Strains and Culture Condition

One species of the periodontal pathogen was used in this study, which consists of four strains, Aggregatibacter actinomycetemcomitans Y4 (serotype b), SUNY 75 (serotype a), OMZ 534 (serotype e), and ATCC 29524. The periodontal pathogens were obtained from Kyushu Dental University, Japan. On the other hand, five probiotic strains of genus Lactobacillus were L. casei subspecies rhamnosus NBRC 3831, L. fermentum JCM 1137, L. fermentum NBRC 15885, L. casei NBRC 15883, and L. johnsonii NBRC 13952. All probiotics strains were obtained from the Kyushu Institute of Technology, Japan. All strains were stored at -80 ºC in appropriate culture media in a sterile 50% glycerol. Then, each strain of periodontal pathogen was stripped onto Brain Heart Infusion (BHI) agar supplemented with 1% yeast extract and incubated at 37 ºC for 48 h in anaerobic conditions. Then, the plate containing A. actinomycetemcomitans was sealed using parafilm and preserved in a chiller at 4 ºC. All probiotic strains were grown in De Man, Rogosa, and Sharpe (MRS) agar under anaerobic conditions at 37 ºC for 48 h using an anaerobic container. The plate containing Lactobacillus sp. was sealed using parafilm and preserved in a chiller at 4 ºC before being used in anti-microbial tests.

Growth Phase Determination

The exponential and stationary phases of Lactobacilli sp. were determined during the preliminary study. First, each strain of periodontal pathogen was cultured into Brain Heart Infusion (BHI) broth supplemented with 1% yeast extract and incubated at 37 ºC for 24 h in anaerobic conditions. Whereas, for the probiotic lactobacilli, all were grown in De Man, Rogosa, and Sharpe (MRS) broth under anaerobic conditions at 37 °C for 48 h by using an anaerobic container. Next, the concentrated stock was set to a new culture media to the initial OD 0.05 with the final volume of 30 mL. Next, the culture media were put into the anaerobic jar and incubated at 37°C. Absorbance was recorded every 2 hours.

Overlay Agar Method

Determination of the anti-microbial activity of each strain of probiotic against each pathogen was evaluated by using an overlay agar method. Two species of Lactobacillus sp. with the most significant and least significant were chosen for the biofilm inhibition assay experiment. First, a single colony of probiotics from the plate was isolated and cultured in De Man, Rogosa, and Sharpe (MRS) broth with a volume of 10 mL. The culture was incubated for 24 h at 37 ºC in anaerobic conditions. This creates an optimal condition for the probiotic lactobacilli to grow and express their capacity to produce anti-A. actinomycetemcomitans components. Next, 3 µL of overnight culture (24 h at 37 ºC) of probiotic strains were spotted two times in distinct on the surface of plates containing 7 mL MRS agar (1.2%) as a first layer agar. Later, the spots were allowed to dry, and the plates were incubated for 24 h at 37 ºC under anaerobic conditions. The following day, a 200 µL sample of an overnight culture (24 h at 37 ºC) of A. actinomycetemcomitans was dropped into the 7 mL of warmed BHI soft agar (0.7%). Immediately, the mixture was poured over the plate surface containing the spots of probiotics to become the second layer of agar. Next, the plate was incubated for 24 h at 37 ºC under anaerobic conditions. For the negative control, A. actinomycetemcomitans was grown on the double-layer agar without the probiotics strain, and for the positive control, antibiotic tetracycline was used for comparison with probiotic Lactobacillus.  Inhibition of A. actinomycetemcomitans growth was determined based on probiotic strains’ inhibition zone formed around the spot. The inhibition of A. actinomycetemcomitans growth diameter was measured, and the petri dish was photographed.        

Biofilm Inhibition Assay

Before this experiment, two species of probiotic lactobacilli cells (exponential and stationary phase) and all four A. actinomycetemcomitans strains (exponential phase) were cultured hours before the experiment following their preliminary growth phase data. Next, a biofilm inhibition assay followed Coffey and Anderson with some modifications [14]. The treatment was prepared by co-culturing 100 µL of each probiotic lactobacilli suspension into 100 µL of A. actinomycetemcomitans suspension in a 96-well plate. For the positive control, 100 µL of A. actinomycetemcomitans suspension was added to 100 µL of BHI broth, whereas for the negative control, the BHI broth without bacteria was used. The 96-well plates were incubated at 37 °C for 24 h, 48 h, and 72 h in anaerobic conditions. For the probiotic lactobacilli’s supernatant preparation, the cultured cells were filtered first using a 0.45 µm syringe filter (Whatman Puradisc 25 Nylon) to separate the supernatant from the cells. Next, the same procedure for the biofilm inhibition assay was applied to the probiotic lactobacilli’s supernatant. Ratio 1:1 of A. actinomycetemcomitans suspension to probiotic lactobacilli’s supernatant was used for the co-culture assay.

Crystal Violet Staining and Biofilm Quantification

Biofilm inhibition was determined using 0.1% crystal violet staining. After incubation, the well was washed with distilled water three times to remove planktonic bacteria and leftover media from each well. The biofilm mass was determined by adding 200 µL of 0.1% crystal violet solution (w/v) to each well and dissolving for 30 min at room temperature [15]. Then, the plate was rinsed with distilled water to remove the crystal violet solution and allowed to air dry in an incubator at 37 °C for 15 min. Next, the stained biofilm was dissolved in 200 µl of 95% ethanol in each well for 30 min to solubilize the dye. Finally, the plate was read using a Tecan Infinite M200 Pro microplate reader at an absorbance of 492 nm. All the experiments were performed in triplicates, and the results were presented in the mean form.

Statistical Analysis

Data from at least three independent experiments were represented as mean ± SD (standard deviation). The comparison was performed using a student t-test using GraphPad Software, and P-value < 0.05 was considered significant.

Figures & Tables

Before the experiment started, preliminary growth curve was determined for all probiotics lactobacilli and A. actinomycetemcomitans strains. The study was focused on the exponential and stationary phases. The growth curve information helps decide the exponential and stationary phase for the probiotic lactobacilli and the exponential phase for A. actinomycetemcomitans strains. The exponential and stationery were chosen because cells in the exponential phase are preferentially used in industrial applications and research [17]. Furthermore, the growth rate increases during the exponential phase, cell doubling occurs at a relatively constant rate, and a uniform metabolic activity is produced. During the stationary phase, the growth rate begins to decline, and growth ceases due to nutrient exhaustion and the accumulation of toxic byproducts. Cells are still metabolically active and create secondary metabolites even while the growth rate is zero. During the stationary phase, the production of particular metabolites is increased due to metabolite deregulation [18]. This phenomenon may occur when the cells are not growing anymore but are metabolically active to secrete the secondary metabolites. Because of that, our interest is to evaluate those conditions’ influence on biofilm inhibition activity.

Another preliminary study was conducted to choose two species out of five lactobacilli cells. The inhibitory effect of Lactobacillus sp. shows a promising inhibition area (strong and very strong antibacterial effect) against A. actinomycetemcomitans, although the area is less than positive control antibiotic tetracycline. Among five species of lactobacilli, L. casei NBRC 15883 showed a very strong antibacterial effect for all A. actinomycetemcomitans strains. L. fermentum JCM 1137 and L. johnsonii NBRC 13952 showed a strong antibacterial effect against all four A. actinomycetemcomitans strains. A study done by Husain et al. [19] showed the diameters of inhibition zones of  B. subtilis strain under the exponential phase (7 hours incubation) and stationary phase (21 hours incubation) had a strong effect against S. aureus and are immensely affect strong against E. coli. At the same time, the effect of tetracycline is categorized as very strong against both E. coli and S. aureus. Prabhurajeshwar and Chandrakanth [20] showed that all the isolated Lactobacillus strains produced the average inhibition (1.5-2.5 cm) pathogens’ growth, whereas T2 (L. fermentum), T4, and T16 were the most effective against the growth of the test pathogens (2.8-3.2 cm). So for this study, L. casei NBRC 15883 and L. casei NBRC 15883 were chosen to determine the range of effectiveness against A. actinomycetemcomitans.

Antibacterial activity is part of the critical options for effective and novel probiotics. Figures 3,4,5, and 6 show that all data showed a significantly different (p < 0.05) between control A. actinomycetemcomitans strains and co-culture treatments with probiotics lactobacilli. One valuable finding in this study was that A. actinomycetemcomitans’s biofilm was inhibited in the presence of lactobacilli which confirmed their high inhibition activity. Moreover, data also confirmed inhibition activity occurred both during the exponential and stationary phases regardless of cells or supernatant. This result could be due to several mechanisms, including producing toxic compounds such as lactic acid, hydrogen peroxide, and bacteriocins [21] enhanced in probiotic lactobacilli. Some lactobacillus produces biosurfactants that can effectively prevent biofilm formation by adsorbing solid surfaces, impairing bacterial adherence [22]. Theoretically, compound production related to inhibitory substances peaked during the exponential and stationary phase of lactobacilli. Rezvani et al. [23] reported that lactic acid production was observed at both exponential and stationary growth phases for five Lactobacillus strains studied. The pH data support the statement since all treatments well during the biofilm inhibition assay were in an acidic medium-ranged between pH 4.33 to 6.38 for cells and 4.14 to 6.41 for supernatant, which correlated with the production of the acidic compound. A study by Prabhurajeshwar and Chandrakanth [20] shows pH values of Lactobacillus isolates in the range of 4.0 to 6.5, which aligns with the current study range. The highest acidity and lowest pH were observed after 72 h incubation at 37 °C for Lactobacillus sp. from all the collected samples. Todorov et al. [21] revealed that organic production increased with the incubation time, and the media’s pH decreased with the increase of acid production. Schultze et al. [24] studied the impact of pH on the biofilm formation of the oral pathogen. In the biofilms representing periodontitis, at 24 and 48 h, the total counts of bacteria (log10 CFU), metabolic activity, and biofilm mass were lowest at pH 5 and 5.5. At the same time, the highest was detected at pH 7-8. This proved that during the acidic condition, periodontal metabolic activity was diminished.

For the comparison between probiotics lactobacilli’s exponential and stationary phase, overall, for cells, there is a significant difference (p < 0.05) between the exponential and stationary phase, especially for L. casei NBRC 15883, which is substantial in all strains of A. actinomycetemcomitans. There is no significant difference (p > 0.05) for supernatant between exponential and stationary. A study by Rezvani et al. [23] showed that lactic acid production of L. fermentum was observed at both exponential and stationary growth phases, where maximum lactic acid concentration was obtained at the end of the stationary phase. A study done by Broeckx et al. [25] showed that Lactobacillus rhamnosus grown to the mid-log phase had a declining pH of 5.27 ± 0.02 to 3.99 ± 0.05 (17 h) and 3.65 ± 0.01 (24 h) during the stationary phase, which corresponds to the increase in lactic acid owing to the bacterial metabolism during growth. High lactic acid concentration might be the reason for the high acidity in the treatment 96-well plate.

For the comparison between cells and supernatant of probiotics lactobacilli, overall, there was a significant difference (p < 0.05) between cells and supernatant. The data showed that lactobacilli supernatant has a higher inhibition activity compared with cells. This finding proved that cells are not the factor inhibiting A. actinomycetemcomitans’s biofilm, but the compound or metabolites produced by lactobacilli. The early production of the biochemical compound by lactobacilli was high enough to inhibit A. actinomycetemcomitans’s biofilm, which was proven by the results where biofilm was inhibited during the first 24 h maintained throughout the 72 h incubation period. The focus now was biochemical compounds or metabolites responsible for inhibiting A. actinomycetemcomitans’s biofilm. Prabhurajeshwar and Chandrakanth’s [20] study found no inhibitory activities of the supernatant of all three isolated Lactobacillus strains after being treated with trypsin and pronase. This finding might suggest that the inhibition effect was caused by the protein-like substance, for instance, bacteriocin produced by the Lactobacillus strains.

To summarize, our finding emphasizes the unique property of probiotic cells that plausible to be clinically effective therapeutics and directly interact with periodontal pathogen biofilms. However, more investigation and focused investigations must be carried out to clarify the mechanisms underlying this action. Comparing how probiotic bacteria and the periodontal pathogen interact with host cells may help develop effective methods to manage infections in vivo.

Author Contributions

Norzawani Jaffar and Kesaven Bhubalan designed the experiment and wrote the manuscript. Syakir Syahiran collected and analyzed the data and wrote the manuscript.

Acknowledgement

This study is part of the research funded by the Fundamental Research Grants Scheme (FRGS) FRGS/1/2017/SKK06/UNISZA/02/1.

Conflict of Interest

1. Nazir MA. Prevalence of periodontal disease, its association with systemic diseases and prevention. International Journal of Health Sciences, (2017); 1(2): 72-80.
2. Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. The Lancet, (2017); 390(1): 1211-1259.
3. Brown SA, Whiteley M. A Novel Exclusion Mechanism for Carbon Resource Partitioning in Aggregatibacter actinomycetemcomitans. Journal of Bacteriology, (2007); 189(17): 6407-6414.
4. Prakasam A, Elavarasu S, Natarajan RK. Antibiotics in the management of aggressive periodontitis. Journal of Pharmacy and Bioallied Sciences, (2012); 4(2): 252-255.
5. Flemming H-C, Wingender J, Szewzyk U, Steinberg P, Rice SA, et al. Biofilms: an emergent form of bacterial life. Nature Reviews Microbiology, (2016); 14(9): 563-575.
6. Kim K-R, Owens G, Kwon S-I, So K-H, Lee D-B, et al. Occurrence and Environmental Fate of Veterinary Antibiotics in the Terrestrial Environment. Water, Air, & Soil Pollution, (2011); 214(1): 163-174.
7. Hashim NT. The Use of Antibiotics vs. Probiotics in Periodontitis Therapy. Journal of Dentistry and Oral Biology, (2020); 5(2): 1166-1170.
8. Karska-Wysocki B, Bazo M, Smoragiewicz W. Antibacterial activity of Lactobacillus acidophilus and Lactobacillus casei against methicillin-resistant Staphylococcus aureus (MRSA). Microbiological Research, (2010); 165(8): 674-686.
9. Coeuret V, Dubernet S, Bernardeau M, Gueguen M, Vernoux JP. Isolation, characterisation and identification of lactobacilli focusing mainly on cheeses and other dairy products. EDP Sciences, (2003); 83(1): 269-306.
10. Samot J, Badet C. Antibacterial activity of probiotic candidates for oral health. Clinical Microbiology, (2013); 19(1): 34-38.
11. Hoque MZ, Akter F, Hossain KM, Rahman MSM, Billah MM, et al. Isolation, Identification and Analysis of Probiotic Propertiesof Lactobacillus Spp. From Selective Regional Yoghurts. World Journal of Dairy & Food Sciences, (2010); 5(1): 39-46.
12. Shimauchi H, Mayanagi G, Nakaya S, Minamibuchi M, Ito Y, et al. Improvement of periodontal condition by probiotics with Lactobacillus salivarius WB21: a randomized, double-blind, placebo-controlled study. Journal of Clinical Periodontology, (2008); 35(10): 897-905.
13. Parvez S, Malik KA, Kang SA, Kim H-Y. Probiotics and their fermented food products are beneficial for health. Journal of Applied Microbiology, (2006); 100(6): 1171-1185.
14. Coffey BM, Anderson GG. Biofilm formation in the 96-well microtiter plate. Methods in Molecular Biology, (2014); 1149(1): 631-641.
15. Sol A, Ginesin O, Chaushu S, Karra L, Coppenhagen-Glazer S, et al. LL-37 Opsonizes and Inhibits Biofilm Formation of Aggregatibacter actinomycetemcomitans at Subbactericidal Concentrations. Infection and Immunity, (2013); 81(10): 3577-3585.
16. Ołdak A, Zielińska D, Rzepkowska A, Kołożyn-Krajewska D. Comparison of Antibacterial Activity of Lactobacillus plantarum Strains Isolated from Two Different Kinds of Regional Cheeses from Poland: Oscypek and Korycinski Cheese. BioMed Research International, (2017); 1-10.
17. Hall BG, Acar H, Nandipati A, Barlow M. Growth rates made easy. Molecular Biology and Evolution, (2014); 31(1): 232-238.
18. Liu S Bioprocess Engineering: Kinetics, Sustainability, and Reactor Design. Chapter: Book Name. 2017 of publication; 1172. Elsevier.
19. Husain DR, Gunawan S, Sulfahri S. Antimicrobial potential of lactic acid bacteria from domestic chickens (Gallus domesticus) from south Celebes, Indonesia, in different growth phases: in vitro experiments supported by computational docking. Iranian Journal of Microbiology, (2020); 12(1): 62-69.
20. Prabhurajeshwar C, Chandrakanth RK. Probiotic potential of Lactobacilli with antagonistic activity against pathogenic strains: An in vitro validation for the production of inhibitory substances. Biomedical Journal, (2017); 40(5): 270-283.
21. D.Todorova S, Prévost H, Lebois M, Dousset X, LeBlanc JG, et al. Bacteriocinogenic Lactobacillus plantarum ST16Pa isolated from papaya (Carica papaya) — From isolation to application: Characterization of a bacteriocin. Food Research International, (2011); 44(5): 1351-1363.
22. Sharma D, Saharan BS. Functional characterization of biomedical potential of biosurfactant produced by Lactobacillus helveticus. Biotechnology Reports, (2016); 24(11): 27-35.
23. Rezvani F, Ardestani F, Najafpour G. Growth kinetic models of five species of Lactobacilli and lactose consumption in batch submerged culture. Brazilian Journal of Microbiology, (2017); 48(2): 251-258.
24. Schultze LB, Maldonado A, Lussi A, Sculean A, Eick S. The Impact of the pH Value on Biofilm Formation. Monographs in Oral Science, (2021); 29(1): 19-29.
25. Broeckx G, Kiekens S, Jokicevic K, Byl E, Henkens T. Effects of initial cell concentration, growth phase, and process parameters on the viability of Lactobacillus rhamnosus GG after spray drying. Drying Technology, (2020); 38(11): 1474-1492.