Phospholipid/Polydiacetylene Vesicle-Based Colorimetric Assay for High-Throughput Screening of Bacteriocins and Halocins

Abstract

The colorimetric assay is phospholipid/polydiacetylene vesicle-based assay used for the detection of membrane-acting peptides. Bacteriocins and halocins are antimicrobial peptides known to kill target cells by membrane disruption. Therefore, the assay was applied for high-throughput (HTP) screening of bacteriocins and halocins produced by lactic acid bacteria and haloarchaea, respectively. The assay consisted of vesicles which were synthesized using four different phospholipids: dipalmitoylphosphatydilcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphoethanolamine (DMPE) and dimyristoylphosphatidylglycerol (DMPG) in combination with diacetylene monomer 10,12-tricosadiy noic acid (TRCDA). These vesicles demonstrated blue colour at 640 nm and turned pink/red after interaction with nisin. DMPE/TRCDA vesicles showed pink colour with the highest colorimetric response (CR %) after treatment with nisin and, therefore, selected for the screening of bacteriocins and halocins. The colour of the vesicles was changed within 5 min in the presence of 5 μM nisin suggesting the sensitivity of assay. The assay was applied on 54 strains of lactic acid bacteria (LAB) and 53 haloarchaea for screening of bacteriocins and halocins, respectively. Out of these strains, three strains of LAB and five strains of haloarchaea were found to be bacteriocin and halocin non-producer, respectively. The other strains demonstrated the presence of bacteriocins and halocins. The colorimetric assay was found to be rapid, specific and reliable for HTP screening of antimicrobial peptides such as bacteriocins and halocins from producer strains isolated from various natural resources.

Responsive Polydiacetylene Vesicles for Biosensing Microorganisms<strong> </strong>

“…This review presents the state-of-the-art of mixed PDA based vesicles formulated in order to mimic cell membranes and how they constitute actual nanosensors for the direct detection of bacteria or The colorimetric assays based on these PDA based vesicles are very cheep and easy to handle. They were applied to the high throughput screening of toxins of natural origin (fungi [52], bacteria [12])…”

“…through the design of a biosensing platform [12]. They were also applied to rapid bacterial detection in food [32,39].…”

“…The antimicrobial properties of metabolites of soil microfungi were tested through a colorimetric assay using PDA-based vesicles [52]. This assay was also applied to the high throughput screening of peptides, bacteriocins, produced by lactic acid bacteria [12]. It appears that for lactic acid bacteria strains LV39 (well 34), LB52 (well 40) and LB64 (well 47) the colour change is weak, compared to that of the other strains.…”

“…Colour change of DMPE/TRCDA vesicles treated with 50 µL cell-free supernatant of 54 lactic acid bacteria strains[12] …”

Modified microassay for the isolation of antimicrobial-producing, spore-forming and nonspore-forming bacteria

“…Based on the preliminary testing in the current study, the method is not suitable for screening spore-forming bacteria for antimicrobial production, because it does not include provisions for inactivating spores or inhibiting their germination and outgrowth. Other high-throughput screening methods required preparation of crude extracts from producing micro-organisms, targeted specific processes using libraries of purified compounds, or examined variants of known compounds (Xu et al 2005;Castiglione et al 2007;Ymele-Hong et al 2009;Ymele-Leki et al 2012;Guralp et al 2013;Baker et al 2016;Nairn et al 2017;Yadav et al 2017).…”