as the lowest concentration of an agent that had no visible bacterial
colonies on the agar plates after 48 hours of incubation (20).
Determination of Minimum Biolm Inhibitory
Concentration and Biolm Formation
The minimum biofilm inhibitory concentration (MBIC) is the
lowest concentration of an agent that inhibits the visible biofilm forma-
tion of a microorganism (21). To determine MBIC, overnight, E.
faecalis and P. gingivalis cultures (106 colony-forming units/mL)
in BHI-YE were treated with 0, 1:10, 1:20, 1:40, 1:80, 1:160,
1:320, 1:1000, 1:2000, 1:4000, 1:8000, 1:16,000, 1:32,000, and
1:64,000 dilutions of Ca(OH)2, TAP, and DAP solutions for 24 hours
in 96-well microtiter plates. Biofilm was gently washed twice with
saline, fixed with 10% formaldehyde (22), washed twice with saline
again, and stained with 0.5% crystal violet for 30 minutes. After
washing the biofilm 3 times with saline, crystal violet was extracted
from the biofilm cells by 200 mL 2-propanol for 1 hour. The extract
was diluted (1:5) with 2-propanol and read at 490 nm with 2-prop-
anol used as a blank control (20). The method of biofilm formation
was similar to the method of MBIC, but the TAP, DAP, and Ca(OH)2
dilutions used in biofilm formation were 0, 1:10, 1:20, 1:40, 1:80,
1:160, and 1:320. Biofilm formation was read at 3 time intervals of
24, 48, and 72 hours to ascertain the ability of the medicaments to
inhibit biofilm over time. Optical absorbance of the diluted crystal
violet stain represents the actual bacterial biofilm mass. A higher
absorbance indicates higher biofilm mass.
1386 Sabrah et al.
Statistical Analysis
Each experimental treatment was conducted in triplicate and
repeated individually at least 3 times. Two-way analysis of variance
and pair-wise comparison were used for statistical analyses. The signif-
icance level was set at .05.
Results
The calculated concentrations of the saturated solution were 16,
96, and 46 mg/mL for Ca(OH)2, TAP, and DAP, respectively.
Ca(OH)2 was not inhibitory in the MIC and MBC assays against E. fae-
calis or P. gingivalis at any dilution used in this study. Visible bacterial
growth was observed in all wells treated with Ca(OH)2. However, the
MBIC values for Ca(OH)2 were 1:10 (1.6 mg/mL) for E. faecalis and
1:80 (0.2 mg/mL) for P. gingivalis. For TAP, the MIC and MBIC values
were 1:32,000 (0.003 mg/mL) against E. faecalis and 1:16,000 (0.006
mg/mL) against P. gingivalis. The MBC values for TAP were 1:320 (0.3
mg/mL) against both bacteria. The MIC and MBIC values for DAP were
1:32,000 (0.001 mg/mL) against E. faecalis and P. gingivalis. The
MBC values for DAP were 1:320 (0.14 mg/mL) against both bacteria.
Biofilm formation was significantly decreased (P < .0001) with TAP
and DAP at all dilutions (Figs. 1 and 2). Furthermore, TAP and DAP
effects on biofilm formation were not significantly different. Ca(OH)2
significantly decreased E. faecalis biofilm formation in a concentra-
tion-dependent gradient (Fig. 1), but it inhibited P. gingivalis biofilm
at only higher dilutions (Fig. 2).
Discussion
In this study, we used a microtiter plate method instead of a root
canal system because of the complexity of the root canal system and the
inaccuracy and contamination commonly associated with the microbi-
ological sampling of root canals (23). The microtiter plate method
allowed examination of the direct effect of tested materials against the
2 bacteria. No MIC and MBC values for Ca(OH)2 were obtained in
this study, which suggests a poor antimicrobial activity of the medica-
ment. This agrees with previous studies that found Ca(OH)2 to be inef-
fective against E. faecalis (2429) and P. gingivalis (27, 29). Low
numbers of E. faecalis bacteria still survived even after exposure to
saturated solution of Ca(OH)2 for 24 hours (26). Mixing Ca(OH)2 paste
with glycerin results in significantly better antibacterial effects (27).
However, Ultracal aqueous Ca(OH)2 paste was used in this study, which
is one of the most common clinically used materials. To confirm our
findings, we used another Ca(OH)2 powder material (Dentonics, Mon-
roe, NC), and the results were identical (data are not shown).
Hoshino et al (9) reported that TAP was effective at a concentration
of 25 mg/mL of each antibiotic, whereas Sato et al (10) found that TAP at
50 mg/mL of each antibiotic was required to sterilize infected root
dentin in situ. Our results demonstrate that TAP was effectively bacte-
ricidal at a concentration of 100 mg/mL of each antibiotic (total of 300
mg/mL) against both E. faecalis and P. gingivalis. The different meth-
odology used explains the variations noted between our reported values
and previous studies. Dilutions of the aqueous saturated solutions were
used in our study to mimic the clinical use of the materials. In the other
2 studies (9, 10), antibiotic powder was used. In this study, DAP was
effectively bactericidal at a concentration of 140 mg/mL against both
bacteria. To the best of our knowledge, no previous studies reported
the antibacterial effectiveness of DAP. An interesting finding of this
study is that both TAP and DAP were effective at dilutions up to 1 in
32,000 (0.0010.003 mg/mL). TAP or DAP diluted solutions might
be further used as efficient antibacterial irrigant during endodontic
regeneration. A previous case report used TAP solution as an irrigant
during endodontic regeneration (3). TAP and DAP demonstrated
JOE Volume 39, Number 11, November 2013
 Basic ResearchBiology

Figure 1. Effect of Ca(OH)2, TAP, and DAP on E. faecalis biofilm formation after (A) 24 hours, (B) 48 hours, and (C) 72 hours. Lowercase letters compare
various materials and the control at each dilution. Uppercase letters compare all materials and the control at various dilutions.

significant reduction in biofilm formation of E. faecalis and P. gingiva-
lis at all tested dilutions during different time periods (24, 48, and 72
hours). Furthermore, there was no significant difference between TAP
and DAP antimicrobial activity against either bacterium. Ca(OH)2 signif-
icantly inhibited the biofilm formation of both bacteria compared with
the control, but it was significantly higher than both TAP and DAP. The
relatively low sensitivity of E. faecalis and P. gingivalis biofilms to
Ca(OH)2 may be explained by the high resistance of some endodontic
bacterial biofilms to an alkaline challenge, which was reported in
previous studies (3032). A recent study suggested that the amount
of hydroxyl ions released from Ca(OH)2 is not high enough to
promote antimicrobial activity against intraoral-infected dentin biofilm
(33). It is worth mentioning that the use of saturated solutions might not
be an accurate representation of the supersaturated pasty medicaments
that are usually used in clinical settings. However, saturated solutions
and their various dilutions were used in the current study to compare
the antibacterial effects of the tested medicaments at relatively low
concentrations. Furthermore, each tested material has a different

JOE Volume 39, Number 11, November 2013 Effectiveness of Antibiotic Medicaments 1387
Basic ResearchBiology

Figure 2. Effect of Ca(OH)2, TAP, and DAP on P. gingivalis biofilm formation after (A) 24 hours, (B) 48 hours, and (C) 72 hours. Lowercase letters compare
various materials and the control at each dilution. Uppercase letters compare all materials and the control at various dilutions.

solubility in distilled water, and this is reflected on the concentrations of
the saturated solutions. Therefore, it is important to highlight that mate-
rials with similar dilutions have different concentrations.
Collectively, this study demonstrated that both TAP and DAP are
effective against endodontic bacteria at high dilutions, which indicates
that low concentrations of antibiotics might be sufficient to obtain the
required antibacterial effect. It might be beneficial to recommend the
use of lower concentrations of antibiotic pastes to avoid the cytotoxic
effect of those medicaments on host stem cells. A recent study suggested
that TAP and DAP concentrations currently used in regenerative
endodontics had a detrimental effect on the survival of human stem cells
of the apical papilla (34). Within the limitations of this in vitro study, it
can be concluded that both TAP and DAP were more effective than
Ca(OH)2 against E. faecalis and P. gingivalis bacteria. DAP can be

1388 Sabrah et al. JOE Volume 39, Number 11, November 2013

considered an effective and comparable antibacterial substitute to TAP
without the discoloration associated with the latter medicament.
Acknowledgments
The authors deny any conflicts of interest related to this study.
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Effectiveness of Antibiotic Medicaments 1389