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.

Antibiofilm activity of antifungal drugs, including the novel drug olorofim, against Lomentospora prolificans

2020 ◽  
Author(s):  
Lisa Kirchhoff ◽  
Silke Dittmer ◽  
Ann-Kathrin Weisner ◽  
Jan Buer ◽  
Peter-Michael Rath ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Antifungal Agents ◽  
Fungal Infections ◽  
Pathogenic Fungus ◽  
Antifungal Drugs ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Antibiofilm Activity

Abstract Objectives Patients with immunodeficiency or cystic fibrosis frequently suffer from respiratory fungal infections. In particular, biofilm-associated fungi cause refractory infection manifestations, linked to increased resistance to anti-infective agents. One emerging filamentous fungus is Lomentospora prolificans. Here, the biofilm-formation capabilities of L. prolificans isolates were investigated and the susceptibility of biofilms to various antifungal agents was analysed. Methods Biofilm formation of L. prolificans (n = 11) was estimated by crystal violet stain and antibiofilm activity was additionally determined via detection of metabolically active biofilm using an XTT assay. Amphotericin B, micafungin, voriconazole and olorofim were compared with regard to their antibiofilm effects when added prior to adhesion, after adhesion and on mature and preformed fungal biofilms. Imaging via confocal laser scanning microscopy was carried out to demonstrate the effect of drug treatment on the fungal biofilm. Results Antibiofilm activities of the tested antifungal agents were shown to be most effective on adherent cells whilst mature biofilm was the most resistant. The most promising antibiofilm effects were detected with voriconazole and olorofim. Olorofim showed an average minimum biofilm eradication concentration (MBEC) of 0.06 mg/L, when added prior to and after adhesion. The MBECs of voriconazole were ≤4 mg/L. On mature biofilm the MBECs of olorofim and voriconazole were higher than the previously determined MICs against planktonic cultures. In contrast, amphotericin B and especially micafungin did not exhibit sufficient antibiofilm activity against L. prolificans. Conclusions To our knowledge, this is the first study demonstrating the antibiofilm potential of olorofim against the human pathogenic fungus L. prolificans.

Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation

2004 ◽  
Vol 53 (7) ◽  
pp. 679-690 ◽  
Author(s):  
Andres Plata Stapper ◽  
Giri Narasimhan ◽  
Dennis E. Ohman ◽  
Johnny Barakat ◽  
Morten Hentzer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Cell System ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Alginate Production ◽  
Green Fluorescent ◽  
Biofilm Development

Extracellular polymers can facilitate the non-specific attachment of bacteria to surfaces and hold together developing biofilms. This study was undertaken to qualitatively and quantitatively compare the architecture of biofilms produced by Pseudomonas aeruginosa strain PAO1 and its alginate-overproducing (mucA22) and alginate-defective (algD) variants in order to discern the role of alginate in biofilm formation. These strains, PAO1, Alg+ PAOmucA22 and Alg− PAOalgD, tagged with green fluorescent protein, were grown in a continuous flow cell system to characterize the developmental cycles of their biofilm formation using confocal laser scanning microscopy. Biofilm Image Processing (bip) and Community Statistics (comstat) software programs were used to provide quantitative measurements of the two-dimensional biofilm images. All three strains formed distinguishable biofilm architectures, indicating that the production of alginate is not critical for biofilm formation. Observation over a period of 5 days indicated a three-stage development pattern consisting of initiation, establishment and maturation. Furthermore, this study showed that phenotypically distinguishable biofilms can be quantitatively differentiated.

scholarly journals Multiple Roles of Biosurfactants in Structural Biofilm Development by Pseudomonas aeruginosa

2007 ◽  
Vol 189 (6) ◽  
pp. 2531-2539 ◽  
Author(s):  
Sünje Johanna Pamp ◽  
Tim Tolker-Nielsen
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Initial Phase ◽  
Laser Scanning ◽  
Multiple Roles ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Structural Development ◽  
Biofilm Development ◽  
Later Phase

ABSTRACT Recent studies have indicated that biosurfactants produced by Pseudomonas aeruginosa play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. Through the use of flow cell technology and enhanced confocal laser scanning microscopy, we have obtained results which suggest that the biosurfactants produced by P. aeruginosa play additional roles in structural biofilm development. We present genetic evidence that during biofilm development by P. aeruginosa, biosurfactants promote microcolony formation in the initial phase and facilitate migration-dependent structural development in the later phase. P. aeruginosa rhlA mutants, deficient in synthesis of biosurfactants, were not capable of forming microcolonies in the initial phase of biofilm formation. Experiments involving two-color-coded mixed-strain biofilms showed that P. aeruginosa rhlA mutants were defective in migration-dependent development of mushroom-shaped multicellular structures in the later phase of biofilm formation. Experiments involving three-color-coded mixed-strain P. aeruginosa biofilms demonstrated that the wild-type and rhlA and pilA mutant strains formed distinct subpopulations on top of each other dependent on their ability to migrate and produce biosurfactants.

scholarly journals Sequential Treatment of Biofilms with Aztreonam and Tobramycin Is a Novel Strategy for Combating Pseudomonas aeruginosa Chronic Respiratory Infections

2016 ◽  
Vol 60 (5) ◽  
pp. 2912-2922 ◽  
Author(s):  
Estrella Rojo-Molinero ◽  
María D. Macià ◽  
Rosa Rubio ◽  
Bartolomé Moyà ◽  
Gabriel Cabot ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Bactericidal Effect ◽  
Cell System ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Antibiofilm Activity ◽  
Content Type ◽  
Inhaled Antibiotics ◽  
Resistant Mutants

ABSTRACTTraditional therapeutic strategies to control chronic colonization in cystic fibrosis (CF) patients are based on the use of a single nebulized antibiotic. In this study, we evaluated the therapeutic efficacy and dynamics of antibiotic resistance inPseudomonas aeruginosabiofilms under sequential therapy with inhaled aztreonam (ATM) and tobramycin (TOB). Laboratory strains PAO1, PAOMS (hypermutable), PAOMA (mucoid), and PAOMSA (mucoid and hypermutable) and two hypermutable CF strains, 146-HSE (Liverpool epidemic strain [LES-1]) and 1089-HSE (ST1089), were used. Biofilms were developed using the flow cell system. Mature biofilms were challenged with peak and 1/10-peak concentrations of ATM (700 mg/liter and 70 mg/liter), TOB (1,000 mg/liter and 100 mg/liter), and their alternations (ATM/TOB/ATM and TOB/ATM/TOB) for 2 (t= 2), 4 (t= 4), and 6 days (t= 6). The numbers of viable cells (CFU) and resistant mutants were determined. Biofilm structural dynamics were monitored by confocal laser scanning microscopy and processed with COMSTAT and IMARIS software programs. TOB monotherapy produced an intense decrease in CFU that was not always correlated with a reduction in biomass and/or a bactericidal effect on biofilms, particularly for the CF strains. The ATM monotherapy bactericidal effect was lower, but effects on biofilm biomass and/or structure, including intense filamentation, were documented. The alternation of TOB and ATM led to an enhancement of the antibiofilm activity against laboratory and CF strains compared to that with the individual regimens, potentiating the bactericidal effect and/or the reduction in biomass, particularly at peak concentrations. Resistant mutants were not documented in any of the regimens at the peak concentrations and only anecdotally at the 1/10-peak concentrations. These results support the clinical evaluation of sequential regimens with inhaled antibiotics in CF, as opposed to the current maintenance treatments with just one antibiotic in monotherapy.

scholarly journals Protozoan impact on bacterial biofilm formation

2010 ◽  
Vol 47 (1) ◽  
pp. 3-10 ◽  
Author(s):  
Krzysztof Rychert ◽  
Thomas Neu
Keyword(s):  
Activated Sludge ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Grazing Pressure ◽  
Bacterial Biofilm ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Protozoan Community ◽  
Biofilm Development ◽  
The Impact

Protozoan impact on bacterial biofilm formationConfocal laser scanning microscopy in combination with digital image analysis was used to assess the impact of protozoa on bacterial colonisation of surfaces. Bacterial biofilms were developed from activated sludge in microscope flow cells and were exposed to the grazing pressure of protozoa. The protozoan community from healthy activated sludge and a culture of flagellateBodo saltanswere used as grazers. Experiments comprised 48-h incubations in 3 treatment variants: bacteria with protozoa, bacteria with protozoa added after some time and bacteria without protozoa. When necessary, the elimination of protozoa from the inoculum was carried out with cycloheximide and NiSO4. Experiments demonstrated that protozoa from healthy activated sludge initially disturbed the biofilm development but later they could stimulate its growth. Similar results could be established in the experiment withBodo saltans(inoculum: 1000 cells/ml), however differences were not statistically significant. The finding that protozoa support biofilm development during specific stages may be relevant for biofilm studies with mixed environmental biofilm communities.

scholarly journals Role of Exopolysaccharides in Pseudomonas aeruginosa Biofilm Formation and Architecture

2011 ◽  
Vol 77 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Aamir Ghafoor ◽  
Iain D. Hay ◽  
Bernd H. A. Rehm
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Bacterial Biofilm ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Content Type ◽  
Biofilm Architecture

ABSTRACTPseudomonas aeruginosais an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslAΔalg8overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture ofP. aeruginosa.

scholarly journals Antibiofilm Activity of Small-Molecule ZY-214-4 Against Staphylococcus aureus

2021 ◽  
Vol 12 ◽  
Author(s):  
Jingyi Yu ◽  
Lulin Rao ◽  
Lingling Zhan ◽  
Yan Zhou ◽  
Yinjuan Guo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Small Molecule ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Pathogenic Bacteria ◽  
Cell Aggregation ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Antibiofilm Activity ◽  
Subinhibitory Concentration

Staphylococcus aureus is the most important pathogenic bacteria in humans. As the resistance of S. aureus to existing antibiotics is increasing, there is an urgent need for new anti-infective drugs. S. aureus biofilms cause persistent infections and resist complete eradication with antibiotic therapy. The present study investigated the inhibitory effect of the novel small-molecule ZY-214-4 (C19H11BrNO4) on S. aureus biofilm formation. At a subinhibitory concentration (4 μg/ml), ZY-214-4 had no effect on the growth of S. aureus strains and also showed no cytotoxicity in human normal bronchial epithelial cells (Bease-2B). The results of a semi-quantitative biofilm test showed that ZY-214-4 prevented S. aureus biofilm formation, which was confirmed by scanning electron microscopy and confocal laser scanning microscopy. ZY-214-4 significantly suppressed the production of polysaccharide intercellular adhesion and prevented cell aggregation, and also inhibited the mRNA expression of icaA and other biofilm-related genes (eno, clfA/B, fnbB, fib, ebpS, psmα, and psmβ) in clinical S. aureus isolates. Thus, at a subinhibitory concentration, ZY-214-4 inhibits biofilm formation by preventing cell aggregation, highlighting its clinical potential for preventing or treating S. aureus infections.

scholarly journals Pseudomonas aeruginosa Psl Is a Galactose- and Mannose-Rich Exopolysaccharide

2007 ◽  
Vol 189 (22) ◽  
pp. 8353-8356 ◽  
Author(s):  
Luyan Ma ◽  
Haiping Lu ◽  
April Sprinkle ◽  
Matthew R. Parsek ◽  
Daniel J. Wozniak
Keyword(s):  
Electron Microscopy ◽  
Pseudomonas Aeruginosa ◽  
Chemical Composition ◽  
Confocal Laser Scanning Microscopy ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Lectin Staining ◽  
Polysaccharide Synthesis

ABSTRACT The Pseudomonas aeruginosa polysaccharide synthesis locus (psl) is predicted to encode an exopolysaccharide which is critical for biofilm formation. Here we used chemical composition analyses and mannose- or galactose-specific lectin staining, followed by confocal laser scanning microscopy and electron microscopy, to show that Psl is a galactose-rich and mannose-rich exopolysaccharide.

scholarly journals Expression of the psl Operon in Pseudomonas aeruginosa PAO1 Biofilms: PslA Performs an Essential Function in Biofilm Formation

2005 ◽  
Vol 71 (8) ◽  
pp. 4407-4413 ◽  
Author(s):  
Jörg Overhage ◽  
Mirle Schemionek ◽  
Jeremy S. Webb ◽  
Bernd H. A. Rehm
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Gene Cluster ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Constitutive Expression ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Coding Region ◽  
Operon Expression ◽  
Rapid Amplification

ABSTRACT The psl gene cluster, comprising 15 cotranscribed genes from Pseudomonas aeruginosa, was recently identified as being involved in exopolysaccharide biosynthesis and biofilm formation. In this study, we investigated the regulation of the psl gene cluster and the function of the first gene in this cluster, the pslA gene. PslA shows strong similarities to UDP-glucose lipid carriers. An isogenic marker-free pslA deletion mutant of P. aeruginosa PAO1 deficient in attachment and biofilm formation was used for complementation studies. The expression of only the pslA gene, comprising a coding region of 1,437 bp, restored the biofilm-forming phenotype of the wild type, indicating that PslA is required for biofilm formation by nonmucoid P. aeruginosa. The promoter region of the psl gene cluster, which encodes PslA-PslO, was identified by rapid amplification of cDNA 5′ ends. Promoter assays using transcriptional fusions to lacZ and gfp indicated a constitutive expression of the psl cluster in planktonic cells and a highly regulated and localized expression in biofilms, respectively. Expression of the psl cluster in biofilms was almost exclusively found in the centers of microcolonies, as revealed by confocal laser scanning microscopy. These data suggest that constitutive expression of the psl operon enables efficient attachment to surfaces and that regulated localized psl operon expression is required for biofilm differentiation.

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.

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