Effects of the twin-arginine translocase on the structure and antimicrobial susceptibility of Escherichia coli biofilms.

In this descriptive study, we used Escherichia coli twin-arginine translocase (tat) mutants to distinguish antibiotic tolerance from the formation of mature biofilm structure. Biofilm formation by wild-type and deltatat strains of E. coli was evaluated using viable cell counts, scanning electron microscopy, and confocal laser-scanning microscopy. Escherichia coli deltatat mutants had an impaired ability to form biofilms when grown in rich or minimal media. These mutants produced disorganized layers and cell aggregates with significantly decreased cell density relative to the wild-type strain. In contrast, wild-type E. coli grown under similar test conditions formed highly structured, surface-adherent communities. We thus determined if this decreased biofilm formation by E. coli deltatat mutants may result in lowered tolerance to antimicrobials. When grown in rich media, planktonic deltatat mutants were hypersensitive to some metals, detergents, and antibiotics. However, the corresponding biofilms were about as resilient as the wild-type strain. In contrast, both planktonic cells and biofilms of the deltatatABC strain grown in minimal media were hypersensitive to many antimicrobials. Remarkably, these biofilms remained up to 365 times more tolerant to beta-lactams than corresponding planktonic cells. Our data suggest that the twin-arginine translocase may play a contributing role in the antimicrobial tolerance, structural organization, and formation of mature E. coli biofilms under nutrient-limited conditions. However, the high tolerance of the deltatatABC strain to bactericidal concentrations of antimicrobials indicates that mature biofilm structure may not be required for surface-adherent E. coli to survive exposure to these lethal factors.

Persister cells mediate tolerance to metal oxyanions in Escherichia coli.

Bacterial cultures produce subpopulations of cells termed 'persisters', reputedly known for high tolerance to killing by antibiotics. Ecologically, antibiotics produced by competing microflora are only one potential stress encountered by bacteria. Another pressure in the environment is toxic metals that are distributed ubiquitously by human pollution, volcanic activity and the weathering of minerals. This study evaluated the time- and concentration-dependent killing of Escherichia coli planktonic and biofilm cultures by the water-soluble metal(loid) oxyanions chromate (CrO4(2-)), arsenate (AsO4(2-)), arsenite (AsO2-), selenite (SeO3(2-)), tellurate (TeO4(2-)) and tellurite (TeO3(2-)). Correlative to previous reports in the literature, control antibiotic assays indicated that a small proportion of E. coli biofilm populations remained recalcitrant to killing by antibiotics (even with 24 h exposure). In contrast, metal oxyanions presented a slow, bactericidal action that eradicated biofilms. When exposed for 2 h, biofilms were up to 310 times more tolerant to killing by metal oxyanions than corresponding planktonic cultures. However, by 24 h, planktonic cells and biofilms were eradicated at approximately the same concentration in all instances. Coloured complexes of metals and chelators could not be generated in biofilms exposed to CrO4(2-) or TeO3(2-), suggesting that the extracellular polymeric matrix of E. coli may have a low binding affinity for metal oxyanions. Viable cell counts at 2 and 24 h exposure revealed that, at high concentrations, all of the metal oxyanions had killed 99 % (or a greater proportion) of the bacterial cells in biofilm populations. It is suggested here that the short-term survival of <1 % of the bacterial population corresponds well with the hypothesis that a small population of persister cells may be responsible for the time-dependent tolerance of E. coli biofilms to high concentrations of metal oxyanions.