Akademik Veri Yönetim Sistemi
33rd International Symposium on Pharmaceutical and Biomedical Analysis, Ankara, Türkiye, 2 - 07 Temmuz 2023, ss.1
ABSTRACT
Oral
diseases are a major cause of morbidity and are among the most prevalent
diseases worldwide. The most prevalent oral diseases globally are dental caries
(tooth decay). Streptococcus mutans is the main etiological agent of
human dental caries, and lives primarily in biofilms on the tooth surfaces
called dental plaque. The main aim of this study is to evaluate the effects of
postbiotics against S. mutans ATCC 25175, a major cariogenic pathogen.
Within this aim Weisella spp. isolated from honey bee (Apis mellifera
L.) pollen microbiota was used as the source of postbiotics. The agar well
diffusion assay was used to determine the anti-microbial activity, and the
crystal violet staining procedure was used to determine the anti-biofilm
activities of postbiotics. According to our results, postbiotics of W.
cibaria 29.1 displayed anti-microbial activity against S. mutans
with the inhibition zone of 20 mm. Following the co-incubation, it was observed
that sub-MIC values of postbiotics inhibited the cariogenic biofilm formation.
In conclusion, postbiotics with their anti-microbial and anti-biofilm
activities can be used as a potential strategy for dental caries.
Keywords:
Dental plaque, cariogenic biofilm, Streptococcus mutans, microbiota,
postbiotic
I. Introduction
Dental caries characterized as a biofilm and sugar-induced disease is a major public
health problem globally and is the most widespread non-scommunicable disease. Streptococcus mutans has been indicated as the main
microorganism related to dental caries development. This microorganism can grow in acidic
environments and metabolize sugars to produce acid that demineralizes
the hard tissues of the teeth (enamel and dentine) as an important factor for caries lesion
progression. Moreover, S. mutans hydrolyze sucrose from diet and
synthesize glucan polymers from the resulting glucose, which contributes to the
three-dimensional architecture of dental biofilms (Bertolini et al. 2022). Therefore, the inhibition
of important factors related with biofilm production mechanisms, and the effect
of these factors on disease progression, also need to be better elucidated. Toothpaste
containing stannous fluoride are in use with their anti-caries effects.
However, consumers tend to use products with natural ingredients, in recent
years. Therefore, biotics has gained
recent interest as some present anti-bacterial activity against oral bacteria,
and potentially reduce side effects, and therefore could emerge as an adjunct
anti-biofilm treatment in the future. Postbiotics known as the microbial fermentation end-products of probiotics can
be a potential candidate with their anti-microbial, anti-inflammatory,
immunomodulatory, anti-hypertensive, anti-carcinogenic, and anti-oxidant
activities. The main aim of
this study is to evaluate the anti-microbial and anti-biofilm effects of
postbiotics against S. mutans ATCC 25175.
II. Material and methods
Weisella spp. isolated from honey bee (Apis
mellifera L.) pollen microbiota were cultured for
24 hours in order to obtain postbiotics. Following the
incubation, centrifugation was performed for 20 minutes at 15,000g at 4ºC. The
cell-free supernatant containing extracellular postbiotics was passed through a
sterile membrane filter and then lyophilized. Anti-bacterial
activity of postbiotics against S. mutans ATCC 25175 was determined
using the agar well diffusion method. Following the incubation period, the
diameters of the inhibition zones around the wells were measured in millimeters.
To find out whether a bacteriocin-like compound or organic acids played a role
in the antibacterial activity against the pathogens, the inhibitory effect was
also analyzed following the treatment of the postbiotics with the 1 mg/mL
proteinase K and neutralization (Todorov and Dicks 2005). Minimum Inhibitory
Concentration (MIC) of the lyophilized postbiotics against S. mutans
ATCC 25175 was determined using microtiter plate assay, according to Clinical
and Laboratory Standards Institute guidelines. Antibiofilm activity of various
postbiotic concentrations was carried out using the crystal violet staining
method (Onbas et al. 2018). All assays were performed with three independent
experiments (biological replicates) and each measurement was carried out in
triplicate (technical replicates). Data were analyzed using SPSS version 22.0
(IBM, New York, NY, USA), by one-way analysis of variance (ANOVA) followed by
Dunnett’s test and unpaired t-test (GraphPad Prism v.3.0, GraphPad Software,
San Diego, CA, USA). All results were presented as a mean ± standard deviation
and p < .05 was used to indicate a significant difference.
III. Results and discussion
Weissella
spp. isolates from honey bee pollen microbiota were tested for their
antibacterial activity against S. mutans ATCC 25175. According to our
results, all isolates showed an inhibitory effect against cariogenic strain.
Among the isolates, Weisella cibaria 29.1 exhibited a remarkable
inhibitory effect with an inhibition zone of 20 ± 1.7 .mm (Fig.1). The effects
of postbiotics were also analyzed in different ways to find out the components,
such as organic acids, or bacteriocin-like compounds responsible for the
antibacterial activity. Although the inhibitory effect was not affected by
proteinase K treatment, the activity disappeared after neutralization (Tab.1),
which may be related to their acidity. MIC value of the postbiotics secreted
into the culture medium of the isolates was determined as 30 mg/mL and 35 mg/mL
as shown in Tab.1 and Fig.2.
The anti-biofilm activity of postbiotics
against S. mutans ATCC 25175 was evaluated within the co-incubation
approach. Following the co-incubation protocol, sub-MIC values of all
postbiotics were found sufficient to inhibit the biofilm formed by S. mutans
ATCC 25175 by more than 90% (Fig.3). Besides, MIC10 value of postbiotics
derived from Weisella cibaria 29.1 was decreased the biofilm formations
as 96.15 ± 0.92%. Additionally, co-treatment with MIC25 values of postbiotics
from Weisella cibaria 18.3 and Weisella confusa 15.1 strains were
able to decrease the formation of biofilms, with the eductions of 95.44 ± 0.91%
and 85.56 ± 0.94%, respectively (Fig.3).
As Streptococci are important cariogenic microorganisms due to their role in dental plaque formation and dental caries development, they remain at the center of the investigations. Moreover, biofilm-associated infectious diseases are hard to control since bacterial cells become resistant to antimicrobial agents and host immune defense mechanisms (Zhang et. al 2011). Owing to the fact that chemical agents used in commercially available products are not sufficient, clinical trials focused on natural compounds, such as plant-derived phytochemicals, and proved that they are effective in preventing dental caries. However, these powerful compounds also showed toxic effects on human cells as well. Therefore, the development of functional antimicrobial agents including novel and effective natural compounds is still needed. Lately, postbiotics are gaining interest for the prevention of dental caries and they are used in this study because their mode of action is mainly based on the production of bioactive compounds such as antimicrobial peptides, organic acids, and hydrogen peroxide [43]. These results showed that postbiotics have the ability to inhibit the activity of pathogenic bacteria while preventing biofilm formation, interfering with biofilm integrity/quality and finally leading to biofilm eradication.
IV. Conclusion
The present study demonstrated that the
postbiotics as the metabolic by-products secreted by Weissella spp.
originating from honey bee pollen microbiota, might be used as a promising
agent for the prevention of dental caries, with its anti-bacterial and
anti-biofilm activity against cariogenic pathogens. However, as the successful
treatment of oral diseases depends on several factors, further studies are
needed to prove the efficacy of postbiotics in managing dental caries, ex
vivo or in vivo.
Acknowledgements
This research was supported by The Scientific and
Technical Research Council of Turkey (TÜBİTAK, project number 119S865).