Escherichia coli K‐12 (pEGFPluxABCDEamp): a tool for analysis of bacterial killing by antibacterial agents and human complement activities on a real‐time basis

Abstract

Photorhabdus luminescens luxCDABE genes were integrated into E. coli K-12 using a high copy number plasmid containing modified luxABCDE genes under the control of the powerful Lac promoter. This strain emitted 10 times higher bioluminescence (BL) than P. luminescens. BL production under different growth conditions was studied. In both bacterial strains, the increase in BL signal correlated with the increase in optical density (OD) in a rich growth medium. However, at the logarithmic growth phase, the BL signal was roughly constant. By contrast, in minimal growth media, there was no substantial growth and the BL/cell was approximately five times higher than in the rich medium. The dynamic measurement range of BL was 10(2) -10(7) colony-forming units (CFU) in E. coli and 10(3) -10(7) CFU in P. luminescens. Because the decrease in the BL signal correlated with the decrease in CFU and OD, i.e. the number of bacterial cells killed, it proved to be very suitable for assessing the antibacterial effects of different antimicrobial agents. Unlike with plate counting, the kinetics of killing can be monitored on a real-time basis using BL measurements. Complement activities in different samples can be estimated using only one serum dilution. The transformed E. coli strain appeared to be superior to P. luminescens in these applications because E. coli was complement sensitive, the detection limit of E. coli was one order lower and the BL-producing system of P. luminescens appeared to be quite unstable.

Antioxidant, antimicrobial and neutrophil-modulating activities of herb extracts.

“…Antimicrobial activity against genetically modified Escherichia coli. In the following experiments transformed E. coli K12 resistant to ampicillin carrying the luxABCDEamp gene was used, expressing bacterial luciferase and its substrate (Atosuo et al, 2013). Bacteria were cultivated in LB medium with 100 µg/ ml ampicillin, washed twice in phosphate buffer (pH 7.0, 133 mM) and adjusted with the phosphate buffer to the final concentration of approximately 330 000 cells per 100 µl before each measurement.…”

“…The wells of microplates contained 180 µl of bacterial suspension and 10 µl (5% concentration) of extracts, buffer was used as a control. The light emission during reaction is positively correlated with bacteria viability (Atosuo et al, 2013). To determine antibacterial properties of extracts, integrals (RLU*s) under kinetics curves were calculated and expressed as percentage of control.…”

Novel electrospun gelatin/oxycellulose nanofibers as a suitable platform for lung disease modeling

“…Inhibitory effect of gelatin, gelatin/CaOC and gelatin/NaOC nanofibers was examined by means of E. coli K-12 bioluminescence [54]. The real-time inhibitory effect was determined from an intensity of the bacteria bioluminescent (BL) signal.…”

Melanin and carotenoid ornaments are related to the individual condition in free-living grey partridges (Perdix perdix)

“…Complement activity was measured using the bioluminescence-based method described in Atosuo et al (2013). The light emission produced by living cells of a bioluminescent strain of Escherichia coli K12 (pEGFPluxABCDEamp) was measured by a luminometer (LM01-T, Immunotech, Prague, Czech Republic) at 37°C.…”