Evaluation of the Probiotic Effects of Bacillus clausii on the Biochemical Profile of Diarrheic Male Rats Experimentally Infected with

Escherichia coli

Almas M. Al-Bayati1, A.H. Awad2, Ahmed Abdulhameed Jabbar3, Al-Salihi Karima Akool4*, Luay Jumaah Jihad5

1Department of Medicine and Preventive, College of Veterinary Medicine, University of Kirkuk, Kirkuk, Iraq; 2Department of Biology, College of Education for Women, University of Kirkuk, Kirkuk, Iraq; 3Department of Kirkuk Health, Kirkuk, Iraq; 4Department of Basic Sciences, College of Dentistry, Al-Iraqia University, Baghdad, Iraq; 5Department of Microbiology, College of Veterinary Medicine, University of Kirkuk, Kirkuk, Iraq.

Received | March 10, 2025; Accepted | April 27, 2025; Published | May 15, 2025

*Correspondence | Al-Salihi Karima Akool, Department of Basic Sciences, College of Dentistry, Al-Iraqia University, Baghdad, Iraq; Email: [email protected]

Citation | Al-Bayati, A.M., Awad AH, Jabbar AA, Akool ASK, Jihad LJ (2025). Evaluation of the probiotic effects of Bacillus clausii on the biochemical profile of diarrheic male rats experimentally infected with Escherichia coli. J. Anim. Health Prod. 13(2): 355-361.

DOI | https://dx.doi.org/10.17582/journal.jahp/2025/13.2.355.361

ISSN (Online) | 2308-2801

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Diarrhea is a common health problem that affects young animals and requires immediate attention to prevent complications (Asma, 2020). The standard definition of diarrhea is a condition that is distinguished by an increase in the number of intestinal motility and passing of loose and watery stools (Karima et al., 2023). Various mechanisms and pathogens cause diarrhea; however, the primary severity of diarrhea depends on the dose, age, and other factors. Symptoms last (1-2) weeks, and the incubation period ranges from 1-7 days (Constable et al., 2017). Many strains of E. coli have developed resistance to standard antibiotics (Hammou et al., 2024) that, result in hard treatment with antibiotic. Most resistance develop from misuse of antibiotic and genetic mutations. The influence of antibiotic resistance is the delay in healing accompanied with long stay in the hospital and raised the mortality rate. Therefore, probiotics are used as substitute or supportive treatment in antibiotics resistance infection to restore normal balance of gut microflora, prevent growth of pathogenic bacteria and improve the immune system (Muteeb et al., 2023).

According to the International Scientific Association for Probiotics, probiotics are defined as live microorganisms that grant health benefits to the host (Hill et al., 2014). Probiotics have shown promising therapeutic effects against colibacillosis through several mechanisms (Priyanka et al., 2013), such as competitive exclusion and the production of antimicrobial compounds. Competitive exclusion refers to the beneficial bacteria in probiotics competing with Escherichia coli for nutrients and attachment sites in the intestinal tract (Hammou et al., 2024). These probiotics form a protective barrier on the intestinal wall, making it harder for pathogenic E. coli to colonize. In addition, many probiotic strains produce natural antimicrobial agents such as organic acids, bacteriocins, and hydrogen peroxide, which can inhibit or kill pathogenic E. coli (Hill et al., 2014). Recently, there has been a growing trend in using probiotics to prevent disease, making it one of the largest sectors in the functional food industry today (Muntingh, 2017).

Bacillus spore-forming bacteria are recognized for being able to create several secondary metabolites including enzymes, antibiotics, and antimicrobial peptides. Furthermore, they play an essential role in host interaction, microbial competition and biotechnology. Metabolites characteristic has contributed to the massive potential of antibiotics in multiple fields of agriculture, industry, and medications (Bahaddad et al., 2023; Danilova and Sharipova, 2020). These species are considered safe and efficacious probiotics, as they do not produce toxic substances and are manageable in terms of their growth requirements and tolerance to high temperatures (Saggese et al., 2021), in addition to classic probiotic traits, that are characterized by high stability towards processing and storage (Bader et al., 2012), and at low production costs. Several modes of action mediate its beneficial effects) (de Simone, 2019; Wieërs et al., 2020). They are widely used in treating many conditions and have found significant use in treating various diseases (Sanders et al., 2019).

Most of the bacterial probiotic strains used in biomedical reviews and studies belong to the genera Bifidobacterium spp. and Lactobacillus spp. Additionally, strains such as Bacillus clausii, Bacillus laterosporus, Bacillus coagulans, Bacillus subtilis, and Bacillus pumilus have also been used as probiotics (Dutta et al., 2011; Jafar et al., 2019).

B. clausii is ubiquitous in nature, mobile rod cell, aerobic, gram-positive bacteria, and endospore-forming isolated from soil. It is also found naturally within the flora of the digestive system of mammals (De Jonghe et al., 2010). The effectiveness and safety of several B. clausii strains have been proven through clinical trials conducted globally (Keya et al., 2015). It is considered the primary causative agent to inhibit the proliferation of pathogenic microorganisms in the gastrointestinal tract. It is frequently used as a probiotic to treat conditions related to diarrhea and enteritis. In Iraq, a previous study approved that the administration of probiotics with standard rehydration therapy reduced the duration of diarrhea in children in Fallujah City, Iraq (Mukhlif and Ali, 2024).

Moreover, an anti-Pseudomonas aeruginosa probiotic extract was investigated for its effectiveness in postoperative wound infections in Al-Najaf province, Iraq. The results showed a synergistic antibacterial inhibition effect between S. boundary and B. clausii (Mohammed et al., 2021).

However, a literature review on the application of B. clausii in experimental animals revealed a limited number of publications. Consequently, this study aims to investigate the potential of B. clausii as a probiotic and explore its therapeutic effects on experimental E. coli-induced diarrhea in rats, focusing on body weight, lipid, and protein parameters.

MATERIALS AND METHODS

Bacterial isolate

The bacterial species used in this study was received from microbiology department, College of Veterinary Medicine, University of Kirkuk. The identification of these bacterial species were carried out at species level by Vitek 2 framework.

Characterization of probiotics

Bile salt and acid tolerance tests were used to assess the extracted probiotic according to the method previously described by Choi and Chang (2015). The survival of probiotic bacteria was determined by evaluating cell survival after 24 hours of exposure to different pH values (2, 4, 6, and 8) and various concentrations of bile salts (0.1%, 0.3%, and 0.5%). The bacterial culture was incubated under aerobic conditions at 37°C for 24 hours.

Experimental animals groups

Fifteen male Albino Sprague-Dawley rats, aged 7-8 weeks and weighing approximately 146-148g, were used in this study. The rats were allowed to acclimatize for 10 days under standard animal house conditions. B. clausii was tested in this study for its potential effects against E. coli, particularly in relation to concerns about antibiotic resistance. All rats had free access to food and water ad libitum. The rats were then randomly divided into three equal groups, each with four replicates, as follows: G1 (healthy group), G2 (infected group with E. coli), and G3 (infected with E. coli and treated with the probiotic B. clausii).

Every day, the rats were administered 2 ml of B. clausii via oral gavage, corresponding to a concentration of 1.5 x 108 CFU/day/rat, based on the standard turbidity of the McFarland solution. The treatment lasted for 28 days. At the end of the treatment period, the rats were subjected to a 20-hour starvation period, after which they were anesthetized with chloroform. Blood samples were collected from all rats and subjected to biochemical assays for lipid profile analysis, including low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol (TC), and triglycerides, using a standard laboratory assay with a lipid analyzer (Multichannel Abbott Spectrum Autoanalyzer, Abbott Laboratories, C4000, USA). The estimation procedures followed the manufacturer’s instructions for lipid profile kits, except for LDL, which was calculated using the Friedewald equation (Friedewald and Levy, 1972). Additionally, serum total protein (TP), globulin, and albumin concentrations were estimated spectrophotometrically using available kits (Biomerex, France).

Statistical analysis

All collected data was alnalysed using SPSS software version 22. The variables were displayed as mean± Standard deviation (SD). Moreover, the General Linear Model and Duncan test were used to estimate the significance of the various factors. Statistically, the significant result was considered at p<0.05.

Results and Discussion

The results of the current study demonstrated the growth of B. clausii probiotic bacteria at various concentrations of pH and bile salts. B. clausii showed growth at pH 4 and 6, while no growth was observed at pH 2. These results are consistent with previous research by Al-Jandal et al. (2024), who reported significant differences in the total number of microorganisms in soft cheese due to the application of bacteriocins. The findings regarding acid tolerance are also in line with those of Nithya and Halami (2013), who observed that cultures of probiotic microorganisms exhibit better acid and bile tolerance. Furthermore, the results showed the growth of B. clausii at bile salt concentrations of 0.1% and 0.3%, while no growth occurred at 0.5%. These findings are consistent with the research of Werdi and Al-Hadidy (2023), who reported a significant increase (P<0.05) in the values of various liver enzymes compared to the control group. Additionally, the results align with the work of Kumar et al. (2022) and Nithya and Halami (2013), further supporting the beneficial properties of Bacillus clausii in terms of its growth under various conditions.

 

Table 1: The growth of B. clausii probiotic bacteria in various pH and different bile salt concentration.

Bile salt %			PH
0.1	0.3	0.5	2	4	6	8
+	+	_	_	+	+	+

 

Table 2: The effect of B. clausii on body weight of the laboratory rats suffered from E.coli induced diarrhea.

Treatments	Initial	Final weight	Weight gain
	G
G1	131.0 b	225.0 b	94.0 b
G2	139.0 b	267.6 a	128.6 a
G3	176.3 a	266.3 a	90.0 b

 

a-b different lettering in a column shows significant difference at probability level (P<0.05). G1: Control; G2: Infected with E. coli; G3: Infected with E. coli and treated with B. clausii.

 

The results of the current study revealed a significant variation in the body weight of rats across the different experimental groups (Table 2). The initial body weights were 131.0 g, 139.0 g, and 176.3 g for G1, G2, and G3, respectively. The results also showed an increase in the body weight of rats in G2, which were exposed to E. coli infection, with a final weight of 267.6 g and a weight gain of 128.6 g (P<0.05). However, the body weight measurement for G3, which was treated with B. clausii, showed a reduction compared to G1 and G2. These results may be attributed to an increase in lipid parameters such as cholesterol, triglycerides, and low-density lipoproteins (LDL). These factors likely contributed to the weight gain observed in the infected animal group, as suggested by Al-Jubouri (2009).

The findings of the current study are in agreement with (Kobyliak et al., 2016), who mentioned that probiotics not only significantly affect weight loss and fat loss processes, but they also have a tendency to decrease the average size of fat cells in all fatty tissues of experimental animals.

The effect of B. clausii on the lipid profile of rats induced by E. coli infection is displayed in Table 3. A significant increase (P<0.05) was observed in the concentrations of cholesterol (73.4 mg/dl), triglycerides (94.0 mg/dl), and LDL-cholesterol (16.4 mg/dl) in G1 (control group). In contrast, there was a significant decrease in the concentrations of HDL-cholesterol (17.4 mg/dl) in G2 (infected group). The concentrations for cholesterol, triglycerides, and LDL-cholesterol in G2 were 74 mg/dl, 96.8 mg/dl, and 18.4 mg/dl, respectively. These values were significantly higher compared to G3 (treated group with B. clausii), which had concentrations of 64.5 mg/dl, 54.6 mg/dl, and 15.13 mg/dl for cholesterol, triglycerides, and LDL-cholesterol, respectively. Additionally, HDL-cholesterol was significantly higher in G3 (36.5 mg/dl) compared to the other groups.

 

Table 3: The effect of B. clausii on Lipid profile of rats.

Treatments	CHOL	TG	LDL	HDL
	mg/dl
G1	73.4 a	94.0 a	16.4 a	38.9a
G2	74 a	96.8 a	18.4 a	17.4 b
G3	64.5 b	54.6 b	12.13 b	36.5 a

 

a-b different lettering in a column shows significant difference at probability level (P<0.05). CHOL: Cholesterol; TG: Triglyceriods; LDL: Low-density lipoprotein; HDL: High-density lipoprotein (HDL); G1: Control; G2: Infected with E. coli; G3: Infected with E. coli and treated with B. clausii.

 

Previous study approved that the high ratio in cholesterol levels is attributed to an increment within the action of the chemical cholesterol acyltransferase, which is sensible for the assimilation of cholesterol (Jumaa and Naji, 2021). It is worth to mentioned that previous studies explained the several mechanisms used by probiotic to lower the cholesterol levels such as the bile salt decongygation, cholesterol assimilation, short chain fatty acid production, and modulation of gut microbiota (Kumar et al., 2012).

Moreover, Hassan and Frank (2001) explained that the decrease in lipid markers may be attributed to disturbances in the lipid digestion system as a result of stress and the formation of lipid peroxides and unsaturated fatty acids. This mechanism can lead to the inhibition of steroid and bile salt secretion and excretion, as well as the occurrence of various absorption and digestion disturbances (Hassan and Frank, 2001).

The effect of probiotics on triglyceride levels varies depending on the type and strain of bacterial agent, and causes of decrease in TG value may be contributed in stimulation of enzyme lipase resulting fatty acids formation that absorbed by fat cells lead to inhibit the synthesis of fatty acids which enter the liver’s procedure for generating fat through the activity of key enzymes preventing (Li et al., 2020).

While, Trieb et al. (2016) found that the levels of HDL decreased because the liver being exposed for some diseases or due to the high concentrations of other lipid profile.

According to previous publication, the causes of decrease in cholesterol concentration, is the cholesterol dehydrogenase enzyme synthesized by probiotic bacteria. This enzyme is activated the converting cholesterol process into Cholest-4-en -3-one, and in turn is used as a mediating factor in converting cholesterol into smaller units Coprostanol, which makes it easier to break it down for use. Accompanied with a decrease in cholesterol retention and a rise within the accompanied with a low value in absorption of cholesterol and a rise within the sum of cholesterol the body excretes (Ahn et al., 2003; Werdi and Al-Hadidy, 2023).

The results of the current study also showed the effects of B. clausii on the protein parameters in E. coli-infected rats. A significant reduction (P<0.05) in total protein and albumin levels was observed in G2, the infection-induced group, with concentrations of 5.00 and 4.20 dL/g for total protein and albumin, respectively. However, the total protein and albumin values in G1 (healthy group) and G3 (treated with B. clausii) were 5.30 and 5.50 dL/g, and 5.9 and 5.10 dL/g, respectively. These results demonstrated a significant increase in total protein and albumin levels in the treatment group with B. clausii, while no significant differences were observed between the groups in globulin values (Table 4).

 

Table 4: The effect of B. clausii on the serum Proteins of infected rats.

Treatments	Total protein	Albumin	Globulin
	g/dL
G1	5.30 b	5.50 a	1.20
G2	5.00 b	4.20 b	1.00
G3	5.9 a	5.10 a	1.20

 

a-b different lettering in a column shows significant difference at probability level (P<0.05). G1: Control; G2: Infected with E. coli; G3: Infected with E. coli and treated with B. clausii.

 

Previous studies (Żołnierkiewicz et al., 2024), explained the reasons of low blood proteins in the infected group. The experimental animal exposed to stress that decrease using of glucose as an energy source and consequently, resort to the use of alternative sources, including proteins, like diabetes. These mechanisms lead to loss of total proteins (Proteinuria) through Urination (Żołnierkiewicz et al., 2024), and several pathological conditions, such as plasma hemolysis, decreased liver protein synthesis, disruption of kidney function, increased concentrations of free radicals, and exposure to oxidative stress were also occurred. However, other researcher showed that the Inhibition of enzymes associated with protein synthesis may be also the reason for the decrease in total protein concentration (Groener et al., 1989).

On the other hand, the results of the current study showed a decrease in albumin levels with no significant variation between the different experimental groups. According to a previous study by Demir et al. (2009), this observation may be attributed to the loss and infiltration of protein into damaged tissues. Additionally, the consumption of certain antioxidants in the body leads to a decrease in calcium concentration, as 50% of calcium is bound to albumin in the blood plasma (Abd El-Hack et al., 2021; Anraku et al., 2011). Low albumin protein values are also considered a clinical sign of severe liver disease, which occurs due to obstruction of its synthesis or glomerulopathy, leading to an increase in albumin loss (Belinskaia et al., 2021). Furthermore, the results of this study agree with previous research that indicated the use of probiotics can lead to an increase in albumin and total protein values (Abd El-Hack et al., 2021).

CONCLUSION

In conclusion, this study revealed the effects of B. clausii bacteria as a probiotic in experimental E. coli infected rats. The results showed significant reduction in the negative effects of E. coli and beneficial effect of B. clausii on lipid and protein parameters in experimentally infected rats, showing a direct link to the modulation of E. coli infection. The authors recommend further studies on B. clausii probiotics to explore their impact on a wider range of physiological and biochemical parameters.

Acknowledgment

Thank you to all of the workers in the internal medicine, microbiology and pharmacology departments of Veterinary Medicine college, Kirkuk University.

Novelty Statement

The novelty of this study is the investigation of the therapeutic potential of Bacillus clause as probiotic to treat the E.coli induce diarrhoea in rat , which have not been extensively studied in vivo. The research contributes to the field by approving the role of Bacillus clause role in modulating gut microbiota and enhancing the immune response.

Author’s Contribution

Almas M. Al-Bayati: conceptualization and experimental study and writing manuscript draft.

Ali H. Awad: experimental study and laboratory analysis and statistical analysis.

Ahmed abdulhameed jabbar : Laboratory analysis and writing draft.

Al Salihi karima Akool : Experimental study and editing the final version of the manuscript.

Luay Jumaah Jihad: experimental study and laboratory analysis and statistical analysis.

Funding

Self-funding.

Ethical statement

This study was carried out in compliance with institutional guidelines and ethical standards stated in the Instructions of the Scientific Research and Higher Education Ministry.

Conflict of interest

The authors have declared no conflict of interest.

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