Abstract

Background: Probiotics are live microorganisms beneficial to human health and may inhibit pathogen colonization and growth. The aims of this work were to develop an assay whereby invasion of an human intestinal cell line by an enteric pathogen could be monitored in real time and to evaluate the effect of the probiotic Escherichia coli strain, Nissle 1917 (EcN), on pathogen invasion. Method: Salmonella enterica sv. Typhimurium MCS5-lite, a bioluminescent reporter organism, was previously transformed with a recombinant plasmid containing the constitutively expressed luxCDABE gene cassette of Photorhabdus luminescens. Aliquots of S. Typhimurium were added to confluent monolayers of human enterocyte-like Caco-2 cells grown in microtitre plates. Following 2 h incubation, colistin (50 μg/mL) was added to inactivate extracellular bacterial cells. After a further 2 h, saponin (0.1 %) was added to some of the wells to lyse the Caco-2 cells. Bioluminescence was monitored for at least 18 h, by automated luminometry. To determine the effect of EcN on salmonella invasion, aliquots of probiotic bacteria were added to Caco-2 monolayers 1 h prior to the addition of the pathogen. Filter-sterilised supernatant from overnight cultures of EcN was also included in some assays, in place of EcN cells. Results: Invasion of S. Typhimurium into Caco-2 cells could be monitored by light output, with bioluminescence increasing gradually over the time period studied. Addition of EcN to monolayers 1 h before addition of S. Typhimurium resulted in lower levels of internalized salmonellae although EcN cell-free supernatant did not affect S. Typhimurium invasion. Conclusion: Invasion into, and growth within, Caco-2 cells by salmonella, and the inhibitory effect of a probiotic species upon invasion, could be monitored non-destructively and in real time using bioluminescence. It is unlikely that a secreted compound was directly inhibiting S. Typhimurium since EcN cell-free supernatant had no effect on invasion.