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INTRODUCTION
In the past decade role of probiotic microorganisms in health promotion and as preventative agents were investigated1,2. Probiotics described as microorganisms which are useful if used in sufficient number3,4. Different bacteria such as Bifidobacterium, Streptococcus, and Lactobacillus introduced as probiotics, and known as generally recognized as safe (GRAS) and non-pathogenic bacteria3,5,6. Recently, Lactobacillus spp. considered as main probiotic in medical and industrial scope1,2. Lactobacillus spp. are heterogeneous group of bacteria including about 145 species of non-spore forming and Gram-positive rods which can be grown in anaerobic or microaerophilic conditions3. Also, Lactobacillus was used in dairy and fermented food for promotion of health quality3. However, Lactobacillus spp. is presented in various places like environment and human body (oral cavity, gastrointestinal and vaginal tract)1,2,5,7.
It was previously demonstrated that consumption of Lactobacillus has several benefits including decrease of time and severity of diarrhea3, antibacterial and antifungal characteristics8, and improvement of vaginal health7.
Inhibitory activity against the pathogens is one of the most characteristics of bacteria which known as probiotics. Probiotic inhibitory activity is related to reduce of pathogenic colonization and causing adverse growth conditions for pathogens by production of hydrogen peroxide (H2O2) and bacteriocin8-10. Moreover, it has recently been demonstrated that Lactobacillus species isolated from human are better probiotics, because these strains were adapted to the human body and also exposure to antibiotics in this place is less than other origins11.
In recent years, the role of oral microbiome in health quality was discussed extensively. Some studies demonstrated correlation between dental caries, oral cancer and inflammation caused by oral microbiome6,12,13. But other study indicated that available Lactobacillus spp. in the oral cavity have useful role in promotion of oral and gastrointestinal health4.
The milk products including yogurt and cheese are important dietary supplements which contain Lactobacillus14. Consumption of these products is main way to probiotic transmission to the host body. Thus, there is probably strong association between diet and commensal microorganism including probiotics bacteria15.
The aims of this study were investigate the inhibitory activity of isolated Lactobacillus from oral cavity and also, determination of association between isolation rates of Lactobacillus and health quality in the host.
MATERIAL AND METHODS
Study population
One hundred healthy persons who use domestic dairy products were recruited in this study. The study was conducted from May to August 2017 at rural area of Bukan, Western Azerbaijan province, Iran. Also, the questioner forms were applied to all persons. These forms were included age, gender, experience of infectious disease, severe gastric pain, herpes, and mental problem (intense anger and depression). The current study was approved by the Research Ethics Committee of Tabriz University of Medical Science; also signed informed consent form was obtained from each patient prior to the initiation of sampling. Participants were subjected for sampling without any hazardous action and participation was completely volunteer. This research has been conducted in full accordance with the World Medical Association Declaration of Helsinki and was done after confirmation of ethic committee of Tabriz University of medical sciences.
Sample collection
According to Kohler and Bratthall (1979), sample collection was performed by modified spatula method16. Briefly, wooden spatula was inserted in oral cavity to moisten it with saliva. Then both sides of spatula placed on Rogosa SL agar (MicroMaster, India). The plates were incubated under anaerobic condition (80% N2, 10% H2, and 10% CO2) provided by Anoxomat (Mart Microbiology BV) at 37°C for 72 h. Then dominant Lactobacillus colonies were selected and stored to further analysis.
Isolation and identification
The suspected colonies, temporary identified as Lactobacillus spp. based on phenotypically tests including colony morphology, wet mount smear, Gram staining, and catalase negative reaction17. DNA extraction was applied by boiling method as previously described18,19. Amplification and sequencing of 16SrRNA was performed to confirmation of Lactobacillus spp. PCR amplification was performed by the following primers: Forward 5-CTCGTTGCGGGACTTAA-3 and Reverse 5 -GCAGCAGTAGGGAATCTTC-3 (Bioneer, Korea)4. The PCR amplification was performed in a total volume of 50 µL containing 25 µL master kit (Ampliqon, Denmark), 5 pmol of each primer and 15 ng DNA. The PCR proceed using a DNA thermal cycler (Master Cycle Gradiant, Eppendrof, Germany) programmed with initial 94°C for 5 min and followed by 35 cycles of 94°C for 30 s, 56°C for 50s and 72°C for 1 min, and with additional extension at 72°C for 10 min. PCR products were electrophoresis in 1.5% agarose gel and after staining with Safestain (Yekta tazhiz, Iran) visualized under UV light20. All PCR products were subjected to sequencing by Bioneer (South Korea). Finally obtained sequences were compared with available sequences in GeneBank (www.ncbi.nlm.nih.gov/blast).
Antimicrobial activity
According to earlier study4, agar well diffusion method was performed to determination of Lactobacillus spp. antibacterial activity against various pathogenic bacteria. In fact, bacteriocin effect against growth of S. typhimurium, K. pneumoniae, S. sonnei, S. dysenteriae, Enterococcus faecalis and Enterococcus faecium was determinate using agar well diffusion assay. For this reason, the Lactobacillus spp. were incubated at anaerobic condition in MRS broth (37°C for 24h). Then, cell free supernatant was prepared by centrifugation at 10,000 g for 10 min4,21.
Different antibacterial compound including organic acid and bacteriocin are produced by Lactobacillus. To remove of the organic acid effect against pathogenic bacteria, pH of supernatant should be neutralized with NaOH (2.5M) and catalase (1 mg/ml, Sigma Aldrich, Germany), respectively4.
Briefly, then 0.5 McFarland was prepared for each pathogen and cultured on MHA (MicroMaster, India). Then 100mL of cell free neutralized supernatant was inoculated in wells and incubated at 37°C for 24 h. Finally Lactobacillus with clear zones <11 mm, 11-16, 17-22 and >22 described as negative (-), mild (+), strong (++), and very strong (+++) inhibitor, respectively. The sterile MRS broth was used as negative control4.
RESULT
Demographic data
In this study, one hundred oral salivary samples were investigated. Demographic data were demonstrated in Table 1. Among one hundred persons, 49 and 51 of them were male and female respectively, with mean age 36+43 years (6-81 years). Lactobacillus strains were isolated from 30 persons including 16 males and 14 females. Mean age in these persons were 34+27 years. The correlation between Lactobacillus isolation and evaluated factors (age, gender, natural or cesarean born, the experience of severe infection disease, herpes infection and gastric pain, and mental problem including intense anger and Depression) were not significant. In the other hand, the incidence of the mental problems in eighteen host L. gasseri significantly was low (4/18) compared to Lactobacillus-negative persons (40/70) (P < 0.05).
Table 1: Demographic data of persons enrolled in this study
| Lactobacillus positive (30) | Lactobacillus negative (70) | Total (100) | P value | |
|---|---|---|---|---|
| Gender (male) | 16 | 33 | 49 | 0.363 |
| Mean age (years) | 34.27 | 37.36 | 36.43 | |
| Natural born | 28 | 66 | 94 | 0.585 |
| Infection disease * | 8 | 19 | 27 | 0.584 |
| Gastric pain * | 10 | 20 | 30 | 0.401 |
| Mental problem * | 12 | 40 | 52 | 0.088 |
| Herpes infection * | 17 | 41 | 58 | 0.516 |
*: number of persons that experience severe infection disease, Gastric pain, mental problem and Herpes infection
Lactobacillus spp.
Totally, thirty Lactobacillus were isolated by phonotypical assay and also, confirmation was performed by PCR and sequencing of 16S rRNA gene. From 30 Lactobacillus spp. eighteen isolates (60%) was identified as L. gasseri. Other isolates were L. vaginalis (n=3), L. salivarius (n=3), L. crispatus (n=2), L. curvatus (n=1), L. fermentum (n=1), L. kitasatonis (n=1) and L. panis (n=1).
Antimicrobial activity
The growth inhibitory activity against S. typhimurium ATCC® 14028™, K. pneumonia ATCC® 13883™, S. sonnei ATCC® 25931™, S. dysenteriae ATCC® 11835™, E. faecalis ATCC® 29212™ and E. faecium ATCC® 19434™ was illustrated in Table 2. Of the 30 Lactobacillus only three isolates of them were included two L. vaginalis and one L. kitasatonis demonstrate ++ and +++ inhibitory zone. These isolates have inhibitory zone 17-22 mm and more than 23 mm against K. pneumonia. Thus, K. pneumonia was the most susceptible investigated species. In fact L. vaginalis was the strain of Lactobacillus that demonstrates strongest activity against K. pneumonia.
Table 2: Antimicrobial activity of cell-free culture supernatants of lactobacillus isolates
| Bacterial isolates | Gram negative | Gram positive | |||||
|---|---|---|---|---|---|---|---|
| Inhibitory range | S. typhimurium | Sh. dysenteriae | Sh. sonnei | K. pneumoniae | E. faecium | E. faecalis | |
| L. gasseri (18) | - | 15 | 10 | 12 | 11 | 7 | 18 |
| + | 3 | 8 | 6 | 7 | 11 | 0 | |
| L. vaginalis (3) | - | 2 | 2 | 3 | 1 | 3 | 3 |
| + | 1 | 1 | - | - | 0 | - | |
| ++ | - | - | - | 1 | - | - | |
| +++ | - | - | - | 1 | - | - | |
| L. salivarius (3) | - | - | - | 3 | - | 3 | 3 |
| + | 3 | 3 | - | 3 | - | - | |
| L. crisptus (2) | - | 2 | 1 | 2 | 1 | 2 | 2 |
| + | - | 1 | - | 1 | - | - | |
| L. curvtus (1) | - | 1 | - | 1 | - | 1 | 1 |
| + | - | 1 | - | 1 | - | - | |
| L. panis (1) | - | 1 | - | 1 | - | 1 | 1 |
| + | - | 1 | - | 1 | - | - | |
| L. fermantus (1) | - | 1 | - | 1 | 1 | - | 1 |
| + | - | 1 | - | - | 1 | - | |
| L. kitasatonis (1) | - | 1 | - | 1 | - | 1 | 1 |
| + | - | 1 | - | - | - | - | |
| ++ | - | - | - | 1 | - | - | |
Antibacterial activity was determined using agar well diffusion assay. Interpretation of Inhibitory range, - : <11 mm (negative); +: 11-16 mm (mild); ++: 17-22 mm (strong); +++: ≥ 23 (very strong).
DISCUSSION
The probiotics bacteria were used for promotion and preservation of health quality for decades5. Lactobacillus species are the most well-known probiotics and the important constituent of human body flora including oral, intestinal and vaginal tract5. One of the main ability of probiotics is host protection against pathogen microorganisms like as Salmonella, Shigella and other invasive pathogens1,22,23. The previous study indicated that, in pregnant women which use from oral probiotics, colonization of Group B Streptococcus was reduced in rectal and vaginal tracts9. Along with listed their advantages, results of previous studies demonstrated possible association of some Lactobacillus species with host unfavorable conditions like as dental caries and glycaemia in human5,24.
In different studies, Lactobacillus species isolated from oral cavity were diverse. In the current study, like of previous studies L. gasseri, L. salivarius, and L. vaginalis were isolated from oral cavity11,25. But predominant isolates recovered from oral cavity in different studies were diverse too. In agreement reported paper26 results of this study demonstrated that, L gasseri was the predominant isolated strain, while in other studies performed by Strahinic et al. and Ahirwar et al, L. rhamnosus and L. fermentum introduced as predominant recovered strains, respectively25,27. It is likely that main reasons related to this diversity are oral health and wealth condition, diet, kind of samples (saliva or dental plaque), the age of patient and performed methods to species identification. For example, in contrast with an earlier study, in this study PCR and sequencing of 16SrRNA (the most reliable method) was performed for species identification and confirmation27. Also, Piwat et al (2010) demonstrated that L. plantarum and L. mucosae were recovered in patients with moderate to high dental caries, but the most frequent species in the low-dental caries group were L. gasseri, L. vaginalis, and L. oris6. However, in accordance with this study, previously reported literature illustrated that, in contrast with dental samples in oral saliva, L gasseri was predominant isolated strain26.
In agreement with previous works, this report demonstrated that Lactobacillus strains were able to inhibit the growth of enteropathogen bacteria9,28. In accordance with previous study, among the tested Enterobacteriaceae, S. typhimurium were the most resistant species against CFS of L. gasseri29. The previous study performed by Koga et al. demonstrated that L. gasseri was unable to inhibit growth of Salmonella species, while in current study few strains of L. gasseri isolates were able to inhibit growth of Enterobacteriaceae including S. sonnei (6/18), S. dysenteriae (8/18), K. pneumoniae (7/18) and S. typhimurium (3/18). This diversity is related to several factors including isolation origin, performed methods and applied Lactobacillus species. For example, Lactobacillus strains with oral and gastrointestinal origin are more effective against Enterobacteriaceae compared to strains with other origins. This is related with the high adaptability of Lactobacillus strains.
Previously showed that antibacterial activities of Lactobacillus strains against S. typhimurium is related to production of different compounds which inhibit motility and penetration of these bacteria into enterocyte-like Caco-2/TC7 cells30.
The Lactobacillus strains have wide range of inhibitory growth effect from absent to very strong against Enterococcus species. Shim et al. (2016) demonstrated mild inhibition effect against E. fecalis31. But in this work, none of Lactobacillus isolates were able to inhibit growth of E. fecalis, while 61% of L. gasseri isolates demonstrate mild inhibitory effect against E. faecium.
In agreement study performed by Zhang et al. (2001), the current work showed that growth of Salmonella was inhibited by treatment of L. salivarius culture supernatants32.
Until now, different studies discussed the impact of Lactobacillus genus on protection and promotion of the human health. Some studies illustrated beneficial effect on human health, but in contrast, some studies have not found these impacts and rejected this theory22,24,33. For example, an earlier study demonstrated relation of dental caries and the prevalence of Lactobacillus strain in oral cavity5. Their ability of acid production by Lactobacillus was expressed for dental caries5. In disagreement this statement, some studies demonstrated that oral health was promoted due to presence of Lactobacillus, and this is related to activity of Lactobacillus against cariogenic Streptococcus, and periodontal pathogens1,5,34.
In this study, any relation between the presence of Lactobacillus and investigated health factors was observed. However, interestingly in accordance with other studies, presence of L. gasseri is contributed to health promotion in current study 9,35,36. This contradictory result is related to the investigated species and showed that L. gasseri has a positive impact on human health in comparison to other species. Until now, it is difficult to explain how the Lactobacillus strains have effects on host health.
In summary: in this study, most Lactobacillus strains isolated from oral cavity able to inhibit growth of gastrointestinal pathogens. This ability is related to high adaptability of Lactobacillus strains. Moreover, non-association between the presence of different Lactobacillus species and investigated health factors was observed. However, only L. gasseri has a positive impact on the mental problem (intense anger and depression) of their hosts. These results indicated that only some species of Lactobacillus genus has the positive impact on their host.
