Bacterial strains, growth media, and culture conditions
L. gasseri H87, isolated from human vaginal was preserved in the China Center for Type Culture Collection (CCTCC) with accession number M 2018477. The vaginal Lactobacillus strains and L. iners ATCC 55195 used in this study were grown in Man-Rogosa-Sharpe (MRS) medium at 37°C under aerobic conditions. Gardnerella vaginalis was grown anaerobically on chocolate agar plates at 37°C. E. coli, Enterococcus faecalis, Streptococcus agalactiae, Staphylococcus aureus, Pseudomonas aeruginosa, and Listeria monocytogenes were grown aerobically on Luria-Bertani (LB) agar plates at 37°C. All strains were maintained as frozen stocks at -80°C in their respective culture media with the addition of 15 to 20% (vol/vol) sterile glycerol.
Preparation of Lactobacillus cell suspension
Cell suspensions used in various experiments, if not indicated otherwise, were prepared by growing Lactobacillus strains at 37°C for 24 h under aerobic conditions in MRS medium. The cells were pelleted by centrifugation (5000 g, 15 min, 4 °C), washed twice with phosphate-buffered saline (PBS; 0.1 M, pH 7.2, containing 0.85 % (w/v) NaCl) and resuspended in phosphate buffer (0.1 M, pH 7) to obtain a cell suspension with OD 600 = 1 and 109 cfu/ml.
Sampling and isolation of Lactobacillus
The study protocol was approved by the ethics review board of The Second Affiliated Hospital of Nanjing Medical University. We have obtained written informed consent from all study participants. All the procedures were performed in accordance with the Declaration of Helsinki and relevant policies in China.
Lactobacilli were isolated from the vaginal microbiome of asymptomatic Chinese women, which were invited to participate in the study during their routine gynecological consultations. MRS agar plates supplemented with 0.05% of L-cysteine were used and anaerobically incubated at 37 °C for 24~48 h. Individual colonies were randomly selected and purified before storing at −80 °C with 20% of glycerol.
Cell-free supernatants from the isolated Lactobacillus strains were screened for antibacterial activity against L. iners ATCC 55195 using the cylinder plate method [13]. The L. iners suspension was diluted to 107 CFU/m L, mixed with the melted solid culture medium cooled to 40°C, poured into a plate, and allowed to solidify. An Oxford cup was gently placed on the solidified plate, after which 200 μL of the lactic acid bacteria fermentation supernatant was added to the cup and incubated at 37℃ for 24 h. The antimicrobial activity was evaluated by measuring the diameter of transparent inhibition zones against the test strain
In vitro antibiotic activity assay
Antibiotic activities of isolated strains were determined using the cylinder plate method as described before [13].
Tolerance to simulated vaginal fluid (SVF) at low pH
One-milliliter overnight-grown bacterial cultures were separately combined with 100 mL of simulated vaginal fluid (SVF) prepared as described by Ahire et al. [3] (pH adjusted with lactic acid), incubated anaerobically at 37 °C and the OD600 measured after 48h. Bacteria grown in MRS were included as a control.
Tolerance to simulated gastric juice (SGJ) and simulated intestinal fluid (SIF)
A sample comprising 100 μl of cell suspension was mixed with 1 ml of SGJ or SIF, and incubated at 37 °C for 4 h. The resistance was determined by measuring the survival rate percentage (SR %), based on the initial (0 h) and final (4 h) number of viable cells enumerated on MRS agar plates after 48 h.
The SGJ was composed of 0.3% pepsin and 0.5% NaCl, with pH adjusted to 2 or 3 adjusted with 1 M HCl. The SIF was composed of 0.1 % pancreatin, 0.5 % bile salts, 0.5 % NaCl, 0.4 % phenol, and pH 8 adjusted with 1 M NaOH [12,14].
Tolerance to bile
Lactobacillus cells (109 cfu/ml) were used to inoculate 5 ml of MRS modified with bile salts (0.3, 0.5 %) at a ratio of 5% (v/v), and incubated at 37 °C for 4 h. The resistance was determined by measuring the survival rate percentage (SR %), based on initial (0 h) and final (3 h) number of viable cells enumerated on MRS agar plates after 48 h.
Aggregation ability
Auto-aggregation assay
The auto-aggregation assay was performed as described by Pithva et al. [12]. A cell suspension (2 ml) was vortexed for 10 s and incubated at 37 °C. Aliquots of 0.1 ml were collected from the upper surface at regular time interval and mixed with 0.9 ml PBS, followed by measurement of the optical density at 600 nm. The auto-aggregation (%) was calculated as [(OD0 − ODt)/OD0] × 100, where OD0 represents the optical density at 0 h and ODt represents the optical density of the cell suspension at 24h.
Co-aggregation assay
Equal volumes of cell suspensions (1 ml = 109 cfu/ml) of Lactobacillus and pathogenic indicator strains were mixed, and incubated at 37 °C. The control contained 2 ml of pure bacterial or yeast cell suspension of the indicator strain. The OD600 of the suspensions was measured at the indicated time intervals. The co-aggregation (%) was calculated using the equation [(ODpat + ODLacto)/2 − ODmix]/[(ODpat + ODLacto)/2]×100 , where ODpat and ODLacto represent the optical densities of the Lactobacillus sp. and the indicator strain, while ODmix represents the optical density of the mixture of Lactobacillus sp. and the indicator strain after 24 h.
Safety assessment
Hemolytic activity
The hemolytic activity was assessed on blood agar plates containing sheep blood according to the method reported by Pino et al. [14], with minor modifications as follows. Lactobacillus strains were streaked onto blood agar plates containing sheep blood, and incubated at 37 °C for 24 h under anaerobic conditions. The hemolytic activity was visually detected and distinguished as β-hemolysis, α-hemolysis, or γ-hemolysis based on the appearance of a clear zone, green halo or no zones around colonies, respectively. Bacillus cereus was used as a positive control.
Antibiotic susceptibility
The antibiotic resistance pattern of Lactobacillus strains was assessed according to the standard protocol of the European Food Safety Authority [15,16].
Identification of the antibacterial substance
In order to exclude the inhibitory effect of organic acids, cell-free supernatants (CFS) were adjusted to pH 6.5 using NaOH. To clarify whether the detected antimicrobial activity is caused by the production of H2O2, 2600 IU/ml of catalase were added to 1 ml CFS of LAB, and incubated for 24 h at 30 °C. In order to determine the biological nature of the antimicrobial activity of the bacteria, CFS (pH 6.0) of selected Lactobacillus isolates, incubated in MRS broth at 30 °C for 24 h, were tested for their sensitivity to proteolytic enzymes. One milliliter of CFS was treated for 2 h at 30 °C with 1 mg/ml final concentration of pepsin, trypsin, and proteinase K. The remaining antimicrobial activity was assessed using the cylinder plate method with L. iners as the indicator strain. Untreated cell-free supernatants were used as controls.
For the bile salt hydrolysis test, fresh cultures were streaked onto MRS agar plates containing 0.5% (w/v) taurodeoxycholic acid. The hydrolysis effect was indicated by different colony morphology (partial hydrolysis recorded as 1) from the control MRS plates, after 48 h of anaerobic incubation at 37 °C [17].
Bile salt hydrolase (BSH) activity was determined using the method previously reported by Caggia et al. [18]. The appearance of a precipitate around colonies was considered as a positive sign and, based on the confluence of the precipitate, each strain was classified as ‘+++’ for heavy; ‘++’ for intermediate; ‘+’ for low; and ‘−’ for no precipitation.
SDS-PAGE
During the purification process, the RPC-FPLC eluted fractions of GasE were analyzed in duplicate by Tris-Tricine SDS-PAGE, using an 18 % acrylamide resolving gel [19]. After electrophoresis at 100 mV for 2 h, one gel was silver stained while the other was used to detect the inhibitory activity in an overlay assay as described previously [20].