Attached bacterial communities can generate three-dimensional (3D) physicochemical gradients that create microenvironments where local conditions are substantially different from those in the surrounding solution. Given their ubiquity in nature and their impacts on issues ranging from water quality to human health, better tools for understanding biofilms and the gradients they create are needed. Here we demonstrate the use of functional tomographic imaging via confocal fluorescence microscopy of ratiometric core-shell silica nanoparticle sensors (C dot sensors) to study the morphology and temporal evolution of pH microenvironments in axenic Escherichia coli PHL628 and mixed-culture wastewater biofilms. Testing of 70-, 30-, and 10-nm-diameter sensor particles reveals a critical size for homogeneous biofilm staining, with only the 10-nm-diameter particles capable of successfully generating high-resolution maps of biofilm pH and distinct local heterogeneities. Our measurements revealed pH values that ranged from 5 to >7, confirming the heterogeneity of the pH profiles within these biofilms. pH was also analyzed following glucose addition to both suspended and attached cultures. In both cases, the pH became more acidic, likely due to glucose metabolism causing the release of tricarboxylic acid cycle acids and CO 2 . These studies demonstrate that the combination of 3D functional fluorescence imaging with well-designed nanoparticle sensors provides a powerful tool for in situ characterization of chemical microenvironments in complex biofilms.

In humans, uropathogenic Escherichia coli (UPEC) is the most common etiological agent of uncomplicated urinary tract infections (UTIs). Cranberry extracts have been linked to the prevention of UTIs for over a century; however, a mechanistic understanding of the way in which cranberry derivatives prevent bacterial infection is still lacking. In this study, we used a fliC-lux reporter as well as quantitative reverse transcription-PCR to demonstrate that when UPEC strain CFT073 was grown or exposed to dehydrated, crushed cranberries or to purified cranberry-derived proanthocyanidins (cPACs), expression of the flagellin gene (fliC) was inhibited. In agreement with these results, transmission electron microscopy imaging of bacteria grown in the presence of cranberry materials revealed fewer flagella than those in bacteria grown under control conditions. Furthermore, we showed that swimming and swarming motilities were hindered when bacteria were grown in the presence of the cranberry compounds. Because flagellum-mediated motility has been suggested to enable UPEC to disseminate to the upper urinary tract, we propose that inhibition of flagellum-mediated motility might be a key mechanism by which cPACs prevent UTIs. This is the first report to show that cranberry compounds inhibit UPEC motility via downregulation of the fliC gene. Further studies are required to establish whether these inhibitors play a role in vivo.There are more than 150 million cases of urinary tract infection (UTI) in the world every year, with correspondingly significant morbidity and health care costs (12). Uncomplicated UTIs, i.e., infections that occur in healthy, immunocompetent individuals, are caused over 80% of the time by uropathogenic Escherichia coli (UPEC) (44). Among UPEC strains, E. coli CFT073, a strain isolated from the blood and urine of a woman diagnosed with acute pyelonephritis (34), is one of the most prevalent clonal lines (21,22). The majority of UTIs develop in an ascending manner (1, 44) that commences when bacteria inoculate the periurethral area and then the bladder (20,35,46). The bacteria may then ascend to the upper urinary tract and kidneys and establish a secondary infection (44). Once in the kidneys, the bacteria can access the bloodstream, causing bacteremia and (potentially) death (44).The bacterial flagellum is a molecular machine driven by a motor which rotates a long, curved filament (2). This filament extends from the basal body outward and is a polymer of flagellin subunits encoded by the fliC gene (6, 31). Mutations in fliC result in loss of flagellation and motility (31). Flagellummediated motility has been suggested to contribute to virulence by enabling UPEC to disseminate to the urinary tract, to escape host immune responses, and to disperse to new sites within the urinary tract (24). Even though this hypothesis remains to be demonstrated, several groups have shown that fliC mutants are outcompeted by motile, wild-type strains during experimental cochallenge of mice (24,40,45), thereby demonstrating tha...

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuousflow drip system.

Transcriptional profiles of uropathogenic Escherichia coli CFT073 exposed to cranberry-derived proanthocyanidins (PACs) were determined. Our results indicate that bacteria grown on media supplemented with PACs were iron deprived. To our knowledge, this is the first time that PACs have been shown to induce a state of iron limitation in this bacterium.