Therapeutic Potential of Probiotic Lactobacillus Plantarum MYS94 Against Campylobacter Jejuni
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Int.J.Curr.Microbiol.App.Sci (2016) 5(12): 869-883 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 5 Number 12 (2016) pp. 869-883 Journal homepage: http://www.ijcmas.com Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.512.095 Therapeutic Potential of Probiotic Lactobacillus plantarum MYS94 against Campylobacter jejuni K. Poornachandra Rao1, N.K. Hemanth Kumar2 and M.Y. Sreenivasa1* 1Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysore, Karnataka, India-570006 2Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore, Karnataka, India-570006 *Corresponding author ABSTRACT Campylobacter jejuni is one of the most common intestinal pathogen causes of human gastroenterocolitis thereby leading to diarrhea and other serious post- K e yw or ds infectious complications. Controlling the C. jejuni infection is critical for reducing campylobacteriosis for a healthy gut. In the current study, Lactobacillus plantarum Lactobacillus MYS94 was isolated from traditional fermented wine and characterized for its plantarum MYS94, probiotics properties and therapeutic potential against C. jejuni.The probiotic Gastrointestinal attributes revealed that, the strain could resist the gut pH, bile salt tolerance, tract, adherence ability to chicken crop epithelial cells, sensitivity to penicillin, C.jejuni, amoxicillin, bacitracin and erythromycin, further the strain also exhibited inhibitory Probiotics. potential against common food pathogens. The neutralized cell-free supernatant of Lp MYS94 exhibited 62.04% inhibition of Pseudomonas aeruginosa MTCC 7903 Article Info Accepted: biofilm. The study also evaluated the cholesterol-lowering property of the Lp 12 November 2016 MYS94 using hen egg yolk as the cholesterol source. The cholesterol in hen egg Available Online: yolk was assimilated by 62.18% and 31.42% by Lp MYS94and its CFS 10 December 2016 respectively. In addition, Lp MYS94 -CFS tested against C. jejuni by well diffusion and time kill assay revealed a significant effect on C. jejuni with strong inhibitory activity. Collectively, the results suggests that L. plantarum MYS94 could be useful as potential probiotic strain suppressing C. jejuni. Introduction Campylobacter jejuni is one of the leading enterocolitis generally treated with antibiotic causes of the enteric bacterial infections that interventions and supportive therapy can can interfere with normal functions of lead to the development of antibiotic- gastrointestinal tract (GIT). This pathogen resistant strains. Studies revealed that causes gastroenteritis in humans probiotic therapy is proved to be attractive characterized by fever, abdominal cramps control strategy for campylobacter infections and diarrhea (Nishiyama et al., 2014). (Bratz et al., 2014). Dietary probiotics in the Enteric infections like human gastro- form of traditional fermented foods would 869 Int.J.Curr.Microbiol.App.Sci (2016) 5(12): 869-883 be of better choice targeting the beneficial sauerkraut, gut of mammals, etc. (Smelt et effects. Traditional fermented foods are the al., 2013). Food sources, including Marcha, rich source for probiotic lactic acid bacteria an ethnic fermented beverage (Das and (LAB) and are involved with many Goyal, 2014), traditional Japanese fermented therapeutic attributes (Kumar et al., 2013). food (Kanno et al., 2012), traditional Among LAB, Lactobacillus plantarum Chinese fermented foods (Kuda et al., perform an vital role in the production and 2010), traditional Iranian cheeses (Hashemi, preservation of traditional fermented foods. 2016) and traditional sorghum based Several probiotic Lactobacillus strains have fermented food (Rao et al., 2015) have been been documented for their probiotic screened for their potential probiotic L properties such as tolerance to acidic pH and plantarum strains. However, there is a need bile salt, resistance to gastric transit and on the exploration of probiotic attributes as desirable antibiogram (Hashemi et al., well as therapeutic properties such as, 2014). Other probiotic attributes include, cholesterol assimilation, antibiofilm, adherence to the intestinal epithelium of the antimicrobial property and anti- host and inhibition of the pathogenic campylobacter activity. With this bacteria by competitive exclusion and their background, the present study was carried capability in promoting the food safety and out to screen traditional fermented paddy contribute health benefits. wine, collected from Coorg region of Karnataka state, India, for the presence of In addition to the nutritional values, potential probiotic L. plantarum strains and ingestion of LAB through fermented foods studied their therapeutic potential against C. has been suggested to confer a range of jejuni. therapeutic effects including immune- modulation, increased resistance to Materials and Methods infectious illness and malignancy (Soccol et al., 2010). Probiotic supplementation could Isolation of LAB from fermented wine: restore the microbial imbalance caused by Nell wine, prepared from the natural antibiotic treatment in the gut. Studies fermentation of paddy, was collected from revealed that Lact. plantarum exerts the Coorg region of Karnataka, India. LAB therapeutic potential with regard to were isolated by serial dilution and plating important biological functions such as anti- on de Man Rogosa Sharpe (MRS, Hi-Media) inflammatory activity, enhancing the gut agar plates supplemented with 0.25 % (w/v) immune system, antagonistic effects against L-cystine (Paolillo et al., 2009). The plates C. jejuni (gastro-enterocolitis) and were incubated for 48 h at 37 ºC. The well Salmonella enterica (food poisoning) have grown, individual colonies were enumerated been investigated (Bratz et al., 2015; Pattani and the subcultured isolates were stored at - et al., 2013). LAB strains may receive 20 oC in 40 % glycerol for further analysis. (either manufacturer's self-proclaimed or governmental agency affirmed) GRAS Phenotypic characterization of LAB: status regardless of their probiotic Biochemical characteristics were studied as characteristics. per the standard protocol of Cappuccino and Sherman (2004). The isolates were tested for L plantarum is a heterogeneous, indigenous Gram reaction, catalase activity, ammonia species that has been isolated from diverse production from arginine, bile salt hydrolase environmental niches such as human saliva, activity, carbohydrate fermentation with human intestine, human stool, milk, cheese, different carbohydrates viz., glucose, D- 870 Int.J.Curr.Microbiol.App.Sci (2016) 5(12): 869-883 fructose, L-arabinose, mannitol, maltose, In vitro probiotic attributes of Lact. raffinose, D-xylose and sorbitol (Hi-Media). plantarum MYS94 The isolates were assessed for survival ability at different temperatures (10, 37 and Acidic pH tolerance: The pH tolerance of 45oC) and salinity (3, 5 and 7 % NaCl the strain MYS94 was determined as concentrations). Gram-positive and catalase described by Sahadeva et al., (2011). One negative colonies were screened as mL of Lact. plantarum MYS94 (106 presumptive LAB. CFU/mL) was inoculated into three test tubes containing 9 mL of phosphate buffer Molecular identification: The isolate saline (PBS) with pH 2, 3 and 6, mixed MYS94 was grown in 5 mL MRS broth thoroughly, and incubated for 0, 90 and 180 supplemented with 0.25 % (w/v) L-cystine min. After incubation, plating was carried (Paolillo et al., 2009) at 37 ºC overnight in a out, and the inoculated plates were rotary shaker. The genomic DNA was incubated anaerobically at 37 ºC for 48 h. extracted by conventional phenol- The uninoculated MRS agar plates were chloroform method and the 16S rRNA gene served as controls. Acid tolerance of the was amplified using the primer 27F strain MYS94 was determined by comparing (5'AGAGTTTGATCMTGGCTCAG 3') and the cell numbers on test MRS agar plates 1492R (5' TACGGYTACCTTGTT with control plates. ACGACTT 3') (Sahadeva et al., 2011). PCR amplification reactions were carried out in a Bile salt hydrolase assay: Bile salt 25 µL reaction mixture. 1 µL of the DNA hydrolysis ability of the Lact. plantarum was amplified with 2.5 µL of 10X PCR MYS94 was determined by direct plate buffer, 2.5 µL of 25 mM MgCl2, 2.0 µL of 2 assay (Kumar et al., 2012) with minor mM dNTPs, 1.0 µL of 20 pmol primer 27F, modifications. Briefly, overnight culture of 1.0 µL of 20 pmol primer 1492R, 0.125 µL Lact. plantarum MYS94 was added onto of LA Taq and sterile deionized water up to sterile discs (Hi-Media, Mumbai) placed on 14.875 µL. PCR conditions were as follows: the MRS agar plates supplemented with 0.5 initial denaturation at 94 °C for 5 min, % (w/v) taurodeoxycholic acid (TDC) and o followed by 30 cycles of denaturation at 94 0.037 % of CaCl2 and incubated at 37 C for °C for 30 sec, annealing for 57 sec at 45 °C, 72 h. MRS agar plates without extension at 72 °C for 1: 30 min, followed supplementation of TDC were served as by final extension at 72 °C for 7 min. control. The presence of precipitated bile Amplicons were confirmed by agarose gel acid around the culture discs was considered (1 %) electrophoresis and documented using positive for bile salt hydrolysis. Syngene G-box gel documenting system. Antibiotic susceptibility test: Antibiotic Nucleotide sequence accession number: susceptibility of Lact. plantarum MYS94 Complete sequence of the 16S rRNA gene was tested against 8 selected antibiotics was confirmed with NCBI BLAST database. using antibiotic discs