Bacterial biofilm formation is a complex developmental process involving cellular differentiation and the formation of intricate 3D structures. Here we demonstrate that exposure to ferric chloride triggers rugose biofilm formation by the uropathogenic Escherichia coli strain UTI89 and by enteric bacteria Citrobacter koseri and Salmonella enterica serovar typhimurium. Two unique and separable cellular populations emerge in iron-triggered, rugose biofilms. Bacteria at the air-biofilm interface express high levels of the biofilm regulator csgD, the cellulose activator adrA, and the curli subunit operon csgBAC. Bacteria in the interior of rugose biofilms express low levels of csgD and undetectable levels of matrix components curli and cellulose. Iron activation of rugose biofilms is linked to oxidative stress. Superoxide generation, either through addition of phenazine methosulfate or by deletion of sodA and sodB, stimulates rugose biofilm formation in the absence of high iron. Additionally, overexpression of Mn-superoxide dismutase, which can mitigate iron-derived reactive oxygen stress, decreases biofilm formation in a WT strain upon iron exposure. Not only does reactive oxygen stress promote rugose biofilm formation, but bacteria in the rugose biofilms display increased resistance to H 2 O 2 toxicity. Altogether, we demonstrate that iron and superoxide stress trigger rugose biofilm formation in UTI89. Rugose biofilm development involves the elaboration of two distinct bacterial populations and increased resistance to oxidative stress.functional amyloid | biofilm matrix | wrinkled colony

Physiological resistance to antibiotics confounds the treatment of many chronic bacterial infections, motivating researchers to identify novel therapeutic approaches. To do this effectively, an understanding of how microbes survive in vivo is needed. Though much can be inferred from bulk approaches to characterizing complex environments, essential information can be lost if spatial organization is not preserved. Here, we introduce a tissue-clearing technique, termed MiPACT, designed to retain and visualize bacteria with associated proteins and nucleic acids in situ on various spatial scales. By coupling MiPACT with hybridization chain reaction (HCR) to detect rRNA in sputum samples from cystic fibrosis (CF) patients, we demonstrate its ability to survey thousands of bacteria (or bacterial aggregates) over millimeter scales and quantify aggregation of individual species in polymicrobial communities. By analyzing aggregation patterns of four prominent CF pathogens, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus sp., and Achromobacter xylosoxidans, we demonstrate a spectrum of aggregation states: from mostly single cells (A. xylosoxidans), to medium-sized clusters (S. aureus), to a mixture of single cells and large aggregates (P. aeruginosa and Streptococcus sp.). Furthermore, MiPACT-HCR revealed an intimate interaction between Streptococcus sp. and specific host cells. Lastly, by comparing standard rRNA fluorescence in situ hybridization signals to those from HCR, we found that different populations of S. aureus and A. xylosoxidans grow slowly overall yet exhibit growth rate heterogeneity over hundreds of microns. These results demonstrate the utility of MiPACT-HCR to directly capture the spatial organization and metabolic activity of bacteria in complex systems, such as human sputum.

Chapter Summary Escherichia coli (E. coli) is one of the world’s best-characterized organisms, as it has been extensively studied for over a century. However, most of this work has focused on E. coli grown under laboratory conditions that do not faithfully simulate its natural environments. Therefore, the historical perspectives on E. coli physiology and life cycle are somewhat skewed toward experimental systems that feature E. coli growing logarithmically in a test tube. Typically a commensal bacterium, E. coli resides in the lower intestines of a slew of animals. Outside of the lower intestine, E. coli can adapt and survive in a very different set of environmental conditions. Biofilm formation allows E. coli to survive, and even thrive, in environments that do not support the growth of planktonic populations. E. coli can form biofilms virtually everywhere; in the bladder during a urinary tract infection, on in-dwelling medical devices, and outside of the host on plants and in the soil. The E. coli extracellular matrix, primarily composed of the protein polymer named curli and the polysaccharide cellulose, promotes adherence to organic and inorganic surfaces, and resistance to desiccation, the host immune system and other antimicrobials. The pathways that govern E. coli biofilm formation, cellulose production, and curli biogenesis will be discussed in this book chapter, which concludes with insights into the future of E. coli biofilm research and potential therapies.

Microbial biofilms are encased in a protein, DNA and polysaccharide matrix that protects the community, promotes interactions with the environment, and helps cells to adhere together. The protein component of these matrices is often a remarkably stable, β-sheet-rich polymer called amyloid. Amyloids form ordered, self-templating fibers that are highly aggregative, making them a valuable biofilm component. Some eukaryotic proteins inappropriately adopt the amyloid fold and these misfolded protein aggregates disrupt normal cellular proteostasis, which can cause significant cytotoxicity. Indeed, until recently amyloids were considered solely the result of protein misfolding. However, research over the past decade has revealed how various organisms have capitalized on the amyloid fold by developing sophisticated biogenesis pathways that coordinate gene expression, protein folding, and secretion so that amyloid-related toxicities are minimized. How microbes manipulate amyloids, by augmenting their advantageous properties and by reducing their undesirable properties, will be the subject of this review.

cEnteric bacteria, such as Escherichia coli, are exposed to a variety of stresses in the nonhost environment. The development of biofilms provides E. coli with resistance to environmental insults, such as desiccation and bleach. We found that biofilm formation, specifically production of the matrix components curli and cellulose, protected E. coli against killing by the soil-dwelling nematode Caenorhabditis elegans and the predatory bacterium Myxococcus xanthus. Additionally, matrix-encased bacteria at the air-biofilm interface exhibited ϳ40-fold-increased survival after C. elegans and M. xanthus killing compared to the nonmatrix-encased cells that populate the interior of the biofilm. To determine if nonhost Enterobacteriaceae reservoirs supported biofilm formation, we grew E. coli on media composed of pig dung or commonly contaminated foods, such as beef, chicken, and spinach. Each of these medium types provided a nutritional environment that supported matrix production and biofilm formation. Altogether, we showed that common, nonhost reservoirs of E. coli supported the formation of biofilms that subsequently protected E. coli against predation.