Bioluminescent high-throughput assay for the bacteria adherence to the tissue culture cells.

The goal of this study was develop a rapid high-throughput method for the assessment of the bacterial adhesion to tissue culture cells and test this method by investigation of the adhesion and growth of pathogenic and non-pathogenic Escherichia coli strains in the presence of HeLa human epithelial cells. Fifteen strains of E. coli were transformed with a plasmid carrying the entire lux operon of Photorhabdus luminescens to make them bioluminescent. By using the Time-to-Detection approach and bioluminescence imaging in microplate format, the adherence and growth of bacteria in tissue culture medium in the presence of HeLa cells was monitored. It was observed that Eagle's minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) significantly inhibited growth of E. coli. However, in the presence of HeLa cells the detected growth of E. coli was similar to the growth observed in LB medium. It was established that the initial number of E. coli cells present in the microplate directly correlated with the time necessary for the bioluminescence signal to reach the threshold level, hence allowing the accurate assessment of the adhered cells within 8-10 h. Neither bacterial adherence nor growth kinetics correlated with the pathogenicity of the strain though they were strain-specific. The developed approach provided new information on the interaction of E. coli with epithelial cells and could be used for both pathogenicity research and for the screening of potential therapeutic agents for the ability to minimize pathogen colonization of human tissues.

Bacteriophage-based biosorbents coupled with bioluminescent ATP assay for rapid concentration and detection of Escherichia coli.

Wild type T4 bacteriophage and recombinant T4 bacteriophages displaying biotin binding peptide (BCCP) and cellulose binding module (CBM) on their heads were immobilized on nano-aluminum fiber-based filter (Disruptor), streptavidin magnetic beads and microcrystalline cellulose, respectively. Infectivity of the immobilized phages was investigated by monitoring the phage-mediated growth inhibition of bioluminescent E. coli B and cell lysis using bioluminescent ATP assay. The results showed that phage immobilization resulted in a partial loss of infectivity as compared with the free phage. Nevertheless, the use of a biosorbent based on T4 bacteriophage immobilized on Disruptor filter coupled with a bioluminescent ATP assay allowed simultaneous concentration and detection of as low as 6 x 10(3)cfu/mL of E. coli in the sample within 2h with high accuracy (CV=1-5% in log scale). Excess of interfering microflora at levels 60-fold greater than the target organism did not affect the results when bacteriophage was immobilized on the filter prior to concentration of bacterial cells.

Oriented immobilization of bacteriophages for biosensor applications.

A method was developed for oriented immobilization of bacteriophage T4 through introduction of specific binding ligands into the phage head using a phage display technique. Fusion of the biotin carboxyl carrier protein gene (bccp) or the cellulose binding module gene (cbm) with the small outer capsid protein gene (soc) of T4 resulted in expression of the respective ligand on the phage head. Recombinant bacteriophages were characterized in terms of infectivity. It was shown that both recombinant phages retain their lytic activity and host range. However, phage head modification resulted in a decreased burst size and an increased latent period. The efficiency of bacteriophage immobilization with streptavidin-coated magnetic beads and cellulose-based materials was investigated. It was shown that recombinant bacteriophages form specific and strong bonds with their respective solid support and are able to specifically capture and infect the host bacterium. Thus, the use of immobilized BCCP-T4 bacteriophage for an Escherichia coli B assay using a phage multiplication approach and real-time PCR allowed detection of as few as 800 cells within 2 h.