Oral Lactobacillus species and their probiotic capabilities in patients with periodontitis and periodontally healthy individuals

Abstract Objectives This study aimed to identify oral Lactobacillus species and characterize their adhesion properties and antibacterial activity in patients with periodontitis compared with periodontally healthy individuals. Materials and Methods Three hundred and fifty‐four isolates from the saliva, subgingival, and tongue plaque of 59 periodontitis patients and 59 healthy individuals were analyzed. Oral Lactobacillus species were identified through the culture method in the modified MRS medium and confirmed by molecular testing. Moreover, the radial diffusion assay and cell culture methods were used to determine the antibacterial activities of oral strains against oral pathogens and their adhesion activity in vitro. Results 67.7% of the cases and 75.7% of the control samples were positive for the Lactobacillus species. Lacticaseibacillus paracasei and Limosilactobacillus fermentum were the dominant species in the case group, whereas Lacticaseibacillus casei and Lactiplantibacillus plantarum were dominant in the control group. Lactobacillus crispatus and Lactobacillus gasseri had higher antibacterial effects against oral pathogens. Moreover, Ligilactobacillus salivarius and L. fermentum demonstrated the highest ability to adhere to oral mucosal cells and salivary‐coated hydroxyapatite. Conclusion L. crispatus, L. gasseri, L. fermentum, and L. salivarius can be introduced as probiotic candidates since they demonstrated appropriate adherence to oral mucosal cells and salivary‐coated hydroxyapatite and also antibacterial activities. However, further studies should be conducted to assess the safety of probiotic interventions using these strains in patients with periodontal disease.


| INTRODUCTION
Dental caries and periodontitis are infectious diseases associated with dysbiosis of the microorganisms in dental plaque biofilm.
Porphyromonas gingivalis is the key factor in periodontal disease development (Hirasawa & Kurita-Ochia, 2020).Periodontitis can have a negative effect on quality of life (Ferreira et al., 2017) and is associated with systemic disorders such as cardiovascular disease (Sanz et al., 2020), metabolic syndromes (Pirih et al., 2021), and respiratory infections (Kelly et al., 2021).Changes to the population of indigenous bacteria balance in the oral cavity are associated with the development of periodontitis, with an increase in the population of pathogenic bacteria and a decrease in the population of beneficial bacteria (Curtis et al., 2020).New bacterial treatments can be used as alternative treatments for infections caused by pathogens, which regulate the oral microbiota and remove pathogenic bacteria (Bosch et al., 2012).
Probiotics have demonstrated promising results as antiinflammatory, anticancer, antimicrobial, antioxidant, and immunomodulating agents (Lu et al., 2021;Sankarapandian et al., 2022) and have been considered to balance the natural microbiome in the human body, such as the urogenital tract, the respiratory system, skin, and the oral cavity (Shimauchi et al., 2008).In addition, they can effectively prevent and treat several infectious diseases in the oral cavity, including periodontitis, tooth decay, and halitosis (Bustamante et al., 2020;Shimauchi et al., 2008;Teughels et al., 2013).Bifidobacteria and lactic acid bacteria (LAB) are the most common probiotic strains, such as Lactobacillus, which is the most crucial group of probiotics that produces lactic acid in the gastrointestinal system (Shokryazdan et al., 2014).As probiotics, Lactobacilli have antibacterial activities and interfere with the growth of surrounding microbiota.In addition, they produce organic acids such as lactic acid and acetic acid, leading to a low pH (Cuozzo et al., 2000;Hirasawa & Kurita-Ochia, 2020).Lactobacillus species can reside in various parts of the oral cavity, such as oral mucosa, hard tissue, saliva, tongue, and supra-and subgingival plaques (Terai et al., 2015).
One of the most critical characteristics of lactobacilli is its ability to adhere to epithelial cells and produce antibacterial substances (Na et al., 2020;Terai et al., 2015).The duration of transfer of foods into the oral cavity is shorter than that in other areas of the gastrointestinal system.The oral bacteria are transferred into the stomach together with the saliva.Therefore, oral probiotics must be capable of adhering to the oral tissues.In addition, the antibacterial activities hinder the growth of pathogenic bacteria through antibacterial substances in the microbial supernatant (Kolenbrander et al., 2002).With the ever-increasing use of these bacteria in treating oral diseases, it is essential to determine the properties of oral probiotics to select the appropriate strain and optimize the results of bacterial treatments (Terai et al., 2015).
More knowledge about oral lactobacilli could help understand oral dysbiosis and might provide measures for novel therapeutic agents.There is considerable evidence on lactobacilli and their probiotic potential (Abdel-Daim et al., 2012;Bosch et al., 2012;Hirasawa & Kurita-Ochia, 2020).With the current interest in probiotics (Raghuwanshi et al., 2015), this is the first study to identify and characterize oral Lactobacillus species from the samples collected from periodontitis patients and periodontally healthy individuals in an Iranian population.Moreover, this study aimed to investigate the Lactobacillus adhesion activity to oral mucosal cells and salivary-coated hydroxyapatite (S-HA), besides the antibacterial activity of these strains against oral pathogens.

| Study subjects
Patients seeking periodontal or dental care in the Alborz Dental School were screened, and 118 volunteers were assigned to two groups (with similar age and gender distribution, age range 25-70 years).(1) Case group: patients with periodontitis classified as moderate to severe (Caton et al., 2018), with at least four sites with probing pocket depth (PPD) > 3 mm, clinical attachment loss, bleeding on probing, bone loss, and (2) Control group: healthy subjects with at least 24 natural teeth (excluding third molars), probing depth (PD) ≤ 3 mm, and without oral predisposing factors causing local irritation or plaque retention (Gomes-Filho et al., 2018;Kuru et al., 2017).Before clinical examinations, medical and dental history was obtained.
Exclusion criteria were as follows: history of smoking, history of diabetes, pregnancy, breastfeeding, autoimmune disease, necrotizing periodontal disease, history of periodontal treatment in the past 6 months, been receiving antibiotics or anti-inflammatory drugs in the past 4 months, and patients indicated for prophylactic antibiotics before routine dental treatments.
An informed consent, including the aim and content of the survey, was signed by all the study subjects.The study protocol was approved by the Alborz University of Medical Sciences Ethics Committee and was conducted following the Helsinki Declaration of 1975 (revised 2013).

| Collection of oral specimens and isolation of Lactobacillus from sample cultures
Oral specimens were collected from subgingival plaque, tongue, and saliva.The teeth were isolated using cotton rolls and dried with compressed air to avoid contamination with saliva.Subgingival plaque samples were pooled from the posterior first molars in each quadrant or, in case of the plaque absence on these teeth, from a tooth with the deepest PPD and significant amounts of dental plaque were obtained using sterile Gracey curettes (Teughels et al., 2013).
Microbial tongue samples were collected using a sterile cotton swab rotated five times on an area of 2.16 cm 2 on the left side of the tongue dorsum (Teughels et al., 2013).Unstimulated saliva samples were collected according to a protocol described by Navazesh (1993).Participants were asked to allow saliva to accumulate on the floor of the mouth for 1-2 min, following which they spat 2-3 mL of saliva into a specimen tube (Navazesh, 1993).
The saliva, subgingival, and tongue plaque samples were collected and diluted in Lactobacillus selective (LBS) broth as a transport medium.Then, they were cultured on modified DeMan, Rogosa, and Sharpe (MRS) agar plates (Difco-Merck) and the LBS agar medium for additional confirmation (Vancomycin-HCL, Bromocresol green, and Cysteine Hydrochloride were added to MRS to specifically identify lactobacilli strains).The plates were incubated anaerobically at 37°C for 3 days.Afterward, catalase and oxidase tests were performed, and wet mounts and Gram-stained slides were prepared and examined under a microscope (Olympus Corporation) to ensure the existence of gram-positive, bacillusshaped, and catalase-and oxidase-negative bacteria.Isolated colonies with typical characteristics of lactobacilli were picked from the plates and stored at −80°C in MRS broth containing 20% Glycerol (Patel, 2016).The 24-h microbial suspension pH of the lactobacilli strains ranged between 3.2 and 5.4.

| DNA extraction using the modified salting-out method
The modified salting-out method was used for the extraction of bacterial genomes.First, 100 μL of bacterial cells were resuspended in 250 μL of lysis buffer (1 M Tris-HCl pH = 8, 0.5 M EDTA pH = 8, and 5 M NaCl pH = 8) by vortexing; 100 μL of sodium dodecyl sulfate (SDS) 10%w/v, and 3 μL of proteinase K (Sigma-Aldrich) were added and vortexed gently.It was incubated at 37°C for 24 h.Then, 6 M NaCl was added and centrifuged at 3400 rpm at 10°C for 30 min.The supernatant was transferred to new microtubes, and cold ethanol was added.Next, the tubes were centrifuged at 4000 rpm at 10°C for 17 min.The supernatant was discarded, and 500-1000 μL of 70% ethanol was added to the tube.
Then, the tubes were centrifuged at 12,000 rpm at 10°C for 5 min (repeated twice).Finally, the tubes were air-dried, and 100 μL of elution buffer was added.The tubes were kept at 4-5°C for 2-3 days to dissolve DNA completely.The quality of the extracted DNA was confirmed using electrophoresis on agarose gel 0.8% (wt/vol) and visualized under UV light.Lactobacillus acidophilus and Lactiplantibacillus plantarum were extracted using the DNA extraction kit and used as positive controls (Chacon-Cortes et al., 2012).

| Identification of isolates based on 16S rDNA genes polymerase chain reaction-restrictionfragment-length polymorphism and Sanger sequencing
The isolates were identified at the species level using restriction fragment length polymorphism analysis of polymerase chain reaction-amplified 16S ribosomal DNA genes (16S rDNA polymerase chain reaction-restriction-fragment-length polymorphism) and Sanger sequencing.Therefore, the genomic DNA samples were amplified by polymerase chain reaction using the universal primers: 27F (5′ AGAGTTTGATCMTGGCTCAG 3′) and 1525R (5′ AAGGAGGTGWTCCARCC 3′) (SinaClon) for the 16S rRNA gene.
The polymerase chain reaction was carried out in a thermocycler (Eppendorf).Thirty-two cycles of amplification were carried out in a final volume of 25 μL, including 5 μL of DNA template and amplification mixture, which contained 0.25 μL of each primer, 0.3 μL of dNTPs, 2.5 μL of 10× amplification buffer, 0.5 μL of MgCl 2 , and 0.2 μL of Taq DNA polymerase.The polymerase chain reaction amplification program consisted of an initial heating step at 95°C for 5 min, 30 cycles at 95°C for 45 s, 60°C for 1 min, 72°C for 15 min, and a final extension step at 72°C for 12 min.At the end of the incubation, the amplification products were separated by electrophoresis through 1% (w/v) agarose gel in 1× TBE buffer and visualized under UV illumination.A 100-3000 bp ladder (SinaClon) was used to estimate the fragment size of the amplicons generated.
The bp1545 bands indicated that the lactobacilli DNA product was obtained (Nikolic et al., 2008).
The polymerase chain reaction products were digested using the restriction endonucleases Taq I and Hae III (Thermo Fisher Scientific).The products of enzymatic reactions were analyzed by electrophoresis in 1.5% (wt/vol) agarose gels.The isolates were categorized into 10 groups based on the weight and the number of bands obtained; then, 53 representative strains (3-4 isolates from each group with different restriction-fragmentlength polymorphism patterns) were selected, and Sanger sequencing was conducted by the same primers used for polymerase chain reaction on ABI 3500 automated sequencers (Applied Biosystems).The identified sequences were analyzed using BLAST software in GenBank (www.ncbi.nlm.nih.gov).
Following the comparison between the sequencing results and the standard sequences in NCBI, 10 species of Lactobacillus were detected (Aranishi et al., 2005).

| Adhesion of Lactobacillus species to S-HA
The adherence ability of the Lactobacillus species to human S-HA was determined according to Terai et al. study (2015).The 24-h culture of 10 isolated Lactobacillus species was rinsed 2-3 times with a phosphate-buffered saline solution (PBS solution) and adjusted to an OD 550 of 1.Then, 10 mL of human saliva was filtered using a 0.22 μm filter (Merck Millipore).It was incubated at 60°C for 30 min and then centrifuged.The filtered saliva was mixed with the hydroxyapatite powder.Then, 5 mg of S-HA (mixture of HA with saliva) was added to a 2-mL bacteria suspension.It was incubated at 60°C for 1 h in a shaking incubator.
Afterward, 1 mL of the collected supernatant was added and mixed with 0.1 mL of 0.5 M EDTA until the remaining hydroxyapatite particles were dissolved.

| Adhesion of isolated Lactobacillus species to oral mucosal cells
The adherence ability of the Lactobacillus species to oral tissues was determined based on a method proposed by Terai et al. (2015).Oral mucosal cells, the KB/C152 cell line, and the HGF3-PI 53/C502 cell line, which originated from human epidermoid carcinoma and human gingival fibroblasts, respectively, were obtained from the National Cell Bank of Iran (Pasteur Institute).
KB and HGF cells were precultured in Dulbecco's Modified Eagle Medium (D-MEM; GIBCO) and Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich), respectively, supplemented with 10% Fetal bovine serum (FBS), 1% Penicillin/Streptomycin, L-glutamine, and nonessential amino acids (GIBCO).The individual cells were cultured in a growth medium containing carbon dioxide (CO 2 ) with 95% humidity for cell proliferation for 72 h.Before culture, gelatin-coated coverslips were placed at the bottom of each well.Then, 0.5 mL of cell suspension and 1 mL of the medium were poured into each well of a six-well Chamber Slide (Jet Biofil).
It was incubated at 37°C with 5% CO 2 and 95% humidity for 48-96 h.PBS rinse was carried out three times to remove the nonadhering cells.Isolated oral lactobacilli cultured in the MRS broth for 24 h were centrifuged and rinsed three times using a PBS solution.Suspension of the tested bacteria was added to PBS to adjust the OD 600 to 0.1.Afterward, 0.5 mL of the prepared suspension was added to the cell culture plate and incubated at 37°C with 5% CO 2 for 3 h.It was rinsed with PBS three times and then fixed with methanol.After staining with a Gram stain kit, the coverslips were removed and observed under a light microscope (Olympus Corporation).Escherichia coli ATCC 25922 was used as the positive control, and the medium without bacterial inoculum was used as the negative control.
The number of bacteria adhered to the oral cells was randomly counted and averaged in six different fields per well.The strains were classified based on the number of attachments: <100 weak, 100-300 medium, 300-500 good, and >500 excellent using the method proposed by Abdel-Daim et al. (2012).

| Antibacterial activity of Lactobacillus species against oral pathogenic bacteria
The antibacterial spectrum of the cell-free supernatant (CFS) of Lactobacillus species isolated from the oral cavity was studied against two oral pathogens using the radial diffusion assay.The two following bacterial species were chosen as examples of oral pathogens: Aggregatibacter actinomycetemcomitans (A.a) Y4 ATCC 43718, a Gram-negative oral bacterium associated with periodontitis (Damgaard et al., 2021), and Actinomyces naeslundii (A.n) ATCC1201, a Gram-positive bacterium responsible for numerous oral infections, including oral multispecies biofilm development (Mashimo et al., 2016), oral lesions (Suzuki & Delisle, 1984), gingivitis, and periodontitis (Ellen, 1976).
First, the CFS of the lactobacilli cultured in the MRS broth (24 h) and centrifuged at 10,000 rpm was sterilized by a 0.22 µm filter (Merck Millipore).The pH of the bacteria supernatant was adjusted to 7 using 1 N NaOH solution (neutralization).Next, the pathogenic bacteria suspension density was prepared to the half McFarland standard (1.5× 10 8 CFU/mL) in Brain-Heart Infusion (BHI) broth.
Then, it was plated onto BHI agar (in a ratio of 1/100 mL).The agar plates were punched with a diameter of 5 mm, and 100 µL of the lactobacilli supernatants of each species were poured into these punches and incubated at 37°C for 1-2 days.The results of inhibition zones were read after 18-48 h together with a positive control antibiotic (0.05-0.1 mg/mL tetracycline hydrochloride and 0.1 mg/ L Chlorhexidine) (Balouiri et al., 2016).Escherichia coli ATCC 25922 was used as the negative control.

| Statistical analysis
The null hypothesis was that there is no difference between the Lactobacillus species in oral isolates (the tongue, subgingival plaque, and saliva) of periodontitis patients compared with periodontally healthy individuals.
The χ 2 test was utilized to compare the frequency of isolates with Lactobacillus species, and the Shapiro-Wilk test was used to check data distribution.The data distribution according to the studied strains was normal in all the studied variables.Data description was presented as mean and standard deviation.One-way analysis of variance was used to compare the mean adhesion indices, and Tukey's post hoc test was used for pair-by-pair comparison.To achieve the antibacterial effect of nongrowth halo in different species, one-way analysis of variance and Tukey's post hoc test were performed.All statistical analyses were performed using SPSS software (version 25).p Values lower than .05were considered to be statistically significant.

| Isolation and identification of Lactobacillus species
The bacterial colonies were isolated from the tongue, saliva, and subgingival plaque samples of 59 patients with periodontitis (177 oral isolates) with a mean age of 48 ± 10.6 years and 59 healthy subjects (177 oral isolates) with a mean age of 37 ± 10.8 years.Among the 59 patients with periodontitis, 6 suffered from stage III and stage IV periodontitis, and 52 suffered from stage II periodontitis according to the current periodontitis classification (Caton et al., 2018).
Out of the 354 collected oral isolates, 134 isolates (75.7%) in the control group and 120 isolates (67.7%) in the case group were positive for Lactobacillus strains, which was confirmed by polymerase chain reaction-restriction-fragment-length polymorphism.Twelve oral isolates from the case group and 21 isolates ETEBARIAN ET AL.
| 749 from the control group showed no growth for Lactobacillus in all saliva, subgingival, and tongue samples (67 isolates were excluded).
Moreover, 22 oral isolates in the control group and 45 oral isolates in the case group were positive for vancomycin-resistant Streptococci and yeasts (67 isolates were excluded).As a result, among the 354 oral isolates collected from both groups, 254 oral isolates were included in further experiments.
As shown in Table 1, there was a 30.5% lower frequency of Lactobacillus species detection (p = .001) in subgingival samples in patients with periodontitis compared with healthy individuals.In contrast, the number of positive isolates in saliva and tongue samples showed no significant difference in the two groups.
We obtained 10 restriction-fragment-length polymorphism patterns from the oral Lactobacillus species.In most of the oral samples, we had the same cutting patterns.In some cases, the patterns were repeated.According to Figure 1, out of the 254 positive isolates based on the 16S rDNA sequencing, 10 Lactobacillus species were identified in the case group and 9 in the control group.
As can be seen in Figure 2, Lacticaseibacillus paracasei and Limosilactobacillus fermentum were the most frequent species detected in the case group, while Lacticaseibacillus casei and L. plantarum were the most frequent species in the control group.The least frequent species belonged to Limosilactobacillus vaginalis (0.8%) in the case group, which was not detected in the oral samples of the control group.In addition, L. paracasei, L. fermentum, and Ligilactobacillus salivarius were the most abundant strains recovered from subgingival samples of patients with periodontitis, respectively.

| Adhesion of Lactobacillus species to S-HA and oral mucosal cells
In total, 40 samples out of the 10 species of lactobacilli (four strains were tested from each species) were tested for the adherence ability of Lactobacillus species in the case group.Figure 3 shows the Lactobacillus adhesion to oral mucosal cells compared with the positive and negative controls.
According to Table 2, L. salivarius, L. fermentum, and L. plantarum showed the highest potency of adhesion to S-HA in the selected strains.Post hoc analysis showed that L. salivarius had significantly higher adhesion to S-HA than all other species.
In addition, L. salivarius had the highest adherence ability to KB cells, with statistically significant differences from others.Moreover, L. fermentum and L. salivarius had the highest adherence ability to HGF cells.Post hoc analysis showed that L. salivarius, L. fermentum, L. acidophilus, and L. plantarum had significantly higher adhesion to HGF cells than other species (Supporting Information: Figures 4, 5, and 6).Post hoc analysis confirmed these significant differences (p < .001).

| DISCUSSION
The traditional approaches to control dental plaque-related diseases were based on nonspecific mechanical removal of all the beneficial and nonbeneficial plaques (Johnston et al., 2021).However, modern treatment approaches have recently emphasized the inhibition of specific small groups of organisms, single species, or even the main pathogenic agents (Allaker & Stephen, 2017).Furthermore, the increase in antibiotic resistance resulted in the search for alternative products or treatment strategies (Myneni et al., 2020).Several species of Lactobacilli, known as probiotics, have been used recently to treat periodontitis (Kuru et al., 2017;Laleman et al., 2020;Pelekos et al., 2020;Schlagenhauf et al., 2020;Shimauchi et al., 2008;Silva et al., 2022;Teughels et al., 2013).It seems necessary to conduct numerous experiments to identify and test their properties to optimize the results of these specific treatments.This was the first study conducted on oral samples of an Iranian population, comparing periodontitis patients and periodontally healthy individuals.This study revealed that the individuals with periodontitis have a lower relative frequency of oral Lactobacillus species compared with the control group, especially in the subgingival samples, which can be due to the establishment and function of pathogenic bacteria in the periodontal tissues.
Furthermore, different Lactobacillus species were identified from the oral isolates of the case group and the control group.
Among them, L. casei and L. plantarum were the dominant species in the control group, and L. paracasei and L. fermentum were the most frequent species in the case group.Most of the identified species in the two groups were consistent with previously reported findings (Gupta, 2011;Koll-Klais et al., 2005).However, there were differences in the frequency of the species in each group.In the study by Ahrne et al., the most frequent species in the samples of the healthy participants were L.
rhamnosus were the most frequent species in the oral cavity of healthy participants (Colloca et al., 2000).As reported by Koll-Klais et al., the most prevalent strains in the healthy participants were L. gasseri and L. fermentum, and the most frequent strain in the case group was L. plantarum (Koll-Klais et al., 2005).The similarity and differences of species found in oral isolates can be due to patients' various food and dietary habits (Sornplang & Piyadeatsoontorn, 2016).
According to previous reports, the ability to adhere to mucosal host surfaces has always been an essential property among bacterial strains used as probiotics (FAO/ WHO, 2002).This study showed that L. salivarius and L. plantarum had the highest adherence activity to KB, HGF cells, and S-HA.Thus, probiotic bacteria such as L. plantarum and L. salivarius can directly adhere to the oral mucosal cells, develop oral biofilms in the saliva, reside on the tongue surface, and exert healthy effects.According to Bosch et al. (Colloca et al., 2000), 10 and 38 isolates from the salivary strain of healthy children showed higher adherence potential than the commercial species of Streptococcus salivarius K12 and Limosilactobacillus reuteri, respectively.
This proves that probiotics isolated from the oral cavity had a higher capacity to develop biofilms and inhibit the growth of pathogens than commercial probiotic products.This emphasizes the importance of identifying and detecting the probiotic properties of healthy individuals' oral cavity to optimize probiotic treatments.In a study by Terai et al., the adherence ability of Lactobacillus species was evaluated by adhesion to S-HA and oral epithelial cells derived from human buccal mucosa carcinoma and human tongue carcinoma.
Only L. fermentum, L. gasseri, and L. casei showed adhesion to S-HA, and L. crispatus had higher adherence activity to human tongue carcinoma cells (Terai et al., 2015).
In addition to preserving the balance of oral microbiota, probiotics improve oral and periodontal health by producing antibacterial metabolites.In this study, most oral Lactobacillus

3. 3 |
Antibacterial activity of isolated Lactobacillus species against two oral pathogens Table 3 compares the inhibition zones of 10 selected lactobacilli species in the case group supernatants (40 samples in total) against A.a and A.n.The antibiogram results of A.n were read after 24 h, and A.a results were read after 48 h due to the slower growth rate.Most strains had almost similar antibacterial activity and inhibition zones.However, Lactobacillus crispatus and Lactobacillus gasseri showed a larger inhibition zone against A.a, and Lacticaseibacillus rhamnosus and L. acidophilus showed the highest antibacterial activity against A.n.
supernatant showed antibacterial activity against A.n and A.a after neutralization.This proves that organic acids are not the only antimicrobial substances in the supernatant of bacteria, and Lactobacillus might potentially produce bacteriocins or other antibacterial substances in the supernatant.Moreover, L. crispatus and L. gasseri showed higher antibacterial activity against these two pathogens.These findings are in agreement with the study carried out byTerai et al., in which they pointed out that the supernatants of T A B L E 3 Mean inhibition zones of the selected oral Lactobacillus strains against oral pathogens.
Number and frequency of oral Lactobacillus species in different oral samples based on the culture method.