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.

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.

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.

Vancomycin-Loaded Microneedle Arrays against Methicillin-Resistant Staphylococcus Aureus Skin Infections.

Skin and soft tissue infections (SSTIs) caused by methicillin-resistant Staphylococcus aureus (MRSA) are a major healthcare burden, often treated with intravenous injection of the glycopeptide antibiotic vancomycin (VAN). However, low local drug concentration in the skin limits its treatment efficiency, while systemic exposure promotes the development of resistant bacterial strains. Topical administration of VAN on skin is ineffective as its high molecular weight prohibits transdermal penetration. In order to implement a local VAN delivery, microneedle (MN) arrays with a water-insoluble support layer for the controlled administration of VAN into the skin are developed. The utilization of such a support layer results in water-insoluble needle shafts surrounded by drug-loaded water-soluble tips with high drug encapsulation. The developed MN arrays can penetrate the dermal barriers of both porcine and fresh human skin. Permeation studies on porcine skin reveal that the majority of the delivered VAN is retained within the skin. It is shown that the VAN-MN array reduces MRSA growth both in vitro and ex vivo on skin. The developed VAN-MN arrays may be extended to several drugs and may facilitate localized treatment of MRSA-caused skin infections while minimizing adverse systemic effects.

Colonization of dermal arterioles by Neisseria meningitidis provides a safe haven from neutrophils.

The human pathogen Neisseria meningitidis can cause meningitis and fatal systemic disease. The bacteria colonize blood vessels and rapidly cause vascular damage, despite a neutrophil-rich inflammatory infiltrate. Here, we use a humanized mouse model to show that vascular colonization leads to the recruitment of neutrophils, which partially reduce bacterial burden and vascular damage. This partial effect is due to the ability of bacteria to colonize capillaries, venules and arterioles, as observed in human samples. In venules, potent neutrophil recruitment allows efficient bacterial phagocytosis. In contrast, in infected capillaries and arterioles, adhesion molecules such as E-Selectin are not expressed on the endothelium, and intravascular neutrophil recruitment is minimal. Our results indicate that the colonization of capillaries and arterioles by N. meningitidis creates an intravascular niche that precludes the action of neutrophils, resulting in immune escape and progression of the infection.

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.

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.

Neutrophil Recruitment to Noninvasive MRSA at the Stratum Corneum of Human Skin Mediates Transient Colonization.

Staphylococcus aureus is a leading cause of skin and soft issue infection, but paradoxically, it also transiently, and often harmlessly, colonizes human skin. An obstacle to understanding this contradiction has been a shortage of in vivo models reproducing the unique structure and immunology of human skin. In this work, we developed a humanized model to study how healthy adult human skin responds to colonizing methicillin-resistant S. aureus (MRSA). We demonstrate the importance of the outer stratum corneum as the major site of bacterial colonization and how noninvasive MRSA adhesion to corneocytes induces a local inflammatory response in underlying skin layers. This signaling recruits neutrophils to the skin, where they control bacterial numbers, mediating transiency in colonization. This work highlights the spatiotemporal aspects of human skin colonization and demonstrates a subclinical inflammatory response to noninvasive MRSA that allows human skin to regulate the bacterial population at its outer surface.

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.

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.

Adhesion to nanofibers drives cell membrane remodeling through one-dimensional wetting.

The shape of cellular membranes is highly regulated by a set of conserved mechanisms that can be manipulated by bacterial pathogens to infect cells. Remodeling of the plasma membrane of endothelial cells by the bacterium Neisseria meningitidis is thought to be essential during the blood phase of meningococcal infection, but the underlying mechanisms are unclear. Here we show that plasma membrane remodeling occurs independently of F-actin, along meningococcal type IV pili fibers, by a physical mechanism that we term 'one-dimensional' membrane wetting. We provide a theoretical model that describes the physical basis of one-dimensional wetting and show that this mechanism occurs in model membranes interacting with nanofibers, and in human cells interacting with extracellular matrix meshworks. We propose one-dimensional wetting as a new general principle driving the interaction of cells with their environment at the nanoscale that is diverted by meningococci during infection.

Nondestructive, real-time determination and visualization of cellulose, hemicellulose and lignin by luminescent oligothiophenes.

Enabling technologies for efficient use of the bio-based feedstock are crucial to the replacement of oil-based products. We investigated the feasibility of luminescent conjugated oligothiophenes (LCOs) for non-destructive, rapid detection and quality assessment of lignocellulosic components in complex biomass matrices. A cationic pentameric oligothiophene denoted p-HTEA (pentamer hydrogen thiophene ethyl amine) showed unique binding affinities to cellulose, lignin, hemicelluloses, and cellulose nanofibrils in crystal, liquid and paper form. We exploited this finding using spectrofluorometric methods and fluorescence confocal laser scanning microscopy, for sensitive, simultaneous determination of the structural and compositional complexities of native lignocellulosic biomass. With exceptional photostability, p-HTEA is also demonstrated as a dynamic sensor for real-time monitoring of enzymatic cellulose degradation in cellulolysis. These results demonstrate the use of p-HTEA as a non-destructive tool for the determination of cellulose, hemicellulose and lignin in complex biomass matrices, thereby aiding in the optimization of biomass-converting technologies.

Microbial pathogenesis revealed by intravital microscopy: pros, cons and cautions.

Intravital multiphoton imaging allows visualization of infections and pathogenic mechanisms within intact organs in their physiological context. Today, most organs of mice and rats are applicable to in vivo or ex vivo imaging, opening completely new avenues for many researchers. Advances in fluorescent labeling of pathogens and infected cells, as well as improved small animal models for human pathogens, led to the increased application of in vivo imaging in infectious diseases research in recent years. Here, we review the latest literature on intravital or ex vivo imaging of viral and bacterial infections and critically discuss requirements, benefits and drawbacks of applied animal models, labeling strategies, and imaged organs.

Real-time optotracing of curli and cellulose in live Salmonella biofilms using luminescent oligothiophenes.

Extracellular matrix (ECM) is the protein- and polysaccharide-rich backbone of bacterial biofilms that provides a defensive barrier in clinical, environmental and industrial settings. Understanding the dynamics of biofilm formation in native environments has been hindered by a lack of research tools. Here we report a method for simultaneous, real-time, in situ detection and differentiation of the Salmonella ECM components curli and cellulose, using non-toxic, luminescent conjugated oligothiophenes (LCOs). These flexible conjugated polymers emit a conformation-dependent fluorescence spectrum, which we use to kinetically define extracellular appearance of curli fibres and cellulose polysaccharides during bacterial growth. The scope of this technique is demonstrated by defining biofilm morphotypes of Salmonella enterica serovars Enteritidis and Typhimurium, and their isogenic mutants in liquid culture and on solid media, and by visualising the ECM components in native biofilms. Our reported use of LCOs across a number of platforms, including intracellular cellulose production in eukaryotic cells and in infected tissues, demonstrates the versatility of this optotracing technology, and its ability to redefine biofilm research.

Humanized mouse model to study bacterial infections targeting the microvasculature.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Assessment of the interplay between blood and skin vascular abnormalities in adult purpura fulminans.

RATIONALE: Purpura fulminans in adults is a rare but devastating disease. Its pathophysiology is not well known. OBJECTIVES: To understand the pathophysiology of skin lesions in purpura fulminans, the interplay between circulating blood and vascular alterations was assessed. METHODS: Prospective multicenter study in four intensive care units. Patients with severe sepsis without skin lesions were recruited as control subjects. MEASUREMENTS AND MAIN RESULTS: Twenty patients with severe sepsis and purpura fulminans were recruited for blood sampling, and skin biopsy was performed in deceased patients. High severity of disease and mortality rates (80%) was observed. Skin biopsies in purpura fulminans lesions revealed thrombosis and extensive vascular damage: vascular congestion and dilation, endothelial necrosis, alteration of markers of endothelial integrity (CD31) and of the protein C pathway receptors (endothelial protein C receptor, thrombomodulin). Elevated plasminogen activating inhibitor-1 mRNA was also observed. Comparison with control patients showed that these lesions were specific to purpura fulminans. By contrast, no difference was observed for blood hemostasis parameters, including soluble thrombomodulin, activated protein C, and disseminated intravascular coagulation markers. Bacterial presence at the vascular wall was observed specifically in areas of vascular damage in eight of nine patients tested (including patients with Streptococcus pneumoniae, Neisseria meningitidis, Escherichia coli, and Pseudomonas aeruginosa infection). CONCLUSIONS: Thrombi and extensive vascular damage with multifaceted prothrombotic local imbalance are characteristics of purpura fulminans. A "vascular wall infection" hypothesis, responsible for endothelial damage and subsequent skin lesions, can be put forward.