Abstract

Background: Multi-antibiotic resistance, which has increased in recent years, poses a serious societal threat as it makes the fight against deadly infection-causing pathogens even more complex and difficult. As such, the search for naturally resistant probiotic microorganisms and metabolic products obtained from these organisms to prevent infections, as an alternative to antibiotics, is crucial. In this context, preventing the quorum sensing (QS) mechanism that provides communication among bacteria is considered a mechanism that can prevent the colonization and progression of deadly infections.
Aims: To determine the QS mechanism and the immunological effects and various biological and biochemical characterizations of exopolysaccharide (EPS) obtained from the Lactobacillus paracasei L1 strain isolated from the vaginal microflora of healthy women.
Study Design: Experimental laboratory study.
Methods: The antibacterial ability, the antibiofilm and QS forming activities, and interferon (IFN)-γ and interleukin (IL)-10 production capacities of EPS were determined. The total antioxidant capacity (TAC), the surface morphology of EPS by scanning electron microscopy (SEM), the presence of functional groups, and the monosaccharide composition were determined by gas chromatography-mass spectrometry (GC-MS).
Results: L. paracasei L1-EPS demonstrated a strong antibiofilm activity on Escherichia coli (65.14%), Staphylococcus aureus (63.27%), and Pseudomonas aeruginosa (54.21%) at a concentration of 5.0 mg/ml. The anti-QS activity of EPS was found to be quite high at 10 mg/ml EPS concentration. In the study performed with human peripheral blood mononuclear cells (hPBMC), the immunostimulatory IFN-γ value was higher (45 ± 0.0.3) than that in the experimental group, while the IL-10 value was lower than that in the control group (36 ± 0.05). The TAC value of L. piracies L1- EPS was found to be 76 μg/ml at 1,000 μg concentration. According to the GC-MS analysis results, glucose constituted 13.80% of the monosaccharide composition of EPS, while alpha-D-galactose constituted 13.89%.
Conclusion: Interestingly, EPSs of L. paracasei L1 strain, which have not been reported previously, demonstrated high anti-QS and antibiofilm properties, making EPSs a prospective compound for application in the pharmaceutical and food industries owing to their strong antimicrobial and antioxidant capacities.

INTRODUCTION

Exopolysaccharides (EPSs) are microbial polysaccharides produced by various bacteria in response to biotic and abiotic stress factors or to adapt to harsh environmental conditions. EPSs have the capacity to form a capsule that can be released into the external environment as slime EPS by the enzymes associated with the cell wall or to adhere to a bacteria’s outer cell wall.^1-3 EPS production during or at the end of the logarithmic developmental stage⁴ facilitates physiological tasks such as phagocytosis, phage attacks, antibiotics, toxic metal ions, and protection against osmotic stress.⁵     

EPSs have attracted the significant interest of researchers at the food institute due to their various industrial properties and physiological characteristics.⁶ In addition, EPS offers various physiological functions such as antioxidant, anti-cancer, immunomodulatory, antibacterial, low glycemic index, anti-hypertensive, anticholesterol effects, and biofilm formation.^7,8 Therefore, the structure-function relationship and the various biological properties of EPS continue to be an important research topic.¹

Quorum sensing (QS) is a system that enables the communication between bacteria through extracellular signaling molecules after the bacterial population density reaches a certain critical point.^9,10 Through QS, bacteria modulate the virulence gene expression and biofilm formation and interact with other microbes. In addition, bacteria affect cellular processes such as spore formation, toxin production, and disinfectant resistance. The inhibition of the QS mechanism appears to be a good mediator in preventing the formation of a biofilm layer by pathogens, thereby controlling the bacterial infection.¹¹

Studies that determined the effectiveness of EPS obtained from lactic acid bacteria on the QS mechanism are limited in the literature³ and there is no study published on the biological characterization of EPS obtained from L. paracasei L1 strain, which has a particularly strong probiotic characteristic. Therefore, the results of this study can be considered preliminary for future applications.

This study aimed to determine the anti-microbial, anti-QS, and antibiofilm activity of EPSs obtained from probiotic Lactobacillus paracasei L1 species isolated from the vaginal flora as well as to determine the IFN-γ- and IL-10-production capacities and the various biochemical characterizations of EPS.

MATERIALS AND METHODS

Obtaining and Culture Conditions of Bacteria

L. paracasei L1 strain was isolated from the vaginal flora of healthy women. Wholesome women who applied during 2016-2017 were selected for this study. The subjects were of age 18-45 years, non-menopausal, used contraception for protection, and had not used antibiotics in the last three months.¹² L. paracasei L1 was developed and activated in solid media and Man Rogosa Sharpe (MRS), Merck broth. The cultures were incubated in an anaerobic jar at 37 °C.¹²

The strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Bacillus cereus ATCC 14579, Listeria monocytogenes ATCC 19115, Staphylococcus aureus ATCC 25923, Enterobacter aerogenes ATCC 1304 ve Shigella dysenteriae ATCC 11456) used in this study were sourced from the biotechnology laboratory’s stock culture. Clinical samples (P. aeruginosa, E. coli, Klebsiella pneumoniae, Enterococcus faecalis ve methicillin-resistant S. aureus [MRSA]) were obtained from the patients hospitalized in the intensive care unit. The clinical and type strains were grown in tryptone soy broth (TSB) medium at 37 ^oC for 24 h.

Purification of Exopolysaccharides

To obtain pure EPS from the tested bacteria, some changes were applied to the methods described by Onbas et al.¹³ before use. In this study, the test bacteria were activated at the appropriate temperature the night before. After being heated to 100 °C for 15 min, the activated cultures were centrifuged for 15 min at 13,000 rpm. The next stages of the study were conducted as stated by Onbas et al.¹³ Pure EPS products were stored at 4 °C before being used in subsequent analyzes.

Antimicrobial Activity of L. paracasei L1-EPS

The antimicrobial property of EPS produced from the L. paracasei L1 strain was investigated by using the agar well-diffusion method. The cultures of the pathogen test bacteria were homogenized into Mueller Hinton Agar (Merck) media spread after activation in TSB medium at 37 °C for 18 h in the appropriate media. The antimicrobial activity study was conducted as indicated by Kiray, 2021.¹⁴ The study was evaluated by taking an average of three tests.

Antibiofilm Activity of L. paracasei L1-EPS

Various biofilm-producing pathogenic microorganisms were used to determine the antibiofilm activity of pure EPS extract from L. paracasei L1. At 37 °C, the pathogenic bacteria were grown overnight in the TSB medium. After serial dilutions, 100 ml of pure EPS extracts (0.2, 0.5, 1.0, 2.0, and 5.0 mg/ml) and 100 ml of bacterial cultures (OD₆₀₀ 0.132) were transferred into a 96-well polystyrene microtiter plate following serial dilutions and heating to 37 °C. The plate was incubated for 24 h at 37 °C. The antibiofilm study described by Theodora et al.¹⁵ was performed as stated. The measurement of the wells was performed at 595 nm with the ELISA reader (Epoch2 BioTek, USA). Bacterial cultures without EPS were used as positive control and the TSB medium as blank. The formula given below was used to calculate the percentage of biofilm inhibitory potential. The experiment was repeated thrice.¹⁵

Antibiofilm activity (%) = (1 - OD sample/OD control ) x100

Antiquorum Sensing Activity of L. paracasei L1-EPS

In our study, C. violaceum ATCC 12472 was used as a monitor strain to determine the anti-QS activity of EPS extracts obtained from L. paracasei L1 strain, which we used as the test bacteria C. violaceum was cultivated in 50 ml of the TSB culture and incubated for 48 h at 28 °C. A sterilized Drigalski spatula was used to spread 100 ml of C. violaceum (OD₆₀₀ 0.132) on TSA. Wells with a diameter of 6 mm were opened on the cultivated culture plates and EPS extracts (100 ml) were applied to the wells at the concentrations of 10 mg/ml and 5 mg/ml. Through the zone generated in the violacein pigment background, the anti-QS activity was measured.¹⁶

The Immunomodulatory Effect of L. paracasei L1-EPS

According to Vissers’ instructions, healthy volunteer hPBMCs were isolated, and LAB treatment was performed as previously explained. Human PBMCs (2 x 10⁵ cells/well) were seeded in a 96-well tissue culture plate for 48 h at 37 °C and 5% CO₂ with 2 x 10⁶ colony-forming unit LAB PBMCs (LAB ratio 1:10). E. coli lipopolysaccharide (1 µg/ml) was used as a control in the study. IFN-γ and IL-10 concentrations were determined in accordance with the manufacturer’s instructions depending on the enzyme.¹⁷

Determination of the Total Antioxidant Capacity of L. paracasei L1-EPS

The test solution was prepared by dissolving 1,235 g of 4 mM ammonium molybdate [0.9942 g of sodium sulfate (28 mM) and 45 ml of sulfuric acid (0.6 M) in 250 ml of distilled water] and then tested to assess its overall antioxidant capability. EPS of various concentrations (50-1,000 µg) was dissolved in 1 ml of antioxidant and then incubated for 15 min. Post-incubation absorbance values were measured at 695 nm [UV-VIS spectrophotometer (Shimadzu 1601)]. Ascorbic acid was used as the standard.¹⁸ The test was conducted thrice.

Electron Microscope Analysis of L. paracasei L1-EPS

EPSs obtained from the L. paracasei L1 strain were glued to aluminum studs and sprayed with gold. The surface morphologies of the EPSs were then visualized with field emission SEM (Quanta FEG-250 SEM) at 2-KV acceleration voltage.¹⁹

Fourier Transform Infrared (FTIR) Spectroscopy

The FTIR spectra were determined by using the Thermo Scientific (Nicolet 6700 FTIR) spectrometer according to the method reported by Shang et al.²⁰ so as to detect the presence of various functional groups in EPS.

Monosaccharide Composition of L. paracasei L1-EPS

The monosaccharide content of EPS was ascertained by using gas chromatography-mass spectrometry (GC-MS). In the study, 2 ml of purified EPS and 2 ml of 2 M trifluoroacetic acid were hydrolyzed at 120 °C for 2 h. After reduction with potassium borohydride dissolved in the hydrolysates, it was methylated with ammonium hydroxide (NH₄OH) and acetic anhydride (CH₃CO)₂O. The procedure for determining the monosaccharide composition was performed as specified by Kanamarlapudi and Muddada.¹⁸

Statistical analysis

Unless stated otherwise, all assays were repeated at least thrice on separate occasions, and the mean and standard deviations were calculated. The SPSS (Ver23, Chicago, IL, USA) package program was used to perform the statistical analysis for the study. All findings are expressed as the mean standard deviation. Student’s t-test was used to compare the reference and test groups to determine the antimicrobial, immunomodulatory, and antioxidant capacities of EPS. EPS’s antibiofilm test data were analyzed by two-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test. p = 0.05 was considered to indicate the significant difference between the groups.

RESULTS

Antimicrobial Activity of L. paracasei L1-EPS

The antimicrobiological action of EPS obtained from the L. paracasei L1 strain, which possesses strong probiotic properties, was determined. EPS, which exhibits an inhibitory effect on both gram-negative and gram-positive bacteria, is found on P. aeruginosa ATCC 27853 and E. aerogenes ATCC 1304 strains, which are especially of high clinical importance from gram-negative bacteria, and S. aureus ATCC 25923 and B. cereus ATCC 14579 strains were determined to have a strong antimicrobial effect. The antimicrobial zone diameters of EPS are listed in Table 1.

Antibiophilim Activity of L. paracasei L1-EPS

According to Figure 1, the antibiofilm activity of EPS obtained from L. paracasei L1 on three pathogens (E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. aureus ATCC 25923) was investigated and their inhibitory activities on biofilm formation were determined by increasing the concentration accordingly. In parallel with the increase in the EPS concentration (0.2-5.0 mg/ml), an increase in biofilm inhibition was also observed. At a concentration of 5.0 mg/ml, EPS showed the highest biofilm inhibitions for E. coli ATCC 25922 (84.17%), S. aureus ATCC 25,923 (88.15%) and P. aeruginosa ATCC 278,533 (68.31%). EPS products demonstrated strong inhibitory effects on E. coli and S. aureus biofilm, but lower activity on P. aeruginosa. These results indicate that EPS from L. paracasei L1 can be used in the food industry against various biofilm-forming microorganisms to control microbial biofilm formation.

Antiquorum Sensing Activity of L. paracasei L1-EPS

C.violaceum ATCC 12472 was used as an indicator strain to determine the anti-QS activity of EPS obtained from L. paracasei L1. In the study performed at different concentrations, the anti-QS activity of EPS was found to be quite high at 10 mg/ml and 5 mg/ml concentrations. These results were expressed in centimeters of the blurred zone created against the background of the violacein pigment. As shown in Figure 2, EPS formed a zone diameter of 24 cm at the concentration of 20 mg/ml, with a zone diameter of 18 cm at the concentration of 10 mg/ml.

The Immunomodulatory Effect of L. paracasei L1-EPS

PBMCs obtained from healthy participants were cultured in tissue culture plates for 48 h with pure EPS obtained from the L. paracasei L1 strain. In the study, the levels of immunostimulatory IFN-γ and immunomodulatory cytokine production were measured by ELISA As seen in Table 2, pure EPS produced higher IFN-γ than controls and the IL-10 concentrations were lower than the corresponding control values. The anti-inflammatory response of EPS from L. paracasei appeared to be strong. The test was conducted thrice.

Determination of Total Antioxidant Capacity of L. paracasei L1-EPS

In this study, the total antioxidant capacity (TAC) of EPS obtained from L. paracasei L1 strain demonstrated dose-dependent activity in the concentration range of 36-76 μg/ml. As seen in Figure 3, the antioxidant capacity increased with an increase in the EPS concentration.

SEM Analysis of L. paracasei L1-EPS

It is a common practice to apply SEM to examine the morphology of macromolecules. EPS images of the L. paracasei L1 strain are given in Figure 4. Large numbers of free hydroxyl groups were exposed by the very porous structures, as shown by SEM, in the polysaccharides, which improved hydration and water-retention capacities.¹⁹

FTIR Analysis of L. paracasei L1-EPS

FTIR spectra were determined in the absorbance mode of 4,000 to 400 cm to further investigate the functional groups of purified EPS. In the FTIR analysis of EPSs of L. paracasei L1 strain, a dense and wide band of approximately 3342.67 cm^-1 was obtained. These bands belong to the OH group. The band at around 1636.37 cm^-1 appeared as bands of C = O bonds in a monosaccharide structure. Bands near 1,100 cm¹ are associated with the C-O tensions of carbohydrates and aromatic groups (Figure 5). This observation was made in accordance with the EPS spectra provided in the literature.²³

Monosaccharide Composition of L. paracasei L1-EPS

The composition of EPS monosaccharides was established by using the GC-MS data. According to Figure 6, GCMS analysis of bacterial EPS peaks revealed a matching glucose concentration of 13.80% and an alphaD-galactose concentration of 13.89%.

DISCUSSION

In various studies, EPSs obtained from Lactobacillus strains have demonstrated antimicrobial activities. The antimicrobial activity of EPS obtained from the L. plantarum S123 strain was performed on E. coli ATCC25922 and S. aureus ATCC29213 strains showed a higher effect on E. coli.²¹ In another study, EPS obtained from L. rhamnosus strain was found in E. coli, Salmonella typhimurium, and Staphylococcus petrasii subsp. pragensis KY196531, with different degrees of antimicrobial activities in each.²² In an additional study, EPS belonging to the Lactobacillus brevis strain demonstrated a strong antimicrobial effect on E. coli and S. aureus strains. These results obtained were similar to our study data.²³

The biofilm-inhibiting ability of EPS obtained from L. fermentum S1 strain (0.25-2 mg/ml) on E. coli and S. aureus was investigated, and the highest inhibition rate on E. coli was found to be 32.25%, while the highest inhibition rate on S. aureus was found to be 42.77%.¹⁹ In our study, EPS obtained from L. paracasei L1 strain at a concentration of 5.0 mg/ml revealed the highest biofilm inhibitions in E. coli ATCC 25922 (84.17%), S. aureus ATCC 25923 (88.17%), and P. aeruginosa ATCC 278533 (68.31%). EPS products demonstrated a strong inhibitory effect on E. coli and S. aureus biofilm, albeit with lower activity on P. aeruginosa. These results suggested that EPS obtained from L. paracasei L1 can be used in the food industry as a food-grade biofilm inhibitor against various biofilm-forming microorganisms to control microbial biofilm formation.

The physiological formations applied by probiotic microorganisms to prevent the biofilm formation process remain to be fully clarified. In some in vitro studies, the autoaggregation and coaggregation abilities of probiotic microorganisms prevented the biofilm formation as well as QS formation, and the various virulence effects were affected by probiotic microorganisms.^24,25 In addition, some probiotic microorganisms possessed properties that prevented the physiological results of bacteria because of QS. Gόmez et al.²⁶ demonstrated comparable inhibiting effects on E. coli O157:H7, Salmonella typhimurium, and L. monocytogenes. In our previous study, various probiotic Lactobacillus spp. (L. paracasei and L. rhamnosus, L. gasseri, L. crispatus, L. acidophilus, and L. acidophilus) the strain was determined to have anti-QS characterization, and the L. paracasei L1 strain used in our study demonstrated strong anti-QS activity.¹⁴

In this study, EPS obtained from L. paracasei L1 strain demonstrated strong anti-QS activity at the concentrations of 10 mg/ml and 5 mg/ml, which is consistent with our previous findings. According to the results obtained, a feature that enabled the probiotic Lactobacillus strains to display anti-QS activities is the production of EPS. There are limitations studies in this area. In the literature, no similar study has been reported on EPS obtained from the vaginal microflora-derived L. paracasei strain. We believe that the present results will facilitate future work in this direction.

In this research, we compared the cytokine profiles, IL-10-production capabilities, and antibiofilm and anti-QS properties of strains with known probiotic relevance. Comparisons between the immunomodulatory impact of L. paracasei EPS on hPBMC cytokine profiles and proliferative response revealed that L. paracasei L1 EPS produced higher IFN-γ than the controls, and the IL-10 concentrations were lower than the control concentrations. The production of inflammatory cells by Lactobacillus spp. is a pathway that demonstrates the helpful influence of healthy bacteria on immune function.²⁷

One of the most significant macrophages with cytokine-stimulating abilities is IFN-. It is crucial for both acquired immunity and innate immune response. Notably, in several studies, lactic acid secreted by vaginal Lactobacillus species was found to inhibit the production of IFN- in T- and natural killer cells. This production is a factor that protects against bacterial vaginosis.^10,28 The immunomodulatory effect of EPSs obtained from L. paracasei has not been investigated so far. In this regard, our study is expected to set the standard for other researches.

Functional units of EPS obtained from the L. paracasei L1 strain were detected by FTIR spectroscopy. This analysis identified a dense and wide band around 3342.67 cm^-1. These bands indicate a significant number of hydroxyl groups, representing the characteristic absorption band of carbohydrates. The present data conform to those reported previosuly.¹⁸

Owing to their capacity to scavenge free radicals, bind metal ion catalysts, and engage in reduction activities, natural antioxidants can perform preventive roles against various illnesses.²⁹ The antioxidant activity of EPS obtained from L. paracasei L1 under in vitro conditions is illustrated in Figure 3. The antioxidant capacity of EPS suggested varying activity depending on the dose increase in the concentration range of 36-76 μg/ml. The 76.25% capacity determined in the present study was obtained from EPS belonging to the

L. paracasei strain isolated from TAC sauerkraut with a similar rate of 76.34%.³⁰ Past studies in this area have reported that EPSs obtained from various lactic acid bacteria demonstrate a similarly strong antioxidant activity.³¹

In this study, glucose, which has been accepted as a valuable product for the food industry, was found at 13.80%, while alpha-D-galactose was found at the rate of 13.89%. The information gleaned from the study’s GC-MS analysis is compatible with those of previous reports on EPS. In previous studies, EPSs obtained from Lactobacillus strains contained both glucose and galactose, generally with the same 1:1 molar ratios.³⁰

In this study, EPS derived from L. paracasei L1 strain, which was previously proven to possess a strong probiotic characteristic,¹² demonstrated strong properties in terms of various biological activities. It has been determined that EPS, which exhibits strong antimicrobial, antibiofilm, and anti-QS activities, exhibits high antioxidant activity and strong anti-inflammatory response to the immune system. Considering the outcomes of this study, we concluded that L. paracasei L1-EPS can disperse E. coli and S. aureus biofilms and inhibit biofilm formation by inhibiting the QS mechanism involved in biofilm formation. In this study, the heteropolysaccharide structure of EPS was determined by FTIR and GC-MS analysis, and its porous structure was identified by SEM images. Owing to its potent antibacterial antibiofilm, anti-QS, and antioxidant activities, it could optimize the production and composition of antioxidant and antibiofilm agents in food and pharmaceutical industries for potential applications.

Ethics Committee Approval: The Kırıkkale University Faculty of Medicine Ethics Committee gave its approval to the protocol. Testing was performed three times.

Data Sharing Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Authorship Contributions: Concept- E.K., N.M.R.; Design- E.K., N.M.R.;  Data Collection or Processing- E.K., N.M.R.;  Analysis or Interpretation- E.K., N.M.R.;  Literature Search- E.K., N.M.R.;  Writing- E.K., N.M.R.

Conflict of Interest: No conflict of interest was declared by the authors.

Funding: The authors declared that this study received no financial support.

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