ORIGINAL ARTICLE

Efficacy of Antimicrobial Peptide GH12 on a Multispecies Endodontic

Biofilm Model: An In-vitro Study
Aishi SINHA,1 Sonali TANEJA,1 Devi Charan SHETTY,2 Vidhi Kiran BHALLA1
1
Department of Conservative Dentistry and Endodontics, I.T.S Centre for Dental Studies and Research,
Ghaziabad, India
2
Department of Oral Pathology, I.T.S Centre for Dental Studies and Research, Ghaziabad, India

ABSTRACT
Objective: This study aimed to evaluate the antibacterial efficacy of different concentrations of GH12 on a
simulated multispecies biofilm comprising Enterococcus faecalis, Streptococcus mutans, Fusobacterium nuclea-
tum and Porphyromonas gingivalis.
Methods: Single rooted teeth were decoronated, cut into 1.5 mm sections to obtain dentine discs which were
randomly allocated into five groups: (n=12 each), Group 1: Phosphate Buffered Solution (PBS) - negative con-
trol, Group II: 5% Sodium hypochlorite (NaOCl) - positive control, Group III: Minimum Inhibitory Concentration
(MIC) of GH12, Group IV: 2x MIC of GH12, Group V: 4x MIC of GH12. Colony forming units, Crystal violet assay
and scanning electron microscopy examinations were performed. One-way ANOVA and Turkey’s test were
applied for statistical analysis using the SPSS software version 22.0.
Results: Group II (NaOCl) showed maximum reduction in bacterial load followed by Group V (GH12 16mg/mL)
with no statistically significant difference (p=1.000). On comparing the mean CFU reduction, the maximum
reduction was identified for S. mutans and the least was for P. gingivalis. There was marked erosion observed
in the NaOCl group whereas the GH12 group showed no erosive changes in the morphology and no bacterial
colonies was identified.
Conclusion: The findings revealed that GH12 at higher concentrations inhibits and disrupts the growth of
multispecies endodontic biofilm comparable to NaOCl but without erosive effects to the dentine, further
highlighting its potential to be used as an antimicrobial solution.
Please cite this article as: Keywords: Antimicrobial action, GH12, multispecies biofilm, peptide, root canal
Sinha A, Taneja S, Shetty DC, Bhalla
VK. Efficacy of Antimicrobial Peptide
Gh12 on a Multispecies Endodontic
Biofilm Model: An In-vitro Study. HIGHLIGHTS
Eur Endod J 2024; 9: 411-7
• Antimicrobial efficacy of higher concentration of GH12 peptide was comparable to Sodium
Address for correspondence:
Sonali Taneja hypochlorite in combatting a multispecies biofilm along with no evident erosive changes
Department of Conservative • F. nucleatum and P. gingivalis species showed resistance to elimination compared to S. mu-
Dentistry and Endodontics, I.T.S tans and E. faecalis.
Centre for Dental Studies and
Research, Ghaziabad, India
E-mail: drsonali_taneja@[Link]
INTRODUCTION self-produced matrix making them resistant to
Received : November 03, 2023,
Revised : December 18, 2023, The primary causative factor in pulpal and pe- antimicrobial agents, thereby posing a signifi-
Accepted : January 20, 2024 riapical pathology is a bacterial infection of the cant clinical challenge (2).
root canal system. Dense multi-species biofilm
Published online: November 25, 2024 Endodontic therapy aims to remove biofilm and
communities are formed when microorganisms
DOI 10.14744/eej.2024.75983
infiltrate the anatomic complexities such as ac- eliminate bacteria from the canal space by thor-
This work is licensed under cessory canals, canal ramifications, isthmuses ough chemical disinfection using irrigants (3).
a Creative Commons Sodium Hypochlorite (NaOCl) is the most po-
Attribution-NonCommercial
and other morphological irregularities (1). Th-
4.0 International License. ese communities persist by embedding in a tent irrigating solution because of its effective
Sinha et al. GH12 Against Multispecies Endodontic Biofilm EUR Endod J 2024; 9: 411-7

antimicrobial action and excellent organic tissue dissolving 100.0±0.12 respectively (9). G*Power version [Link] was used
ability (4). However, NaOCl is toxic to the host tissue, especially as follows, power 95% and α 5% the effect size was 2.90619.
at high concentrations (5). Also, its high surface tension lim- The sample size was estimated to be 5 for each group and the
its its ability to penetrate and disinfect the irregularities of the actual power was 97.91%. However, in this study, 12 samples
root canal system (6). were allocated for each group.
Antimicrobial peptides (AMP) represent a diverse class of Sampling and Specimen Preparation
biomolecules that offer early-stage defense against encroach- Single-rooted teeth with one canal from patients requiring ex-
ing microbes and are suggested as potential substitutes for tractions for orthodontic purposes were chosen for this study.
traditional pharmaceuticals in the fight against multidrug- The freshly extracted teeth were rinsed with the phosphate
resistant pathogens and infections caused by biofilms (7). buffer solution, followed by the removal of gingival or peri-
Recently, a cationic peptide GH12 has been developed with odontal tissue tags using an ultrasonic scaler. The inclusion
broad-spectrum antibiofilm activity. In laboratory study mod- criteria were intact root, completely formed apices, unrestored
els, it has been demonstrated to have excellent antimicrobial teeth and non-carious teeth. Teeth with open apices, root re-
efficacy against cariogenic bacteria and mono-species Entero- sorption, calcification, root canal treated, anatomical devel-
coccus faecalis biofilm (8, 9). opmental anomalies and fractured tooth were excluded. The
teeth were decoronated and sixty sections of 1.5 mm thick-
An infected root canal system harbours multiple microbial
ness were obtained from the middle portion of the teeth. The
species. Amongst them, the most common species cultured
smear layer was removed by placing the dentine blocks in an
from persistent endodontic infections is Enterococcus faecalis
ultrasonic bath containing 5.25% NaOCl and 17% EDTA (Aarc
(10). However, considering that endodontic infections are typ-
Dental, India) for 1 min each following which the dentine discs
ically polymicrobial in nature, this study developed a multi-
were placed in a Brain Heart Infusion (BHI) broth (HiMedia Lab,
species endodontic biofilm comprising Enterococcus faecalis,
Mumbai) and autoclaved for 20 min at 121°C.
Streptococcus mutans, Fusobacterium nucleatum and Porphy-
romonas gingivalis microorganisms. Peptide Synthesis and Storage
GH12 peptide was synthesised and purified from Grey Matter
There is a limited literature on the effectiveness of antimicrobial
Foundation, India. The peptide was stored in an airtight con-
peptides on endodontic biofilms. To the best of knowledge,
tainer until usage, as per the recommendations of the man-
there are no research studies analysing the antibiofilm efficacy
ufacturer. A strict aseptic protocol was ensured by wearing
of different concentrations of GH12 against a multi-species en-
Latex free gloves at all times when working with peptides to
dodontic biofilm. Hence, this study aimed to test the antibac-
avoid cross contamination. Also, while handling the peptide, it
terial efficacy of this peptide in varying concentrations as an
was ensured that all the instruments are sterilized.
antibacterial agent on a simulated in-vitro multi-species model.
Bacterial Culture
The first null hypothesis was that there was no difference in
Four bacterial strains of E. faecalis ATCC 29212, F. nucleatum
the amount of bacterial reduction in the multi-species en-
ATCC 25586, P. gingivalis ATCC 33277 and S. mutans ATCC
dodontic biofilm after irrigating with different concentrations
25175 were procured from HiMedia, India. They were culutred
of GH12 and NaOCl peptide solution when compared with
on BHI agar plates to form subcultures. A 0.5 McFarland stan-
Phosphate Buffer Solution (PBS) solution. The second null hy-
dard suspension was prepared in BHI broth and then diluted
pothesis was that there was no difference in the surface mor-
4-fold to obtain an initial bacterial suspension of approxi-
phology of the dentin e pecimens after irrigating with differ-
ent concentrations of GH12 and NaOCl peptide solution when mately 1.5×104 colony forming units per millilitre (CFU/mL).
Minimum inhibitory concentration (MIC) and Minimum bacte-
compared with Phosphate Buffer Solution (PBS) solution. The
ricidal concentration (MBC) for each bacterium was obtained
third null hypothesis tested was that there was no difference
in the biofilm biomass after irrigating with different concen- using the broth dilution method.
trations of GH12 and NaOCl peptide solution when compared Formation of a Multispecies Biofilm
with Phosphate Buffer Solution (PBS) solution. Bacterial susceptibility assay was performed for all the bacte-
MATERIALS AND METHODS rial strains using the broth microdilution assay and absorbance
The study was conducted in the I.T.S Centre for Dental Studies at 600 nm was recorded using a spectrophotometer (Lab India
and Research, Ghaziabad in the Department of Conservative Analytical, India). To establish the multi-species biofilm, under
Dentistry and Endodontics in collaboration with the Advanced sterile conditions, the bacterial suspensions were co- cultured
Research Centre after obtaining institutional ethical clearance in a [Link] ratio in a glass jar. 100µL of each of the four bacte-
under the protocol number ITSCDSR/IIEC/2020-23/CONS/01 rial cultures were taken and incubated in BHI media (HiMedia
on October 12, 2021. The study was conducted in accordance Lab, Mumbai ) for 24 hr anaerobically. The prepared bacterial
with the Declaration of Helsinki. suspension containing the four species was distributed into
Eppendorf tubes (HiMedia, Mumbai) with 100µL of the sus-
Sample Size Calculation pension using sterile micro-pipettes. The tooth substrate was
It was revealed from the literature that the expected mean±SD inoculated into these Eppendorf tubes containing the bacte-
of parameters between the two groups were 99.75±0.02 and rial suspension and was incubated under anaerobic conditions
EUR Endod J 2024; 9: 411-7 Sinha et al. GH12 Against Multispecies Endodontic Biofilm

TABLE 1. Intergroup comparison of mean reduction in bacterial load

Subgroup PBS NaOCl 4mg/mL 8mg/mL 16mg/mL
GH12 GH12 GH12

E. faecalis 1.42×105 1.79×106 1.69×106 1.70×106 1.76×106

S. mutans 1.66×105 2.07×106 2.01×106 2.03×106 2.05×106
F. nucleatum 1.54×105 2.12×106 1.88×106 2.09×106 2.10×106
P. gingivalis 1.52×105 2.21×106 2.08×106 2.09×106 2.20×106
PBS: Phosphate Buffered Solution, NaOCl: Sodium hypochlorite

TABLE 2. Pairwise comparison of mean reduction in bacterial load TABLE 3. Intergroup comparison of mean absorbance based on
among the five groups A595 quantitation
Subgroups E. faecalis S. mutans F. nucleatum P. gingivalis Group Mean reduction SD p

Gr I vs Gr II <0.001* <0.001* <0.001* <0.001* Group 1 (PBS) 1.367 0.002 <0.001*

Gr I vs Gr III <0.001* <0.001* <0.001* <0.001* Group 2 (NaOCl) 0.203 0.002
Gr I vs Gr IV <0.001* <0.001* <0.001* <0.001* Group 3 (GH12) 0.546 0.002
Gr I vs Gr V <0.001* <0.001* <0.001* <0.001* Group 4 (2×GH12) 0.339 0.002
Gr II vs Gr III 0.010* 0.906 <0.001* <0.001* Group 5 (4×GH12) 0.205 0.003
Gr II vs Gr IV 0.022* 0.982 0.988 0.519
One-way ANOVA test. *: Indicates significant difference at p≤0.05. SD: Standard
Gr II vs Gr V 0.850 0.998 0.994 1.000
debiation, PBS: Phosphate Buffered Solution, NaOCl: Sodium hypochlorite
Gr III vs Gr IV 0.998 0.998 <0.001* 1.000
Gr III vs Gr V 0.117 0.979 <0.001* 0.515
Gr IV vs Gr V 0.214 0.9999 1.000 0.594 Crystal Violet Assay
5mL of the bacterial suspension after treating with the re-
Post-hoc Turkey test. *: Indicates significant difference at p≤0.05
spective test solutions were placed in a test tube. 1mL of 0.2%
crystal violet stain (HiMedia, Mumbai, India) was added to
for 21 days. The anaerobic conditions were maintained using the test tubes and left for 5 minutes. The tubes were gently
an incubator at 37°C. The incubator was rendered anaerobic washed and 100% ethanol was added to them. Absorbance
by infusing with 10% hydrogen, 10% CO2 and 80% Nitrogen. was recorded for the samples at 590 nm using a spectropho-
tometer (Lab India Analytical, India).
Grouping
Sixty samples were randomly divided into five groups (n=12 Statistical Analysis
each) according to the antimicrobial treatment being used. All the results were calculated and entered in MS Excel. SPSS
Group 1: PBS solution - negative control, Group 2: 5%NaOCl - (Statistical Package for Social Sciences) Version 22.0 (IBM Corp
positive control, Group 3: MIC of GH12 (4mg/ml), Group 4: 2x 2013, New York, USA) software was used for the statistical anal-
MIC of GH12 (8 mg/ml), Group 5: 4x MIC of GH12(16 mg/ml). ysis. For comparison of the reduction in colony forming units
of specific bacteria and biomass reduction among the groups,
The Eppendorf tubes containing the biofilm were taken out af- one-way ANOVA was applied and Turkey’s test for pairwise
ter 21 days of contamination in the incubator and pre-quantifi- comparison of means of different groups.
cation of the biofilm was done using a digital colony counter.
The level of significance and the confidence interval were set
Anti-biofilm Treatment at 5% and 95% respectively.
1 mL of the test solution (PBS, NaOCl and GH12) according to
the groups were added to the samples and left for 10 minutes. RESULTS
Samples were taken from the dentine surface and plated into Group II (NaOCl) showed maximum reduction in bacterial
specific media plates and were grown anaerobically for 24 hrs. load followed by Group V (GH12 16 mg/mL) with no statis-
tically significant difference (p=1.000) (Table 1, Table 2, Fig.
Bacterial Sampling and Colony Forming Unit Count (CFU) 1). On comparing the mean CFU reduction for specific bac-
After incubation, serial dilutions were done and 1µL of the di- teria, a similar pattern was observed in all the five groups,
luted bacterial suspension was placed in specific agar plates to in increasing order: S. mutans > E. faecalis > F. nucleatum > P.
form colonies and the number of colonies was recorded with gingivalis. Maximum reduction was seen for S. mutans and
the help of a digital colony counter. the least was seen for P. gingivalis.
Scanning Electron Microscopy (SEM) The SEM images revealed the presence of biofilm colonies in
The dentine samples were fixed with serial dilutions of 2.5% the PBS group (Fig. 2). There was a marked erosion observed
glutaraldehyde and then sputter coated with gold and viewed in the NaOCl group whereas the GH12 group showed no such
under SEM (JEOL JSM-6610LV, Tokyo, Japan), to observe the erosive changes in the morphology and the presence of any
morphological changes. bacterial colonies was also not visible.
Sinha et al. GH12 Against Multispecies Endodontic Biofilm EUR Endod J 2024; 9: 411-7

Figure 1. Intergroup comparison of mean reduction in bacterial load

PBS: Phosphate Buffered Solution, NaOCl: Sodium hypochlorite

a b c d e

Figure 2. SEM images presenting morphological changes when treated with (a) PBS solution ( b) NaOCl (c) 4mg/ml GH 12 (d) 8 mg/ml GH12 (e)
16 mg/ml GH12
SEM: Scanning electron microscope, PBS: Phosphate Buffered Solution, NaOCl: Sodium hypochlorite

The absorbance values revealed that Group II (NaOCl) showed

maximum biomass reduction and Group I (PBS) showed the
minimum reduction (Table 3, Fig 3).

DISCUSSION
Biofilm is a microbial community adhered to a solid surface
and embedded with a polysaccharide or a glycocalyx matrix
which exhibits increased antibiotic resistance and the ability to
evade host immune defense mechanism by showing unique
traits in terms of gene expression, protein synthesis, growth
rate, and metabolic processes (11, 12). The present study in-
vestigated the antibiofilm activity of 5% Sodium Hypochlorite
(NaOCl) and different concentrations of GH12 antimicrobial
peptide on a multispecies biofilm model.

A significant reduction in bacterial load was observed after irri-

gation with NaOCl and different concentrations of GH12 when
compared to PBS solution thereby rejecting the first null hypoth-
esis. The antibacterial activity of NaOCl may be attributed to the
release of chlorine ion which interferes with the cellular metabo- Figure 3. Intergroup comparison of absorbance
lism and causes inhibition of essential bacterial enzymes (4). PBS: Phosphate Buffered Solution, NaOCl: Sodium hypochlorite

GH12 when used at a higher concentration of 16 mg/mL, GH12 makes it a broad-spectrum antimicrobial agent (13). In
showed lower but statistically comparable results to NaOCl. the first step, the positively charged peptide accumulates on
The design of GH12 is such that it is positively charged and the negatively charged bacterial membrane by means of elec-
has a high propensity to fold into an amphipathic alpha-heli- trostatic interactions. After which, the hydrophobic groups
cal structure in hydrophobic environments. Such a structure of of the antimicrobial peptide insert into the lipid bilayers and
EUR Endod J 2024; 9: 411-7 Sinha et al. GH12 Against Multispecies Endodontic Biofilm

cause direct disruption of the bacterial membrane via several species to hypochlorite treatment (23, 24). Wang et al. (25)
mechanisms hypothetically proposed in the literature such as stated that P. gingivalis has a strong proteolytic action which
barrel-stave, toroidal pore, aggregate, and carpet models. This has caused the degradation of the AMP, thereby affecting the
eventually results in the leakage of intracellular metabolites antimicrobial efficacy of the peptide (25).
and essential ions causing subsequent cell death (14).
The bacterial reduction achieved with lower concentrations (4
When comparing the mean CFU reduction, none of the bac- mg/mL and 8 mg/mL) of GH12 was less than that with NaOCl
terial groups were completely eradicated, although all the in all the bacterial subgroups. The lower antibacterial action of
bacteria were significantly reduced. S. mutans is a facultative 4 mg/mL GH12 when compared to NaOCl could be attributed
gram-positive bacterium, with a semi-permeable lipid bilayer to the fact that 4 mg/mL of GH12 is the minimum inhibitory
membrane. Upon interaction with NaOCl, it causes the oxida- concentration (MIC) of the peptide against single-species
tion of outer membrane proteins and inner with subsequent planktonic bacteria. When the individual bacterial species are
cell death, thereby significantly reducing the bacterial colony combined to form a multispecies biofilm, the microorganisms
count. These results are in accordance with two studies (15, coaggregates and undergo complex interactions that play a
16). GH12 directly affects the activity of F1F0-ATPase located key role in their increased pathogenicity and virulence (26).
on the cell wall of S. Mutans and inhibits the synthesis of water- Studies have reported that these coaggregated cells may have
soluble EPS which in turn causes cytoplasmic acidity inhibiting a combined metabolic advantage over single cells, and hence,
the normal process of glycolysis and further triggers a cascade the cells show resistance to elimination with reduced concen-
of molecular reactions which leads to disruption of microbial tration of antimicrobial agents (24, 27).
cells. These results are in accordance with the study by Jiang et
al. (8), wherein GH12 was reported to be effective against the There was a significant difference in the biofilm biomass after ir-
cariogenic virulence factor of S. mutans (8). rigating with different concentrations of GH12 peptide solution
when compared with NaOCl and PBS solution, thus rejecting
For S. mutans, NaOCl caused the oxidation of the outer mem- the second null hypothesis. The reason for the superior biomass
brane. The lipid bilayer membrane composed of proteins un- reduction ability of NaOCl can be attributed to the bactericidal
dergoes oxidation causing subsequent cell death and a sig- activity against microbial cells as well as its ability to act on the
nificant reduction in bacterial colony count. The incomplete extracellular matrix of proteins and polysaccharides (28).
eradication of NaOCl could be attributed to the invasion of E.
faecalis into the dentinal tubules because of its small size (4). As the concentration of GH12 increased, a reduction in
Also, E. faecalis has adaptive capabilities and display increased biomass was observed. This could be attributed to the re-
resistance to antimicrobial action (17). These results are in ac- duction of bacterial load because of its antibacterial activity
cordance with two studies (18, 19). In the current study, E. fae- which has been previously explained and also to its ability to
calis was co- cultured with four other bacterial species, allow- degrade the polysaccharide and biofilm matrix (25). It may be
ing for intricate bacterial interactions that possibly resulted in speculated that lower concentrations of GH12 peptide might
increased antimicrobial resistance in the community. have been insufficient to penetrate the EPS matrix. Also, the
contact time in this current study was 10 minutes, which might
Studies have shown that E. faecalis has specific genes which facil- not be enough to allow sufficient interaction to eliminate the
itate primary attachment and biofilm formation. Gelatinase, an biomass (29). This is in accordance with the study by Li et al (9),
extracellular zinc-containing metalloproteinase that is encoded which showed that as the contact time was increased, greater
by these genes, facilitates hydrolysis of bioactive compounds antimicrobial efficacy was observed (9).
such as gelatin and collagen, which makes E. faecalis more im-
mune and thereby harder to eradicate by endodontic medica- The dentine samples treated with 5% NaOCl showed erosive
ments. GH12 acts against E. faecalis, making it susceptible to en- changes. Higher concentrations of NaOCl have a significantly
dodontic treatment by suppressing these pathogenic genes (9). deeper penetration (up to 300 µm) into the dentinal tubules
and thereby leading to irregular erosive changes in the per-
The antibacterial efficacy of GH12 against F. nucleatum and P. itubular and intertubular dentine resulting from the loss of or-
gingivalis showed inferior results when compared with NaOCl, ganic matrix. These erosive changes have also been reported
though it was non-significant. The long rod shape of F. nuclea- by two studies (29–31).
tum makes it a bridge organism and enables its interaction
with many other microbial cells. It has the ability to co-aggre- The SEM images of the GH12 peptide, even when used at higher
gate with P ginigvalis which enhances further colonization concentrations (16mg/mL), revealed no erosive changes with
and biofilm advancement, which makes them resistant to no evident bacterial aggregation on the dentine surface. Th-
complete elimination by antimicrobial treatment, this syner- ese results can be corroborated with the study by Li et al. (9),
gistic effect between the two bacterial species has been em- which reported no apparent damage to the root canal dentine
phasized in previous studies (20, 21). According to Altman et and further highlighted the advantage and potential of using
al. (22), the virulence factors that allow them to survive in hos- GH12 as a root canal irrigating solution (9). Since there was a
tile environments could be the most probable reason for the significant difference in the surface morphology of the den-
resistance to elimination of these two bacterial species after tine specimens after irrigating with different concentrations of
treatment with NaOCl. These findings are in accordance with GH- 12 peptide solution when compared with NaOCl and PBS
two studies that reported similar resistance of these microbial solution, thus rejecting the third null hypothesis.
Sinha et al. GH12 Against Multispecies Endodontic Biofilm EUR Endod J 2024; 9: 411-7

The high production cost has been recognised as one of the Use of AI for Writing Assistance: No AI technologies utilized.
limitations of peptides especially in clinical settings. Another Financial Disclosure: The authors declared that this study received no finan-
limitation of this study design could be that endodontic cial support.
biofilm consists of more than 1000 bacterial species and the Peer-review: Externally peer-reviewed.
interaction of only four bacterial strains was considered in our
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