The Halotolerant Probiotic Bacterium Enterococcus lactis ASF-2 from Al-Asfar Lake, Saudi Arabia, Reduces Inflammation in Carrageenan-Induced Paw Edema

Abstract

Inflammation-related diseases are major causes of mortality and disability worldwide. This study aimed to identify and investigate probiotic bacteria that could be present in Al-Asfar Lake in Al-Ahsa City, Saudi Arabia to prevent the inflammatory responses of carrageenan-induced paw edema. In total, seven active strains were isolated, and three isolates (ASF-1, ASF-2, and ASF-3) exhibited a positive Gram stain and viable growth at 20% NaCl salinity; they also lacked catalase and hemolytic activities and had high levels of cell surface hydrophobicity (CSH). They also demonstrated potent antibacterial activity against Salmonella typhi and Staphylococcus aureus. These results revealed that ASF-2 had probiotic qualities, and it was selected for further research. ASF-2 demonstrated significant anti-inflammatory effects in an experimental model of carrageenan-induced paw edema; the experimental model showed decreased levels of pro-inflammatory markers, such as interleukin 6 (IL-6), interleukin 17 (IL-17), and transforming growth factor-β (TGF-β), and an increased level of an anti-inflammatory marker (interferon gamma (IFN-γ)). Animals in the control group saw a 45% decrease in edema when compared to mice in the carrageenan group. When comparing tissue damage and infiltration in the ASF-2-treated and non-treated mice, the histological examination of the sub-planar tissues of the hind leg revealed that the inflamed tissues had healed. The 16S rRNA sequencing method was utilized to establish that ASF-2 is, in fact, Enterococcus lactis with a 99.2% sequence similarity. These findings shed further light on ASF-2's potential as a biocompatible anti-inflammatory medication.

Keywords: Al-Asfar Lake; Enterococcus lactis; halotolerant bacteria; inflammatory disorders.

Figure 1

Screening of hydrophobicity and gastric and bile juice tolerance. Evaluation of hydrophobicity for ASF-2 and ASF-3 was carried out using heptane as the nonpolar solvent. (A) Cell survival in log10X7 for hydrophobicity and bile tolerance. (B) Tubes showing hydrophobicity and acid and bile salt tolerance of ASF-2 where cells tend to aggregate to the upper hydrophobic layer compared to the control. Notice the orange color of the ASF-2 tube indicating well-grown cells compared to the control. Acid and bile tolerance were on all selected isolates using bile sale 0.3% and pH 2.0 of MRS broth.

Figure 2

Potential probiotic strains were used to assess the antimicrobial properties using the well diffusion method. The isolates ASF-2 and Asf-3 were used in this study. The cell-free extracts were prepared and tested against Salmonella typhi and Staphyllococcus aureus (ATCC strains). Scale bar 2 cm.

Figure 3

Evolutionary relationships of taxa. The evolutionary history was inferred using the UPGMA. Enterococcus lactis OR121375 had 99.2% similarity with E. lactis NR117562. Evolutionary analyses were conducted in MEGA5. * Indicates the lactic acid bacteria isolated in this experiment.

Figure 4

Antioxidant activity of probiotic cell-free extracts. ABTS and DPPH were used to evaluate the antioxidant activity. Gallic acid was used as the positive control of the experiment. Values represent mean ± SEM for triplicate. * Values decreased significantly from control.

Figure 5

Effect of various drugs on impairment in motility associated with carrageenan-induced inflammation. Edema was induced by injecting 0.1 mL of a 1% solution of carrageenan into the subplantar surface of the right hind paw. The probiotic was administered orally 30 min after injecting the inflammagen. Pathology score and tissue damage were assessed using the H&E staining method. Data are expressed as mean ± standard error of seven mice per group. * Values decreased significantly from control. ** Values decreased in highly significant manner from the control. Group A: control mice; Group B: carrageenan control; Group C: carrageenan with ASF2-fed mice; Group D: ASF2-fed mice.

Figure 6

ffect of probiotic bacteria on carrageenan-induced paw edema. (A): Size of paw edema (B): responses of pro-inflammatory markers, such as interleukin 6 (IL-6), interleukin 17 (IL-17), and transforming growth factor-β (TGF-β), and an increased level of an anti-inflammatory marker (interferon gamma (IFN-γ). PBS: control mice with buffer solution; CIE: carrageenan-induced edema; CIE+ASF-2: carrageenan with ASF2-fed mice; ASF-2: ASF2-fed mice. The results are presented as the mean ± SD (n = 5). * Values decreased significantly from control. Effect of probiotic bacteria on carrageenan-induced paw edema. (A,B) The results are presented as the mean ± SD (n = 5). * Values decreased significantly from control.