Subject selection and sampling
The study was approved by Institutional Ethics Committees of Bharati Vidyapeeth (Deemed to be) University Medical College (BVMC) (Ref: BVDU/MC/57), and B. J. Government Medical College and Sassoon General Hospitals (BJGMCSGH), Pune (Ref No. BJGMC/IEC/Pharmac/ND-Dept 0119007-007). Vaginal swabs from healthy (n꓿45); low grade squamous intraepithelial lesion, LSIL (n=1); high grade squamous intraepithelial lesion, HSIL (n=1) and invasive cervical carcinoma ICC (n꓿6) patients (aged between 18-55 years) were collected by the clinicians at the respective clinical sites. All the patients provided informed written consent. The patients were screened for eligibility criteria based upon Pap test, vaginal cytology or colposcopy findings and were categorized under healthy, LSIL, HSIL and ICC groups. Post collection, the samples were immediately placed on ice and taken to IRSHA for further analysis within 2 hours of the collection.
Culture and reagents
The clinical strain, Pseudomonas aeruginosa MCC 2081, was procured from National Centre for Microbial Resource (NCMR), NCCS, Pune. All the media components such as de Man, Rogosa and Sharpe (MRS), Tryptone Soya (TSA) or Brain Heart Infusion (BHI), L-cysteine, Bile, Sheep blood agar plates were purchased from HiMedia Laboratories, Mumbai, India. AnaeroGasPak, antibiotics discs and sterile cotton swabs were procured from Himedia Laboratories, Mumbai, India. Pepsin and pancreatin were purchased from Sigma‑Aldrich (St. Louis, MO, USA). Plasticware was purchased from Tarson Products Private Limited, India. Ethanol (99% v/v) was procured from Changsu Hongsheng Fine Chemical Co. Ltd. (China).
Isolation and cultivation of microorganisms
For isolation of microflora, aliquots from the collected swabs were plated on de Man, Rogosa and Sharpe (MRS), Tryptone Soya (TSA) or Brain Heart Infusion (BHI) media. Based on the morphology and biochemical tests, Lactobacillus and non-Lactobacillus colonies were separated and purified. All the Lactobacillus strains grown on MRS media, were supplemented with 0.05% L-cysteine at 37°C for 48 h in anaerobic jars, supplemented with anaeroGasPak (Himedia, Mumbai). Non-Lactobacillus strains were grown either on TSA or BHI media and incubated at 37°C for 24 h. The pure colonies were stored in 20% glycerol at -80oC until further use.
All the isolates were determined for hemolytic activity by streaking the strains on sheep blood agar plates (HiMedia, Mumbai) and incubated at 37°C for 24-48 h. The hemolytic activity was detected by observing either a clear zone of hydrolysis around the colonies (beta hemolysis), or a greenish zone (alpha hemolysis), or no zone (gamma hemolysis).
Acid and bile tolerance
The tolerance assay was done as reported earlier (Azat et al. 2016; Ehrmann 2002). Briefly, the isolates were grown, harvested and the pellet was washed and resuspended in 1X PBS (pH 3) with a final concentration of 8-9 logCFU/ml or PBS (pH 7.4) containing 0.3 % bile, followed by incubation at 37°C for 3 h. Appropriate dilutions from each sample were spread plated on the respective agar plates and incubated anaerobically using GasPack system at 37°C for 24-48 h. The tolerance was evaluated by total viable count method (log CFU/ml) after 3 h of incubation. The survival rate (%) was calculated as per the following equation:
Survival rate (%) = ,
where, N0 and N1 are the total viable counts of the selected strains before and after treatment, respectively.
MALDI-TOF MS analysis
The identification of isolates by MALDI-TOF MS was carried out at National Centre for Microbial Resources (NCMR), National Centre for Cell Sciences (NCCS), Pune. Sample preparation for MALDI-TOF MS analysis was performed as described earlier (Kurli et al. 2018). The samples were loaded onto the MALDI-TOF MS instrument (AUTOFLEX speed, Bruker Daltonics, GmbH, Germany) and MALDI biotyper software 3.1 (Bruker Daltonik GmbH, Germany) was used to identify the isolates and visualize the mass spectra. The strain showing ≥1.7 log value with the strain in the database were confirmed as the member of that genus and strains showing ≥2.0 log values were confirmed to be the member of that species.
Preparation of cell-free supernatant (CFS)
The LAB strains were grown for 24-48 h and centrifuged at 3000 rpm for 15 min. The CFS was collected and filter-sterilized with 0.22 µm syringe filter. The resultant CFS was used for antimicrobial assays and for LCMS, lyophilized powder was stored until further use.
The antibacterial activity of CFS of the selected isolates was determined by agar well diffusion method (Reuben et al. 2019). Briefly, cultures of overnight grown indicator pathogens were adjusted to OD600nm of 0.5 McFarland. 100 µL adjusted pathogen culture was aseptically spread on the nutrient agar plates. Wells of 8 mm diameter were cut out with sterile cork borer and 150 µl CFS was loaded. The plates were kept at 4ºC for diffusion and incubated at 37°C for 24 h. After incubation, the diameter (mm) of the zone of inhibition (ZOI) around the well was measured. Pseudomonas aeruginosa MCC 2081 was used as the standard clinical pathogen. The pathogens (with beta and alpha hemolysis) used in this study were isolated from cervical cancer patients and identified by MALDI TOF MS method. Antibacterial activity of the eight standard antibiotics [carbencillin (100𝜇g), cefoxitin (30𝜇g), clindamycin (2 𝜇g), chloramphenicol (30 𝜇g), erythromycin (15𝜇g), metronidazole (5𝜇g), penicillin G (10 unit) and tetracycline (30 𝜇g)] was also evaluated against pathogens by disc diffusion method (Bayer et al. 1966).
Liquid chromatography mass spectroscopy (LCMS)
LCMS analysis of the lyophilized CFS of the selected probiotics was carried out at Center for Applications of Mass Spectrometry (CAMS), Venture Centre, Pune. Samples were analyzed by non‑targeted LCMS QTOF using Agilent 1290 HPLC system as described previously (Aphale et al. 2018). Briefly, 8 µl of sample was injected onto an Agilent 1290 HPLC system having Zorbax Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 µm particle sizes). The mobile phases consisted of (A) water and (B) acetonitrile (LCMS grade, J. T. Baker) with flow rate of 0.3 ml/min and 95:5 acetonitrile/water. Both mobile phases were modified with 0.1% (v/v) formic acid for MS analysis in positive mode and with 5 mm ammonium acetate for analysis in negative mode. The chromatographic conditions included first 18 min run of B from 95% to 5% gradient, applied from 18 to 30 min, followed by 3 min isocratically at 100%. MS analysis was performed on an Agilent 6530 Quadrupole time‑of‑flight spectrometer fitted with an electrospray ionization source in both positive and negative mode. Data were analysed by using Mass Hunter Qualitative Analysis Software Package (Agilent Technologies) and online database Metlin. Compound lists were screened against online mass databases; METLIN Metabolomics Database and MassBank Database.
16S rRNA gene sequencing and phylogenetic analysis
The identification of isolates was carried out at the sequencing facility of NCMR, Pune. The genomic DNA was isolated by the standard phenol/chloroform extraction method (Sambrook et al.1989), followed by PCR amplification of the 16S rRNA gene using universal primers 16F27 [5'-CCA GAG TTT GAT CMT GGC TCA G-3'] and 16R1492 [5'-TAC GGY TAC CTT GTT ACG ACT T-3']. The amplified 16S rRNA PCR product was purified by PEG-NaCl precipitation and directly sequenced on ABI® 3730XL automated DNA sequencer (Applied Biosystems, Inc., Foster City, CA) as per the manufacturer’s instructions. Assembly was carried out using Lasergene package followed by identification using the EzBioCloud database (Yoon et al. 2017). The phylogenic trees were constructed using the neighbor-joining method (Tamura et al. 2013) with MEGA6 software.
Tolerance to simulated gastrointestinal conditions
The ability of Lactobacilli to tolerate the simulated gastric conditions was determined as described previously (Pino et al. 2019; Pithva et al. 2014). Briefly, simulated gastric juice (SGJ) consisting of 0.3% pepsin in NaCl solution was adjusted to pH 2.5 by adding 1 M HCl. On the other hand, simulated intestinal fluid (SIF) comprising of 0.1% (w/v) pancreatin and 0.3% (w/v) bile in NaCl solution was adjusted to pH 8 by adding 1 M NaOH. Both the solutions were prepared immediately before use and sterilized using 0.22 µm filter. Probiotics were grown for 48 h, centrifuged for 3000 × g for 15 min. The pellet was washed with saline, resuspended in gastric fluid and incubated at 37°C for 3 h. After incubation, the cells were pelleted down by centrifugation, washed with 1X PBS and resuspended in SIF, followed by incubation at 37 °C for 3 h. The cells were washed with 1X PBS, serially diluted and plated on MRS agar for determining viability by total viable count method. The survival rate (%) was calculated as described for the acid and bile tolerance test.
Hydrophobicity and auto-aggregation properties
Hydrophobicity (H) (Kang et al. 2018) and auto‐aggregation (A) (Juárez Tomás et al. 2005) of the selected probiotic strains were determined as described earlier. Overnight grown bacterial suspensions in MRS broth were harvested by centrifugation at 3000 rpm for 15 min at room temperature. The cells were washed twice with 1X PBS and adjusted to an optical density (OD) of 0.5 ± 0.1 (A0) at 600 nm. To determine the hydrophobicity of the cell surface, xylene was used as a solvent. It was added to each bacterial suspension in the ratio of 1:1 and the mixtures were vortexed for 1 min and incubated for 5 h at 37°C. After phase stabilization and separation, the aqueous phase was removed, followed by measurement of absorbance (At) at 600 nm. Hydrophobicity percentage was calculated from the formula, H(%) =A0 − At/A0×100, where, A0 and At are the optical densities before and after extraction with xylene, respectively. For the auto-aggregation assay, each bacterial suspension (initial OD600nm = 0.5 ± 0.1) was vortexed for 10 s and incubated at 37°C for 5 h without agitation. The absorbance (At) was measured at 600 nm in microplate reader (Epoch, BioTek Instruments, Inc, USA). The percentage of auto-aggregation was calculated from the formula A(%) =A0 − At/A0×100, where, A0 is the OD at initial time (0 h) and At is the OD at final time (5 h) of the assay.
For biofilm formation (Terraf et al. 2012), the selected probiotic strains were inoculated in MRS media at 37°C for 24 h. Around 200 μl of the grown bacterial culture (OD 0.5) was added into the each well of 96-well plate and incubated at 37 °C for 72 h. The biofilm formed in the wells was washed twice with 200 μl 1X PBS and dried for 30 min at 37°C, followed by incubation with 200 μl of 0.1% (w/v) crystal violet for 30 min at room temperature (RT). The well was washed twice with 200 μl distilled water, dried for 10 min at RT. The residual crystal violet was dissolved in 200 μl solution containing 95% ethanol and 0.1% acetic acid in water, followed by measurement of the absorbance at 570 nm in multiplate reader.
Susceptibility of the selected probiotics to antibiotics was determined by the agar diffusion method by using two different antibiotics disks (Combi II and Combi III, Himedia, Mumbai). A total of 16 antibiotics were tested that included carbencillin (100𝜇g), cefoxitin (30𝜇g), clindamycin (2 𝜇g), chloramphenicol (30 𝜇g), erythromycin (15𝜇g), metronidazole (5𝜇g), penicillin G (10 unit), tetracycline (30 𝜇g), ampicillin (10 𝜇g), norfloxacin (10 𝜇g), nitrofurantoin (300 𝜇g), nalidixic acid (30𝜇g), gentamicin (10 𝜇g), cotrimoxazole (25𝜇g), cefalotin (30 𝜇g), and cefotaxime (30𝜇g).
The data has been presented as mean ± standard deviation (SD) and was analyzed for statistical significance by one-way and two-way analysis of variance (ANOVA) by using Tukey's multiple comparisons test. Statistical significance level was defined at p value < 0.05. All the statistical analyses were performed by using Graph Pad Prism version 9 (GraphPad, San Diego, USA).
The nucleotide sequences of 16S rRNA of the four strains were deposited at the GenBank database under the following accession numbers: Lactobacillus delbrueckii P31Mcs (OM049479), Lactobacillus gasseri P36Mops (OM049480), Limosilactobacillus fermentum P37Mws (OM049481) and Enterococcus faecium P15Mcm (OM049482).