Spatial patterns and mechanisms of antibiotic tolerance in Escherichia coli biofilms. Role of extracellular matrix and c-di-GMP

OBANDO ROJAS, MAYRA; SERRA, DIEGO O.

Chapadmallal

Congreso; XVIII Congreso Argentino de Microbiología General (SAMIGE 2023); 2023

Sociedad Argentina de Microbiología General (SAMIGE)

Biofilms are communities that bacteria build by embedding themselves in an extracellular matrix (ECM). While it is recognized that cells within biofilms exhibit tolerance to antibiotics, it remains unknown in which internal zones bacteria tolerate these agents more effectively and which molecular mechanisms they deploy to achieve this resilience. In prior studies with agar-grown macrocolony biofilms, we discovered a precise and highly regulated spatial pattern of physiological differentiation among E. coli cells. This arrangement yields distinct upper and lower strata, with ECM production confined solely to the upper stratum. Building upon this insight, our main aim was to uncover the spatial patterns of antibiotic susceptibility/tolerance within these strata. Additionally, we aimed to study how the ECM and the second messenger c-di-GMP influence these patterns. To achieve this, we carried out studies that included pre-growing E. coli macrocolonies, subjecting them to aminoglycoside treatment, differentially labelling viable and non-viable cells, thin-sectioning the treated biofilms, and imaging labelled cells. For our analysis, we defined three regions along the macrocolony radius: the border (designated as region 1), the transitional region that links the border with the central area (referred to as region 2), and the central core region of the biofilm (recognized as region 3). Each of these regions was analysed to identify viable and non-viable cells across the two strata (upper and lower). Upon analysing E. coli wild-type macrocolonies, we observed that in the border (region 1), aminoglycosides effectively eradicated the bacteria. This is in line with the fact that this region mainly consists of actively growing cells, which are likely to be more susceptible to the antibiotics. Remarkably, within region 2, 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. These internal zones are referred as “susceptibility zones” and “tolerance zones”, respectively. Progressing from region 2 to 3, the count of cells that perish within the "susceptible zones" in both strata gradually diminishes until nearly all cells survive at the biofilm center (region 3). Analysing antibiotic-treated biofilms of E. coli strains unable of producing ECM components or exhibiting altered c-di-GMP levels, we found that the absence of ECM, combined with elevated c-di-GMP levels, renders E. coli cells in the “tolerance zone” of the upper stratum, particularly in the region 3, highly susceptible to antibiotics. In summary, our studies unveil intricate internal patterns of cell death and survival within antibiotic-treated E. coli biofilms. The "tolerance zones" can serve as discrete reservoirs for surviving cells, including persisters. Moreover, our results support a strikingly localized role for ECM and c-di-GMP in promoting antibiotic tolerance within biofilms.