مقالات پذیرفته شده در سومین کنگره بین المللی زیست پزشکی
Antibacterial Activity of Bacteriocin from native Lactobacilli Isolates Against clinical Gram-Positive Pathogenic Bacteria
Antibacterial Activity of Bacteriocin from native Lactobacilli Isolates Against clinical Gram-Positive Pathogenic Bacteria
Shahrbanoo Asgarian,1,*
1. Tarbiat Modares University, Faculty of Medical Sciences, Department of Bacteriology
Introduction: Several probiotic species such as lactobacillus strains have direct antimicrobial effects that include inhibiting the growth of pathogenic bacteria through the production of antimicrobial factors.
The spread of multiple antibiotic resistance is a significant major challenge for healthcare. Lactobacilli, is that the largest cluster of lactic acid bacteria, turn out amounts of antimicrobial metabolites like fatty acids, hydrogen peroxide, organic acids, diacetyl, ammonia, and bacteriocins. These bacteriocins are biologically active peptides against both gram-positive and negative pathogenic bacteria, protozoa, yeast, fungi, and viruses. Thus, there is an urgent need to develop natural alternatives to antibiotics. This study aimed to determine the antibacterial activity of bacteriocin produced against Bacillus cereus, Bacillus subtilis, and Staphylococcus epidermidis.
Methods: Kohgiluyeh and Boyer-Ahmad Province in the South West of Iran
Tribal areas as pristine areas and enable farms were selected for sampling and isolated Lactobacilli from yogurt samples. Isolated Lactobacilli from yogurt samples on MRS Agar was done in anaerobic conditions. Features such as the appearance of colonies, bacterial morphology, catalase and biochemical tests, growth temperatures in 37 o C and 45 o C were studied. The DNA extraction was performed by the Sina clon kit for the isolation of DNA from Gram-positive bacteria. Designed for the qualitative detection of lactobacilli spp. was done by Lactobacillus spp. PCR Detection Kit from Iranian Gene Fanavar Institute.
The disc diffusion method was used for screening antibacterial effects of Lactobacillus against clinical isolates of Bacillus cereus, Bacillus subtilis, and Staphylococcus epidermidis.
Bacteriocin production was done by inoculating each culture of LAB to MRS broth. It is a medium used to produce bacteriocins that bacteriocins can be released into the medium. LAB cultures in MRS broth were incubated at 32 ° C for 24 hours and will turn turbid due to the release of antimicrobial agents. For Bacteriocin Production, cultures were inoculated into MRS broth, then vortexed after that incubated at 32 ° C for 24 hours. The liquid culture was centrifuged at a speed of 10,000 rpm at 4°C for 15 minutes with a refrigerated micro-centrifugation device. The filtrate was neutralized to pH 7.0 using a pH meter by adding 0.1N NaOH solution. The filtrate was sterilized by bacteria filter with a diameter of 0.22 μm, into a sterile tube to obtain an antibacterial supernatant.
The antibacterial activity test was done by using the disc diffusion method. A 6 mm diameter sterile disc paper was immersed in a bacteriocin supernatant. Disc papers were placed on
Muller Hinton Agar culture medium contained test bacteria. The zone diameter produced around the disc paper was measured using a caliper after incubation for 24 hours at 37° C.
Results: 158 yogurt samples were examined in different areas of the temperate and tropical province. Lactobacilli isolates were sorted out which were divided into three groups: obligate homo-fermentative, facultative homo-fermentative, and obligate hetero-fermentative. Fifty-one Lactobacillus isolates from traditional yogurt showed strong antibacterial activity against the clinical isolates of Bacillus cereus, Bacillus subtilis, and Staphylococcus epidermidis. Lactobacilli produces the optimum bacteriocin at the incubation time of 24 hours and the maximum incubation time is 48 hours. The maximum time in the growth cycle for bacteriocin production depends on the type of bacteria and can occur from the log phase to the initial stationary phase.
For example inhibition zone produced from L. brevis, L. casei and L. plantarum against B. cereus were 15.30, 16.73 and 14.60 mm, against B. subtilis were 13.48, 14.30 and 12.63 mm and against S. epidermidis were 11.47, 14.75 and 12.65 mm respectively. The activity of each bacteriocin was active in the pH range of 3-10 and heating temperature of 45-120oC. The statistical test showed that the variation of pH and heating had significant differences (p<0.05).
Conclusion: Clinical isolates have been shown that lactobacilli killed pathogens by pore formation or inhibition of cell wall synthesis. Given the diversity of the lactobacilli and maintenance of native strains in areas of animal husbandry could lead to the enrichment of native reserves.
Therefore, further studies need to be conducted to include in vitro and in vivo analyses, human trials or animal model studies. These results and recent successes are supportive for the rational design of multi-strain lactobacilli and their bacteriocin's dose for clinical applications and drug formulation.