In-vitro and in-vivo antibacterial activity of potential probiotic Lactobacillus paracasei against Staphylococcus aureus and Escherichia coli

Abstract

Previous studies documented that Lactobacillus paracasei has obvious in vitro cholesterol-lowering abilities. In this study, initially, L. paracasei was tested in terms of antibacterial properties as well as antibiogram profile. Then, the safety of the mentioned strain was evaluated in rats. Evaluation of antibiotic susceptibility revealed that the L. paracasei strain had high antibiotic resistance to several antibiotics as well as a great ability to autoaggregation. After identification of the probiotic aptitude, six groups of six rats from both sexes were used (three groups of each sex). L. paracasei was administered to the experimental groups via drinking water for 28 days (1 × 108 and 1 × 109, respectively). The negative control group received only tap water during this period. Hematological indicators, serum liver enzyme activity including (alanine transaminase (ALT), alkaline phosphatase (ALP), and aspartate transaminase (AST)) as well as serum creatinine and urea were evaluated at the end of 28 days. The blood and serum factors were not changed significantly during the 28 days. The only noticeable difference was the increase of blood urea in both sexes which was in a normal range. Furthermore, the evaluation of antagonistic properties revealed that L. paracasei had antibacterial aptitude against Escherichia coli and Staphylococcus aureus. In conclusion, this strain has good cholesterol-lowering and antibacterial properties and is a safe supplement in Wistar rats.

1. Introduction

Probiotics are living bacteria that can control the immune system of the host and benefit the host when properly administered [1]. Probiotics have become a hot research topic in recent years due to their safety, nutritional and health value, and antimicrobial function. Probiotic is a Greek word that originally meant “for life,” but over time, the meaning has changed. The development of the probiotic word over time has been strongly correlated with the rise in popularity of using supplements containing live bacteria and the advancements in our understanding of their mechanisms of action [2]. These microorganisms are widespread and can be found in soil, water, vegetable products, meats, fermented and cooked meats, dairy products, and other microorganisms as well as in the gastrointestinal tracts of humans, mammals, and other animals [3]. Nowadays, Lactobacillus and Bifidobacterium genera comprise the majority of probiotic bacteria. Probiotic microorganisms, however, additionally include species from the genera Lactococcus, Enterococcus, Saccharomyces, and Propionibacterium [4,5]. The most important effect of probiotics is their replacement in the small intestine, which stimulates and clears it, thus preventing pathogens from sticking and inhibiting the toxic effects of toxins.

Public health and the food business continue to suffer from the effects of foodborne illnesses and food spoilage microbes. In addition to a high fatality rate, foodborne disease outbreaks have a significant financial burden due to the high cost of healthcare. The following bacteria are linked to the most frequent causes of foodborne diseases in the European Union: Listeria monocytogenes, Salmonella enterica, Escherichia coli, Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, and viral pathogens like noroviruses and rotaviruses are all examples of bacterial pathogens [6]. According to the Council for Agricultural Science and Technology, between 6.5 million and 33 million incidents of human illnesses are related to food, with 9000 recorded fatalities per year in the United States (Food and Agriculture Organization) [7]. Moreover, by 2050, it is predicted that 10 million people worldwide will die each year from foodborne illnesses if immediate action is not taken. The first line of treatment for these infectious intestinal diseases is antibiotic therapy. However, since the microbiota remains out of balance as a result of the use of broad-spectrum antibiotics, recurrence is common, especially in cases of antibiotic-associated diarrhea (AAD). A systematic approach to the eradication, prevention, and reduction of harmful bacteria in foods by the application of innovative antimicrobial drugs is therefore necessary given the current global prevalence of foodborne infection rates [6].

Numerous studies have shown the alleviation of some diseases such as AAD with probiotics [[8], [9], [10]]. The effectiveness of several probiotic strains, particularly those from the Bifidobacterium and Lactobacillus species, raises the possibility that they share traits that could improve patient health in certain pathological conditions. Several possible mechanisms of probiotics have been researched and may be responsible for the observed health advantage, including organic acid, hydrogen peroxide, carbon dioxide, diacetyl, bacteriocin, and adhesion inhibitors. but in terms of translational research, this is a clear flaw that prevents the creation of better treatments [11,12]. Romero-Luna et al., (2020) demonstrated that the cell-free supernatant of L. paracasei had antimicrobial, antifungal, and antioxidant capacity in kefir grains [13]. In a study, L. paracasei had antimicrobial activity against Staphylococcus aureus above 19 mm [14]. Therefore in this study, a new isolate of L. paracasei, which was isolated from camel milk, was examined for antibacterial activity and potential probiotic properties.

2. Materials and methods

2.1. Bacterial growth and culture

L. paracasei was prepared by the Agricultural Biotechnology Research Institute of Iran (ABRII). After preparation, the strain was seeded on de Man Rogosa and Sharpe (MRS) broth and incubated at 37 °C for 24 h in the warehouse. The strain was then stocked for short-term and long-term use with 25% glycerol and stored in the refrigerator at −20 °C and −80 °C, respectively.

2.2. Antibacterial properties of L. Paracasei

The well diffusion method and determining the diameter of the growth inhibition zone were used. For this purpose, fresh overnight culture was prepared from the strains used in the experiment (Müller-Hinton Broth culture medium was used for the culture of pathogenic strains, and MRS broth was used for the culture of L. paracasei). After that, the cell-free supernatant from strain L. Paracasei was prepared. In the next step, each of the pathogenic strains in the study (including Escherichia coli (PTCC 1276), Shigella Flexneri (ATCC 9199), Listeria monocytogenes (PTCC 1163), Klebsiella pneumoniae (ATCC 43816), Staphylococcus aureus (PTCC 1764), Yersinia enterocolitica (ATCC 23715), and Bacillus cereus (PTCC 1539) were cultured in separate plates. Then, using the circular section of a pasteurized pipette, wells with a diameter of 6 mm were made on the culture medium. Next, under aseptic conditions, 100 μL of cell-free supernatant was inoculated into each of the wells. The plates were incubated at 37 °C for 24 h in the last step. The diameter of the growth inhibition zone due to the antibacterial activity of the cell-free supernatant was measured by a digital caliper.

2.3. Antibiotic susceptibility

For this purpose, fresh overnight culture of L. Paracasei was prepared in a test tube containing liquid MRS medium. The strain was also cultured overnight in 8 cm sterile plates containing MRS Agar culture medium. Then, the sensitivity to standard antibiotics was determined by using the antibiogram disk diffusion method and determining the diameter of the growth inhibition zone.

2.4. Hydrophobicity assessment

To measure the surface hydrophobicity of bacteria, the bacterial binding test to xylene hydrocarbon was used. First, the bacterial suspension was prepared by absorbing light at 0.08 to 0.1 at a wavelength of 600 nm (A₀). Then 3 mL of the suspension was added to 0.5 mL of hydrocarbon. After stirring for 2 min, the suspension was placed in a fixed place for 15 min. After this time, the aqueous phase optical absorption was recorded at 600 nm (A₁), and the percentage of hydrocarbon-attached cells was calculated using the following formula [15].

Hydrophobicity (%) = (1 − A₁/A₀) × 100 (1)

2.5. Autoaggregation assessment

The autoaggregation ability of L. paracasei was performed according to the method described by Angmo et al., (2016). Autoaggregation percentage was measured using the following formula.

Autoaggregation (%) = 1 − (A_t/A₀) × 100 (2)

where A₀ represents absorbance at t = 0 and at represents absorbance at time t.

2.6. Cholesterol assimilation

The cholesterol absorption capacity of strain L. Paracasei was measured in an MRS broth medium. Thus, PEG 600 cholesterol was added to MRS broth to reach a final concentration of 100 μg/mL. Then, 1% (v/v) of the above probiotic strain (which was cultured overnight) was inoculated and incubated for 48 h at 37 °C. Survival viability during incubation was assessed by standard colony counting methods. The probiotic suspension was centrifuged at 4 °C for 10 min for cholesterol analysis, and the cholesterol-free supernatant was collected. Finally, cholesterol uptake was measured by the Rudel and Morris protocol [16].

2.7. Hemolysis activity

A hemolysis test is used to evaluate the ability of this strain to lubricate red blood cells. In this method, a blood agar culture medium is used. The probiotic strain is cultured as a zigzag line on the culture plate and incubated at 37 °C for 24 h. Finally, a possible discoloration of the culture medium is recorded.

2.8. Biofilm formation

To evaluate the phenotypic ability of biofilm production by L. Paracasei in the laboratory, a modified violet crystal plate microtiter method was used according to Stepanovic et al. [17] was used. Based on this, overnight culture in an ELISA plate (three replications), 0.1 dilutions in the final volume of 200 μL was prepared. Fresh MRS broth medium was used to prepare this dilution, and then the plate was heated at 37 °C overnight. After this time, the contents of the plate were emptied, and the plate was washed with sterile saline. After three washes, 200 μL of methanol was added to each well to stabilize the possible structure formed and allowed to stand for 15 min. After this period, the plate was emptied and placed at room temperature to dry. Crystal Violet 2% was used to stain the possible stabilized structure so that 200 μL of crystal violet were poured into each cavity and kept at room temperature for 5 min. After this time, the drain paint and plate were washed with distilled water and placed in the medium until dry. Then, to suspend the possible structure in each cavity, 200 μL of ethanol-acetone (80–20) were poured into each cavity and given for 15 min. Then, the amount of optical density (OD) of the holes was read at 100 nm using the ELISA reader. E. coli (ATCC 25922) was purchased from the Center for Genetic Resources and used as a positive control. Finally, the probiotic strain in terms of biofilm production in four levels of harmful, poor producer, medium producer, and Strong manufacturers were evaluated.

2.9. Evaluation of the cumulative effect of L. Paracasei against pathogens

The ability of the cumulative effect of L. paracasei against B. cereus, E. coli, Y. enterocolitica, and L. monocytogenes was examined by spectrophotometer. For this purpose, overnight culture was prepared from the studied probiotic strain in MRS broth culture medium and pathogenic microbes in appropriate culture media. After 24 h, the bacteria were removed by centrifugation for 10 min at a speed of 4300×g. Rinsing was performed twice with PBS solution, and a suspension of bacteria was prepared with 10 mM phosphate buffered saline (PBS). In the next step, bacterial suspensions at a wavelength of 600 nm in light absorption were set. Each pathogenic bacterium was piled on top and incubated at 37 °C without mixing. Suspensions were sampled to measure adsorption at intervals of 1, 4, and 20 h after incubation, and their adsorption was estimated at a wavelength of 600 nm [18]. Finally, the cumulative effect ratio was measured using the following formula:

$$\mathrm{C}\mathrm{o}\mathrm{a}\mathrm{g}\mathrm{g}\mathrm{r}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\%=\frac{\left[\left(Ax+Ay\right)/2-A\left(x+y\right)\right]}{\left(Ax+Ay\right)/2}$$ (3)

Ax = Adsorption of each pathogen strain before mixing, Ay = Adsorption of L. Paracasei before mixing, A (x + y) = Absorption of L. paracasei and each of the pathogen strains after mixing.

2.10. In vivo safety evaluation

2.10.1. Animal feeding and grouping

Thirty-six Wistar rats (18 male and 18 female rats) were purchased (Pasteur institute of Iran) and transported to the animal house at the University of Maragheh. The animals had a weight range of 230–260 g at the beginning of the experiments. A standard housing condition was established during adaptation period (one week) and throughout the tests as follows: temperature 22 ± 2 °C, 12 h of light (from 8 a.m. to 8 p.m.) and 12 h of darkness, and free access to water and food. All rats were weighed daily at a specific hour. After adaptation period, the rats randomly were assigned into six groups. Grouping of the animals was based on the quantity of the target bacteria in their drinking water. 1. The negative control groups were 6 male rats and 6 female rats that did not receive any bacteria for 28 days. 2. Experimental group 1: There were 6 male rats and 6 female rats that received a final concentration of 1.2 × 9¹⁰ CFU of the target bacteria for 28 days. 3. Experimental group 2: included 6 male rats and 6 female rats, which were given 1.5 × 8¹⁰ CFU daily for 28 days.

During this period, probiotic suspension for feeding the rats was calculated. To this end, L. paracasei was cultured in MRS broth for 24 h at 37 °C containing 5% of CO₂. Cultured bacteria were separated from the supernatant by centrifuging for 10 min at 5000 rpm at 4 °C and were washed with tap water 2 times. Finally, sufficient drinking water was prepared for the rats for 24 h for each of the experimental groups, using 4 McFarland and 0.5 McFarland solutions.

2.10.2. Blood sampling and analysis of blood and serum factors

One day after the last treatment, all rats were anesthetized by injecting a combination of ketamine (80 mg/kg B.W) and xylazine (20 mg/kg B.W). Then, two blood samples were taken directly from the heart using a syringe. One of the blood samples immediately was transferred to the tube containing EDTA anticoagulant for analyzing blood factors. The other sample was transferred to a tube containing clot activator. The sample was used for serum extraction to measure serum liver enzymes, urea, and creatinine.

3. Results and discussion

3.1. Antibacterial effects of L. paracasei

Table 1 showed the results of the antibacterial ability of L. paracasei against seven tested indicator bacteria. In the present study, we investigated the antibacterial activity of cell-free supernatant (CFS) of L. paracasei against seven important pathogenic bacteria. It was observed that all pathogenic strains listed in Table 1 are susceptible to CFS. The most inhibitory effect of L. paracasei was observed against S. aureus (33.2 ± 0.6 mm), while the least inhibitory effect was observed against L. monocytogenes (5.3 ± 0.6 mm).

Table 1.

Results of antibacterial effect of cell-free supernatant L. Paracasei against model pathogenic strains (mm).

Strain	Pathogenic bacteria
	E. coli (ATCC 25922)	St. aureus (ATCC 25923)	Sh. flexneri (ATCC 1234)	Kl. pneumoniae (ATCC 1053)	Y. enterocolitica (ATCC 23715)	B. subtilis (ATCC 19652)	L. monocytogenes (ATCC 13932)
L. paracasei	29 ± 0.4	33.2 ± 0.6	17 ± 0.5	8.3 ± 0.8	9.1 ± 0.7	21 ± 0.9	5.3 ± 0.6

Values are mean ± standard error of triplicates.

In general, it was found that the inhibitory effects of potential probiotic strains against Gram-positive pathogenic bacteria are more common than Gram-negative pathogenic bacteria (except its high growth inhibitory effect on E. coli). This result is quite understandable considering the relative inherent resistance of most Gram-negative bacteria against acidic conditions as well as against bacteriocins produced by lactobacilli [19]. Lactobacilli exert protective or therapeutic effects by producing antimicrobial compounds, lowering pH, competing at binding sites against pathogens, stimulating immune-modulatory cells, and competing with pathogens in food [20]. The role of inhibiting the CFS growth of probiotic lactobacilli has been proven in many previous studies [[21], [22], [23], [24], [25]].

The common feature of most Lactobacillus probiotic strains is the ability of these bacteria to produce metabolites and various antimicrobial compounds, including small peptides, bacteriocins, and organic acids, such as butyric, acetic, and lactic acid [26,27]. Indeed, the production of the above compounds, both in vitro and in vivo, can cause growth inhibitory effects against pathogenic microbial strains. Based on the results, this strain showed the inhibitory effect against all seven tested pathogenic microbes.

3.2. Antibiotic susceptibility test

For this purpose, after three repetitions, the mean diameter of the growth inhibition zone (in millimeters) was obtained and recorded in Table 2. One of the valuable features that a potential probiotic strain can have to be used more in the industry is its lack of resistance to standard antibiotics. Since if the strain is resistant to a particular antibiotic, this resistance is likely to be transmitted to other bacteria, especially pathogenic bacteria [28]. The present study found that the studied strain is resistant to the antibiotics vancomycin, colistin, tetracycline, and erythromycin. It is also sensitive to the antibiotics streptomycin, gentamicin, cefepime, and ciprofloxacin and has moderate resistance to ampicillin, kanamycin, and sulfamethoxazole antibiotics. Thus, it could be concluded that the L. paracasei strain has almost high antibiotic resistance, and this indicates the need for further research on this bacterial strain to determine whether resistance genes are transmissible.

Table 2.

Antibiotic susceptibility results.

Strain	Antibiotics
	V	CL	S	FEB	CFM	SXT	K	CP	TE	E	AM	GM
L. paracasei	R	R	S	S	S	I	I	S	R	R	I	S

V: vancomycin; CL: colistin; S: streptomycin; FEB: cefepime; SXT: sulfamethoxazole; K: kanamycin; CP: ciprofloxacin; TE: tetracycline; E: erythromycin; AM: ampicillin; GM: gentamycin.

Erythromycin results based on R ≤ 13 mm; I: 13–23 mm; S ≥ 23 mm.

Gentamycin results based on R ≤ 6 mm; I: 7–9 mm; S ≥ 10 mm.

Vancomycin results based on R ≤ 12 mm; I: 12–13 mm; S ≥ 13 mm.

I: Intermediate (zone diameter, 12.5–17.4 mm); R: resistant (zone diameter, ≤12.4 mm); S: susceptible (zone diameter, ≥17.5).

3.3. Hydrophobicity

The hydrophobicity capacity of L. Paracasei was 61 ± 0.8% (Table 3), indicating moderate and suitable hydrophobicity of the tested strain. One of the essential characteristics of a potential probiotic bacterial strain is its high hydrophobicity to be established in the intestine and gastrointestinal tract under conditions consumed by the individual and could colonize. Fortunately, the strain studied in this study had good hydrophobicity. The hydrophobicity index of Lactobacillus is a variable factor, and the articles contain different values [29,30].

Table 3.

Results of hydrophobicity tests, autoaccumulation, cholesterol uptake, hemolysis and biofilm formation ability of L. paracasei.

Strain	Hydrophobicity (%)	Autoaggregation (%)	Cholesterol Assimilation (%)	Hemolysis	Biofilm Formation
L. paracasei	61 ± 0.8	78 ± 1.6	74 ± 2.3	γ	Strong

Values are mean ± standard error of triplicates.

3.4. Auto-aggregation

After performing the auto-aggregation test, the amount of auto-aggregation of the strain L. Paracasei was approximately 78% (Table 3). The results of this study showed that the tested strain has a high ability for auto-aggregation. With hydrophobicity, the values for the ability to accumulate spontaneously vary between different probiotic strains, for example, Jena et al. (2013) assessed the three strains of Lactobacillus intestinalis (CS4, CS7, and CS3) and found that the ability to auto-accumulate the above strains was 47.2%, 45%, and 33%, respectively, indicating the highest ability. The researchers also concluded that the overall temperature increases the ability of auto-aggregation [26], whereas the L. paracasei in the present study showed a 78% auto-aggregation ability. In this respect, it has higher auto-aggregation ability than many potential probiotic bacterial strains, which is an advantage for the strain under investigation. Of course, potential probiotic strains with much higher auto-aggregation potential than the current strain have also been identified. For example, Bautista-Gallego et al. (2013) showed that four strains including GG1ST0-4, HM2T0-63, GM2FT1-129, and HG2T1-138 had the auto-aggregation ability of above 90% [31].

3.5. Cholesterol uptake

Table 3 shows the L. paracasei cholesterol assimilation capacity, about 74 ± 2.3%. Moreover, the tested strain could also absorb high cholesterol, indicating a potentially effective probiotic candidate for use in the food sector to produce low-cholesterol products that exhibit hypocholesterolaemic activity in the host.

A number of different mechanisms have explained the hypocholesterolaemic effects of probiotics. Theoretically, cholesterol-lowering effects of probiotics include the following mechanisms: activity of BSH for bile deconjugation [32,33] binding of cholesterol to probiotic cells and incorporation of cholesterol molecules into the probiotic cellular membrane [34]. Production of short-chain fatty acids (SCFAs) from oligosaccharides, deconjugated bile co-precipitates with cholesterol [35], and coprostanol production from converted cholesterol [35]. Meanwhile, the activity of BSH has been identified as the most important probiotic cholesterol-lowering mechanism, despite the existence of other hypocholesterolemic probiotic pathways. Moreover, it has been proposed that lacking BSH activity, the bacteria are unable to eliminate the cholesterol. According to Lambert, Bongers, de Vos, and Kleerebezem (2008), the hypocholesterolemic effects of probiotics are primarily attributed to their interference with micelle formation for intestinal absorption and their inhibition of bile salt hydrolase activity (BSH), which can be found in all strains of lactobacilli and bifidobacteria. It has been discovered that L. paracasei BY2 (LP-BY2) can lower cholesterol levels by controlling the expression of key liver genes involved in cholesterol synthesis (HMGCR and SREBP-2), uptake (LDLR), and outflow (LXR-, ABCA1, ABCG5, ABCG8, and CYP7A1). The EPS of L. paracasei M7 also demonstrated good functional traits, including hypocholesterolemia (70.78%), antioxidant activity (DPPH radical scavenging activity 78.09%; hydroxyl radical scavenging activity 74.64%), and antibiofilm potential (59–64%) against several human diseases.

3.6. Hemolysis test

The tested strain L. paracasei on blood agar medium did not show any color change, and it was found that the above strain is gamma hemolysis (Table 3). The inability to lubricate red blood cells is one of the most basic requirements for using a potential probiotic strain. Only gamma hemolysis strains could be used as having the potential to be used as probiotics. The studies performed on strain L. Paracasei found that this strain lacked the ability to lysis red blood cells in an agar culture medium and displayed gamma hemolysis.

3.7. Biofilm formation ability

Based on the results, it was found that the above strain has a high ability to form biofilms at the edge of the test tube or plate (Table 3). Suppose the probiotic strain can form a biofilm. In that case, it can establish itself more strongly in the intestinal wall and gastrointestinal tract or the female genitourinary system, so the more significant the ability of the newly discovered probiotic strain to form a biofilm, it is the advantage [36]. Fortunately, experiments on the ability to form a biofilm strain, L. Paracasei was performed, and it was found that the above strain can form a high and robust biofilm (Table 3).

3.8. In vivo assay

3.8.1. Investigation of L. paracasei effect on serum liver enzymes

Aminotransferases (including alanine aminotransferase or ALT and aspartate aminotransferase (ALP) along with alkaline phosphatase or ALP are three of the most important enzymes that are an indicator of the health of the liver. Noteworthy, an increase in the concentration of each of the above enzymes in the serum blood, either single or double, or the increase of all three enzymes can be a sign of a specific type of liver damage ranging from cholestasis to necrosis of liver tissue, fibrosis, cirrhosis, etc. According to Fig. 1, we found that the consumption of L. paracasei had no significant effect in raising any of the liver enzymes ALT, AST and ALP compared to the control groups in both gender. Therefore, this demonstrates the lack of hepatotoxicity of the above probiotic strain in the laboratory in both male and female rats.

Fig. 1.

The effect of Lactobacillus paracasei consumption on the amount of ALP, ALT and AST enzymes in rat serum. Panels A and B: ALT in female and male. Panels C and D: AST in female and male. Panels E and F: ALP in female and male. G1: 1 × 10⁸ CFU/mL; G2: 1 × 10⁹ CFU/mL.

Many studies have investigated the possible effect of probiotics on the levels of liver enzymes. Most of the research has concluded that administration of different probiotic strains to experimental animals not only does not induce the increase of serum liver enzymes, but also it reduces the amounts of the above enzymes. This indicates the hepatoprotective role of probiotics [[37], [38], [39], [40], [41]]. Furthermore, evaluation of serum liver enzymes of the tested rats revealed that they are not gender-dependent among the rats fed by L. paracasei. Similar results verify the present findings. Jantararussamee et al. (2021) have shown that probiotic lactic acid bacteria (mixture of L. paracasei, Lactobacillus casei, and Weissella confusa) on thioacetamide (TAA)–induced liver fibrosis in rats lead to noticeably reduced levels of the serum enzymes, less inflammation, and less fibrosis. Interestingly, the group that received TAA + probiotics had less liver damage [42]. In a study, B. animalis subsp. lactis KV9 considerably reduced 22.12% activity of AST and 27.53% activity of ALT in mice serum (p 0.05) while dramatically increasing nearly 56.69% activity of ADH and nearly 68.02% activity of ALDH in the liver. The authors suggested that supplementing with B. animalis subsp. lactis KV9 significantly reduced hepatic inflammation, liver damage, and improved alcohol metabolism [43]. It was found that consumption of four weeks of potential probiotic Lactobacillus acidophilus 5LA in doses of 2 × 10¹⁰ to 1 × 12¹⁰ colony forming units per kilogram of weight. Their body did not have a significant effect on the change in the activity level of serum liver enzymes (including ALT, ALP, and AST).

4. Conclusion

In this study, the L. paracasei strain isolated in previous studies was examined for antibacterial properties and standard antibiogram profiles. After performing the auto-aggregation test, the amount of auto-aggregation of the strain L. Paracasei was approximately 78%. Also, after performing the test, the cholesterol absorption class was about 74%. In addition, after testing, it was found that the above strain has a high ability to form biofilms. Also, in the present study, it was found that the studied strain is resistant to the antibiotics vancomycin, calcitonin, tetracycline, and erythromycin. It is also sensitive to the antibiotics streptomycin, gentamicin, cefepime, and ciprofloxacin and has moderate resistance to ampicillin, kanamycin, and sulfamethoxazole antibiotics. Thus, it could be concluded that the strain under study has almost high antibiotic resistance, and this indicates the need for further research on this bacterial strain to determine whether resistance genes are transmissible.

Author contribution statement

Shadi Shahverdi: Performed the experiments; Wrote the paper.

Amir Abbas Barzegari: Analyzed and interpreted the data.

Reza Vaseghi Bakhshayesh: Contributed reagents, materials, analysis tools or data.

Yousef Nami: Conceived and designed the experiments.

Funding statement

This work was supported by the Agricultural Biotechnology Research Institute of Iran [3-05-0551-88020].

Data availability statement

No data was used for the research described in the article.

Additional information

No additional information is available for this paper.

Declaration of interest's statement

The authors declare no conflict of interest.