Study Design
Experiments was conducted using completely randomized design with control groups using female mice (Mus musculus) as test units. The study was divided in two phases. In phase one, colonization of GV and L. reuteri were checked in immunocompromised mice. In phase 2 effectiveness of prophylactic application of L. reuteri was studied by challenging animals with GV.
Study Samples
Clinical signs of the disease, reisolation of L. reuteri and GV from vaginal washes, bacterial load and expression of Immune markers in vaginal tissue and histological changes in vaginal sections were taken into consideration.
Characterization of microbes used in study
L. reuteri and GV were isolated from healthy women and patients diagnosed with Bacterial vaginosis (BV) respectively in a previous study. In in vitro setting L. reuteri was confirmed for its probiotic and antagonistic ability through in vitro procedures. it displays good adherence ability, self-aggregation and co-aggregation, acid, bile salt, NaCl and lysozyme tolerance as well as antagonistic activity against G. vaginalis, E. faecalis, E. coli, S. aureus and Streptococcus spp. (Data not shown). Antibiotic profile shows that strain was sensitive to erythromycin (E15) streptomycin (S10), and gentamicin (CN10) and resistant to ampicillin/sulbactum (SAM20), oxacillin (OX5) and Methicillin (MET5).
GV is Gram variable, pleomorphic rod shaped and show small, transparent colonies on GV selective media (Columbia agar containing nalidixic acid (5mg/L), gentamicin sulfate (10mg/L) and human blood (5%). It shows β-hemolysis on human blood agar but not on sheep’s blood. GV was negative for oxidase, catalase, nitrate reduction and Voges Proskauer and ferment starch, sucrose, fructose and maltose but not mannitol. In addition, it was resistant to ampicillin/sulbectum (SAM20).
Phase 1: (Establishment of GV murine model)
Animal housing and grouping
Two-month-old female BALB/c mice (n=15) weighing 20-30g were purchased from stocks of Government College University Lahore. Mice were housed in standard sized cages (12″×18″) at 28-33°C temperature and 40% humidity and they were fed conventional balanced diet and water ad libitum. After initial weighing, mice were equally distributed into three groups (A-C). Group A was maintained as negative control, group B and C were exposed to GV and L. reuteri respectively.
Initial screening of mice
Vaginal endogenous flora of each mice was collected by flushing vagina with 50 µl sterile Phosphate buffer saline (PBS) using micropipettes (Dragon LAB, China). Each vaginal sample obtained was cultured on MRS and GV selective media. Each isolated colony grown on these agar plates was studied for colony morphology, Gram staining. oxidase catalase, nitrate reduction, Voges Proskauer and antibiotic profile to distinguish vaginal flora of mice from probiotic L. reuteri and etiological agent GV.
Preparation of mice for Bacterial colonization
All mice were prepared for bacterial colonization (24). Briefly, mice were injected subcutaneously β-estradiol 17-valerate weekly throughout the experiment. Dexamethasone sodium phosphate injection was also given daily 2 and 4 days prior to exposure to L. reuteri and GV respectively and continued up to 6 days post exposure to GV. A dose of 5 × 105 CFU ml-1 of G. vaginalis and 1 × 106 CFU ml-1 of L. reuteri was intravaginally inoculated in mice of groups B and C respectively. Details of experimental treatments are given in Table 1.
Monitoring of infection
(a) Clinical signs
Animals were monitored for clinical signs of morbidity such as animal irritation, riffled fur, vaginal itching, discharge and redness which may be associated with GV induced vaginosis.
(b) Re-isolation of reuteri and GV
Animals were screened for the presence of L. reuteri and GV for 9 days post exposure to GV. Five vaginal washes were collected on alternative days and spread on MRS and GV selective agar plates for re-isolation of inoculated L. reuteri and GV respectively. These plates were incubated for 24-48h under anaerobic condition. After incubation, plates were observed for presence of G. vaginalis and L. reuteri. Colonies on Columbia agar plates were reconfirmed as GV using biochemical procedures (Negative oxidase, catalase, nitrate reduction and Voges Proskauer tests). L. reuteri was detected on basis of colony morphology, Gram staining and matching antibiotic susceptibility profile. On 10th dpi, mice were euthanized following ULAM guide lines of euthanization by intraperitoneal injection of ketamine (200 mg/kg of body weight). Vaginal tissues were removed to quantify bacterial load of L. reuteri and GV (CFU/g).
Phase:2 Protective effects of L. reuteri against GV
Animals
Two-month-old female BALB/c mice, weighing 20-30g were procured from stocks of Government College University Lahore. Mice were housed in cages as mentioned in phase 1. A total of 32 mice were randomly divided into four groups I-IV (n = 8). Experimental treatments to each group are summarized in Table 2.
Exposure to L. reuteri
After confirming the absence of L. reuteri and GV in experimental mice, Estrogen and dexamethasone injections were given to all mice as described in phase 1. Exposure to probiotic was given following (25) with slight modification. Freshly cultured L. reuteri was re-suspended in agarized peptone (0.1%, 0.5% w/v peptone and agar respectively) at a concentration of 1×106 CFU ml-1. Two-days post β-estradiol administration, group III and IV were intravaginally inoculated with 50 µl of L. reuteri suspension with an average inter-inoculation period of twelve hours.
Exposure to G. vaginalis
On 5th day of experiment, the mice were exposed to GV in 50 µl inoculum (5 × 105 CFU ml-1) as mentioned in Table 2. These doses of L. reuteri and GV were selected following (25) with slight modifications and initial application in phase 1.
Monitoring of G. vaginalis infection
(a) Recording of clinical signs/symptoms
Mice were observed for clinical signs of BV including irritation, riffled fur, vaginal itching, discharge and redness. All symptoms were given equal weightage. Animals were scored from 0-1 depending upon mean number of symptoms present e.g. animal showing two of the five symptoms was scored as 0.4. After scoring, each animal mean score of each group was used for comparison of general health condition of each group.
(b) Re-isolation of reuteri and G. vaginalis
Vaginal washes were collected and L. reuteri and GV were confirmed on the basis of biochemical characteristics and antibiotic profile (as mentioned in phase 1).
Sampling of tissue
On 17th day of experiment, mice were euthanized by using over dose of ketamine (200 mg/kg), and vaginal tissues were harvested. A part of vaginal tissue was preserved by placing in liquid nitrogen later on these were stored at -80°C till further use. Similarly, 100 -150 mg of tissue was used for determination of bacterial load and rest of the tissue was fixed in 10% formalin for histological studies.
Bacterial load
Vaginal tissue (bisected longitudinally) was weighed and homogenized in sterile saline and 100 µl of suspension was spread on MRS and GV selective agar plates at 37°C for 24-48h under anaerobic condition. After incubation period, colonies were enumerated and expressed as log10 CFU/ g of vaginal tissue (26).
Histopathological examination
In brief, paraffin-embedded vaginal sections (5µm) were stained with hematoxylin and eosin (H &E) and observed under light microscope to assess histopathological changes. Images were captured and degree of thickness and exfoliation of epithelium were observed. The pathology of vaginal tissue was recorded following (7).
RNA extraction and cDNA preparation
Total RNA was extracted from stored tissues at -80°C using TRIZOL RNA extraction protocol (27). RNA extracted was quantified using Nanodrop spectrophotometer followed by complementary DNA (cDNA) synthesis using 500 ng of RNA with Superscript TM IV First-Strand cDNA Synthesis Kit according to manufacturer’s protocol. Quantity of cDNA was measured using aforementioned Nanodrop spectrophotometer.
Real-Time PCR
The PCR reaction mixture having a final volume of 25 µl containing 12.5 µl SYBR green Dye, 1 µl cDNA, 1 µl forward and reverse primer each of final concentration of 10 Pmole and 9.5 DNase - and RNase-free water. Details of the primers along with annealing temperatures are given in Table 3. Real- Time PCR was carried out on a Bio-Rad CFX Real-Time PCR system for 40 cycles using the following conditions: denaturation at 95 °C for 3 min, annealing at 54-59 °C for 10s and elongation at 72°C for 30s. Average quantification cycle (Cq) values from duplicate measurements were used to determine mRNA expressions. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) was used as Normalization control (28). It was normalized with the average Cq value of the control group.
Normalized expression ratio (NER) calculation
Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) was used as normalization control (Eissa et al., 2016). The degree of expression of all markers was evaluated by difference in Ct value (ΔCt value). The Ct values were normalized by both the “Ct value of GAPDH” and “Ct value of control group” to get ΔΔCt values. Mean of ΔΔCt values were compared between groups II, III and IV.
Statistical analysis
Data of bacterial load and inflammatory markers was parametric while Clinical symptoms and degree of epithelial exfoliation data was non parametric. Parametric data was analyzed by Independent sample T-test and ANOVA following post-Hoc Tukey’s test. Kruskel wallis H-test was applied to non- parametric data. The p value < 0.05 was considered significant. Data was analyzed using software IBM SPSS version 21 Chicago IL, USA.