Could Bioluminescent Bacteria be Used in the Search for New Plant-derived Antibacterial Substances?

Abstract

Introduction. Currently, the search for new antibacterial substances is an urgent task due to the growing resistance of pathogens to existing antibiotics. One of the key directions in this area is the expansion of scientific research of medicinal plants, as new sources of therapeutic agents. This article examines the possibility of using highly sensitive bioluminescent test bacteria for these purposes, which can quickly detect non-specific antimicrobial activity and can be adapted to highly effective pharmaceutical screening technologies. Aim. To study the applicability of bioluminescent bacteria for the analysis of the antibacterial activity of biologically active substances (BAS) of plant origin. Materials and methods. BAS quercetin, 8-hydroxyquinoline, gallic acid and thymoquinone, which are often found in medicinal plant raw materials and with which its antibacterial properties are associated, were used in the work. Bacteria with constitutive bioluminescence Aliivibrio fischeri F1 and Escherichia coli (pXen7), as well as recombinant bioreporter strains with inducible luminescence were used as test-objects: E. coli (pRecA-lux), E. coli (pColD-lux), reacting to nucleic acid damage; E. coli (pKatG-lux) and E. coli (pSoxS-lux), sensitive to oxidative stress. Results and discussion. It was found that the nonspecific antimicrobial activity of the studied BAS is manifested in the inhibition of bacterial bioluminescence of test-strains with constitutive glowing. It was noted that the marine test-bacteria A. fischeri F1 have significantly greater sensitivity to the action of BAS, compared with the recombinant strain of E. coli (pXen7). It has been shown that their inhibitory effect begins at concentrations of 2 mcg/ml, and bactericidal activity occurs at concentrations of more than 20 mcg/ml. The results obtained are compared with the data on MIC and MBC of gram(+) and gram(–) pathogens. The study of the induction of bioluminescence of recombinant bioreporter strains showed that the antibacterial effect of the BAS is accompanied by oxidative stress. Also, quercetin caused activation of luminescence in E. coli (pRecA-lux) and E. coli (pColD-lux), which may indicate its participation in damage to nucleic acids. Analysis of the induction factors of bioreporter strains indicates that the revealed mechanisms of antibacterial activity are not major, but may be of a secondary nature. Conclusion. It has been shown that the intensity of the glow of natural and recombinant bioluminescent bacteria can be an indicator of the antibacterial activity of BAS of natural origin. The high sensitivity of A. fischeri F1 bacteria to the action of substances such as quercetin, 8-hydroxyquinoline, gallic acid and thymoquinone has been shown. Considering that bioluminescence analysis is a quantitative instrumental method, it can be easily adapted for high-throughput pharmaceutical screening. It has been shown that the luminescence intensity of natural and recombinant bioluminescent bacteria can be an indicator of the antibacterial activity of BAS of natural origin. The high sensitivity of A. fischeri F1 to the action of substances such as quercetin, 8-hydroxyquinoline, gallic acid and thymoquinone has been established. Taking in an account that bioluminescent analysis is a quantitative instrumental method, it can be easily adapted for high-throughput pharmaceutical screening.