ABSTRACT
Extended-spectrum ß-lactamase (ESBL)-producing bacteria pose a big challenge in clinical practices, warranting a new therapeutic strategy. In this study, methanol extract of the marine cyanobacterium Oscillatoria acuminata NTAPC05 was fractionated under bioassay guidance and the fractions were tested against three well-characterized ESBL-producing bacteria Escherichia coli U655, Stenotrophomonas maltophilia B929 and Enterobacter asburiae B938. Out of the four HPLC fractions, fraction 2 showed bactericidal activity against all the three ESBL producers much more efficiently (MIC 100 µg ml-1) than the fourth-generation cephalosporin (MIC >125 µg ml-1). The active fraction was subjected to time-kill test at concentrations of 1/2 × MIC, 1 × MIC and 2 × MIC, and the results substantiated the bactericidal property of the fraction against the ESBL producers. Spectral analysis revealed monogalactosyldiacylglycerol containing a palmitoyl (MGDG-palmitoyl), being reported for the first time, as the active fraction, and its bactericidal property against ESBL producers was determined. The active fraction appears to damage the bacterial membrane leading to lysis of the cell, as revealed in confocal laser scanning microscopy (CLSM) analysis, that was confirmed in scanning electron microscopic analysis. Cytotoxicity assay revealed the O. acuminata compound to be safe to a normal cell line HEK293 (human embryonic kidney cell). The in silico analysis of MGDG-palmitoyl revealed two successive H-bonding interactions with Leu198 of TEM1 ß-lactamase. Taken together, the MGDG-palmitoyl from O. acuminata NTAPC05 offers potential to develop analogs as a therapeutic for bacteremia caused by ESBL producers.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Enterobacter/drug effects , Escherichia coli/drug effects , Oscillatoria/metabolism , Stenotrophomonas maltophilia/drug effects , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Cephalosporins/pharmacology , Computer Simulation , Galactolipids/chemistry , HEK293 Cells , Humans , Microbial Sensitivity Tests , Microscopy, Confocal , Microscopy, Electron, Scanning , beta-Lactamases/metabolismABSTRACT
In this study, 27 strains of Lactic acid bacteria (LAB) were isolated and identified from different milk sources. All the isolates were biochemically characterized and screened for their ability to produce exopolysaccharides (EPS), among which two isolates namely Lactobacillus plantarum NTMI05 (197mg/L) and Lactobacillus plantarum NTMI20 (187mg/L) showed higher EPS production. Both the isolates were molecular characterized and tested for their probiotic properties. The chemical composition of EPS from L. plantarum NTMI05 and NTMI20 revealed the presence of 95.45% and 92.35% carbohydrates, 14±0.1and 11±0.15mg/L lactic acid, 10.5±0.2 and 9±0.1mg/mL of reducing sugar, respectively. HPLC analysis showed galactose at the retention time of 2.29.The maximum EPS yield was optimized for the media components like glucose (20g/L), yeast extract (25g/L) and ammonium sulphate (2g/L) using Central Composite Design and Response Surface Methodology (RSM). Under optimum conditions the predicted maximum EPS production was 0.891g/L, 0.797g/L, while the actual experimental value was 0.956g/L and 0.827g/L for L. plantarum NTMI05 and NTMI20, respectively. The antioxidant capacity was also evaluated by DPPH and reducing power assay proving the potentiality of these organisms in food and dairy industries.
