Effects of Mixed Probiotic Spores of Bacillus Coagulans and Bacillus Subtilis on Gut Microbiota and Intestinal and Liver Damage of Rats Infected with Salmonella Typhimurium


 Background: Although the intestinal lumen is a major way for the entrance of pathogens such as Salmonella, they face a dense microbiota in the gut lumen. This community confers health benefits. The probiotics enhance the colonization resistance and direct inhibitory effects against pathogens, which are important in reducing the incidence, duration of gastroenteritis and also histopathological effects. Methods: In this study spore of two probiotic bacteria, Bacillus subtilis and Bacillus coagulans were fed to rats for three weeks through their daily water intake after which Salmonella Typhimurium was gavaged to the rats. On days 1, 3, 5 and 7 after gavaging, intestinal microbiota and days 1, 3, 5 and 7 after gavaging, gut and liver histopathological changes were evaluated. Results: In comparison to the control group, Salmonella caused no change in spore and aerobic bacteria population but it caused anaerobic and coliform bacteria increase and LAB reduction. But B. subtilis and B. coagulans accompaniment with salmonella reduced the anaerobe and coliform bacteria and decreased the LAB population following Salmonella infection. Also, the histopathological evaluation showed that these probiotic attenuated shortening and fusion of villi, mononuclear cells infiltration, submucosal edema and epithelial necrosis in the intestine, hepatocyte degeneration and mononuclear cells infiltration in the liver which occurred due to Salmonella infection.Conclusion: B. subtilis and B. coagulans help the maintenance of the gut microflora population and reduce the histopathological effects when exposing to the Salmonella, which, causes the reduction of this pathogen effects and early recovery.


Background
Salmonella infection remains a distressing public health concern worldwide [1]. Salmonella strains are adapted into various environments, including human, animal and non-animal hosts due to their genetic make-up [2]. Non-Typhoidal Salmonella (NTS) refers to Salmonella strains other than S. Typhi and S. Paratyphi which are found in animal reservoirs. In humans, NTS infections cause gastroenteritis or 'stomach u', which is accompanied by symptoms such as non-bloody diarrhea, vomiting, nausea, headache, abdominal cramps, and myalgia and sometimes hepatomegaly and splenomegaly [3]. Among NTS, S. Typhimurium and S. Enteritidis are recognized as important foodborne pathogens [4,5]. Nowadays Salmonella spp. are becoming resistant to antibiotics, which makes the treatment so di cult [6,7].
Although the intestinal lumen is a major way for pathogens entrance such as Salmonella, the pathogens face a dense microbiota in the gut lumen. This community confers health bene ts [8], including the prevention of enteropathogen colonization and growth through competition for nutrients [9,10]. Some microbiota strains secrete inhibitory compounds, preventing the growth of invading pathogens [11]. Other microbiota strains may compete for binding sites on the mucous surface of the epithelium. Also, the resident microbiota causes an oxygen-depleted environment, which reduces the growth of facultative anaerobic pathogens like Salmonella spp. [11,12].
Probiotics are de ned by the Food and Agriculture Organization of the United Nations (FAO)/WHO as "live microorganisms which when administered in adequate amounts confer a health bene t on the host". The mechanism of probiotic action is likely to be multifactorial. The enhancement of colonization resistance and direct inhibitory effects against pathogens are important in reducing the incidence and duration of gastroenteritis. They also slow down the pathogens through the production of inhibitory compounds (such as bacteriocins), reduction of luminal pH through the production of short-chain fatty acid, competition for nutrients and adhesion sites on the intestinal mucosa and submucosa, modulation of the immune response and regulating of gene expression (for instance, the expression of mucin genes) [13,14]. The mechanism used by probiotics within the human gut microbiota is unclear [15]. Spore-former probiotics are so important due to their high vitality against gastric acid and bile salts [16]. Spores of Bacillus spp. commercially used as probiotics, and unlike Lactobacillus spp., they are dormant [17]. Consequently, this study was planned to evaluate the preventive effects of spores of Bacillus subtilis and Bacillus coagulans as a probiotic on gut microbiota population and intestinal and liver damage of rats infected with Salmonella Typhimurium.

Study settings
In this experimental study 80 Spargue-Dawley rats weighting 170-180 gr were procured from the Department of animal lab, Shiraz University of Medical Sciences, Iran. They were housed in plastic cages and kept under 12-hour light/ dark condition, a temperature of 20-25 °C, humidity of 50-60 %, and free access to food (commercial standard pellets) and water in order to adaptation to the new environment.
After an acclimatization period of 1 week, they were randomly divided into four main groups, each containing ve subgroups (four rats in each subgroup) and treated as follows: 1. Blank Control group: Receiving drinking water containing 0.1% dextrose for 4 weeks and intragastric gavage of 0.5 ml normal saline and 0.5 ml 6% (w/v) NaHCO 3 (to neutralize the gastric acid) in day 22 of the study.
The concentration of probiotic spores fed to the rats was determined based on our previous study [18] November 24, 1986, about the animal protection, was entirely deliberated for test trials. Effects of mixed probiotic spores of Bacillus coagulans and Bacillus subtilis on gut microbiota and intestinal and liver damage of rats infected with Salmonella Typhimurium were evaluated.

Preparation of probiotic bacteria
Spray-dried spore of B. coagulans (PRM101) and B. subtilis (PRM102) in dextrose powder (1×10 11 spore per gram) was donated separately by the Pardis Roshd Mehregan Company, Iran. To con rm the spore concentrations of the probiotics, one gram of each powder was dissolved in 100 ml tap water (1×10 9 each spore/ml) and heated at 80 °C for 15 min, to kill the vegetative cells, and surface plating on Brilliant Green agar (BG agar, Merck, Germany) after tenfold serial dilution was done.

Preparation of Salmonella
In order to prepare the solution for intragastric gavage, a pure colony of Salmonella enterica serovar Typhimurium (S. Typhimurium) (ATCC 14028) was cultured in 100 ml of Tryptic Soy Broth (TSB) (Merck, Germany) for 24 h at 37 °C. After incubation, it was centrifuged at 5000 ×g for 10 min at 4 °C and then the pellet was re-suspended in 50 ml physiological saline. Plating was performed for the suspension to con rm the concentration of 2×10 9 CFU/ml [22].
Salmonella-free con rmation For con rmation that the rats are Salmonella-free, on days 0, 7, 14 and 21 of the study, fecal samples from each subgroup were taken and examined. One gram of pooled fecal sample from each subgroup was added into 9 ml lactose broth (Merck, Germany) and incubated at 37 °C for 24 h. After incubation, 1 ml of that was added to 10 ml Selenite Cystine broth (Merck, Germany) and in parallel, 0.1 ml was added to 10 ml Rappaport Vassiliadis broth (Merck, Germany).
They were incubated at 37 °C and 42 °C, respectively, for 24 h. After which a loop of the selective enrichment broth was cultured on Xylose Lysine Deoxycholate agar (XLD agar, Merck, Germany) and Brilliant Green agar (BG agar, Merck, Germany). The plates were incubated at 37 °C for 24 h and checked for Salmonella colonies [21].
Intestinal micro ora enumeration One day before and days 1, 3, 5 and 7 after intragastric gavage of S. Typhimurium, a pooled fecal sample from the rats in each subgroup was taken. Just after collection, each sample was weighed, homogenized in sterile phosphate buffer saline (PBS) (pH: 7.2), and tenfold serial dilutions were made in PBS. Aerobic and anaerobic microorganisms were cultured in Plate Count agar (Merck, Germany). LAB were cultured in MRS (de Man, Rogosa, Sharpe) agar (Merck, Germany) and the Coliforms were cultured in VRBL (Violet Red Bile Lactose) agar (Merck, Germany). Anaerobes and LAB medium plates were placed in an anaerobic jar. All plates were incubated at 37 °C for 48 h. The micro ora enumeration was expressed as CFU/gr . In order to count bacterial spores in fecal samples, the homogenized samples in PBS were put into a water bath at 80 °C for 10 min and cooled immediately. Then the samples were serially diluted in PBS and cultured in Plate Count agar (Merck, Germany). The plates were incubated at 37 °C for 48 h. The spore enumeration was expressed as spores/gr. All the samples were cultured in duplicate [23 -25].

Histopathological evaluation
On days 3, 5 and 7 after intragastric gavage of S. Typhimurium, one rat was chosen randomly from each subgroup (replicated independently ve times from each treatment group) and euthanized (by CO 2 from commercially available gas cylinders , according to European Communities instructions 1997). The euthanized rats were dissected and their liver and intestine (ileum and cecum) were removed. All samples were xed in 10% neutral buffered formalin, embedded in para n wax, sectioned at 5 µm, stained with hematoxylin and eosin and examined for histopathological changes by an ordinary light microscope (Olympus, Tokyo, Japan). Histopathological indexes included shortening and fusion of villi, mononuclear cell in ltration, necrosis and submucosal edema in the intestine, and hepatocyte degeneration and necrosis with mononuclear cell in ltration. We used a semi-quantitative scoring method based on the ordinal method [26]. Four grades were considered in scoring as follows: no pathological changes (0), mild, moderate and severe pathological changes scored 1, 2, and 3, respectively.

Statistical analysis
The results were analyzed using a one-way analysis of variance and the statistical signi cance of differences between mean values was analyzed by Duncan's multiple range tests. The Histopathological evaluation were compared by Kruskal-wallis nonparametric test. P-values less than 0.05 (P < 0.05) were considered statistically signi cant. The analysis was performed using the Statistical Package for Social Sciences (SPSS) software (SPSS 16 for windows, SPSS Inc, Chicago, IL, USA).

Results
Intestinal micro ora enumeration The spore former probiotics (B. coagulans and B. subtilis) signi cantly increased the number of fecal total aerobics, LAB and spores and reduced the number of coliforms (P < 0.05). By gavaging S. Typhimurium, no change was observed in the total aerobic and spore count of feces, while it increased the number of coliforms and anaerobics (P < 0.05) and decrease the lactic acid bacteria population (P < 0.05) in the feces. These observed changes in Salmonella group were different in comparison to the Blank Control group untill 7 th day after gavaging S. Typhimurium (P < 0.05). Using probiotics before gavaging, reduced the number of coliforms and anaerobic bacteria and increased the number of total aerobics, LAB and spores count in comparison to the Salmonella group (P < 0.05) (Figs. 1-5).

Histopathological evaluation
The mean ± SD of histopathological parameters are represented in Tables 1 and 2. No lesion was observed in the Blank Control ( Figs. 6A and 7A) and Probiotic Control groups.
On the 3 rd day after gavaging, the histopathological lesions in Salmonella group were mild to moderate shortening and fusion of intestinal villi with mild to moderate mononuclear cells in ltration as well as mild hepatocyte degeneration with mild to moderate mononuclear cells in ltration. While the mild fusion of intestinal villi was observed in the Probiotic+Salmonella group.
In the 5 th day after gavaging, the histopathological lesions in Salmonella group were mild to moderate shortening and fusion of intestinal villi with moderate to severe mononuclear cells in ltration, mild to moderate submucosal edema, and mild hepatocyte degeneration with mild to moderate mononuclear cells in ltration. Besides, moderate epithelial necrosis of the intestine was observed in a rat. Concurrent use of probiotic and salmonella signi cantly reduced these complications and a mild fusion of intestinal villi and mild mononuclear cells in ltration in the liver were observed (P < 0.05).
In the 7 th day after gavaging, the histopathological lesions in Salmonella group were moderate to severe shortening and fusion of intestinal villi, mononuclear cells in ltration and submucosal edema (Fig. 6B), and moderate hepatocyte degeneration and necrosis ( Fig. 7B) with moderate to severe mononuclear cells in ltration (Fig. 7C). Also, there was severe necrosis of intestine in a rat (Fig. 6C). Probiotic+Salmonella group showed mild fusion of the intestinal villi ( Fig.  6D) and mild mononuclear cells in ltration in the liver (Fig. 7D).

Discussion
Colonization is the rst step of any infection. But enteropathogenic bacteria face a dense microbial community called microbiota. The microbiota provides an e cient barrier against infections that must be overcome by enteropathogenic bacteria. In this study, we concluded that gavaging S. Typhimurium, didn't change the total aerobic and spore count, but it reduced the population of LAB and increased the coliform and anaerobe count. The use of probiotics for three weeks before the rats became infected with Salmonella, changed the gut micro ora population so that the population of bene cial LAB increased and harmful ones such as coliform decreased. Therefore, they prepared the intestinal lumen to ght the pathogen and decreased its colonization. LAB send signals to activate immune cells and induce good mucosal immunostimulation without inducing side effects such as bacterial translocation or a strong in ammatory immune response [27]. As mentioned before, the intestinal microbiota limits the pathogens such as Salmonella infection, one of the mechanisms referred to as colonization resistance [28]. Salmonella (as a gastrointestinal pathogen) must overcome the host defenses in order to colonizing and resulting in disease [29], but the usage of B. coagulans and B. subtilis spore before Salmonella entrance created a strong barrier that limited Salmonella mucosal penetration. Also, Thirabunyanon and Thongwittaya (2012) showed that usage of B. subtilis signi cantly reduced S. Enteritidis invasion of intestinal epithelial cells. They expressed that high tolerance of the B. subtilis to low pH and bile salt conditions helps them work well [30].
Cecum and small intestine are two parts of the GI tract that showed in ammatory changes in infection with Salmonella [31,32]. In our study, histopathological lesions in Salmonella group increased in the 3 rd , 5 th and 7 th days after gavaging Salmonella. Salmonella caused shortening and fusion of intestinal villi, mononuclear cells in ltration, epithelial necrosis, and submucosal edema. While probiotic bacteria, B. coagulans and B. subtilis, reduced the hepatic and intestinal lesions induced by S. Typhimurium. Invasive S. Typhimurium form a gap in the follicle associated epithelium by M cell destruction, resulting in organisms invasion of enterocytes adjacent to the dead cell. Furthermore, replicating Salmonella enters both the apical and basolateral surfaces of enterocytes adjacent to infected M cells [33]. Probiotics reduce this process by a decrease of Salmonella adhesion and colonization. Histopathological effect of S. Typhimurium decreased in intestine and liver, using Lactobacillus acidophilus [34]. Gancarcikova et al (2018) observed that prominent in ammatory changes and the destruction of hepatocytes with S. Typhimurium result in infection [35]. The marked alleviation of overall in ammation and hepatocyte necrosis represented treat with Lactobacillus reuteri (LAB). They recorded a signi cantly reduced mean area of nodules in the livers in LAB group in comparison with an infected control group, probably resulting from a decrease of bacterial load. In the other study, severe mucosal damages occurred by loss of villi, permeation of red blood and in ammatory cells in the mucosa and submucosa in the induced group by pathogenic bacteria Salmonella. Mice receiving Lact. acidophilus, Lact. bulgaricus and Lact. casei had the typical structure of the small intestine tissue with lesser in ltration of in ammatory cells in special areas [36]. Manuel et al, (2017) showed probiotic supplementation (Lact. casei and Lact. paracasei), in the chronic stress model, improved the length and cellularity of the intestinal villi, increased phagocytic activity of peritoneal and spleen macrophages and the humoral response, and protected against Salmonella infection [37].

Conclusion
Using B. coagulans and B. subtilis before exposure to Salmonella Typhimurium helps the maintenance of the gut micro ora population. They inhibit the coliform and anaerobic bacteria count increase and lactic acid bacteria decrease which happens following Salmonella Typhimurium infection. Also, they cause a reduction in Salmonella histopathological effects in the liver and intestine. It can be concluded that using these probiotics leads to a reduction of pathogen effects and early recovery. University. The authors also thank Dr. Javad Sajedianfard, Department of Basic Sciences, School of Veterinary Medicine, Shiraz University for supporting this study, Pardis Roshd Mehregan Company, Iran for donating the probiotic bacteria.

Funding
This work was supported by funds from Shiraz University, Shiraz, Iran (1395/9234308). The funder had no role in study design, data collection and analysis, decision to publish or preparation of manuscript.
Availability of data and materials The datasets used and/or analyzed during this study are available from the corresponding author on reasonable request.
Authors' contributions SSS, SB and FN conceived and designed the study and critically revised the manuscript. SM and FZK contributed to sample collection and performed laboratory tests. SM analyzed the data and drafted the manuscript. All the authors have reviewed and approved the nal manuscript.
Ethics approval and consent to participate All methods were performed following the relevant guidelines and regulations by Ethical Committees of Shiraz University and approved by the governing body (1395/9234308).
coagulans and intragastric gavage of 1×10 9 CFU of S. Typhimurium on day 22.  The logarithm of LAB count in feces samples of rats with different treatments before and after intragastric gavage of S.

Figure 3
The logarithm of spore count in feces samples of rats with different treatments before and after intragastric gavage of S.  The logarithm of anaerobic bacteria count in feces samples of rats with different treatments before and after intragastric gavage of S. Typhimurium. Values are mean of 5 independent replicates. Blank Control group: Receiving water; Probiotic Control group: Receiving water containing 5×107 spore/ml B. coagulans and 5×107 spore/ml B. subtilis ; Salmonella group: Receiving water and intragastric gavage of 1×109 CFU S. Typhimurium; Probiotic+Salmonella group: Receiving water containing 5×107 spore/ml B. coagulans and 5×107 spore/ml B. subtilis and intragastric gavage of 1×109 CFU S. Typhimurium. Different letters indicate signi cant differences between groups in each day (P < 0.05).

Figure 5
The logarithm of colifom bacterial count in feces samples of rats with different treatments before and after intragastric gavage of S.