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Free-living bacteria can assemble into multicellular structures called biofilms. Biofilms help bacteria tolerate multiple stresses, including antibiotics and the host immune system. Nontuberculous mycobacteria are a group of emerging opportunistic pathogens that utilize biofilms to adhere to household plumbing and showerheads and to avoid phagocytosis by host immune cells. Typically, bacteria regulate biofilm formation by controlling expression of adhesive structures to attach to surfaces and other bacterial cells. Mycobacteria harbor a unique cell wall built chiefly of long-chain mycolic acids that confers hydrophobicity and has been thought to cause constitutive aggregation in liquid media. Here we show that aggregation is instead a regulated process dictated by the balance of available carbon and nitrogen. Understanding that mycobacteria utilize metabolic cues to regulate the transition between planktonic and aggregated cells reveals an inroad to controlling biofilm formation through targeted therapeutics., Nontuberculous mycobacteria (NTM) are emerging opportunistic pathogens that colonize household water systems and cause chronic lung infections in susceptible patients. The ability of NTM to form surface-attached biofilms in the nonhost environment and corded aggregates in vivo is important to their ability to persist in both contexts. Underlying the development of these multicellular structures is the capacity of mycobacterial cells to adhere to one another. Unlike most other bacteria, NTM spontaneously and constitutively aggregate in vitro, hindering our ability to understand the transition between planktonic and aggregated cells. While culturing a model NTM, Mycobacterium smegmatis, in rich medium, we fortuitously discovered that planktonic cells accumulate after ∼3 days of growth. By providing selective pressure for bacteria that disperse earlier, we isolated a strain with two mutations in the oligopeptide permease operon (opp). A mutant lacking the opp operon (Δopp) disperses earlier than wild type (WT) due to a defect in nutrient uptake. Experiments with WT M. smegmatis revealed that growth as aggregates is favored when carbon is replete, but under conditions of low available carbon relative to available nitrogen, M. smegmatis grows as planktonic cells. By adjusting carbon and nitrogen sources in defined medium, we tuned the cellular C/N ratio such that M. smegmatis grows either as aggregates or as planktonic cells. C/N-mediated aggregation regulation is widespread among NTM with the possible exception of rough-colony Mycobacterium abscessus isolates. Altogether, we show that NTM aggregation is a controlled process that is governed by the relative availability of carbon and nitrogen for metabolism.

Uropathogenic Escherichia coli (UPEC), which causes the majority of urinary tract infections (UTI), uses pilus-mediated adherence to initiate biofilm formation in the urinary tract. Oxygen gradients within E. coli biofilms regulate expression and localization of adhesive type 1 pili. A transposon mutant screen for strains defective in biofilm formation identified the ubiI (formerly visC ) aerobic ubiquinone synthase gene as critical for UPEC biofilm formation. In this study, we characterized a nonpolar ubiI deletion mutant and compared its behavior to that of wild-type bacteria grown under aerobic and anoxic conditions. Consistent with its function as an aerobic ubiquinone-8 synthase, deletion of ubiI in UPEC resulted in reduced membrane potential, diminished motility, and reduced expression of chaperone-usher pathway pili. Loss of aerobic respiration was previously shown to negatively impact expression of type 1 pili. To determine whether this reduction in type 1 pili was due to an energy deficit, wild-type UPEC and the ubiI mutant were compared for energy-dependent phenotypes under anoxic conditions, in which quinone synthesis is undertaken by anaerobic quinone synthases. Under anoxic conditions, the two strains exhibited wild-type levels of motility but produced diminished numbers of type 1 pili, suggesting that the reduction of type 1 pilus expression in the absence of oxygen is not due to a cellular energy deficit. Acute- and chronic-infection studies in a mouse model of UTI revealed a significant virulence deficit in the ubiI mutant, indicating that UPEC encounters enough oxygen in the bladder to induce aerobic ubiquinone synthesis during infection. IMPORTANCE The majority of urinary tract infections are caused by uropathogenic E. coli , a bacterium that can respire in the presence and absence of oxygen. The bladder environment is hypoxic, with oxygen concentrations ranging from 4% to 7%, compared to 21% atmospheric oxygen. This work provides evidence that aerobic ubiquinone synthesis must be engaged during bladder infection, indicating that UPEC bacteria sense and use oxygen as a terminal electron acceptor in the bladder and that this ability drives infection potential despite the fact that UPEC is a facultative anaerobe.

Enteric 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 ∼40-fold-increased survival after C. elegans and M. xanthus killing compared to the non-matrix-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.

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., IMPORTANCE The advent of metagenomic and metatranscriptomic analyses has improved our understanding of microbial communities by empowering us to identify bacteria, calculate their abundance, and profile gene expression patterns in complex environments. We are still technologically limited, however, in regards to the many questions that bulk measurements cannot answer, specifically in assessing the spatial organization of microbe-microbe and microbe-host interactions. Here, we demonstrate the power of an enhanced optical clearing method, MiPACT, to survey important aspects of bacterial physiology (aggregation, host interactions, and growth rate), in situ, with preserved spatial information when coupled to rRNA detection by HCR. Our application of MiPACT-HCR to cystic fibrosis patient sputum revealed species-specific aggregation patterns, yet slow growth characterized the vast majority of bacterial cells regardless of their cell type. More broadly, MiPACT, coupled with fluorescent labeling, promises to advance the direct study of microbial communities in diverse environments, including microbial habitats within mammalian systems.

Escherichia coli is one of the world’s best-characterized organisms, because 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 (ECM), 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 article, which concludes with insights into the future of E. coli biofilm research and potential therapies.

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.