The maturation of native uropathogenic Escherichia coli biofilms seen through a non-interventional lens.

Urinary tract infections (UTI) caused by uropathogenic Escherichia coli (UPEC) are a significant global health challenge. The UPEC biofilm lifestyle is believed to play an important role in infection recurrency and treatment resistance, but our understanding of how the extracellular matrix (ECM) components curli and cellulose contribute to biofilm formation and pathogenicity is limited. Here, we study the spatial and temporal development of native UPEC biofilm using agar-based detection methods where the non-toxic, optically active fluorescent tracer EbbaBiolight 680 reports the expression and structural location of curli in real-time. An in vitro screen of the biofilm capacity of common UPEC strains reveals significant strain variability and identifies UPEC No. 12 (UPEC12) as a strong biofilm former at 28 °C and 37 °C. Non-interventional microscopy, including time-lapse and 2-photon, reveal significant horizontal and vertical heterogeneity in the UPEC12 biofilm structure. We identify region-specific expression of curli, with a shift in localization from the bottom of the flat central regions of the biofilm to the upper surface in the topographically dramatic intermediate region. When investigating if the rdar morphotype affects wettability of the biofilm surface, we found that the nano-architecture of curli guided by cellulose, rather than the rdar macrostructures, leads to increased hydrophobicity of the biofilm. By providing new insights at exceptional temporal and spatial resolution, we demonstrate how non-interventional analysis of native biofilms will facilitate the next generation of understanding into the roles of ECM components during growth of UPEC biofilms and their contribution to the pathogenesis of UTI.

High-Resolution Large-Area Image Analysis Deciphers the Distribution of Salmonella Cells and ECM Components in Biofilms Formed on Charged PEDOT:PSS Surfaces.

Biofilms, comprised of cells embedded in extracellular matrix (ECM), enable bacterial surface colonization and contribute to pathogenesis and biofouling. Yet, antibacterial surfaces are mainly evaluated for their effect on bacterial cells rather than the ECM. Here, a method is presented to separately quantify amounts and distribution of cells and ECM in Salmonella biofilms grown on electroactive poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS). Within a custom-designed biofilm reactor, biofilm forms on PEDOT:PSS surfaces electrically addressed with a bias potential and simultaneous recording of the resulting current. The amount and distribution of cells and ECM in biofilms are analyzed using a fluorescence-based spectroscopic mapping technique and fluorescence confocal microscopy combined with advanced image processing. The study shows that surface charge leads to upregulated ECM production, leaving the cell counts largely unaffected. An altered texture is also observed, with biofilms forming small foci or more continuous structures. Supported by mutants lacking ECM production, ECM is identified as an important target when developing antibacterial strategies. Also, a central role for biofilm distribution is highlighted that likely influences antimicrobial susceptibility in biofilms. This work provides yet a link between conductive polymer materials and bacterial metabolism and reveals for the first time a specific effect of electrochemical addressing on bacterial ECM formation.

Fluorescent optotracers for bacterial and biofilm detection and diagnostics.

Effective treatment of bacterial infections requires methods that accurately and quickly identify which antibiotic should be prescribed. This review describes recent research on the development of optotracing methodologies for bacterial and biofilm detection and diagnostics. Optotracers are small, chemically well-defined, anionic fluorescent tracer molecules that detect peptide- and carbohydrate-based biopolymers. This class of organic molecules (luminescent conjugated oligothiophenes) show unique electronic, electrochemical and optical properties originating from the conjugated structure of the compounds. The photophysical properties are further improved as donor-acceptor-donor (D-A-D)-type motifs are incorporated in the conjugated backbone. Optotracers bind their biopolymeric target molecules via electrostatic interactions. Binding alters the optical properties of these tracer molecules, shown as altered absorption and emission spectra, as well as ON-like switch of fluorescence. As the optotracer provides a defined spectral signature for each binding partner, a fingerprint is generated that can be used for identification of the target biopolymer. Alongside their use for in situ experimentation, optotracers have demonstrated excellent use in studies of a number of clinically relevant microbial pathogens. These methods will find widespread use across a variety of communities engaged in reducing the effect of antibiotic resistance. This includes basic researchers studying molecular resistance mechanisms, academia and pharma developing new antimicrobials targeting biofilm infections and tests to diagnose biofilm infections, as well as those developing antibiotic susceptibility tests for biofilm infections (biofilm-AST). By iterating between the microbial world and that of plants, development of the optotracing technology has become a prime example of successful cross-feeding across the boundaries of disciplines. As optotracers offers a capacity to redefine the way we work with polysaccharides in the microbial world as well as with plant biomass, the technology is providing novel outputs desperately needed for global impact of the threat of antimicrobial resistance as well as our strive for a circular bioeconomy.

Dynamic single cell analysis in a proximal-tubule-on-chip reveals heterogeneous epithelial colonization strategies of uropathogenic Escherichia coli under shear stress.

The urinary tract is a hydrodynamically challenging microenvironment and uropathogenic Escherichia coli (UPEC) must overcome several physiological challenges in order to adhere and establish a urinary tract infection. Our previous work in vivo revealed a synergy between different UPEC adhesion organelles, which facilitated effective colonization of the renal proximal tubule. To allow high-resolution real-time analysis of this colonization behavior, we established a biomimetic proximal-tubule-on-chip (PToC). The PToC allowed for single-cell resolution analysis of the first stages of bacterial interaction with host epithelial cells, under physiological flow. Time-lapse microscopy and single-cell trajectory analysis in the PToC revealed that while the majority of UPEC moved directly through the system, a minority population initiated heterogeneous adhesion, identified as either rolling or bound. Adhesion was predominantly transient and mediated by P pili at the earliest time-points. These bound bacteria initiated a founder population which rapidly divided, leading to 3D microcolonies. Within the first hours, the microcolonies did not express extracellular curli matrix, but rather were dependent on Type 1 fimbriae as the key element in the microcolony structure. Collectively, our results show the application of Organ-on-chip technology to address bacterial adhesion behaviors, demonstrating a well-orchestrated interplay and redundancy between adhesion organelles that enables UPEC to form microcolonies and persist under physiological shear stress.

An optotracer-based antibiotic susceptibility test specifically targeting the biofilm lifestyle of Salmonella.

Antimicrobial resistance is a medical threat of global dimensions. Proper antimicrobial susceptibility testing (AST) for drug development, patient diagnosis and treatment is crucial to counteract ineffective drug use and resistance development. Despite the important role of bacterial biofilms in chronic and device-associated infections, the efficacy of antibiotics is determined using planktonic cultures. To address the need for antibiotics targeting bacteria in the biofilm lifestyle, we here present an optotracing-based biofilm-AST using Salmonella as model. Our non-disruptive method enables real-time recording of the extracellular matrix (ECM) components, providing specific detection of the biofilm lifestyle. Biofilm formation prior to antibiotic challenge can thus be confirmed and pre-treatment data collected. By introducing Kirby-Bauer discs, we performed a broad screen of the effects of antibiotics representing multiple classes, and identified compounds with ECM inhibitory as well as promoting effects. These compounds were further tested in agar-based dose-response biofilm-AST assays. By quantifying the ECM based on the amount of curli, and by visualizing the biofilm size and morphology, we achieved new information directly reflecting the treated biofilm. This verified the efficacy of several antibiotics that were effective in eradicating pre-formed biofilms, and it uncovered intriguing possible resistance mechanisms initiated in response to treatments. By providing deeper insights into the resistances and susceptibilities of microbes, expanded use of the biofilm-AST will contribute to more effective treatments of infections and reduced resistance development.

Optotracing for live selective fluorescence-based detection of Candida albicans biofilms.

Candida albicans is the most common fungal pathogen in humans, implicated in hospital-acquired infections, secondary infections in human immunodeficiency virus (HIV) patients, and is a significant contributor to the global antimicrobial resistance (AMR) burden. Early detection of this pathogen is needed to guide preventative strategies and the selection and development of therapeutic treatments. Fungal biofilms are a unique heterogeneous mix of cell types, extracellular carbohydrates and amyloid aggregates. Perhaps due to the dominance of carbohydrates in fungi, to date, few specific methods are available for the detection of fungal biofilms. Here we present a new optotracing-based method for the detection and analysis of yeast and biofilms based on C. albicans SC5314 as a model. Using commercial extracts of cell wall carbohydrates, we showed the capability of the optotracer EbbaBiolight 680 for detecting chitin and ß-glucans. The sensitivity of this tracer to these carbohydrates in their native environment within fungal cells enabled the visualization of both yeast and hyphal forms of the microbe. Analysis of optotracer fluorescence by confocal laser scanning microscopy revealed extensive staining of fungi cell walls as well as the presence of intracellular amyloid aggregates within a subpopulation of cells within the biofilm. Further analysis of the photophysical properties of bound tracers by spectroscopy and spectral imaging revealed polymorphisms between amyloid aggregates within yeast and hyphal cells and enabled their differentiation. With exceptional spatial and temporal resolution, this assay adds a new technique that facilitates future understanding of fungal biofilms and their formation, and enables direct, unbiased diagnostics of these medically relevant biofilms, as well as the development of antifungal strategies.

Effect of anticoagulant and platelet inhibition on the risk of bacteremia among patients with acute pyelonephritis: a retrospective cohort study.

BACKGROUND: An increasing number of patients are being prescribed anticoagulants and platelet inhibitors (antithrombotic treatment). Basic research has suggested an association between antithrombotic treatment and bacteremia during kidney infection. Here, we investigated the association between antithrombotic treatment, bacteremia and acute kidney injury in patients with acute pyelonephritis. METHODS: A retrospective cohort study was conducted in a large university hospital in Sweden. Data were retrieved from electronic medical records for adult patients with acute pyelonephritis in 2016. The main outcome was bacteremia and secondary outcome acute kidney injury. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated through multiple logistic regression. Treatment with different groups of antithrombotic agents were compared to no antithrombotic treatment. RESULTS: 1814 patients with acute pyelonephritis were included, in whom bacteremia developed in 336 (18.5%). Low-molecular-weight heparin (LMWH) at prophylactic doses was associated with a lower risk of bacteremia, compared to no antithrombotic treatment (OR 0.5; 95% CI 0.3-0.7). Other antithrombotic treatments were not associated with a risk of bacteremia. Additionally, patients with prophylactic doses of LMWH had a lower risk of acute kidney injury (OR 0.5; 95% CI 0.3-0.8). CONCLUSIONS: We found no association between antithrombotic treatment and an increased risk of bacteremia during acute pyelonephritis. Conversely, patients with prophylactic doses of LMWH had a slightly reduced risk of bacteremia. LMWH at prophylactic doses was also associated with a lower risk of acute kidney injury. Our results suggest that it is safe to continue antithrombotic treatment during acute pyelonephritis, in regards to bacteremia and acute kidney injury risk.

Structural Properties Dictating Selective Optotracer Detection of Staphylococcus aureus.

Optotracers are conformation-sensitive fluorescent tracer molecules that detect peptide- and carbohydrate-based biopolymers. Their binding to bacterial cell walls allows selective detection and visualisation of Staphylococcus aureus (S. aureus). Here, we investigated the structural properties providing optimal detection of S. aureus. We quantified spectral shifts and fluorescence intensity in mixes of bacteria and optotracers, using automatic peak analysis, cross-correlation, and area-under-curve analysis. We found that the length of the conjugated backbone and the number of charged groups, but not their distribution, are important factors for selective detection of S. aureus. The photophysical properties of optotracers were greatly improved by incorporating a donor-acceptor-donor (D-A-D)-type motif in the conjugated backbone. With significantly reduced background and binding-induced on-switch of fluorescence, these optotracers enabled real-time recordings of S. aureus growth. Collectively, this demonstrates that chemical structure and photophysics are key tunable characteristics in the development of optotracers for selective detection of bacterial species.

A semi high-throughput method for real-time monitoring of curli producing Salmonella biofilms on air-solid interfaces.

Biofilms enable bacteria to colonize numerous ecological niches. Bacteria within a biofilm are protected by the extracellular matrix (ECM), of which the fibril-forming amyloid protein curli and polysaccharide cellulose are major components in members of Salmonella, Eschericha and Mycobacterium genus. A shortage of real-time detection methods has limited our understanding of how ECM production contributes to biofilm formation and pathogenicity. Here we present optotracing as a new semi-high throughput method for dynamic monitoring of Salmonella biofilm growth on air-solid interfaces. We show how an optotracer with binding-induced fluorescence acts as a dynamic fluorescent reporter of curli expression during biofilm formation on agar. Using spectrophotometry and microscopic imaging of fluorescence, we analyse in real-time the development of the curli architecture in relation to bacterial cells. With exceptional spatial and temporal precision, this revealed a well-structured, non-uniform distribution of curli organised in distally projecting radial channel patterns. Dynamic monitoring of the biofilm also showed defined regions undergoing different growth phases. ECM structures were found to assemble in regions of late exponential growth phase, suggesting that ECM forms on site after bacteria colonize the surface. As the optotracer biofilm method expedites screening of curli production, providing exceptional spatial-temporal understanding of the surface-associated biofilm lifestyle, this method adds a new technique to further our understanding of bacterial biofilms.

UPEC kidney infection triggers neuro-immune communication leading to modulation of local renal inflammation by splenic IFNγ.

Bacterial infection results in a veritable cascade of host responses, both local and systemic. To study the initial stages of host-pathogen interaction in living tissue we use spatially-temporally controlled in vivo models. Using this approach, we show here that within 4 h of a uropathogenic Escherichia coli (UPEC) infection in the kidney, an IFNγ response is triggered in the spleen. This rapid infection-mediated inter-organ communication was found to be transmitted via nerve signalling. Bacterial expression of the toxin α-hemolysin directly and indirectly activated sensory neurons, which were identified in the basement membrane of renal tubules. Nerve activation was transmitted via the splenic nerve, inducing upregulation of IFNγ in the marginal zones of the spleen that led to increasing concentrations of IFNγ in the circulation. We found that IFNγ modulated the inflammatory signalling generated by renal epithelia cells in response to UPEC infection. This demonstrates a new concept in the host response to kidney infection; the role of nerves in sensing infection and rapidly triggering a systemic response which can modulate inflammation at the site of infection. The interplay between the nervous and immune systems is an exciting, developing field with the appealing prospect of non-pharmaceutical interventions. Our study identifies an important role for systemic neuro-immune communication in modulating inflammation during the very first hours of a local bacterial infection in vivo.

Optotracing for selective fluorescence-based detection, visualization and quantification of live S. aureus in real-time.

Methods for bacterial detection are needed to advance the infection research and diagnostics. Based on conformation-sensitive fluorescent tracer molecules, optotracing was recently established for dynamic detection and visualization of structural amyloids and polysaccharides in the biofilm matrix of gram-negative bacteria. Here, we extend the use of optotracing for detection of gram-positive bacteria, focussing on the clinically relevant opportunistic human pathogen Staphylococcus aureus. We identify a donor-acceptor-donor-type optotracer, whose binding-induced fluorescence enables real-time detection, quantification, and visualization of S. aureus in monoculture and when mixed with gram-negative Salmonella Enteritidis. An algorithm-based automated high-throughput screen of 1920 S. aureus transposon mutants recognized the cell envelope as the binding target, which was corroborated by super-resolution microscopy of bacterial cells and spectroscopic analysis of purified cell wall components. The binding event was essentially governed by hydrophobic interactions, which permitted custom-designed tuning of the binding selectivity towards S. aureus versus Enterococcus faecalis by appropriate selection of buffer conditions. Collectively this work demonstrates optotracing as an enabling technology relevant for any field of basic and applied research, where visualization and detection of S. aureus is needed.

Bacterial Sensing and Biofilm Monitoring for Infection Diagnostics.

Recent insights into the rapidly emerging field of bacterial sensing and biofilm monitoring for infection diagnostics are discussed as well as recent key developments and emerging technologies in the field. Electrochemical sensing of bacteria and bacterial biofilm via synthetic, natural, and engineered recognition, as well as direct redox-sensing approaches via algorithm-based optical sensing, and tailor-made optotracing technology are discussed. These technologies are highlighted to answer the very critical question: "how can fast and accurate bacterial sensing and biofilm monitoring be achieved? Following on from that: "how can these different sensing concepts be translated for use in infection diagnostics? A central obstacle to this transformation is the absence of direct and fast analysis methods that provide high-throughput results and bio-interfaces that can control and regulate the means of communication between biological and electronic systems. Here, the overall progress made to date in building such translational efforts at the level of an individual bacterial cell to a bacterial community is discussed.

Nitrate Metabolism Modulates Biosynthesis of Biofilm Components in Uropathogenic Escherichia coli and Acts as a Fitness Factor During Experimental Urinary Tract Infection.

To successfully colonize a variety of environments, bacteria can coordinate complex collective behaviors such as biofilm formation. To thrive in oxygen limited niches, bacteria's versatile physiology enables the utilization of alternative electron acceptors. Nitrate, the second most favorable electron acceptor after oxygen, plays a prominent role in the physiology of uropathogenic Escherichia coli (UPEC) and is abundantly found in urine. Here we analyzed the role of extracellular nitrate in the pathogenesis of the UPEC strain CFT073 with an initial focus on biofilm formation. Colony morphotyping in combination with extensive mutational, transcriptional, and protein expression analyses of CFT073 wild-type and mutants deficient in one or several nitrate reductases revealed an association between nitrate reduction and the biosynthesis of biofilm extracellular matrix components. We identified a role for the nitrate response regulator NarL in modulating expression of the biofilm master regulator CsgD. To analyze the role of nitrate reduction during infection in vivo, we tested wild-type CFT073 and a nitrate reductase null mutant in an ascending urinary tract infection (UTI) model. Individually, each strain colonized extensively, suggesting that nitrate reduction is expendable during UTI. However, during competitive co-infection, the strain incapable of nitrate reduction was strongly outcompeted. This suggests that nitrate reduction can be considered a non-essential but advantageous fitness factor for UPEC pathogenesis. This implies that UPEC rapidly adapts their metabolic needs to the microenvironment of infected tissue. Collectively, this work demonstrates a unique association between nitrate respiration, biofilm formation, and UPEC pathogenicity, highlighting how the use of alternative electron acceptors enables bacterial pathogens to adapt to challenging infectious microenvironments.

A Cinematic View of Tissue Microbiology in the Live Infected Host.

Tissue microbiology allows for the study of bacterial infection in the most clinically relevant microenvironment, the living host. Advancements in techniques and technology have facilitated the development of novel ways of studying infection. Many of these advancements have come from outside the field of microbiology. In this article, we outline the progression from bacteriology through cellular microbiology to tissue microbiology, highlighting seminal studies along the way. We outline the enormous potential but also some of the challenges of the tissue microbiology approach. We focus on the role of emerging technologies in the continual development of infectious disease research and highlight future possibilities in our ongoing quest to understand host-pathogen interaction.

Conjugated Oligo- and Polymers for Bacterial Sensing.

Fast and accurate detection of bacteria and differentiation between pathogenic and commensal colonization are important keys in preventing the emergence and spread of bacterial resistance toward antibiotics. As bacteria undergo major lifestyle changes during colonization, bacterial sensing needs to be achieved on different levels. In this review, we describe how conjugated oligo- and polymers are used to detect bacterial colonization. We summarize how oligothiophene derivatives have been tailor-made for detection of biopolymers produced by a wide range of bacteria upon entering the biofilm lifestyle. We further describe how these findings are translated into diagnostic approaches for biofilm-related infections. Collectively, this provides an overview on how synthetic biorecognition elements can be used to produce fast and easy diagnostic tools and new methods for infection control.

High Resolution Intravital Imaging of the Renal Immune Response to Injury and Infection in Mice.

We developed an experimental set up that enables longitudinal studies of immune cell behavior in situ in the challenged as well as unchallenged kidney of anesthetized mice over several hours. Using highly controlled vacuum to stabilize the kidney, the superficial renal cortex could continuously be visualized with minimal disruption of the local microenvironment. No visible changes in blood flow or neutrophils and macrophages numbers were observed after several hours of visualizing the unchallenged kidney, indicating a stable tissue preparation without apparent tissue damage. Applying this set up to monocyte/macrophage (CX3CR1GFP/+) reporter mice, we observed the extensive network of stellate-shaped CX3CR1 positive cells (previously identified as renal mononuclear phagocytes). The extended dendrites of the CX3CR1 positive cells were found to bridge multiple capillaries and tubules and were constantly moving. Light induced sterile tissue injury resulted in rapid neutrophil accumulation to the site of injury. Similarly, microinfusion of uropathogenic Escherichia coli into a single nephron induced a rapid and massive recruitment of neutrophils to the site of infection, in addition to active bacterial clearance by neutrophils. In contrast, the kidney resident mononuclear phagocytes were observed to not increase in numbers or migrate toward the site of injury or infection. In conclusion, this model allows for longitudinal imaging of responses to localized kidney challenges in the mouse.

Protective vascular coagulation in response to bacterial infection of the kidney is regulated by bacterial lipid A and host CD147.

Bacterial infection of the kidney leads to a rapid cascade of host protective responses, many of which are still poorly understood. We have previously shown that following kidney infection with uropathogenic Escherichia coli (UPEC), vascular coagulation is quickly initiated in local perivascular capillaries that protects the host from progressing from a local infection to systemic sepsis. The signaling mechanisms behind this response have not however been described. In this study, we use a number of in vitro and in vivo techniques, including intravital microscopy, to identify two previously unrecognized components influencing this protective coagulation response. The acylation state of the Lipid A of UPEC lipopolysaccharide (LPS) is shown to alter the kinetics of local coagulation onset in vivo. We also identify epithelial CD147 as a potential host factor influencing infection-mediated coagulation. CD147 is expressed by renal proximal epithelial cells infected with UPEC, contingent to bacterial expression of the α-hemolysin toxin. The epithelial CD147 subsequently can activate tissue factor on endothelial cells, a primary step in the coagulation cascade. This study emphasizes the rapid, multifaceted response of the kidney tissue to bacterial infection and the interplay between host and pathogen during the early hours of renal infection.

Rapid diagnostic assay for detection of cellulose in urine as biomarker for biofilm-related urinary tract infections.

The ability of uropathogenic Escherichia coli (UPEC) to adopt a biofilm lifestyle in the urinary tract is suggested as one cause of recurrent urinary tract infections (UTIs). A clinical role of UPEC biofilm is further supported by the presence of bacterial aggregates in urine of UTI patients. Yet, no diagnostics exist to differentiate between the planktonic and biofilm lifestyle of bacteria. Here, we developed a rapid diagnostic assay for biofilm-related UTI, based on the detection of cellulose in urine. Cellulose, a component of biofilm extracellular matrix, is detected by a luminescent-conjugated oligothiophene, which emits a conformation-dependent fluorescence spectrum when bound to a target molecule. We first defined the cellulose-specific spectral signature in the extracellular matrix of UPEC biofilm colonies, and used these settings to detect cellulose in urine. To translate this optotracing assay for clinical use, we composed a workflow that enabled rapid isolation of urine sediment and screening for the presence of UPEC-derived cellulose in <45 min. Using multivariate analysis, we analyzed spectral information obtained between 464 and 508 nm by optotracing of urine from 182 UTI patients and 8 healthy volunteers. Cellulose was detected in 14.8% of UTI urine samples. Using cellulose as a biomarker for biofilm-related UTI, our data provide direct evidence that UPEC forms biofilm in the urinary tract. Clinical implementation of this rapid, non-invasive and user-friendly optotracing diagnostic assay will potentially aid clinicians in the design of effective antibiotic treatment.

Erratum: Author Correction: Redox-active conducting polymers modulate Salmonella biofilm formation by controlling availability of electron acceptors.

[This corrects the article DOI: 10.1038/s41522-017-0027-0.].