Tracking Escherichia coli infection of lettuce by dual-fluorescent labelling of bacterial cells

DUBLAN M DE LOS A; LETT L; CURATTI L

Tucuman

Congreso; VI Reunion Anual; 2011

Sociedad Argentina de Microbiologia

Escherichia coli is a major cause of hemorrhagic colitis and the hemolytic-uremic syndrome worldwide. Although most outbreaks have been associated to undercooked ground beef and dairy products, also fresh vegetables are vectors of food-borne illness. It is of special public-health concern that as few as 10-100 cells are required for this pathogen to cause illness in humans. The mechanisms by which the pathogen is introduced into plants are not fully understood; however, it is presumed that plants become contaminated when grown in fields fertilized with improperly treated manure, watering with contaminated irrigation water and(or) improper post-harvest manipulation..This work aimed at better understanding the infection pathway of lettuce by E. coli . We have prepared E. coli reporter strains expressing green- or red-fluorescent proteins to visually track the infection process and made viable-cell counting determinations to confirm fluorescence microscopy information. In roots, we have observed a preferred localization of bacterial cells clusters in zones of elongation of lateral roots and the apoplastic space between parenquima cells. Later on during plantlet development, infection was more prominent in parenquima cells of leaf tissue, especially in young (still folded) leaves once the shoot have developed 2 to 4 unfolded true- leaves. Using this infection system, E. coli typically formed cell-patches in leaves surrounding the vascular tissue. The general infection pathway was modified according to the nutritional status of the plants: while a richer medium favored proliferation of bacteria in the leaves, starvation changed the pattern towards root colonization. By simultaneous infection with reporter E. coli strains with difference fluorescence properties we only observed mixtures of green- or red-fluorescent cell patches in leaves (no mixed patches), strongly suggesting that they are true micro-colonies developed from single cells that might reach the leaf parenquima through the vascular tissue. Conversely, cells in the apoplast of roots were more randomly arranged, similarly as they remained in the culturing substrate. A similar approach was used to investigate whether bacterial cells that reach the leaves were more infective than cells maintain in culture medium for enterobacteria. Thus, reciprocal competition experiments show that in planta cultured cells were nearly 10-fold more infective to lettuce.Additionally, we have evaluated the effect of the commonly used growth-promoting rhizobacterium Azospirillum brasilense on lettuce infection by E. coli. We found that the infection was reduced by several orders of magnitude both in roots and leaves.These results may help understanding the infection and colonization mechanisms of fresh produce by E. coli and to model agricultural practices towards food-born illness-risk amelioration. CONICET, ANPCyT, FIBA.