Estimation of the bacteriocin ColE7 conjugation-based "kill" - "anti-kill" antimicrobial system by real-time PCR, fluorescence staining and bioluminescence assays.

The efficiency of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system, was assessed using real-time PCR, flow cytometry and bioluminescence. The ColE7 antimicrobial system consists of the genetically modified Escherichia coli strain Nissle 1917 harbouring a conjugative plasmid (derivative of the F-plasmid) encoding the "kill" gene (ColE7 activity gene) and a chromosomally encoded "anti-kill" gene (ColE7 immunity gene). On the basis of traJ gene expression in the killer donor cells, our results showed that the efficiency of the here studied antimicrobial system against target E. coli was higher at 4 than at 24 h. Flow cytometry was used to indirectly estimate DNase activity of the antimicrobial system, as lysis of target E. coli cells in the conjugative mixture with the killer donor strain led to reduction in cell cytosol fluorescence. According to a lux assay, E. coli TG1 (pXen lux+ Apr ) with constitutive luminescence were killed already after 2 h of treatment. Target sensor E. coli C600 with DNA damage SOS-inducible luminescence showed significantly lower SOS induction 6 and 24 h following treatment with the killer donor strain. Our results thus showed that bioluminescent techniques are quick and suitable for estimation of the ColE7 bacterial conjugation-based antimicrobial system antibacterial activity.

SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial antimicrobial resistance is worldwide rising and causing deaths of thousands of patients infected with multi-drug resistant bacterial strains. In addition, there is a lack of efficient alternative antimicrobial agents. The significance of our research is the use of a number of methods (real-time PCR, flow cytometry and bioluminescence-based technique) to assess the antibacterial activity of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system. Bioluminescent techniques proved to be rapid and suitable for estimation of antibacterial activity of ColE7 bacterial conjugation-based antimicrobial system and possibly other related systems.