Enhanced antibacterial metabolite production through the application of statistical methodologies by a Streptomyces nogalater NIIST A30 isolated from Western Ghats forest soil.

Streptomyces strains isolated from Nelliyampathy forest soil of Western Ghats, Kerala, India were evaluated for their antibacterial efficacy against two indicator pathogenic bacteria (Escherichia coli and Staphylococcus aureus). Among 140 strains tested, sixteen recorded potent antibacterial properties and were further screened against eleven bacterial pathogens. A strain identified as Streptomyces nogalater and designated as NIIST A30 exhibited maximum inhibition against all the test pathogens. Among the eight fermentation media tested, inorganic salts starch broth recorded the best for antibacterial production. The ethyl acetate crude extract exhibited antioxidant properties with IC50 value of 30 μg/mL and had no cytotoxicity towards L6, H9c2 and RAW 264.7 cell lines up to a concentration of 50 μg/mL. Maximum metabolite production was achieved in pH 7.0 at 35°C after 7 days incubation. The significant media components for maximum metabolite production were optimized through response surface methodology employing Plackett-Burman and Box-Behnken designs. The composition of the final optimized medium was soluble starch, 14.97g; (NH4)2SO4, 2.89g; K2HPO4, 2.07g; MgSO4.7H2O, 1g; NaCl, 1g, CaCO3, 2g; FeSO4.7H2O, 1mg; MnCl2.7H2O, 1mg; and ZnSO4.7H2O, 1mg per litre of distilled water. The optimization resulted an antibacterial activity of 28±1.5mm against S. epidermidis which was in close accordance with the predicted value of 30 mm. It is also evident from the result that an increase of 86.66% antibacterial production was recorded in optimized media. The chosen method was economical, efficient and useful for future antibacterial drug discovery from a broad spectrum metabolite producer like Streptomyces nogalater NIIST A30

<i>In vitro</i> antibacterial activity of NIIST A30 against test pathogens.

<p><i>In vitro</i> antibacterial activity of NIIST A30 against test pathogens.</p

Statistical analysis of effects of variables (media components) on antibacterial activity employing Plackett-Burman design.

<p>Statistical analysis of effects of variables (media components) on antibacterial activity employing Plackett-Burman design.</p

Box-Behnken response surface design.

<p>Box-Behnken response surface design.</p

(A) Pareto chart showing the effect of different variables (media components) on antibacterial metabolite production against <i>S</i>. <i>epidermidis</i>; (B) Response surface contour and 3D plots showing individual and interactive effects of variables on antibacterial activity of NIIST A30.

<p>(a) and (b) Effects of (NH₄)₂SO₄ and starch on antimicrobial activity(c) and (d) Effects of K₂HPO₄ and (NH₄)₂SO₄ on antimicrobial activity (e) and (f) Effects of K₂HPO₄ and starch on antimicrobial activity.</p

The effect of crude extract on normal cell line viabilities.

<p>The effect of crude extract on normal cell line viabilities.</p

Analysis of variance of fitted quadratic model.

<p>Analysis of variance of fitted quadratic model.</p

Scanning electron microscopic image of spore arrangement.

<p>Scanning electron microscopic image of spore arrangement.</p

Phylogenetic tree showing the evolutionary relationship of the strain <i>Streptomyces nogalater</i> NIIST A30 with its related species.

<p>Phylogenetic tree showing the evolutionary relationship of the strain <i>Streptomyces nogalater</i> NIIST A30 with its related species.</p

Antibacterial activity of NIIST A30 crude extracts from different broths.

<p>Antibacterial activity of NIIST A30 crude extracts from different broths.</p