Strains and growth conditions
Lactobacillus strains were grown in de man, Rogosa, Sharpe (MRS, Oxoid, Hampshire, UK) medium and incubated at 37 °C under microaerophilic conditions (5% CO2) for 24–48 h. S. mutans ATCC 25175 was grown in brain heart infusion medium (BHI, Oxoid, Hampshire, UK) at 37 °C under microaerophilic conditions. Porphyromonas gingivalis GIM1.851 and Fusobacterium nucleatum CGMCC 1.2528 were grown at 37 °C anaerobically in TSB medium and PYG medium, respectively.
Lactobacilli isolation and identification from human saliva and dental plaques
Human saliva and dental plaques samples of 10 healthy donors (five female and five male; mean age 24 ± 3 years) were obtained from Wuxi No. 2 People’s Hospital (Wuxi, Jiangsu Province, China) according to the method reported by Krajden [30]. Lactobacillus strains were isolated by plating the serial diluted samples on MRS agar and cultivated at 37 °C under microaerophilic conditions. Genomic DNA of Lactobacillus strains were extracted using EZNA genomic DNA isolation kits (Omega Bio-Tek, Doraville, USA). Strains were identified by analysis of 16S rDNA (amplified by primers 16SF 5′-TACGGYTACCTTGTTACGACTT-3′ and 16SR 5′-AGAGTTTGATCMTGGCTCAG-3′) and pheS (amplified by primers pheSF 5′-TTCCCATTTACGGAGCCTTCTG-3′ and pheSR 5′-GCACCATACCGGCACCTAACAC-3′) sequences and blasted against the NCBI database.
Antimicrobial activity assay
Antimicrobial activities of isolates against S. mutans ATCC 25175 were evaluated by the agar well-diffusion method [31]. Briefly, 10 mL of base agar (1.5%, w/v) was plated on a Petri dish and allowed to cool before placing on sterile Oxford Cups. S. mutans ATCC 25175 (final concentration in agar: 106 CFU/mL) was mixed with 10 mL of semi-solid BHI agar (0.8%, w/v) and overlaid on the base agar. Oxford Cups were removed when the upper layer set. Finally, 100 μL of supernatant of bacterial culture or heat-inactivated supernatant (control) was added to wells and incubated at 37 °C (5% CO2) for 24 h. The inhibition zones represent the growth inhibition of S. mutans ATCC 25175 by corresponding strains.
Analysis of acid production
Lactobacillus strains and S. mutans were inoculated in MRS broth (devoid of carbon source) supplemented with single carbon source (equal moles of glucose, sucrose, lactose, and fructose) and grown at 37 °C and 5% CO2 for 24 h. Following a 24-h incubation, the pH value of the broth was measured. Production of lactic acid by these strains grown in MRS at 37 °C (5% CO2) for 24 h was determined with high performance liquid chromatography using a modified method [32]. Strains were removed by centrifugation at 10,000 rpm for 10 min. Corresponding supernatants were passed through a 0.22-μm membrane filter, and 20 μL of the filtrate or its dilution was injected onto HPLC for lactic acid quantification. Separation was performed using an HPLC organic acid analysis column (300 mm × 7.8 mm, BIO-RAD, Hercules, CA, USA) at 40 °C, using 0.05 mM sulfuric acid as the mobile phase (flow rate 0.6 mL/min, 30 min). Lactic acid was detected at 210 nm.
Detection of acid resistance of Lactobacillus
L. brevis BBE-Y52, L. casei DSM 20011 and L. paracasei XJ02 (a strain isolated from oral cavity, this study) were grown in MRS broth at 37 °C. Cells of these strains collected during the exponential growth phase were harvested by centrifugation (8000 rpm, 5 min), and then washed three times with phosphate buffer saline (PBS, pH 7.2) before suspended in PBS or PBS adjusted to pH 2.0 and 3.0 using 5 M HCl, and incubated at 37 °C. Samples of each solution were taken at different time intervals, diluted and plated on MRS agar. Cell numbers were counted and used for calculating the survival rate.
Detection of extracellular polysaccharides (EPS) and hydrogen peroxide
Bacterial strains were grown on MRS agar containing 10% (w/v) sucrose, and the plates were incubated at 30 °C for 48 h. Strains that appeared as viscous colonies were considered to be EPS producers [33]. For quantitative analysis of EPS, strains were grown at 30 °C for 48 h in MRS broth containing 10% (w/v) sucrose. Proteins in cultures were precipitated with trichloroacetic acid (Sigma–Aldrich, St. Louis, MO, USA) and stirred for 30 min on ice. Following centrifugation at 15,000 g for 30 min at 4 °C, cold ethanol (three times the volume) was added to the supernatant and stored overnight at 4 °C to precipitate EPS. The sediment was collected by centrifugation at 15,000 g for 30 min at 4 °C and dissolved in distilled water. EPS was determined by phenol–sulfuric acid method using D-glucose as a standard [34].
To measure hydrogen peroxide production, bacterial strains were grown in MRS broth and incubated at 37 °C (5% CO2) for 24 h. Hydrogen peroxide in cell-free supernatant of corresponding bacterial cultures was measured using semi-quantitative test strips (Merck, Poole, UK), which detect hydrogen peroxide at 0 to 25 mg/L.
Lysozyme, hydrogen peroxide, and antibiotic susceptibility
The susceptibility of lactobacilli (107 CFU/mL) to lysozyme (Thermo Fisher, Waltham, MA, USA) was examined by an agar well-diffusion method [28]. Susceptibility to hydrogen peroxide (Sigma–Aldrich) was evaluated by the survival of the corresponding strain when exposed to hydrogen peroxide [35]. The Lactobacillus strain was cultivated in MRS at 37 °C (5% CO2) for 24 h. Cells were harvested by centrifugation (8000 rpm, 15 min) and rinsed twice with phosphate-buffered saline (PBS, pH 7.0) before re-suspending (108 CFU/mL) in PBS containing 0–2 mM hydrogen peroxide at 37 °C (5% CO2) for 1 h. Corresponding cultures were plated in MRS agar, and viable cells were counted and compared with the control (0 mM hydrogen peroxide). Survival rate was calculated to evaluate the resistance to hydrogen peroxide. Antibiotic susceptibility of lactobacilli was determined by the disk diffusion method [36]. Strains were classified as resistant or susceptible by comparing the minimum inhibitory concentrations of antibiotics (Sigma–Aldrich) with European Food Safety Authority standards [37].
Aggregation assays
Bacterial aggregation of single strain (auto-aggregation) and two strains were examined [38, 39]. Cells of S. mutans ATCC 25175, P. gingivalis GIM1.851 and F. nucleatum CGMCC 1.2528 collected during the exponential growth phase were harvested, cells were washed twice with PBS, and suspended in PBS to an OD600 value of 0.5 (A0). Absorbance at 600 nm of the upper suspension (At) was measured after 1–3 h incubation. Coaggregations of oral lactobacilli with the foregoing strains were determined by combining equal volumes (500 μL) of the adjusted suspensions of isolates (Aprobio) and oral pathogens (Apat). Coaggregation was calculated using the formula described by Collado [38].
Auto-aggregation (%) = (A0 − At)/A0 × 100.
Coaggregation (%) = [(Apat + Aprobio)/2 –(Amix)/ (Apat + Aprobio)/2] × 100.
where Amix represents absorbance at 600 nm of culture of two strains.
Biofilm formation assays
The formation of biofilm by oral isolates and S. mutans ATCC 25175 was assessed using the microtiter plate method [40]. Lactobacillus strains and S. mutans were cultivated in MRS or BHI at 37 °C (5% CO2) for 24 h. Subsequently, 125 μL of cell suspensions (OD600 = 1.0) of individual strains were added to wells of a 24-well plate containing 875 μL BHI broth (750 μL BHI broth for two strains), and incubated at 37 °C (5% CO2) for 12–36 h. Cell suspensions were removed by careful pipetting. Wells were rinsed three times with PBS and air-dried for 30 min. Wells were stained with crystal violet (1%, w/v) for 20 min, rinsed with PBS, and air-dried for 30 min. Ethanol (1 mL, 95%) was added, and absorbance at 570 nm was measured.
Adhesion assays
The oral epithelial carcinoma cell line (KB) was used to assess the adhesion of oral lactobacilli [41, 42]. KB cells (provided by Prof. Jian Jin, and purchased from ATCC, Manassas, VA, USA) were grown in minimum essential medium (MEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (Invitrogen) at 37 °C (5% CO2). A concentration of 3.0 × 105 KB cells was seeded in 35-mm-diameter dishes (Corning, New York, NY, USA) and incubated until the formation of a complete monolayer. The KB cells monloayer was washed twice with PBS, and an aliquot of 2 mL minimum essential medium (MEM, Invitrogen) was added to the plates and incubated at 37 °C (5% CO2) for 30 min. The bacterial cultures were washed and suspended in MEM (108 CFU/mL). Bacterial suspension (120 μL) was seeded onto the KB cells monolayer and incubated for 1 h at 37 °C (5% CO2). The KB cells monolayer were subsequently washed to remove unattached bacteria. Methanol (3 mL) was added to fix the cells (10 min) and removed prior to staining with 3 mL Giemsa stain for 30 min. For detecting the adhesion of lactobacilli to KB cells, the dishes were rinsed, dried at 37 °C for 16 h, and examined by microscopy [42]. To quantify the adhesion capability of bacteria to KB cells, the monolayers were lysed with distilled water. Corresponding solutions were plated on MRS, and viable adherent bacteria were counted. Adhesive capacity was determined according to the number of strains that adhered to one KB cell [41].
Effects of lactobacilli on cytokine production by human peripheral blood mononuclear cells (PBMCs)
PBMCs were isolated from the donors’ peripheral blood by Ficoll gradient centrifugation. PBMCs (2 × 106 cell/mL) were incubated at 37 °C (5% CO2) for 2 h and then incubated alone (the control) or with either S. mutans ATCC 25175 (107 CFU/mL) or oral lactobacilli (107 CFU/mL) for 12 h at 37 °C (5% CO2). The suspensions in the wells were aspirated and washed with RPMI 1640 medium (Thermo Fisher, Shanghai, China). For the double-challenge, S. mutans ATCC 25175-stimulated PBMCs (2 × 106 cell/mL) were incubated either alone or with oral lactobacilli (108 CFU/mL) for 24 h prior to cytokine analysis [11]. The production of anti-inflammatory IL-10 cytokine and pro-inflammatory IL-12p70 cytokine were determined by enzyme linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA).
Statistical analysis
All experiments were performed in triplicate. Results were analyzed using Student’s t-test by SPSS 19.0. Statistical significance was set at P < 0.05.
