Intermittent Pili-Mediated Forces Fluidize Neisseria meningitidis Aggregates Promoting Vascular Colonization.

Neisseria meningitidis, a bacterium responsible for meningitis and septicemia, proliferates and eventually fills the lumen of blood capillaries with multicellular aggregates. The impact of this aggregation process and its specific properties are unknown. We first show that aggregative properties are necessary for efficient infection and study their underlying physical mechanisms. Micropipette aspiration and single-cell tracking unravel unique features of an atypical fluidized phase, with single-cell diffusion exceeding that of isolated cells. A quantitative description of the bacterial pair interactions combined with active matter physics-based modeling show that this behavior relies on type IV pili active dynamics that mediate alternating phases of bacteria fast mutual approach, contact, and release. These peculiar fluid properties proved necessary to adjust to the geometry of capillaries upon bacterial proliferation. Intermittent attractive forces thus generate a fluidized phase that allows for efficient colonization of the blood capillary network during infection.

Physical Sequestration of Bacillus anthracis in the Pulmonary Capillaries in Terminal Infection.

The lung is the terminal target of Bacillus anthracis before death, whatever the route of infection (cutaneous, inhalational, or digestive). During a cutaneous infection in absence of toxins, we observed encapsulated bacteria colonizing the alveolar capillary network, bacteria and hemorrhages in alveolar and bronchiolar spaces, and hypoxic foci in the lung (endothelial cells) and brain (neurons and neuropil). Circulating encapsulated bacteria were as chains of approximately 13 µm in length. Bacteria of such size were immediately trapped within the lung capillary network, but bacteria of shorter length were not. Controlling lung-targeted pathology would be beneficial for anthrax treatment.

Sepsis impairs microvascular autoregulation and delays capillary response within hypoxic capillaries.

INTRODUCTION: The microcirculation supplies oxygen (O2) and nutrients to all cells with the red blood cell (RBC) acting as both a deliverer and sensor of O2. In sepsis, a proinflammatory disease with microvascular complications, small blood vessel alterations are associated with multi-organ dysfunction and poor septic patient outcome. We hypothesized that microvascular autoregulation-existing at three levels: over the entire capillary network, within a capillary and within the erythrocyte-was impaired during onset of sepsis. This study had three objectives: 1) measure capillary response time within hypoxic capillaries, 2) test the null hypothesis that RBC O2-dependent adenosine triphosphate (ATP) efflux was not altered by sepsis and 3) develop a framework of a pathophysiological model. METHODS: This was an animal study, comparing sepsis with control, set in a university laboratory. Acute hypotensive sepsis was studied using cecal ligation and perforation (CLP) with a 6-hour end-point. Rat hindlimb skeletal muscle microcirculation was imaged, and capillary RBC supply rate (SR = RBC/s), RBC hemoglobin O2 saturation (SO2) and O2 supply rate (qO2 = pLO2/s) were quantified. Arterial NOx (nitrite + nitrate) and RBC O2-dependent ATP efflux were measured using a nitric oxide (NO) analyzer and gas exchanger, respectively. RESULTS: Sepsis increased capillary stopped-flow (p = 0.001) and increased plasma lactate (p < 0.001). Increased plasma NOx (p < 0.001) was related to increased capillary RBC supply rate (p = 0.027). Analysis of 30-second SR-SO2-qO2 profiles revealed a shift towards decreased (p < 0.05) O2 supply rates in some capillaries. Moreover, we detected a three- to fourfold increase (p < 0.05) in capillary response time within hypoxic capillaries (capillary flow states where RBC SO2 < 20 %). Additionally, sepsis decreased the erythrocyte's ability to respond to hypoxic environments, as normalized RBC O2-dependent ATP efflux decreased by 62.5 % (p < 0.001). CONCLUSIONS: Sepsis impaired microvascular autoregulation at both the individual capillary and erythrocyte level, seemingly uncoupling the RBC acting as an "O2 sensor" from microvascular autoregulation. Impaired microvascular autoregulation was manifested by increased capillary stopped-flow, increased capillary response time within hypoxic capillaries, decreased capillary O2 supply rate and decreased RBC O2-dependent ATP efflux. This loss of local microvascular control was partially off-set by increased capillary RBC supply rate, which correlated with increased plasma NOx.

Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood-brain barrier.

Neisseria meningitidis is a commensal bacterium of the human nasopharynx. Occasionally, this bacterium reaches the bloodstream and causes meningitis after crossing the blood-brain barrier by an unknown mechanism. An immunohistological study of a meningococcal sepsis case revealed that neisserial adhesion was restricted to capillaries located in low blood flow regions in the infected organs. This study led to the hypothesis that drag forces encountered by the meningococcus in the bloodstream determine its attachment site in vessels. We therefore investigated the ability of N. meningitidis to bind to endothelial cells in the presence of liquid flow mimicking the bloodstream with a laminar flow chamber. Strikingly, average blood flows reported for various organs strongly inhibited initial adhesion. As cerebral microcirculation is known to be highly heterogeneous, cerebral blood velocity was investigated at the level of individual vessels using intravital imaging of rat brain. In agreement with the histological study, shear stress levels compatible with meningococcal adhesion were only observed in capillaries, which exhibited transient reductions in flow. The flow chamber assay revealed that, after initial attachment, bacteria resisted high blood velocities and even multiplied, forming microcolonies resembling those observed in the septicemia case. These results argue that the combined mechanical properties of neisserial adhesion and blood microcirculation target meningococci to transiently underperfused cerebral capillaries and thus determine disease development.

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.

Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells requires protein kinase C-alpha activation.

Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of C. neoformans. Our previous studies have shown that the CPS1 gene is required for C. neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C. neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant-negative form of PKCalpha in HBMEC. During C. neoformans infection, phosphorylation of PKCalpha was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKCalpha isoform might play a crucial role during C. neoformans invasion. Immunofluorescence microscopic images showed that induced phospho-PKCalpha colocalized with beta-actin on the membrane of HBMEC. In addition, cytochalasin D (an F-filament-disrupting agent) inhibited fungus invasion into HBMEC in a dose-dependent manner. Furthermore, blockage of PKCalpha function attenuated actin filament activity during C. neoformans invasion. These results suggest a significant role of PKCalpha and downstream actin filament activity during the fungal invasion into HBMEC.

Secondary flow mixing due to biofilm growth in capillaries of varying dimensions.

Using a magnetic resonance microscopy (MRM) technique, velocity perturbations due to biofouling in capillaries were detected in 3D velocity maps. The velocity images in each of the three square capillary sizes (2, 0.9, and 0.5 mm i.d.) tested indicate secondary flow in both the x- and y-directions for the biofouled capillaries. Similar flow maps generated in a clean square capillary show only an axial component. Investigation of these secondary flows and their geometric and dynamic similarity is the focus of this study. The results showed significant secondary flows present in the 0.9 mm i.d. capillary, on the scale of 20% of the bulk fluid flow. Since this is the "standard 1 mm" size capillary used in confocal microscopy laboratory bioreactors to investigate biofilm properties, it is important to understand how these enhanced flows impact bioreactor transport.

Gut microbiota - architects of small intestinal capillaries.

The commensal gut microbiota is an environmental factor that exerts manifold effects on host physiology. One obvious trait is the impact of this densely colonized ecosystem on small intestinal mucosal vascularization. At present, the microbiota-triggered signaling pathways influencing small intestinal renewal, angiogenesis, and vascular remodeling are largely unexplored. While the interplay of gut microbial communities with pattern recognition receptors, such as Toll-like receptors, in intestinal homeostasis is increasingly understood, it is unresolved how commensal microbiota affect the signaling pathways responsible for the formation of capillary networks in the intestinal mucosa. It is evident that intestinal vascular remodeling and renewal is disturbed in case of dysbiosis of this densely colonized microbial ecosystem, in particular under conditions of intestinal inflammation, but the effects of individual components of the gut microbiota are elusive. This review article provides an overview on the revealed microbiota-host interactions, influencing angiogenesis and vascular remodeling processes in the small intestine.

Borrelia burgdorferi adhere to blood vessels in the dura mater and are associated with increased meningeal T cells during murine disseminated borreliosis.

Borrelia burgdorferi, the causative agent of Lyme disease, is a vector-borne bacterial infection that is transmitted through the bite of an infected tick. If not treated with antibiotics during the early stages of infection, disseminated infection can spread to the central nervous system (CNS). In non-human primates (NHPs) it has been demonstrated that the leptomeninges are among the tissues colonized by B. burgdorferi spirochetes. Although the NHP model parallels aspects of human borreliosis, a small rodent model would be ideal to study the trafficking of spirochetes and immune cells into the CNS. Here we show that during early and late disseminated infection, B. burgdorferi infects the meninges of intradermally infected mice, and is associated with concurrent increases in meningeal T cells. We found that the dura mater was consistently culture positive for spirochetes in transcardially perfused mice, independent of the strain of B. burgdorferi used. Within the dura mater, spirochetes were preferentially located in vascular regions, but were also present in perivascular, and extravascular regions, as late as 75 days post-infection. At the same end-point, we observed significant increases in the number of CD3+ T cells within the pia and dura mater, as compared to controls. Flow cytometric analysis of leukocytes isolated from the dura mater revealed that CD3+ cell populations were comprised of both CD4 and CD8 T cells. Overall, our data demonstrate that similarly to infection in peripheral tissues, spirochetes adhere to the dura mater during disseminated infection, and are associated with increases in the number of meningeal T cells. Collectively, our results demonstrate that there are aspects of B. burgdorferi meningeal infection that can be modelled in laboratory mice, suggesting that mice may be useful for elucidating mechanisms of meningeal pathogenesis by B. burgdorferi.

Evidence of Rickettsia spp. infection in Sweden: a clinical, ultrastructural and serological study.

Sweden is an area potentially endemic for spotted fever rickettsioses. Rickettsia helvetica has been isolated from its tick vector Ixodes ricinus, and in a handful of cases linked to human disease. This study demonstrates for the first time in Sweden the transmission of rickettsial infection after a tick bite and the attack rate in an endemic area. We present three cases of documented rickettsial infection and a prospective serological study of Swedish recruits who were trained in the area where the patients lived and showed seroconversion to spotted fever rickettsiae. All patients showed a four-fold increase in antibody titer to the spotted fever rickettsia, R. helvetica, and immunohistochemical examination revealed rickettsia-like organisms in the walls of skin capillaries and veins. Electron microscopy showed organisms resembling R. helvetica and immunogold labeling with two anti-rickettsial antibodies demonstrated specific labeling of the rickettsial organisms in the skin biopsy specimens. Eight of the thirty-five recruits showed a four-fold increase in IgG titer reflecting a high rate of exposure. The results of this study demonstrate that spotted fever rickettsioses should be taken into consideration in the diagnosis of tick-transmitted infections in Sweden.

Immunohistochemical detection of Yersinia pestis in formalin-fixed, paraffin-embedded tissue.

Yersinia pestis infection usually is limited to lymph nodes (bubo); rarely, if bacteria are aerosolized, pneumonic plague occurs. We developed an immunohistochemical assay using a monoclonal anti-fraction 1 Y pestis antibody for formalin-fixed tissues. We studied 6 cases using this technique. Respiratory symptoms were prominent in 2 cases; histologically, one showed intra-alveolar inflammation, and the other had alveolar hemorrhage and edema. By using the immunohistochemical assay, we found intact Yersinia and granular bacterial antigen staining in alveoli, bronchi, and blood vessels. Of the remaining cases, 2 had septicemia and 2 had a bubo. Pathologic changes included lymphocyte depletion, necrosis, edema, and foamy macrophages in lymph nodes; multiple abscesses in the spleen; fibrin thrombi in glomeruli; and unremarkable lungs. By using the immunohistochemical assay, we identified intact bacteria inside monocytes and granular antigen staining in blood vessels. The immunohistochemical assay provided a fast, nonhazardous method for diagnosing plague. The immunohistochemical assay localizes bacteria, retaining tissue morphologic features, and can help define transmission mechanisms.

Electron microscopy of herpes simplex hepatitis with hepatocyte pulmonary embolization in kwashiorkor.

We report a case of herpes simplex hepatitis in a child with edematous malnutrition. Electron microscopy showed virus in parenchymal cells, with pulmonary embolization of necrotic, infected hepatic cell fragments. Systemic dissemination of herpes simplex may be related both to the profound immunoincompetence associated with kwashiorkor and to a reduction in the circulating and fixed polyanions that normally inhibit viral attachment to cells.

A clinical and bacteriological examination of Mycobacterium leprae in the epidermis and cutaneous appendages of patients with multibacillary leprosy.

In the specimens examined at Ryukyu University Hospital, acid-fast bacilli (AFB) were observed in the epidermis, cutaneous appendages and endothelial cells of capillaries. These specimens were taken from non-ulcerating skin lesions of patients with multibacillary leprosies such as LL and borderline lepromatous leprosy (BL). Of the 211 specimens examined, 23 (10.9%) were AFB-positive [AFB (+)] in the above mentioned skin regions. These AFB (+) samples were taken from nine leprosy patients; six cases (17 samples) of LL, two cases (5 samples) of BL, and one case (one sample) of BB. The AFB positive rate [AFB (+)-rate] in the above mentioned skin regions was high in the unmedicated LL sample (50.0%, 7/14) and low in the medicated mid-borderline leprosy (BB) samples (0.0%, 0/10). Particularly in the intraepidermal eccrine sweat duct (acrosyringium), a relatively high number of AFB were observed. The AFB (+)-rate appears likely to be higher in non-ulcering skin lesions with minor inflammation or in lesions with leprosy reaction than typical skin lesions such as papules, nodules, and infiltrated punched out skin lesions. Although the possibility that viable bacilli could be excreted from non-ulcerating skin lesions appeared to be small, these lesions were suspected of being a possible source of infection.

Possible death of a buffalo calf (Syncercus caffer) due to suspected heartwater (Ehrlichia ruminantium).

Rickettsial organisms resembling Ehrlichia ruminantium (the causative organism of heartwater) were demonstrated in brain smears and formalin-fixed brain sections derived from a buffalo calf that died on a private game reserve in northern KwaZulu-Natal. The possibility that the tick-free environment of a quarantine boma may have affected the calf's immunity, is discussed. These findings suggest that monitoring heartwater in wild ruminants and making brain smears as a routine during post mortem evaluations of wild ruminants, should be encouraged.

[Ultrastructural analysis of Pseudomonas aeruginosa invasion into the blood capillaries of the subcutaneous fat]. / Ul'trastrukturnyi analiz invazii sinegnoinoi palochki v krovenosnye kapilliary podkozhnoi kletchatki.

Wound wall soft tissues from 14 male rats were studied electron-microscopically 5 hours after subcutaneous implantation of a gauze piece with 3 X 10(10) organisms of Ps. aeruginosa on it. In a special series of experiments done on 75 rats, 78% of the animals have demonstrated a generalized pseudomonal infection, i. e. sepsis. The bacterial invasion into the blood stream occurred on the microcirculatory level due to inadequacy of the phagocytic barrier caused by bacterial damage of micro and macrophages. Vascular wall component destruction and capillary haemostasis (stasis, thrombosis) appeared to predispose the microbial invasion into the vascular lumen. The bacteria entered the circulation by the microembolic mechanism.

Filamentous Escherichia coli cells swimming in tapered microcapillaries.

This study analyzed the swimming characteristics of filamentous Escherichia coli cells inside tapered capillaries with a diameter decreasing from 700 µm to 4 µm and a mean body length of 27.8 µm ± 11.9 µm. Cells that were pre-oriented towards the narrower diameter section of the tapered capillary swam with high directional persistence, following conical-helix trajectories along the capillary wall. The confinement of the tapered capillary significantly diminished the mean swimming speed of filamentous cells when compared to their unrestricted mean swimming speed. The cell body rotation of individual filamentous bacteria decreased along the tapered direction, likely due to increased steric interactions with the capillary wall. Filamentous cells that swam under imposed flow rates ranging from 0.2 µl/min to 0.8 µl/min showed positive rheotaxis inside the 150 µm-350 µm diameter region of the tapered capillary. Depending on the imposed flow rate, none of the bacteria could advance beyond a critical diameter in the tapered capillary. This critical diameter is likely to be the position of the maximum shear rate they can tolerate without being flushed away. This work showed experimental evidence of how a simple flow constriction such as a tapered tube forms a hydrodynamic barrier that can deter the advance of bacterial rheotaxis.

Capsule independent uptake of the fungal pathogen Cryptococcus neoformans into brain microvascular endothelial cells.

Cryptococcosis is a life-threatening fungal disease with a high rate of mortality among HIV/AIDS patients across the world. The ability to penetrate the blood-brain barrier (BBB) is central to the pathogenesis of cryptococcosis, but the way in which this occurs remains unclear. Here we use both mouse and human brain derived endothelial cells (bEnd3 and hCMEC/D3) to accurately quantify fungal uptake and survival within brain endothelial cells. Our data indicate that the adherence and internalisation of cryptococci by brain microvascular endothelial cells is an infrequent event involving small numbers of cryptococcal yeast cells. Interestingly, this process requires neither active signalling from the fungus nor the presence of the fungal capsule. Thus entry into brain microvascular endothelial cells is most likely a passive event that occurs following 'trapping' within capillary beds of the BBB.

Modeling bacterial clearance using stochastic-differential equations.

Capillary - tissue fluid exchange is controlled by the blood pressure in the capillary and the osmotic pressure of blood (pressure of the tissue fluid outside the capillaries). In this paper, we develop a mathematical model to simulate the movement of bacteria into and within a capillary segment. The model is based on Fokker-Planck equation and Navier-Stocks equations that accounts for different boundary conditions. Also, we model the transportation through capillary walls by means of anisotropic diffusivity that depends on the pressure difference across the capillary walls. By solving the model with a numerical method, it was possible to predict the concentration of bacteria at points within the capillary. However, numerical analysis consumes computational time and resources. To efficiently simulate the bacterial clearance, we propose a segmentation model that is based on breaking the capillary network into smaller sections with pre-defined properties in order to reduce the overall computational time. The proposed model shows a great reduction in computational time and provides accurate results when compared to the numerical analysis.