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Original Research Article

Antibiofilm efficacy of biosynthesized silver

nanoparticles against endodontic‑periodontal
pathogens: An in vitro study
Kiran R. Halkai, Rahul Halkai1, Jayashree A. Mudda2, Vasundhara Shivanna3, Vandana Rathod4
Departments of Conservative Dentistry and Endodontics and 2Periodontics, HKE’s SN Institute of Dental Sciences and Research,
4
Department of Microbiology, Gulbarga University, Kalaburgi, 3Department of Conservative Dentistry and Endodontics, College of Dental
Sciences, Davangere, Karnataka, India, 1Department of Conservative Dentistry and Endodontics, College of Dentistry, Gulf Medical
University, Ajman, United Arab Emirates

Abstract
Background: Endodontic‑periodontal pathogens exist as biofilms which are difficult to eliminate. Biosynthesized silver
nanoparticles (AgNPs) emerged as newer antimicrobial agents with potential benefits.
Aim: The aim of this study is to determine the minimum inhibitory concentration (MIC); evaluate the antibiofilm efficacy of
fungal‑derived AgNPs against Porphyromonas gingivalis, Bacillus pumilus, and Enterococcus faecalis.
Materials and Methods: MIC of AgNPs against test pathogens was determined using micro broth dilution method. Serial
dilutions of AgNPs ranging from 80 to 1 µg/ml concentration were added to wells containing 10 µl of bacterial inoculum
in culture media and control group without AgNPs. For biofilm models, 120 dentin blocks were prepared, sterilized, and
contaminated for 2 weeks with (n = 40 each). Group 1: B. pumilus, Group 2: E. faecalis, and Group 3: P. gingivalis and each
group is divided into four subgroups (n = 10 each) and treated with distilled water, AgNPs, 2% and 0.2% chlorhexidine (CHX).
Colonies counted after 24 h of incubation and statistically analyzed using one‑way ANOVA and post hoc Tukey tests.
Results: MIC for B. pumilus was determined as 20 µg/ml and 30 µg/ml for E. faecalis and P. gingivalis. AgNPs were effective
as 2% CHX against all biofilms compared to control group. Post hoc Tukey test (P < 0.0001) shows no significant difference
between groups.
Conclusion: Fungal‑derived AgNPs are effective against endo‑perio pathogens.
Keywords: Antimicrobial agents; bacterial resistance; biofilm; biosynthesized silver nanoparticles; endo‑perio lesions;
minimum inhibitory concentration

INTRODUCTION resistance to host defense mechanisms and decreased

susceptibility to conventional antimicrobials.[1]
Most of the periodontal and endodontic infections are
biofilm mediated and have been the subject of intensive Porphyromonas gingivalis is a Gram‑negative, black pigmented,
research for many years. Bacteria in biofilm exhibit rod‑shaped obligate anaerobic microorganism which causes
several periodontal diseases and endodontic infections
Address for correspondence:
Dr. Kiran R. Halkai, Department of Conservative Dentistry and periodontitis.[2] Enterococcus faecalis is a Gram‑positive,
Endodontics, HKE’s SN Institute of Dental Sciences and Research, facultative organism most commonly found in persistent
Kalaburgi, Karnataka, India. apical or lateral periodontitis and endodontic reinfections.
E‑mail: drkiranhalkai@gmail.com
Date of submission : 25.05.2018 This is an open access journal, and articles are distributed under the terms
Review completed : 31.07.2018 of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0
Date of acceptance : 29-08-2018 License, which allows others to remix, tweak, and build upon the work
non‑commercially, as long as appropriate credit is given and the new
Access this article online creations are licensed under the identical terms.
Quick Response Code: For reprints contact: reprints@medknow.com
Website:
www.jcd.org.in
How to cite this article: Halkai KR, Halkai R, Mudda JA,
Shivanna V, Rathod V. Antibiofilm efficacy of biosynthesized
DOI: silver nanoparticles against endodontic-periodontal pathogens:
10.4103/JCD.JCD_203_18
An in vitro study. J Conserv Dent 2018;21:662-6.

662 © 2018 Journal of Conservative Dentistry | Published by Wolters Kluwer - Medknow

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Halkai, et al.: Biosynthesized silver nanoparticles against biofilms of endo perio pathogens
It can survive deep in the dentinal tubules and cementum[3]
It possesses several virulence factors which makes it the
most resistant microbe [3] Bacillus pumilus is a Gram‑positive,
aerobic, spore‑bearing rod‑shaped microorganism found in
both periodontal pockets, and root canals associated with
severe infections[4] The endo‑perio pathogens E. faecalis, P.
gingivalis, and B. pumilus forms the biofilms either single or
multispecies which are difficult to eradicate.[5,6]
Recently, biosynthesized silver nanoparticles (AgNPs)
have emerged as novel antimicrobial agents owing to
their unique chemical and physical properties, smaller
size and shape, high surface area to volume ratio, and
no bacterial resistance. Hence, show an insight for the
management of endodontic, periodontal, or the combined
lesions.[7] In our previous study, AgNPs were biosynthesized
using endophytic fungi fusarium semitectum and showed
efficient antibacterial activity against P. gingivalis,
B. pumilus, and E. faecalis[8] The present study aims at
evaluating the minimum inhibitory concentration (MIC)
of biosynthesized AgNPs and antibacterial efficacy against
endo‑periopathogens P. gingivalis, B. pumilus, and E. faecalis
in biofilm form using dentin blocks.
MATERIALS AND METHODS
AgNPs were biosynthesized using endophytic fungi
Fusarium semitectum and characterized by ultraviolet
spectrum, transmission electron microscopy and Fourier
transform infrared spectroscopy as described earlier were
used in the present study.[8,9] Briefly, the fungi isolated
from healthy leaves of Withania Somnifera (Ashwagandha)
were allowed to grow on potato dextrose agar plates.
Fresh fungal cultures were inoculated in 100 ml malt
glucose yeast peptone broth and incubated at 29°C for
72 h. The fungal biomass was filtered using Whatman
filter paper number 1, washed with sterile distilled water
and 25 g of fungal biomass was then added to flasks
containing 100 ml of sterile distilled water and incubated
for 48 h. The cell‑free fungal filtrate was mixed with
100 ml of 1 mM concentration AgNO3 solution and kept
in the dark at 29°C for 24 h and pH adjusted to 7 was
used further.
The American type culture collection strains ([ATCC],
Manassas, VA, USA) of B. pumilus (27142) and
E. faecalis (29212) strains were sub‑cultured on trypticase
soy agar (TSA) plates and incubated at 35°C for 24 h.
P. gingivalis (33277) strains were subcultured on sterilized
TSA supplemented with 5% sheep blood, hemin (5 µg/ml),
vitamin K1 (0.5 µg/ml), and yeast extract (10 mg/ml) and
incubated at 37°C for 72 h under anaerobic conditions.
MIC of AgNPs against test organisms was determined by
micro broth dilution technique as per Clinical and Laboratory
Journal of Conservative Dentistry | Volume 21 | Issue 6 | November-December 2018
Standards guidelines.[10] Each bacterial inoculum was adjusted
to 1–2 × 105 cells/ml and 10 µl bacterial inoculum of each
test organism was added to wells of microtiter plates with
respective culture medium containing 100 µl of series of
dilutions of AgNPs ranging from 80 µg/ml to 1 µg/ml. Culture
media without AgNPs was used as control group and 10 µl
Alamar blue (0.7 mg/ml) was added to all the wells. The treated
test plates were incubated at 37°C for 24 h and optical density
at 600 OD was measured in plate reader (BioTek, PowerWave
XS2, Vermont, USA). The lowest concentration of AgNPs that
inhibits the growth of test organisms in comparison to the
control group was defined as the MIC value. The experiments
were performed in triplicates [Figure 1a‑c].
The antibacterial efficacy of biosynthesized AgNPs against
test microorganisms in biofilm form was evaluated using
the dentin blocks according to Nascimento et  al.[11] with
slight modifications. The study was approved by the
Institutional Ethical Committee. Overnight grown cultures
of test organisms were centrifuged at 6000 rpm for 10 min
at 4°C. The supernatants were discarded, and the cells were
washed twice in sterile deionized water and suspended in
5 ml of TSA broth for B. pumilus and E. faecalis and in TSA
supplemented broth for P. gingivalis and incubated for 4 h
at 37°C. Turbidity was adjusted to 1 × 107 cells/ml using a
spectrophotometer.
One hundred and twenty dentin blocks were prepared
using sixty human extracted single‑rooted teeth. The
crown and the apical part of the root were sectioned
using a rotary diamond disc to obtain the middle
third of the root which was vertically sectioned into
two halves, and cementum was removed using the
diamond bur to obtain the dentin blocks measuring
5 mm × 5 mm × 1 mm (width × length × thickness).
The dentin blocks were treated in an ultrasonic bath
of 17% EDTA (Prime Dental Products, Mumbai, India) for
a b c
Figure 1: Micro broth dilution test plate for minimum
inhibitory concentration determination (a) Bacillus pumilus,
(b) Enterococcus faecalis and (c) Porphyromonas gingivalis
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Halkai, et al.: Biosynthesized silver nanoparticles against biofilms of endo perio pathogens

5 min to remove the smear layer followed by 3% sodium
hypochlorite (Prime Dental Products, Mumbai, India) for
5 min to remove the organic and inorganic debris and
distilled water for another 5 min to remove all the chemicals
used and sterilized by autoclaving. The dentin blocks
were randomly divided into three groups (n = 40 each) as
group 1: B. pumilus (ATCC 27142), Group 2: E. faecalis (ATCC
29212), and Group 3: P. gingivalis (ATCC 33277).
For Groups 1 and 2, each dentin block was placed in
individual test tubes containing 1 ml of TSA broth and for
Group 3 the dentin blocks were placed in individual test
tubes containing TSA supplemented broth and subjected
to the second cycle of sterilization.
For bacterial contamination of the dentin blocks, about 50 µl
of the bacterial inoculum was transferred to pre‑sterilized
individual test tubes (n = 40 each) containing dentin blocks
in respective broth. The purity of the cultures was checked
by subculturing 5 µl from the incubated dentin blocks on
respective culture plates. The broth was replenished every
alternate day for a period of 2 weeks. After incubation,
dentin blocks were washed twice with 5 ml of sterile
distilled water. Biofilm models of dentin blocks in each
group were randomly divided into four subgroups (n = 10
each) and treated as follows:
• Group 1: B. pumilus (ATCC 27142) (n = 40)
• Subgroup 1A: Sterile distilled water (Control)
• Subgroup 1B: 0.2% CHX
• Subgroup 1C: 2 % CHX
• Subgroup 1 D: 20 µg/ml of AgNPs (MIC dose).
• Group 2: E. faecalis (ATCC 29212) (n = 40)
• Subgroup 2 A: Sterile distilled water (Control)
• Subgroup 2B: 0.2% CHX
• Subgroup 2C: 2 % CHX
• Subgroup 2D: 30 µg/ml of AgNPs (MIC dose).
• Group 3: P. gingivalis (ATCC 33277) (n = 40)
• Subgroup 3A: Sterile distilled water (Control)
• Subgroup 3B: 0.2% CHX
• Subgroup 3C: 2 % CHX
• Subgroup 3D: 30 µg/ml of AgNPs (MIC dose).
After 24 h incubation, each dentin block was rinsed in saline
and transferred to micro centrifuge tubes and shaken for
60 s (vortex AP 56). Serial decimal dilutions were prepared,
and 50 µl of the dilution were placed on TSA plates for
Groups 1 and 2 specimens and on TSA supplemented plates
for Group 3 specimens and incubated for 24 h followed by
colony forming units (cfu/ml) counting. Data collected was
statistically analyzed using One‑way ANOVA and Post Hoc
Tukey multiple comparison tests [Figure 2].
RESULTS
All the test microorganisms were sensitive to biosynthesized
AgNPs. The mean value of the three readings was calculated
as MIC. MIC of AgNPs for B. pumilus was determined
as 20 µg/ml and for E. faecalis and P. gingivalis 30 µg/ml
respectively.

Figure 2: Biofilm plates of cfu/ml: Group  1  (Bacillus pumilus) 1a: Control, 1b: 0.2% chlorhexidine, 1c: 2% chlorhexidine and
1d: Silver nanoparticles. Group 2 (Enterococcus faecalis) 2a: Control, 2b: 0.2% chlorhexidine, 2c: 2% chlorhexidine and 2d: Silver
nanoparticles. Group  3  (Porphyromonas gingivalis) 3a: Control, 3b: 0.2% chlorhexidine, 3c: 2% chlorhexidine and 3d: Silver
nanoparticles

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Halkai, et al.: Biosynthesized silver nanoparticles against biofilms of endo perio pathogens

For antibacterial efficacy in biofilm mode, the mean colony
count (cfu/ml [107]) for all the groups was evaluated and
shown in Tables 1 and 2. The results of the present study
indicate biosynthesized AgNPs are effective against all the
three test microbes when compared to the control group.
There is no statistically significant difference between the
three groups when compared between groups by Post hoc
Tukey multiple comparison test.
Different bacterial species have varying MICs. In vitro
detection of MIC acts as a guideline for clinical application[13]
Although antimicrobial activity of biosynthesized AgNPs
was proven on many pathogenic bacteria;[8,9,14] Literature
is scarce for application of biosynthesized AgNPs for
determining MIC and antibacterial efficacy of fungal‑derived
AgNPs using tooth models against P. gingivalis, B. pumilus,
and E. faecalis.[15]

DISCUSSION The results of the present study show that AgNPs are
effective against test microorganisms with MIC values of
Microorganisms grown in biofilms are difficult to 30 µg/ml against P. gingivalis and E. faecalis and 20 µg/ml
eradicate with existing antimicrobial agents and exhibit against B. pumilus. MIC values of AgNPs synthesized using
resistance.[12] Since endodontic‑periodontal infections are Chamaemelum nobile extract against Escherichia coli,
biofilm mediated and resistant to existing antimicrobial Staphylococcus aureus and Bacillus subtilis was found to be
agents, the present study aims to evaluate the antibacterial 7.8, 31.2, and 15.6 μg/ml.[16] MIC of AgNPs‑derived from
efficacy of biosynthesized AgNPs against the biofilms of Garcinia imberti against E. faecalis was observed maximum
B. pumilus, E. faecalis and P. gingivalis. at a concentration of 62.5 mg/ml[17] MIC of fungal‑derived
AgNPs against E. faecalis biofilm was determined 30 µg/ml
MIC is the lowest concentration of a drug that inhibits the with effective antibacterial efficacy.[15] The results of the
visible growth of the test organism, used to determine the present study showed effective antibacterial activity
against all the test microbes.
in vitro antibacterial activity of new antimicrobial agents.
In the present study, the MIC dose of AgNPs was
Table 1: One‑way ANOVA analysis for within groups of
all three groups for antibiofilm efficacy standardized to evaluate the antibacterial efficacy against
test pathogens in biofilm form because it is the minimum
Groups n Mean SD F P
dose which inhibits the visible bacterial growth[13]
Group 1: Bacillus pumilus
Control (distilled water) 10 6.49 0.0738 7729.29 <0.00001
therefore used to evaluate the efficacy of AgNPs at a
0.2% CHX 10 2.60 0.0816 (S) minimal dose.
2% CHX 10 2.30 0.0667
AgNPs 10 2.35 0.0707 Biosynthesised AgNPs have emerged as newer antimicrobial
Group 2: Enterococcus
faecalis agents with effective antimicrobial activity against several
Control (distilled water) 10 6.77 0.1160 6960.76 <0.00001 pathogens.[8,18]
0.2% CHX 10 3.20 0.0667 (S)
2% CHX 10 2.40 0.0667 Chlorhexidine (CHX) is the most effective antimicrobial
AgNPs 10 2.47 0.0483
Group 3: Porphyromonas agent; its effectiveness can be attributed to its substantivity
gingivalis within the oral cavity. It is recommended to use 0.2%–0.12%
Control (distilled water) 10 6.50 0.1155 5805.47 <0.00001 CHX for plaque control and 2% CHX is recommended as
0.2% CHX 10 2.00 0.0667 (S)
root canal irrigant;[19,20] hence, both 0.2 and 2% CHX were
2% CHX 10 2.00 0.0667
AgNPs 10 2.11 0.1101 used in the present study.
CHX: Chlorhexidine, AgNPs: Silver nanoparticles, SD: Standard deviation,
S: Significant Antimicrobial studies on planktonic culture models do
not correlate well with clinical findings.[21] Therefore; to
Table 2: P value by one‑way ANOVA and post hoc ascertain the antibacterial efficacy of AgNPs effectively
Tukey test to compare antibiofilm efficacy between biofilm model using dentin blocks was evaluated. Human
groups permanent teeth were used to prepare the dentin blocks as
Groups Distilled 0.2% CHX 2% CHX AgNPs they simulate clinical conditions.[22]
water
Group 1 and 0.0010 (S) 0.0010 (S) 0.0065 (S) 0.0029 (S) E. faecalis biofilm was more resistant to CHX than the
Group 2
Group 1 and 0.8999 (IS) 0.0010 (S) 0.0010 (S) 0.0010 (S) same strain grown in planktonic suspension and 2% CHX
Group 3 eliminated most of the E. faecalis biofilms.[23,24] AgNPs
Group 2 and 0.0010 (S) 0.0010 (S) 0.0010 (S) 0.0010 (S) against E. faecalis biofilm using dentin blocks showed less
Group 3 number of viable bacteria for AgNPs compared to CHX.[18]
F 23.49 94.29 97.50 63.00
P <0.00001 <0.00001 <0.00001 <0.00001 The results of the present study show effective antibacterial
(S) (S) (S) (S) activity of biosynthesized against B. pumilus, E. faecalis
CHX: Chlorhexidine, AgNPs: Silver nanoparticles, S: Significant, IS: Insignificant and P. gingivalis biofilm model at a dose ranging from 20

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Halkai, et al.: Biosynthesized silver nanoparticles against biofilms of endo perio pathogens
to 30 µg/ml indicating they can be used as antimicrobial
agents in periodontal, endodontic and combined lesions.
Results of the present study show an insight for application
of biosynthesized AgNPs for the treatment of endo‑perio
lesions; can be used as root canal irrigants, intracanal
medicaments, root canal sealers, can be incorporated
for periodontal therapy alternative to topical antiseptics,
antimicrobial agents and for local drug delivery.[7]
CONCLUSION
The present in vitro study helps to design and develop
intervention approaches for controlling periodontal,
endodontic or combined lesions incorporating
biosynthesized silver nanoparticles. However, in vitro
values of MIC may not hold for in vivo conditions as the
bacterial growth varies from in vitro and in vivo conditions.
In vitro determination of MIC serves as an attempt for
quantifying drug activity against the test microorganisms,
and the results of the study show biosynthesized AgNPs
are effective against biofilms of P. gingivalis, B. pumilus and
E. faecalis.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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