scholarly journals Autolysin-Independent DNA Release inStreptococcus pneumoniae in vitroBiofilms

2018 ◽  
Author(s):  
Mirian Domenech ◽  
Ernesto García
Keyword(s):  
Extracellular Matrix ◽  
Quorum Sensing ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Lytic Enzymes ◽  
Microbial Biofilms ◽  
Dna Release

ABSTRACTBiofilms are defined as layers of cells of microorganisms adhered to the surface of a substrate and embedded in an extracellular matrix and provide an appropriate environment for increased genetic exchange. Extracellular DNA (eDNA) is an essential component of the extracellular matrix of microbial biofilms, but the pathway(s) responsible for DNA release are largely unknown. Autolysis (either spontaneous or phage-induced) has been proposed the major event leading to the appearance of eDNA. The ‘suicidal tendency’ ofStreptococcus pneumoniaeis well-known, with lysis mainly caused by the triggering of LytA, the major autolytic amidase. However, the LytC lysozyme and CbpD (a possible murein hydrolase) have also been shown involved. The present work examines the relationship between eDNA, autolysins, and the formation and maintenance ofin vitropneumococcal biofilms, via fluorescent labelling combined with confocal laser scanning microscopy, plus genetic transformation experiments. Bacterial DNA release mechanisms other than those entailing lytic enzymes were shown to be involved by demonstrating that horizontal gene transfer in biofilms takes place even in the absence of detectable autolytic activity. It had been previously suggested that the quorum sensing systems ComABCDE and LuxS/AI-2 are involved in the production of eDNA as a response to the accumulation of quorum sensing signals, although our immunofluorescence results do not support this hypothesis. Evidence that the release of DNA is somehow linked to the production of extracellular vesicles byS. pneumoniaeis provided.

scholarly journals Alginate Oligosaccharide-Induced Modification of the lasI-lasR and rhlI-rhlR Quorum-Sensing Systems in Pseudomonas aeruginosa

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Alison A. Jack ◽  
Saira Khan ◽  
Lydia C. Powell ◽  
Manon F. Pritchard ◽  
Konrad Beck ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Laser Scanning ◽  
Homoserine Lactone ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Mass Spectrometry Analysis ◽  
Confocal Laser ◽  
Steric Interaction ◽  
Content Type

ABSTRACT Pseudomonas aeruginosa plays a major role in many chronic infections. Its ability to readily form biofilms contributes to its success as an opportunistic pathogen and its resistance/tolerance to antimicrobial/antibiotic therapy. A low-molecular-weight alginate oligomer (OligoG CF-5/20) derived from marine algae has previously been shown to impair motility in P. aeruginosa biofilms and disrupt pseudomonal biofilm assembly. As these bacterial phenotypes are regulated by quorum sensing (QS), we hypothesized that OligoG CF-5/20 may induce alterations in QS signaling in P. aeruginosa . QS regulation was studied by using Chromobacterium violaceum CV026 biosensor assays that showed a significant reduction in acyl homoserine lactone (AHL) production following OligoG CF-5/20 treatment (≥2%; P < 0.05). This effect was confirmed by liquid chromatography-mass spectrometry analysis of C 4 -AHL and 3-oxo-C 12 -AHL production (≥2%; P < 0.05). Moreover, quantitative PCR showed that reduced expression of both the las and rhl systems was induced following 24 h of treatment with OligoG CF-5/20 (≥0.2%; P < 0.05). Circular dichroism spectroscopy indicated that these alterations were not due to steric interaction between the AHL and OligoG CF-5/20. Confocal laser scanning microscopy (CLSM) and COMSTAT image analysis demonstrated that OligoG CF-5/20-treated biofilms had a dose-dependent decrease in biomass that was associated with inhibition of extracellular DNA synthesis (≥0.5%; P < 0.05). These changes correlated with alterations in the extracellular production of the pseudomonal virulence factors pyocyanin, rhamnolipids, elastase, and total protease ( P < 0.05). The ability of OligoG CF-5/20 to modify QS signaling in P. aeruginosa PAO1 may influence critical downstream functions such as virulence factor production and biofilm formation.

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 and Comparison of Biofilm Development by Pathogenic and Commensal Isolates of Histophilus somni

2007 ◽  
Vol 189 (22) ◽  
pp. 8179-8185 ◽  
Author(s):  
Indra Sandal ◽  
Wenzhou Hong ◽  
W. Edward Swords ◽  
Thomas J. Inzana
Keyword(s):  
Extracellular Matrix ◽  
Surface Area ◽  
Laser Scanning ◽  
Systemic Disease ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Histophilus Somni ◽  
Biofilm Development

ABSTRACT Histophilus somni (Haemophilus somnus) is an obligate inhabitant of the mucosal surfaces of bovines and sheep and an opportunistic pathogen responsible for respiratory disease, meningoencephalitis, myocarditis, arthritis, and other systemic infections. The identification of an exopolysaccharide produced by H. somni prompted us to evaluate whether the bacterium was capable of forming a biofilm. After growth in polyvinyl chloride wells a biofilm was formed by all strains examined, although most isolates from systemic sites produced more biofilm than commensal isolates from the prepuce. Biofilms of pneumonia isolate strain 2336 and commensal isolate strain 129Pt were grown in flow cells, followed by analysis by confocal laser scanning microscopy and scanning electron microscopy. Both strains formed biofilms that went through stages of attachment, growth, maturation, and detachment. However, strain 2336 produced a mature biofilm that consisted of thick, homogenous mound-shaped microcolonies encased in an amorphous extracellular matrix with profound water channels. In contrast, strain 129Pt formed a biofilm of cell clusters that were tower-shaped or distinct filamentous structures intertwined with each other by strands of extracellular matrix. The biofilm of strain 2336 had a mass and thickness that was 5- to 10-fold greater than that of strain 129Pt and covered 75 to 82% of the surface area, whereas the biofilm of strain 129Pt covered 35 to 40% of the surface area. Since H. somni is an obligate inhabitant of the bovine and ovine host, the formation of a biofilm may be crucial to its persistence in vivo, and our in vitro evidence suggests that formation of a more robust biofilm may provide a selective advantage for strains that cause systemic disease.

scholarly journals Characterization of Enterococcus faecalis in different culture conditions

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Mi-Ah Kim ◽  
Vinicius Rosa ◽  
Kyung-San Min
Keyword(s):  
Enterococcus Faecalis ◽  
Laser Scanning ◽  
Colorimetric Assay ◽  
Culture Conditions ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Qrt Pcr ◽  
Microscopy Analysis

AbstractThe aim of this study was to investigate how carbohydrates (glucose or sucrose) affect the characteristics of Enterococcus faecalis (E. faecalis) planktonic and biofilm in vitro. For this study, E. faecalis was cultured in tryptone-yeast extract broth with 0% glucose + 0% sucrose, 0.5% glucose, 1% glucose, 0.5% sucrose, or 1% sucrose. Viability of E. faecalis was examined by colony forming unit counting assays. Biofilm formation was assessed by measuring extracellular DNA (eDNA), a component of the biofilm matrix. Quantitative real-time PCR (qRT-PCR) was performed to investigate the expression of virulence-associated genes. Field emission scanning electron microscopy analysis, confocal laser scanning microscopy analysis, and crystal violet colorimetric assay were conducted to study E. faecalis biofilms. E. faecalis showed the highest viability and eDNA levels in 1% sucrose medium in biofilms. The result of qRT-PCR showed that the virulence-associated genes expressed highest in 1% sucrose-grown biofilms and in 1% glucose-grown planktonic cultures. E. faecalis showed highly aggregated biofilms and higher bacteria and exopolysaccharide (EPS) bio-volume in sucrose than in 0% glucose + 0% sucrose or glucose. The results indicate that the production of eDNA and EPS and expression of virulence-associated genes in E. faecalis are affected by the concentration of carbohydrates in biofilm or planktonic culture.

scholarly journals Investigating Extracellular DNA Release in Staphylococcus xylosus Biofilm In Vitro

2021 ◽  
Vol 9 (11) ◽  
pp. 2192
Author(s):  
Sabine Leroy ◽  
Isabelle Lebert ◽  
Carine Andant ◽  
Pierre Micheau ◽  
Régine Talon
Keyword(s):  
Laser Scanning ◽  
Amino Sugar ◽  
Cell Lysis ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Staphylococcus Xylosus ◽  
Oxidative Stresses ◽  
Dnase Treatment

Staphylococcus xylosus forms biofilm embedded in an extracellular polymeric matrix. As extracellular DNA (eDNA) resulting from cell lysis has been found in several staphylococcal biofilms, we investigated S. xylosus biofilm in vitro by a microscopic approach and identified the mechanisms involved in cell lysis by a transcriptomic approach. Confocal laser scanning microscopy (CLSM) analyses of the biofilms, together with DNA staining and DNase treatment, revealed that eDNA constituted an important component of the matrix. This eDNA resulted from cell lysis by two mechanisms, overexpression of phage-related genes and of cidABC encoding a holin protein that is an effector of murein hydrolase activity. This lysis might furnish nutrients for the remaining cells as highlighted by genes overexpressed in nucleotide salvage, in amino sugar catabolism and in inorganic ion transports. Several genes involved in DNA/RNA repair and genes encoding proteases and chaperones involved in protein turnover were up-regulated. Furthermore, S. xylosus perceived osmotic and oxidative stresses and responded by up-regulating genes involved in osmoprotectant synthesis and in detoxification. This study provides new insight into the physiology of S. xylosus in biofilm.

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 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 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 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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