Isolation,Selection And Identification Of Antagonistic Marine Bacteria And Safety Assessment For Their Potential Application As Probiotics

Antimicrobial resistance burden and hazard associated with chemical treatment ofinfections demanded for new antimicrobial natural alternative. Oceanic hunt by marineorganism enforces to produce contemporary and novel strategies to compete, surviveand reproduce their population. This fact facilitates to find new solutions to controlpathogenic bacteria. In this study we have isolated272marine bacteria among them136(50%) were antagonistic to at least one of the four pathogenic strains Listeriamonocytogenes, Vibrio cholerae, E. coli and S. aureus. Only two strains exhibitedantibacterial activity against all four test strains, which were identified by16S rDNAsequencing as Brevibacterium halotolerans DK1-SA11and Vibrio kanaloae DK6-SH8.Antagonistic activity and tolerance assessment showed that strain DK1-SA11hasbroader spectrum of antagonistic activity, including superbug MRSA and pathogenicyeast Candida albicans, and can tolerate boiling temperature and pH2-12, as comparewith strain DK6-SH8.Phenotypic characterization by macroscopic and microscopic studies, biochemicalcharacterization by API kits (API20E, API CORENY, API50CHB and API ZYM),and genotypic classification by DNA recA, gyrA and gyrB genes phylogenetic analysisrevealed that, strain DK1-SA11is a novel strain and does not belongs to genusBrevibacterium. The genotypic data confirmed that strain DK-SA11belongs to speciecluster of Bacillus subtilis subsp. spizizenii with96-99.9%similarity with existingdata of DNA sequences.Probiosis characteristics, safety and tolerance assessment of marine isolate DK1-SA11revealed that, strain DK1-SA11can produce alkaline phosphatase, esterase (C-4), esterase lipase (C-8), lipase (C-14), leucine arylamidase, valine arylamidase, acidphosphatase, phosphohydrolase, α-glucosidase and β-glucosidase with variableconcentrations which was quantified by API ZYM kit assay. Gastrointestinal tracktolerance revealed that there was no significant change (P<0.05) in bacterial countsafter4h at pH2.5, and MIC of conjugated bile acids taurodeoxycholic acid andnonconjugated salt deoxycholic acid were≥1.0mM and0.25mM, respectively. StrainDK1-SA11does not produce non-hemolytic and hemolysin BL enterotoxins andnegative for entFM, bceT, cytK, Nhe and Hbl toxin genes. Moreover, strain is non- hemolytic and negative for plasmids and lecithinase production. Additionally strainDK1-SA11was sensitive to all antibiotics listed in European food safety associationstandards for live bacteria usage guideline for animals and human consumptions.Acute and chronic studies confirmed that there was neither death, nor any treatmentrelated health effects on mice. The oral LD50of strain DK1-SA11was greater than2×1011cfu. Acute toxicity results on zebra fish (Danio rerio) revealed that strain hasneither fatal nor visible toxic effect to fish and the lethal concentration (LC50) of DK1-SA11to the fish was>108cfu.mL-1.Growth optimization of Marine isolate DK1-SA11revealed that, among all testedmedia, marine broth2216E was found to be the best media for bacterial growth andantibacterial compounds production. Moreover, pH-7, Incubation temperature25°Cwith≥180rpm for60-72h was optimized conditions. Out of49differentcarbohydrates D-mannose increases the antibacterial activity by33.3%and D-arabitoldecreases antimicrobial activity by44.4%. Cold storage below-20°C of crude CFSshowed activity even after three months; whereas after boiling for10min CFS was100%active. CFS loses its100%antimicrobial activity after lipase, trypsin and papainenzymatic treatments.Analysis and compound profiling of strain DK1-SA11have shown that maximumamount of antibacterial compounds could be extracted from butanol extraction. Thinlayer chromatography, high performance liquid chromatography and massspectrometry data revealed that all three kind of lipopeptides surfactin, iturin andfengycin are being produce by strain DK1-SA11which have similar mass peaksreported before. Taken together all our data we conclude that strain DK1-SA11is anovel marine Bacillus which is qualified for its application as probiotics. Moreover,strain DK1-S11is a putative source of several antimicrobial compounds with broadspectrum of bioactivity.