Emergence of differential spatial patterns of antibiotic tolerance in Escherichia coli biofilms. Role of cellular stress responses and extracellular matrix.

SERRA, D.O.; SAMBRAILO, A.

Los Cocos - Córdoba

Congreso; XVII Congreso Argentino de Microbiología General; 2022

Sociedad Argentina de Microbiología General

Within self-organized communities known as biofilms, bacteria can tolerate and survive antibiotic treatments, which ultimately facilitates the persistence of the infections, as frequently occurs with those caused by Escherichia coli. Due to the structural complexity of biofilms, it has remained challenging to reveal in which internal zones the bacteria better tolerate the antibiotics and which molecular mechanisms they deploy to tolerate the treatments. Taking advantage of microscopic approaches that allow examining E. coli macrocolony biofilms at single-cell resolution, we previously revealed that cells within these communities physiologically differentiate giving rise to two strata (upper and lower) that include subzones where cells exhibit either vegetative growth or stationary phase physiology. This complex physiological stratification results from the differential activation of cellular responses -such as the General Stress Response (GSR) mediated by the stationary-phase sigma factor RpoS- that integrates nutrient and oxygen gradients generated across the biofilms. Based on this knowledge, we performed studies that combined antibiotic (aminoglycosides) treatment of E. coli macrocolony biofilms, differential labelling of non-viable/viable cells, thin-sectioning of the biofilms and microscopy with the aim of revealing the spatial patterns of survival/death of individual cells within treated biofilms and examining the role of the RpoS-mediated GSR in cell survival. Our studies showed that in young biofilm regions the aminoglycosides killed those bacteria located at the outermost colony border, which is consistent with this region being commonly occupied by actively growing cells that are hence likely to be susceptible to the antibiotics. Remarkably, in more mature biofilm regions (towards the center of the treated macrocolonies) we observed that each stratum exhibits a zone where cells effectively die due to the bactericidal action of the aminoglycosides and a zone where cells survive the treatments (here referred to as “susceptibility zones” and “tolerance zones”, respectively). Susceptibility zones spatially coincide with areas of vegetative growth, whereas tolerance zones, which are the outer zone of the upper stratum (at the interface with the air) and the inner zone of the lower stratum, coincide with areas where cells exhibit active RpoS expression. Deletion of rpoS rendered cells in the outer zone of the upper macrocolony stratum highly susceptible to the aminoglycosides, supporting a role for RpoS, and hence for the GSR, in promoting antibiotic tolerance of cells located in that zone of the biofilms. Overall, our studies revealed for the first time the existence of distinct zones of antibiotic tolerance inside E. coli macrocolony biofilms that could serve as independent reservoirs of surviving cells -including persisters- and demonstrated the involvement of the GSR in the emergence of one such zone.