scholarly journals Characterization of a mucoid-like Pseudomonas aeruginosa biofilm

Fine Focus ◽  
2015 ◽  
Vol 1 (2) ◽  
pp. 121-137
Author(s):  
Brandon M. Bauer ◽  
Lewis Rogers ◽  
Monique Macias ◽  
Gabriella Iacovetti ◽  
Alexander M. Woodrow ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Growth Conditions ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Chronic Infections ◽  
Planktonic Bacteria ◽  
The Difference

Pseudomonas aeruginosa biofilms are implicated in chronic infections. A key element of P. aeruginosapathogenicity is the formation of a biofilm, a community of bacteria encased in an exopolymeric substance (EPS) that shields the bacteria from the host immune response and antibiotic treatment. A crucial step in biofilm production is a switch in motility from freely swimming, planktonic bacteria to twitching movement and then to attached and sedentary bacteria that develop into a mature pillar-shaped biofilm. A mucoid biofilm produces an excess of alginate and is clinically the most pathogenic and the most resistant to antibiotics. Biofilms from patients exhibit a wide variety of structure, motility, and levels of attachment. In vitrobiofilms do not exhibit such a wide variety of structure and physiology. The difference between in vivo and in vitro biofilms has made the translation of in vitro studies into in vivo treatments difficult. Under different growth conditions in our lab, the P. aeruginosa strain PAO1 demonstrates two phenotypes: a non-mucoid and a mucoid-like phenotype. Confocal laser scanning microscopy (CLSM) indicates the mucoid-like phenotype is intermediate in height to the non-mucoid phenotype and biofilms formed in a once-flow-through chamber. Both mucoid-like and non-mucoid phenotypes exhibit a significant increase in twitching between 24 and 72 hours of development. The mucoid-like phenotype had greater attachment at 72 hours compared to non-mucoid phenotype. Therefore, the two phenotypes observed in our lab may represent the effect of environment to stimulate development of two types of biofilms by PAO1.

scholarly journals Interactions between Polymorphonuclear Leukocytes and Pseudomonas aeruginosa Biofilms on Silicone ImplantsIn Vivo

2012 ◽  
Vol 80 (8) ◽  
pp. 2601-2607 ◽  
Author(s):  
Maria van Gennip ◽  
Louise Dahl Christensen ◽  
Morten Alhede ◽  
Klaus Qvortrup ◽  
Peter Østrup Jensen ◽  
...  
Keyword(s):  
Immune Response ◽  
Pseudomonas Aeruginosa ◽  
Polymorphonuclear Leukocytes ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Chronic Infections ◽  
Content Type ◽  
Biofilm Development

ABSTRACTChronic infections withPseudomonas aeruginosapersist because the bacterium forms biofilms that are tolerant to antibiotic treatment and the host immune response. Scanning electron microscopy and confocal laser scanning microscopy were used to visualize biofilm developmentin vivofollowing intraperitoneal inoculation of mice with bacteria growing on hollow silicone tubes, as well as to examine the interaction between these bacteria and the host innate immune response. Wild-typeP. aeruginosadeveloped biofilms within 1 day that trapped and caused visible cavities in polymorphonuclear leukocytes (PMNs). In contrast, the number of cells of aP. aeruginosa rhlAmutant that cannot produce rhamnolipids was significantly reduced on the implants by day 1, and the bacteria were actively phagocytosed by infiltrating PMNs. In addition, we identified extracellular wire-like structures around the bacteria and PMNs, which we found to consist of DNA and other polymers. Here we present a novel method to study a pathogen-host interaction in detail. The data presented provide the first direct, high-resolution visualization of the failure of PMNs to protect against bacterial biofilms.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

scholarly journals Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

2019 ◽  
Vol 75 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Odel Soren ◽  
Ardeshir Rineh ◽  
Diogo G Silva ◽  
Yuming Cai ◽  
Robert P Howlin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Nitric Oxide Donor ◽  
Confocal Laser ◽  
Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy

Development ◽  
1992 ◽  
Vol 114 (2) ◽  
pp. 379-388 ◽  
Author(s):  
M.J. Carette ◽  
M.W. Ferguson
Keyword(s):  
Laser Scanning ◽  
Time Course ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Palatal Fusion ◽  
Morphological Criteria ◽  
Medial Edge ◽  
Mesenchymal Transformation

Fusion of bilateral shelves, to form the definitive mammalian secondary palate, is critically dependent on removal of the medial edge cells that constitute the midline epithelial seam. Conflicting views suggest that programmed apoptotic death or epithelial-mesenchymal transformation of these cells is predominantly involved. Due in part to the potentially ambiguous interpretation of static images and the notable absence of fate mapping studies, the process by which this is achieved has, however, remained mechanistically equivocal. Using an in vitro mouse model, we have selectively labelled palatal epithelia with DiI and examined the fate of medial edge epithelial (MEE) cells during palatal fusion by localisation using a combination of conventional histology and confocal laser scanning microscopy (CLSM). In dynamic studies using CLSM, we have made repetitive observations of the same palatal cultures in time-course investigations. Our results concurred with the established morphological criteria of seam degeneration; however, they provided no evidence of MEE cell death or transformation. Instead we report that MEE cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate. Subsequently these cells become incorporated into the oral and nasal epithelia on the surface of the palate. We hypothesize an alternative method of seam degeneration in vivo which largely conserves the MEE population by recruiting it into the nasal and oral epithelia.

Consequences of topoisomerase II inhibition in early embryogenesis of Drosophila revealed by in vivo confocal laser scanning microscopy

1993 ◽  
Vol 104 (4) ◽  
pp. 1175-1185 ◽  
Author(s):  
P. Buchenau ◽  
H. Saumweber ◽  
D.J. Arndt-Jovin
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Topoisomerase Ii ◽  
Metaphase Plate ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser

The regulation of DNA topology by topoisomerase II from Drosophila melanogaster has been studied extensively by biochemical methods but little is known about its roles in vivo. We have performed experiments on the inhibition of topoisomerase II in living Drosophila blastoderm embryos. We show that the enzymatic activity can be specifically disrupted by microinjection of antitopoisomerase II antibodies as well as the epipodophyllotoxin VM26, a known inhibitor of topoisomerase II in vitro. By labeling the chromatin of live embryos with tetramethylrhodamine-coupled histones, the effects of inhibition on nuclear morphology and behaviour was followed in vivo using confocal laser scanning microscopy. Both the antibodies and the drug prevented or hindered the segregation of chromatin daughter sets at the anaphase stage of mitosis. In addition, high concentrations of inhibitor interfered with the condensation of chromatin and its proper arrangement into the metaphase plate. The observed effects yielded non-functional nuclei, which were drawn into the inner yolk mass of the embryo. Concurrently, undamaged nuclei surrounding the affected region underwent compensatory division, leading to the restoration of the nuclear population, and thereby demonstrating the regulative capacity of Drosophila blastoderm embryos.

scholarly journals Astrogliosis in an Experimental Model of Hypovitaminosis B12: A Cellular Basis of Neurological Disorders due to Cobalamin Deficiency

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2261
Author(s):  
Zuzanna Rzepka ◽  
Jakub Rok ◽  
Justyna Kowalska ◽  
Klaudia Banach ◽  
Justyna Magdalena Hermanowicz ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Cobalamin Deficiency ◽  
Confocal Laser ◽  
In Vivo Model ◽  
Cellular Expression ◽  
Wide Range ◽  
Vitro Model

Cobalamin deficiency affects human physiology with sequelae ranging from mild fatigue to severe neuropsychiatric abnormalities. The cellular and molecular aspects of the nervous system disorders associated with hypovitaminosis B12 remain largely unknown. Growing evidence indicates that astrogliosis is an underlying component of a wide range of neuropathologies. Previously, we developed an in vitro model of cobalamin deficiency in normal human astrocytes (NHA) by culturing the cells with c-lactam of hydroxycobalamin (c-lactam OH-Cbl). We revealed a non-apoptotic activation of caspases (3/7, 8, 9) in cobalamin-deficient NHA, which may suggest astrogliosis. The aim of the current study was to experimentally verify this hypothesis. We indicated an increase in the cellular expression of two astrogliosis markers: glial fibrillary acidic protein and vimentin in cobalamin-deficient NHA using Western blot analysis and immunocytochemistry with confocal laser scanning microscopy. In the next step of the study, we revealed c-lactam OH-Cbl as a potential non-toxic vitamin B12 antagonist in an in vivo model using zebrafish embryos. We believe that the presented results will contribute to a better understanding of the cellular mechanism underlying neurologic pathology due to cobalamin deficiency and will serve as a foundation for further studies.

scholarly journals Calpurnia aurea (Aiton) Benth Extracts Reduce Quorum Sensing Controlled Virulence Factors in Pseudomonas aeruginosa

Molecules ◽  
2020 ◽  
Vol 25 (10) ◽  
pp. 2283
Author(s):  
Sekelwa Cosa ◽  
Jostina R. Rakoma ◽  
Abdullahi A. Yusuf ◽  
Thilivhali E. Tshikalange
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Docking ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Promising Alternative ◽  
The Individual

Pseudomonas aeruginosa is the causative agent of several life-threatening human infections. Like many other pathogens, P. aeruginosa exhibits quorum sensing (QS) controlled virulence factors such as biofilm during disease progression, complicating treatment with conventional antibiotics. Thus, impeding the pathogen’s QS circuit appears as a promising alternative strategy to overcome pseudomonas infections. In the present study, Calpurnia aurea were evaluated for their antibacterial (minimum inhibitory concentrations (MIC)), anti-quorum sensing/antivirulence (AQS), and antibiofilm potential against P. aeruginosa. AQS and antivirulence (biofilm formation, swimming, and swarming motility) activities of plant extracts were evaluated against Chromobacterium violaceum and P. aeruginosa, respectively. The in vitro AQS potential of the individual compounds were validated using in silico molecular docking. Acetone and ethanolic extracts of C. aurea showed MIC at 1.56 mg/mL. The quantitative violacein inhibition (AQS) assay showed ethyl acetate extracts as the most potent at a concentration of 1 mg/mL. GCMS analysis of C. aurea revealed 17 compounds; four (pentadecanol, dimethyl terephthalate, terephthalic acid, and methyl mannose) showed potential AQS through molecular docking against the CviR protein of C. violaceum. Biofilm of P. aeruginosa was significantly inhibited by ≥60% using 1-mg/mL extract of C. aurea. Confocal laser scanning microscopy correlated the findings of crystal violet assay with the extracts significantly altering the swimming motility. C. aurea extracts reduced the virulence of pseudomonas, albeit in a strain- and extract-specific manner, showing their suitability for the identification of lead compounds with QS inhibitory potential for the control of P. aeruginosa infections.

scholarly journals Enhancement of Vancomycin Activity against Biofilms by Using Ultrasound-Targeted Microbubble Destruction

2011 ◽  
Vol 55 (11) ◽  
pp. 5331-5337 ◽  
Author(s):  
Nianan He ◽  
Jian Hu ◽  
Huayong Liu ◽  
Tao Zhu ◽  
Beijian Huang ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Laser Scanning ◽  
Rabbit Model ◽  
Antibiotic Activity ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Content Type ◽  
Implanted Medical Devices

ABSTRACTTreating biofilm infections on implanted medical devices is formidable, even with extensive antibiotic therapy. The aim of this study was to investigate whether ultrasound (US)-targeted microbubble (MB) destruction (UTMD) could enhance vancomycin activity againstStaphylococcus epidermidisRP62A biofilms. Twelve-hour biofilms were treated with vancomycin combined with UTMD. The vancomycin and MB (SonoVue) were used at concentrations of 100 μg/ml and 30% (vol/vol), respectively, in studiesin vitro. After US exposure (0.08 MHz, 1.0 W/cm2, 50% duty cycle, and 10-min duration), the biofilms were cultured at 37°C for another 12 h. The results showed that many micropores were found in biofilms treated with vancomycin combined with UTMD. Biofilm densities (A570values) and the viable counts ofS. epidermidisrecovered from the biofilm were significantly decreased compared with those of any other groups. Furthermore, the highest percentage of dead cells was found, using confocal laser scanning microscopy, in the biofilm treated with vancomycin combined with UTMD. The viable counts of bacteria in biofilms in anin vivorabbit model also confirmed the enhanced effect of vancomycin combined with UTMD. UTMD may have great potential for improving antibiotic activity against biofilm infections.

Cyclodextrin Nanosponge Based Babchi Oil Hydrogel Ameliorates Imiquimod-Induced Psoriasis in Swiss Mice: An impact on Safety and Efficacy

2021 ◽  
Vol 13 ◽  
Author(s):  
Sunil Kumar ◽  
Babu Lal Jangir ◽  
Rekha Rao
Keyword(s):  
Essential Oil ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Epidermal Keratinocytes ◽  
Topical Formulation ◽  
Safe Alternative ◽  
Safety And Efficacy

Background: Psoriasis, a chronic autoimmune disease, involves the integration of biological and molecular events by hyperproliferation of the epidermal keratinocytes and generation of inflammation markers. Due to severe complications of synthetic corticosteroids, there is a strong need for potential and safe alternative . Babchi oil (natural essential oil; BO) may prove as a promising natural agent for psoriasis. Objective: The aim of the present work was to investigate the safety and efficacy of cyclodextrin nanosponge-based babchi oil (BONS) hydrogel on skin annexes. Methods: Babchi oil nanosponge hydrogel (BONS-HG) was fabricated and evaluated. Cell viability studies have been carried out on THP1 cell lines to evaluate cytocompatibility. Irritation potential and in vivo visualization of cutaneous uptake of BONS-HG were carried out using Hen’s Egg Chorioallantoic Membrane Test (HET-CAM) and confocal laser scanning microscopy (CLSM), respectively. The nano hydrogel was tested in vivo using imiquimod-induced psoriasis mouse model. Results: The in vitro irritation potential of BONS-HG indicated no sign of erythema or irritation, suggesting the safety of prepared hydrogel as topical formulation. CLSM studies advocated targeting of BO to epidermis and dermis. Along with histopathological assessment, evaluation of oxidative stress markers revealed the significant antipsoriatic activity (p< 0.001) of the prepared BONS-HG. Conclusion: The present study amalgamated the advantages of natural essential oil with this approach for skin targeting and provided an effective and safe topical alternative for psoriasis.

scholarly journals Glycoconjugate Expression Defines the Origin and Differentiation Pathway of Intestinal M-cells

1997 ◽  
Vol 45 (10) ◽  
pp. 1341-1350 ◽  
Author(s):  
Andreas Gebert ◽  
Wolfgang Posselt
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Lymphoid Tissues ◽  
M Cells ◽  
Glycosylation Pattern ◽  
Clonal Origin ◽  
Computer Based

Intestinal M-cells are specialized epithelial cells located in the domes of the gut-associated lymphoid tissues, which transport antigens from the lumen to the underlying lymphoid tissue, thereby initiating immune reactions. It is assumed that M-cells arise from stem cells in the crypts, from which they migrate to the top of the domes. To study the differentiation pathway of M-cells, we used the rabbit cecal lymphoid patch in which the M-cells express high levels of α1–2-linked fucose and N-acetyl-galactosamine residues in their apical membrane. Dome areas were labeled with fluorescein- and rhodamine-conjugated lectins specific for α1–2-linked fucose and N-acetyl-galactosamine in vivo and in vitro, and were observed with confocal laser scanning microscopy. Ultrathin sections were double labeled with lectin–gold conjugates and the labeling density was quantified by computer-based image analysis. All cecal patch M-cells expressed α1–2-linked fucose and N-acetyl-galactosamine, but the amount of the two saccharides varied considerably depending on the position of the M-cells at the base, flank, or top of the dome. In eight of 18 rabbits studied, radial strips of M-cells with common glycosylation patterns were observed, each strip associated with an individual crypt. Confocal microscopy revealed that lectin-labeled M-cells were not restricted to the dome epithelium but were also detected in the upper third of crypts surrounding the domes. The results show that M-cells are heterogeneous concerning the glycosylation pattern of membrane glycoconjugates. This pattern is modified as the M-cells differentiate and migrate from the base to the top of the dome. Radial strips of M-cells with a common proclivity of glycoconjugate expression suggest that those M-cells that derive from the same crypt have a clonal origin. The presence of (pre-) M-cells in the crypts surrounding the domes indicates that M-cells derive directly from undifferentiated crypt cells and do not develop from differentiated enterocytes.

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