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

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.

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Role of Exopolysaccharides in Pseudomonas aeruginosa Biofilm Formation and Architecture

Applied and Environmental Microbiology ◽

10.1128/aem.00637-11 ◽

2011 ◽

Vol 77 (15) ◽

pp. 5238-5246 ◽

Cited By ~ 207

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.

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

Molecules ◽

10.3390/molecules25102283 ◽

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.

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Dynamics of the Action of Biocides in Pseudomonas aeruginosa Biofilms

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01760-10 ◽

2011 ◽

Vol 55 (6) ◽

pp. 2648-2654 ◽

Cited By ~ 67

Author(s):

A. Bridier ◽

F. Dubois-Brissonnet ◽

G. Greub ◽

V. Thomas ◽

R. Briandet

Keyword(s):

Pseudomonas Aeruginosa ◽

Laser Scanning ◽

Biochemical Analysis ◽

Laser Scanning Microscopy ◽

Diffusion Reaction ◽

Confocal Laser ◽

Staining Procedure ◽

Fluorescence Staining ◽

Content Type ◽

Real Time Visualization

ABSTRACTThe biocidal activity of peracetic acid (PAA) and benzalkonium chloride (BAC) onPseudomonas aeruginosabiofilms was investigated by using a recently developed confocal laser scanning microscopy (CLSM) method that enables the direct and real-time visualization of cell inactivation within the structure. This technique is based on monitoring the loss of fluorescence that corresponds to the leakage of a fluorophore out of cells due to membrane permeabilization by the biocides. Although this approach has previously been used with success with various Gram-positive species, it is not directly applicable to the visualization of Gram-negative strains such asP. aeruginosa, particularly because of limitations regarding fluorescence staining. After adapting the staining procedure toP. aeruginosa, the action of PAA and BAC on the biofilm formed by strain ATCC 15442 was investigated. The results revealed specific inactivation patterns as a function of the mode of action of the biocides. While PAA treatment triggered a uniform loss of fluorescence in the structure, the action of BAC was first localized at the periphery of cell clusters and then gradually spread throughout the biofilm. Visualization of the action of BAC in biofilms formed by three clinical isolates then confirmed the presence of a delay in penetration, showing that diffusion-reaction limitations could provide a major explanation for the resistance ofP. aeruginosabiofilms to this biocide. Biochemical analysis suggested a key role for extracellular matrix characteristics in these processes.

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Autolysin-Independent DNA Release inStreptococcus pneumoniae in vitroBiofilms

10.1101/322818 ◽

2018 ◽

Cited By ~ 1

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.

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Subinhibitory Concentrations of Mupirocin Stimulate Staphylococcus aureus Biofilm Formation by Upregulating cidA

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01912-19 ◽

2020 ◽

Vol 64 (3) ◽

Cited By ~ 3

Author(s):

Ye Jin ◽

Yinjuan Guo ◽

Qing Zhan ◽

Yongpeng Shang ◽

Di Qu ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Laser Scanning ◽

Multidrug Resistant ◽

Laser Scanning Microscopy ◽

Extracellular Dna ◽

Confocal Laser ◽

Content Type ◽

Subinhibitory Concentrations ◽

Biofilm Development

ABSTRACT Previous studies have shown that the administration of antibiotics at subinhibitory concentrations stimulates biofilm formation by the majority of multidrug-resistant Staphylococcus aureus (MRSA) strains. Here, we investigated the effect of subinhibitory concentrations of mupirocin on biofilm formation by the community-associated (CA) mupirocin-sensitive MRSA strain USA300 and the highly mupirocin-resistant clinical S. aureus SA01 to SA05 isolates. We found that mupirocin increased the ability of MRSA cells to attach to surfaces and form biofilms. Confocal laser scanning microscopy (CLSM) demonstrated that mupirocin treatment promoted thicker biofilm formation, which also correlated with the production of extracellular DNA (eDNA). Furthermore, quantitative real-time PCR (RT-qPCR) results revealed that this effect was largely due to the involvement of holin-like and antiholin-like proteins (encoded by the cidA gene), which are responsible for modulating cell death and lysis during biofilm development. We found that cidA expression levels significantly increased by 6.05- to 35.52-fold (P < 0.01) after mupirocin administration. We generated a cidA-deficient mutant of the USA300 S. aureus strain. Exposure of the ΔcidA mutant to mupirocin did not result in thicker biofilm formation than that in the parent strain. We therefore hypothesize that the mupirocin-induced stimulation of S. aureus biofilm formation may involve the upregulation of cidA.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00196-16 ◽

2016 ◽

Vol 60 (5) ◽

pp. 2912-2922 ◽

Cited By ~ 12

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.

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Dynamics of Mutator and Antibiotic-Resistant Populations in a Pharmacokinetic/Pharmacodynamic Model of Pseudomonas aeruginosa Biofilm Treatment

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00617-11 ◽

2011 ◽

Vol 55 (11) ◽

pp. 5230-5237 ◽

Cited By ~ 41

Author(s):

María D. Macià ◽

José L. Pérez ◽

Soeren Molin ◽

Antonio Oliver

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Resistance Development ◽

Antibiotic Resistant ◽

Content Type ◽

Mutant Prevention Concentration ◽

Resistant Mutants

ABSTRACTBiofilm growth, antibiotic resistance, and mutator phenotypes are key components of chronic respiratory infections byPseudomonas aeruginosain cystic fibrosis patients. We examined the dynamics of mutator and antibiotic-resistant populations inP. aeruginosaflow-cell biofilms, using fluorescently tagged PAO1 and PAOMS (mutator [mutS] derivative) strains. Two-day-old biofilms were treated with ciprofloxacin (CIP) for 4 days (t4) at 2 μg/ml, which correlated with the mutant prevention concentration (MPC) and provided an AUC/MIC ratio of 384 that should predict therapeutic success. Biofilms were monitored by confocal laser scanning microscopy (CLSM), and the numbers of viable cells and resistant mutants (4- and 16-fold MICs) were determined. Despite optimized pharmacokinetic/pharmacodynamic (PK/PD) parameters, CIP treatment did not suppress resistance development inP. aeruginosabiofilms. One-step resistant mutants (MexCD-OprJ or MexEF-OprN overexpression) were selected for both strains, while two-step resistant mutants (additional GyrA or GyrB mutation) were readily selected only from the mutator strain. CLSM analysis of competition experiments revealed that PAOMS, even when inoculated at a 0.01 proportion, took over the whole biofilm after only 2 days of CIP treatment outnumbering PAO1 by 3 log at t4. Our results show that mutational mechanisms play a major role in biofilm antibiotic resistance and that theoretically optimized PK/PD parameters fail to suppress resistance development, suggesting that the increased antibiotic tolerance driven by the special biofilm physiology and architecture may raise the effective MPC, favoring gradual mutational resistance development, especially in mutator strains. Moreover, the amplification of mutator populations under antibiotic treatment by coselection with resistance mutations is for the first time demonstratedin situforP. aeruginosabiofilms.

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

Infection and Immunity ◽

10.1128/iai.06215-11 ◽

2012 ◽

Vol 80 (8) ◽

pp. 2601-2607 ◽

Cited By ~ 49

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.

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

Journal of Medical Microbiology ◽

10.1099/jmm.0.47031-0 ◽

2007 ◽

Vol 56 (6) ◽

pp. 738-748 ◽

Cited By ~ 23

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.

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

International Journal of Bioassays ◽

10.21746/ijbio.2017.01.007 ◽

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.

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