Most pathogens form biofilms. Bacterial survival instinct inside the biofilm kicks in altered metabolic activity to harvest energy for persistence. What about their virulence phenotypes and pathogenesis? We used Listeria monocytogenes as a model pathogen to study the infection of biofilm-isolated cells. Listeria, a frequent food-processing plant dweller, forms biofilms and loses its major virulence phenotypes. Does this mean, it is unable to infect if a food is consumed immediately after being contaminated with these cells?
First, we tested infectivity in a mammalian cell culture model. It was alarming to learn that these biofilm isolated cells appeared to be avirulent (Figure 1a). Virulence protein markers especially the ones that are needed for intestinal epithelial cell barrier invasion, Listeria adhesion protein (LAP)1 and Internalin A (InlA)2 are barely detectable (Figure 1b). If we stopped our investigation at this stage, our conclusion would have been – “the biofilm-isolated cells are avirulent and there is a very little food safety concern due to contamination of food with biofilm-isolated cells”.
Figure 1. (a) Biofilm-isolated L. monocytogenes cells were less invasive to Caco-2 cells, and (b) showed reduced expression of LAP and InlA proteins in both cell wall (CW) and intracellular (IC) fractions of biofilm (B) cells compared to planktonic (P) cells.
Such a conclusion is problematic to accept knowing most pathogens form biofilms and cause disease. We continued our investigation in a mouse model. To our surprise, at 12 and 24 h post-infection (hpi), biofilm-isolated cells still showed reduced tissue invasion and systemic spread compared to planktonic counterparts, and data agree with our in vitro cell culture experiment. Are they truly less invasive? It was still an unsettling conclusion to accept! Next, we pushed forward and looked at Listeria tissue invasion after 48 h post-infection. It was to our delight, we observed tissue invasion and systemic spread of biofilm-isolated cells similar to the planktonic cells (Figure 2).
Figure 2. Listeria monocytogenes (Lm) burdens in extra-intestinal organs of mice infected with biofilm-isolated (B) or planktonic (P) cells at 24 and 48 h post-infection (hpi). Bars represent the median values.
Why such a delay in infectivity? Virulence genes especially the genes encoding LAP and InlA that were downregulated during biofilm formation were turned on when Listeria begin to pass through the acidic stomach and oxygen-depriving environment in the intestine.
This study led us to conclude that in vitro cell culture assay is not suitable for assessing the virulence potential of physiologically stressed bacteria such as the biofilm-isolated cells therefore an animal model is essential. The delay in infectivity also implies incubation time and the onset of symptoms may vary depending on the physiological state of the bacteria (biofilm-isolated vs planktonic) consumed with the food.
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