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.

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 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 Diversity of biofilms produced by quorum-sensing-deficient clinical isolates of Pseudomonas aeruginosa

2007 ◽  
Vol 56 (6) ◽  
pp. 738-748 ◽  
Author(s):  
J. Andy Schaber ◽  
Adrienne Hammond ◽  
Nancy L. Carty ◽  
Simon C. Williams ◽  
Jane A. Colmer-Hamood ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Confocal Laser ◽  
Post Inoculation ◽  
Twitching Motility ◽  
Type Iv ◽  
Continuous Culture System

The quorum-sensing (QS) systems control several virulence attributes of Pseudomonas aeruginosa. Five QS-deficient P. aeruginosa clinical isolates (CI) that were obtained from wound (CI-1), tracheal (CI-2, CI-3, CI-4) and urinary tract (CI-5) infections had previously been characterized. In this study, a flow-through continuous-culture system was utilized to examine in detail the biofilms formed by these isolates in comparison with the P. aeruginosa prototrophic strain PAO1. Analysis of the biofilms by confocal laser scanning microscopy and COMSTAT image analysis at 1 and 7 days post-inoculation showed that the isolates produced diverse biofilms. In comparison with PAO1, the CI produced biofilms that scarcely or partially covered the surface at day 1, although CI-1 produced larger microcolonies. At day 7, CI-2 and CI-4 produced mature biofilms denser than that produced by PAO1, while the biofilm formed by CI-1 changed very little from day 1. CI-1 was defective in both swarming and twitching motilities, and immunoblotting analysis confirmed that it produced a reduced level of PilA protein. The twitching-motility defect of CI-1 was not complemented by a plasmid carrying intact pilA. In the 48 h colony biofilm assay, the CI varied in susceptibility to imipenem, gentamicin and piperacillin/tazobactam. These results suggest that: (1) the isolates produced biofilms with different structures and densities from that of PAO1; (2) biofilm formation by the isolates was not influenced by either the isolation site or the QS deficiencies of the isolates; (3) the behaviour of CI-1 in the different biofilm systems may be due to its lack of swarming motility and type IV pilus-related twitching motility.

Unfractionated heparin reduces the elasticity of sputum from patients with cystic fibrosis

2007 ◽  
Vol 293 (5) ◽  
pp. L1240-L1249 ◽  
Author(s):  
Victoria J. Broughton-Head ◽  
Jagdeep Shur ◽  
Mary P. Carroll ◽  
James R. Smith ◽  
Janis K. Shute
Keyword(s):  
Cystic Fibrosis ◽  
Unfractionated Heparin ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Boyden Chamber ◽  
Target Cells ◽  
Maximal Effect ◽  
Dornase Alfa

Mucus obstruction of the airway in patients with cystic fibrosis (CF) reduces lung function, invites infection, and limits delivery of inhaled drugs including gene therapy vectors to target cells. Not all patients respond to presently available mucolytics, and new approaches are needed. Our objectives were to investigate the in vitro effects of unfractionated heparin (UFH) on the morphology and rheology of sputum and the effect of UFH on diffusion of 200-nm nanospheres through sputum from adult CF patients. Confocal laser scanning microscopy was used to image fluorescently stained actin and DNA components of CF sputum, and atomic force microscopy was used to image isolated DNA networks. The viscoelasticity of CF sputum was measured using dynamic oscillatory rheometry. Nanosphere diffusion was measured through CF sputum using a Boyden chamber-based assay. Actin-DNA bundles in CF sputum were disaggregated by UFH at concentrations of 0.1–10 mg/ml, and UFH enhanced the endonuclease activity in sputum from patients on dornase alfa therapy. UFH significantly reduced the elasticity and yield stress, but not the viscosity, of CF sputum from patients not receiving dornase alfa therapy. Heparin dose-dependently significantly increased the diffusion of nanospheres through CF sputum from patients not on dornase alfa therapy from 10.5 ± 2.5% at baseline to 36.9 ± 4.4% at 10 mg/ml but was more potent, with maximal effect at 0.1 mg/ml, in patients who were on dornase alfa therapy. Thus the mucoactive properties of UFH indicate its potential as a new therapeutic approach in patients with cystic fibrosis.

scholarly journals Rhamnolipids from Pseudomonas aeruginosa Rn19a Modifies the Biofilm Formation over a Borosilicate Surface by Clinical Isolates

Coatings ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 136
Author(s):  
Jair Carrazco-Palafox ◽  
Blanca Estela Rivera-Chavira ◽  
Jaime Raúl Adame-Gallegos ◽  
Luz María Rodríguez-Valdez ◽  
Erasmo Orrantia-Borunda ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Confocal Laser ◽  
Dynamic Conditions ◽  
Initial Adhesion ◽  
Static Conditions

Microbial cells are reversibly associated with surfaces in the form of biofilms. Adhesion is the mechanism used by the microorganisms to bind to a surface initially; no biofilm is formed without the initial adhesion. The aim of this work was to evaluate the efficacy of the rhamnolipids of Pseudomonas aeruginosa Rn19a in inhibiting the biofilms formed by the clinical isolates Escherichia coli I5, Pseudomonas aeruginosa E26, Enterococcus faecalis I27 on borosilicate coupons inside a Center for Disease Control and Prevention (CDC) reactor. The isolate E26 (P. aeruginosa) did not show an adverse effect on biofilm formation by the rhamnolipid presence and showed normal growth in all the conditions tested (dynamic and static growth). The Enterococcus faecalis I27 isolate decreased its biofilm formation ability in 2.2 log CFU/cm2 in static conditions by the addition of rhamnolipids and 3.0 log units in dynamic conditions. Finally, the E. coli I5 isolate was more susceptible to the influence of the borosilicate coupon covered with rhamnolipids. E5 reduced its biofilm formation capacity by 3.0 log CFU/cm2 units at static conditions by the rhamnolipid addition and 6.0 log units at dynamic conditions. Biofilm formation was also observed by Confocal Laser Scanning Microscopy. In summary, the application of rhamnolipids may be useful to prevent the initial adhesion of bacteria to borosilicate surfaces. At a minimum, rhamnolipids effectively inhibit or diminish adhesion to surfaces by biofilm-forming isolates that do not belong to the genus Pseudomonas.

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.

Confocal and SEM imaging to demonstrate food pathogen- biofilm biocontrol by pyocyanin from Pseudomonas aeruginosa BTRY1

2016 ◽  
Vol 6 (01) ◽  
pp. 5218
Author(s):  
Laxmi Mohandas ◽  
Anju T. R. ◽  
Sarita G. Bhat*
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Antibiofilm Activity ◽  
Opportunistic Pathogens ◽  
Redox Active ◽  
Sem Imaging ◽  
The Impact

An assortment of redox-active phenazine compounds like pyocyanin with their characteristic blue-green colour are synthesized by Pseudomonas aeruginosa, Gram-negative opportunistic pathogens, which are also considered one of the most commercially valuable microorganisms. In this study, pyocyanin from Pseudomonas aeruginosa BTRY1 from food sample was assessed for its antibiofilm activity by micro titer plate assay against strong biofilm producers belonging to the genera Bacillus, Staphylococcus, Brevibacterium and Micrococcus. Pyocyanin inhibited biofilm activity in very minute concentrations. This was also confirmed by Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM). Both SEM and CLSM helped to visualize the biocontrol of biofilm formation by eight pathogens. The imaging and quantification by CLSM also established the impact of pyocyanin on biofilm-biocontrol mainly in the food industry.

scholarly journals Effect of dissolution rate and subsequent ion release on cytocompatibility properties of borophosphate glasses

2019 ◽  
Vol 5 (1) ◽  
pp. 85-97
Author(s):  
Nusrat Sharmin ◽  
Mohammad S. Hasan ◽  
Md. Towhidul Islam ◽  
Chengheng Pang ◽  
Fu Gu ◽  
...  
Keyword(s):  
Dissolution Rate ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ion Release ◽  
Scanning Calorimetry ◽  
Cell Culture Study ◽  
Borophosphate Glasses ◽  
Mg63 Cells

AbstractPresent work explores the relationship between the composition, dissolution rate, ion release and cytocompatibility of a series of borophosphate glasses. While, the base glass was selected to be 40mol%P2O5-16mol%CaO-24mol%MgO-20mol%Na2O, three B2O3 modified glass compositions were formulated by replacing Na2O with 1, 5 and 10 mol% B2O3. Ion release study was conducted using inductively coupled plasma atomic emission spectroscopy (ICP-AES). The thermal scans of the glasses as determined by differential scanning calorimetry (DSC) revealed an increment in the thermal properties with increasing B2O3 content in the glasses. On the other hand, the dissolution rate of the glasses decreased with increasing B2O3 content. To identify the effect of boron ion release on the cytocompatibility properties of the glasses, MG63 cells were cultured on the surface of the glass discs. The in vitro cell culture study suggested that glasses with 5 mol% B2O3 (P40B5) showed better cell proliferation and metabolic activity as compares to the glasses with 10 mol% (P40B10) or with no B2O3 (P40B0). The confocal laser scanning microscopy (CLSM) images of live/dead stained MG63 cells attached to the surface of the glasses also revealed that the number of dead cells attached to P40B5 glasses were significantly lower than both P40B0 and P40B10 glasses.

scholarly journals Use of electromagnetic stimulation on an Enterococcus faecalis biofilm on root canal treated teeth in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Beatriz H. D. Panariello ◽  
Justin K. Kindler ◽  
Kenneth J. Spolnik ◽  
Ygal Ehrlich ◽  
George J. Eckert ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Root Canal ◽  
Laser Scanning ◽  
Confocal Imaging ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microscopy Imaging ◽  
Electromagnetic Stimulation ◽  
Root Canal Disinfection

AbstractRoot canal disinfection is of utmost importance in the success of the treatment, thus, a novel method for achieving root canal disinfection by electromagnetic waves, creating a synergistic reaction via electric and thermal energy, was created. To study electromagnetic stimulation (EMS) for the disinfection of root canal in vitro, single rooted teeth were instrumented with a 45.05 Wave One Gold reciprocating file. Specimens were sterilized and inoculated with Enterococcus faecalis ATCC 29,212, which grew for 15 days to form an established biofilm. Samples were treated with 6% sodium hypochlorite (NaOCl), 1.5% NaOCl 1.5% NaOCl with EMS, 0.9% saline with EMS or 0.9% saline. After treatments, the colony forming units (CFU) was determined. Data was analyzed by Wilcoxon Rank Sums Test (α = 0.05). One sample per group was scored and split for confocal laser scanning microscopy imaging. There was a significant effect with the use of NaOCl with or without EMS versus 0.9% saline with or without EMS (p = 0.012 and 0.003, respectively). CFUs were lower when using 0.9% saline with EMS versus 0.9% saline alone (p = 0.002). Confocal imaging confirmed CFU findings. EMS with saline has an antibiofilm effect against E. faecalis and can potentially be applied for endodontic disinfection.

scholarly journals Cocktail of carbohydrases from Aspergillus niger: an economical and eco-friendly option for biofilm clearance from biopolymer surfaces

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Arashdeep Kaur ◽  
Sanjeev Kumar Soni ◽  
Shania Vij ◽  
Praveen Rishi
Keyword(s):  
Aspergillus Niger ◽  
Laser Scanning ◽  
Statistical Modelling ◽  
Laser Scanning Microscopy ◽  
Enzyme Treatment ◽  
Confocal Laser ◽  
Abiotic Surfaces ◽  
Enzyme Cocktail ◽  
Natural Variant

AbstractBiofilm formation on both biotic and abiotic surfaces accounts for a major factor in spread of antimicrobial resistance. Due to their ubiquitous nature, biofilms are of great concern for environment as well as human health. In the present study, an integrated process for the co-production of a cocktail of carbohydrases from a natural variant of Aspergillus niger was designed. The enzyme cocktail was found to have a noteworthy potential to eradicate/disperse the biofilms of selected pathogens. For application of enzymes as an antibiofilm agent, the enzyme productivities were enhanced by statistical modelling using response surface methodology (RSM). The antibiofilm potential of the enzyme cocktail was studied in terms of (i) in vitro cell dispersal assay (ii) release of reducing sugars from the biofilm polysaccharides (iii) the effect of enzyme treatment on biofilm cells and architecture by confocal laser scanning microscopy (CLSM). Potential of the enzyme cocktail to disrupt/disperse the biofilm of selected pathogens from biopolymer surfaces was also assessed by field emission scanning electron microscopy (FESEM) analysis. Further, their usage in conjunction with antibiotics was assessed and it was inferred from the results that the use of enzyme cocktail augmented the efficacy of the antibiotics. The study thus provides promising insights into the prospect of using multiple carbohydrases for management of heterogeneous biofilms formed in natural and clinical settings.

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