Mice. Sixty male BALB/c mice (age 6-8 weeks; mean weight 25 ± 0.5 gram) were purchased from the animal house of the Islamic Azad University Isfahan branch and maintained in the animal facility of the pharmacology department of Isfahan University of medical sciences, Isfahan, Iran. Groups of 6 mice were housed in standard polypropylene cages under control (lighting 12 h light/ dark, temperature 24°C, and humidity 55%). They were fed with standard rodent chow during experiments with free excess water and food. The mice maintained a regular diet, and the water bottles were changed with new bottles every three days.
Ethical statement. The experimental procedures were performed following the institutional animal care and use committee of Isfahan University of Medical Sciences (IR.MUI. RESEARCH REC.1400.435). Two mice from each group were selected randomly and euthanized by cervical paralysis to estimate histological changes.
Mice body weights were recorded regularly and expressed as mean ± SD for each group over the experiment.
Feeding of mice and procedure of infection
After an adaptation time of 72 h, sixty mice were set arbitrary to one of the ten following experimental groups of six mice each:
(a) Control group: not be given Lpb. plantarum and L. acidophilus, not challenged. The mice in the control group were fed with standard feedstuff.
(b) Infected group: Not be given Lpb. plantarum and L. acidophilus, infected with E. coli O157:H7.
(c) Pre-infection feeding groups: Given Lpb. plantarum and L. acidophilus on days −7 to 0, infected with E. coli O157:H7.
(d) Pre-infection feeding groups: Given SeNP Lpb. plantarum and SeNP L. acidophilus on days −7 to 0, infected with E. coli O157:H7.
(e) Post-infection feeding groups: Given Lpb. plantarum and L. acidophilus on days 0 to 7, infected with E. coli O157:H7.
(f) Post-infection feeding group: Given SeNP Lpb. plantarum and SeNP L. acidophilus on days 0 to 7, infected with E. coli O157:H7.
Colloidal selenium nanoparticles (analytical grade, 1000 ppm, suspension in H2O, 10-45 nm, Iranian nanomaterials pioneers Cas Number: 7782-49-2, Iran(www.nanosany.com) were added to media in growing probiotics. The number of probiotic supplementation was enumerated at 106–107 CFU/g.
Due to the procedure program, a daily 0.5 ml dose of Lpb. plantarum and L. acidophilus, including 2×109 CFU/ml, were administrated intra-gastrically to mice. At the same time, the infection was induced on day 0 with a single 0.5 ml dose of E. coli O157:H7, included 1×1010 CFU/ml suspended in phosphate-buffered saline (PBS, pH 7.3). Mice in the control group received 0.5 ml of PBS.
Lpb. plantarum, L. acidophilus, and E. coli O157:H7 cell counts were assessed by analyzing feces from two mice per group every two days.
Isolation and preparation of bacterial strains
Feces of mice were obtained by pushing the abdomen of mice, were analyzed by suspending 50 mg in 0.5 ml of 0.1% (w/v) sterile peptone water to get a concentration of 100 mg/ml. The suspensions were serially diluted 10-fold, and proper dilutions were plated on MacConkey sorbitol agar (Hi-media, India) for E. coli O157:H7 and Man Rogosa and Sharpe (MRS) agar (Quelab/UK) for Lpb. plantarum and L. acidophilus MRS plates were incubated aerobically for 72 h and MacConkey plates for 18 h at 37 °C [18].
E.coli O157:H7 strain EDL 933 (Kindly from Prof. Yahya Tahamtan, the Institute of Razi in Shiraz University, Shiraz. Iran), which makes both Stx1 and Stx2, was used as a positive control. It was reactivated in tryptic soy broth (Difco, Le Point de Claix, France) in a 37°C shaker. When the culture reached the exponential phase, 0.5 ml was transferred to an Erlenmeyer flask containing 50 ml TSB and incubated in a 37°C shaker overnight. The bacterial pellet was harvested by washing and re-suspended in PBS to obtain an optimal concentration of 2 ×1011 CFU/ml [19]. The dose was adjusted by reading the bacterial suspension's optical density at 600 nm. Inoculums were checked for every experiment by counting CFU [8].
Lpb. plantarum and L. acidophilus were obtained kindly from Dr. Shahrzad Ahangarzadeh, Isfahan University of Medical Sciences, Isfahan, Iran). All strains were kept in a 20% glycerol supply at −80 °C. MRS media were used for the preparation of fresh viable Lpb. plantarum and L. acidophilus stocks.
Infection of mice
Mice were injected intraperitoneally with approximately 109 E. coli cells in 500 μL of PBS using a fine insulin syringe [19]. Control mice received the same volume of PBS. During the treatment period, mice were observed every 24 h to notice behavior, food, and weight. Stools were diluted in sterile PBS to a concentration of 100 mg/ml, and CFU was determined by plating dilutions onto MacConkey agar plates and incubating at 37°C overnight. Colonies were counted and confirmed by PCR to detect stx1 and stx2 genes [8].
Body weight and Feed intake measurement
Mice's body weight was inspected daily and expressed as a mean±SD for each group over the research. Feed intake was determined by weighting the amount of food residue of mice in each group [18].
Histological assessment
Hematoxylin and eosin (H&E) staining was performed to detect histological changes in the studied mice's small intestine (terminal ileum) and intact kidneys. In brief, the washed organs (with normal saline solution) were fixed in 10% buffered formalin, dehydrated in ethanol, and embedded in paraffin wax. Then the 4-μm thickness sections of the organs were stained with H&E stains. The staining process was performed according to the standard operation used in the histopathology laboratories [20].
Histological evaluation was carried out using the intestine scoring system developed by stewart et al. (2009) for intestinal samples and using the toxic nephropathy and nephritis scoring system designed by chen SM et al. (2002) and Zheng ZY et al. (2005) for kidney samples [21-23].
Experienced histologists evaluated each sample. Briefly, the histological score of bacterial ileitis was based on the level of hemorrhage, inflammation, the extent of necrosis, congestion, edema, and the number of neutrophils [19]. The intestinal Tissues were examined in terms of hemorrhage, villous atrophy, edema, congestion, neutrophilic infiltration, and epithelial necrosis. Also, the kidney tissue was examined in terms of thrombosis, tubular cyst/cast, intestinal inflammation, glomerular cellularity, and glomerular hypertrophy [8, 15, 18].
DNA Extraction and assessment of gut microbiota targeted bacteria and Shiga toxin genes abundance using PCR and qPCR
Bacterial DNA was extracted from 500 mg of the intestine and kidney tissues and 220 mg of stools of mice to evaluate the abundance of stx1 and stx2 genes and gut microbiota targeted bacteria (Supplementary Table S1) according to the procedure of QIAamps (Qiagen, Hilden, Germany) [9, 24, 25]. Lpb. plantarum, L. acidophilus, and E. coli O157:H7 counts were performed by fecal analysis from two mice per group by manually pressing the stomach of mice during the experiment. The fecal samples were suspended in sterile PBS at 100 mg/ml. The suspensions were diluted and assessed with qPCR. All primers and annealing temperatures are detailed in Supplementary Table S1.[26, 27]
The quantity and quality of DNA were assessed based on absorbance at 260 and 280 nm using Qubit™ 4Florometer (Life Technologies, Invitrogen, Singapore).
Quantitative real-time PCR (qPCR)
The qPCR was conducted in duplicate with SYBR® Green PCR Master Mix (Yekta Tajhiz Azma, Tehran, Iran) in Rotor-Gene 6000 (Qiagen Corbett, Hilden, Germany). The proper qPCR primers (0.5 μM of each primer), about 50 ng of DNA, and 1 × SYBR green qPCR master mix were used to estimate the total bacterial load based on the abundance of 16S ribosomal subunit (Supplementary Table S1). The amplification was performed with the program of one cycle at 95°C for 5 min, followed by 40 cycles of 1 min at 95°C and 30 s at the proper annealing temperature and 72°C for 1 min, followed by a final extension step at 72 °C for 5 min [25]. Melting curve analysis was performed after qPCR with a temperature gradient of 0.1°C/s from 72 to 95°C with fluorescence signal measurement at 0.1°C intervals.
The standard curve method was utilized to calculate copy numbers of the targeted bacteria.The reference strains DNA bacteria were applied to make a standard curve. Typical bacterial strains, efficiencies, and coefficients of the standard curves are displayed in Supplementary Table S2.
Statistical analysis
All data were evaluated with SPSS 21 (SPSS, Inc., Chicago, IL, USA) and expressed as the mean ± standard deviation (SD). Pearson's Chi-square and One-way analysis of variance (ANOVA) were used for analyzing categorical and continuous differences, respectively. Bonferroni's and Dunnett's tests were done in multiple comparisons analyzing between the groups. Box-plot graphs showed the average number of bacteria, and the Dunnet test analyzed the results. A significant statistical difference was a P- value of < 0.05.