Abstract
We describe an antibacterial assay based on bioluminescence of two Gram negative bacteria, Photorhabdus luminescens and transformed Escherichia coli, which can be used as a real-time measurement of antibacterial activity in insect haemolymph. This method is based on the production of the bioluminescence signal depending on the viability of bacterial cells. We observed a significant rapid dose-dependent decrease in bioluminescence using both bacterial species, and Bombyx mori or Galleria mellonella haemolymph, which was confirmed by the decrease in bacterial viability determined by plating. The humoral origin of the antibacterial activity observed in whole haemolymph was confirmed for haemolymph plasma without haemocytes. Antibacterial activity directed against Gram negative bacteria was recorded in unaffected insect larvae as well as after septic injury; increased antibacterial activity of haemolymph was detected in the latter case confirming the inducibility of antimicrobial agents. We think it is likely that this method could be widely used for determining antibacterial activity in insects and other invertebrates.
Highlights
Bioluminescence is the production and emission of light by living organisms; it is a naturally occurring form of chemiluminescence in which energy is released in the form of light by an enzymatic reaction
In this study we show that bioluminescent bacteria can be used for determining antibacterial activity of samples of insect haemolymph
The bioluminescence measurements are expressed in terms of counts per second (CPS), which are dependent on bacterial growth and viability
Summary
Bioluminescence is the production and emission of light by living organisms; it is a naturally occurring form of chemiluminescence in which energy is released in the form of light by an enzymatic reaction. In bacteria the expression of genes related to bioluminescence is controlled by lux operon encoding the enzyme luciferase. Luciferases differ among bacteria they all emit light with a maximum at 490 nm and the emission requires both a reduced flavin mononucleotide (FMNH2) and long-chain aliphatic aldehyde (Poinar et al, 1980). Upon entering an insect host nematodes release bacterial cells from their intestinal tract, which quickly establish a lethal septicaemia in the host (ffrench-Constant et al, 2003). When an insect is infected with Photorhabdus bacteria the carcass becomes visibly bioluminescent (Poinar et al, 1980). In addition to the species of Photorhabdus found in nematodes, P. asymbiotica was obtained from human clinical specimens
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