Pseudomonas aeruginosa inhibits in-vitro Candida biofilm development.

In order to shed light on contamination risks along the ready-to-eat chain of fresh commodities by emerging foodborne pathogens, we investigated the biofilm development in vitro of two Pseudomonas aeruginosa strains on fresh-cut lettuce (Lactuca sativa L. var. Iceberg). The experiment was performed employing a floating bioreactor system where modified atmosphere package conditions were mimicked, and fresh-cut lettuce disks of 2 cm2 were put into contact with a 106 CFU/mL of a phenotypic mucoid P. aeruginosa phenotype (muc+) or a non-mucoid one (muc-). Following a simulated 2-day refrigerated-shelf quantitative Real-Time PCR, designed on a target gene region of the 16S rRNA gene, defined the different muc phenotypes behavior on biofilm in lettuce phyllo-plane. Between the two strains, a development difference of nearly 1.0 log CFU/cm2 occurred, with the muc+ phenotype being the most settled and adherent. This result clearly showed a distinct contamination risk according to P. aeruginosa phenotype and the need to develop real-time, specific, fast, and easy to use detection protocols along with specific sanitation systems for modified atmosphere package ready-to-eat commodities.

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Modulation of Pseudomonas aeruginosa Biofilm Dispersal by a Cyclic-Di-GMP Phosphodiesterase with a Putative Hypoxia-Sensing Domain

Applied and Environmental Microbiology ◽

10.1128/aem.01233-10 ◽

2010 ◽

Vol 76 (24) ◽

pp. 8160-8173 ◽

Cited By ~ 103

Author(s):

Shuwen An ◽

Ji'en Wu ◽

Lian-Hui Zhang

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Regulatory Protein ◽

Phosphodiesterase Activity ◽

Low Oxygen ◽

Ggdef Domain ◽

Biofilm Dispersal ◽

Biofilm Development

ABSTRACT Pseudomonas aeruginosa encodes many enzymes that are potentially associated with the synthesis or degradation of the widely conserved second messenger cyclic-di-GMP (c-di-GMP). In this study, we show that mutation of rbdA, which encodes a fusion protein consisting of PAS-PAC-GGDEF-EAL multidomains, results in decreased biofilm dispersal. RbdA contains a highly conserved GGDEF domain and EAL domain, which are involved in the synthesis and degradation of c-di-GMP, respectively. However, in vivo and in vitro analyses show that the full-length RbdA protein only displays phosphodiesterase activity, causing c-di-GMP degradation. Further analysis reveals that the GGDEF domain of RbdA plays a role in activating the phosphodiesterase activity of the EAL domain in the presence of GTP. Moreover, we show that deletion of the PAS domain or substitution of the key residues implicated in sensing low-oxygen stress abrogates the functionality of RbdA. Subsequent study showed that RbdA is involved in positive regulation of bacterial motility and production of rhamnolipids, which are associated with biofilm dispersal, and in negative regulation of production of exopolysaccharides, which are required for biofilm formation. These data indicate that the c-di-GMP-degrading regulatory protein RbdA promotes biofilm dispersal through its two-pronged effects on biofilm development, i.e., downregulating biofilm formation and upregulating production of the factors associated with biofilm dispersal.

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In Vitro Models for Candida Biofilm Development

Methods in Molecular Biology - Candida Species ◽

10.1007/978-1-4939-3052-4_8 ◽

2016 ◽

pp. 95-105 ◽

Cited By ~ 10

Author(s):

Bastiaan P. Krom ◽

Hubertine M. E. Willems

Keyword(s):

In Vitro Models ◽

Biofilm Development ◽

Candida Biofilm

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Rhamnose Binding Protein as an Anti-Bacterial Agent—Targeting Biofilm of Pseudomonas aeruginosa

Marine Drugs ◽

10.3390/md17060355 ◽

2019 ◽

Vol 17 (6) ◽

pp. 355 ◽

Cited By ~ 2

Author(s):

Tse-Kai Fu ◽

Sim-Kun Ng ◽

Yi-En Chen ◽

Yuan-Chuan Lee ◽

Fruzsina Demeter ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Antimicrobial Agents ◽

Lung Epithelial Cells ◽

Bacterial Agent ◽

Lung Epithelial ◽

Biofilm Development

More than 80% of infectious bacteria form biofilm, which is a bacterial cell community surrounded by secreted polysaccharides, proteins and glycolipids. Such bacterial superstructure increases resistance to antimicrobials and host defenses. Thus, to control these biofilm-forming pathogenic bacteria requires antimicrobial agents with novel mechanisms or properties. Pseudomonas aeruginosa, a Gram-negative opportunistic nosocomial pathogen, is a model strain to study biofilm development and correlation between biofilm formation and infection. In this study, a recombinant hemolymph plasma lectin (rHPLOE) cloned from Taiwanese Tachypleus tridentatus was expressed in an Escherichia coli system. This rHPLOE was shown to have the following properties: (1) Binding to P. aeruginosa PA14 biofilm through a unique molecular interaction with rhamnose-containing moieties on bacteria, leading to reduction of extracellular di-rhamnolipid (a biofilm regulator); (2) decreasing downstream quorum sensing factors, and inhibiting biofilm formation; (3) dispersing the mature biofilm of P. aeruginosa PA14 to improve the efficacies of antibiotics; (4) reducing P. aeruginosa PA14 cytotoxicity to human lung epithelial cells in vitro and (5) inhibiting P. aeruginosa PA14 infection of zebrafish embryos in vivo. Taken together, rHPLOE serves as an anti-biofilm agent with a novel mechanism of recognizing rhamnose moieties in lipopolysaccharides, di-rhamnolipid and structural polysaccharides (Psl) in biofilms. Thus rHPLOE links glycan-recognition to novel anti-biofilm strategies against pathogenic bacteria.

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In Situ Growth Rates and Biofilm Development of Pseudomonas aeruginosa Populations in Chronic Lung Infections

Journal of Bacteriology ◽

10.1128/jb.01581-07 ◽

2007 ◽

Vol 190 (8) ◽

pp. 2767-2776 ◽

Cited By ~ 145

Author(s):

Lei Yang ◽

Janus A. J. Haagensen ◽

Lars Jelsbak ◽

Helle Krogh Johansen ◽

Claus Sternberg ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Growth Rates ◽

Growth Dynamics ◽

Chronic Infections ◽

Lung Infections ◽

New Perspective ◽

Biofilm Development

ABSTRACT The growth dynamics of bacterial pathogens within infected hosts are a fundamental but poorly understood feature of most infections. We have focused on the in situ distribution and growth characteristics of two prevailing and transmissible Pseudomonas aeruginosa clones that have caused chronic lung infections in cystic fibrosis (CF) patients for more than 20 years. We used fluorescence in situ hybridization (FISH) directly on sputum specimens to examine the spatial distribution of the infecting P. aeruginosa cells. Mucoid variants were present in sputum as cell clusters surrounded by an extracellular matrix, whereas nonmucoid variants were present mainly as dispersed cells. To obtain estimates of the growth rates of P. aeruginosa in CF lungs, we used quantitative FISH to indirectly measure growth rates of bacteria in sputum samples (reflecting the in vivo lung conditions). The concentration of rRNA in bacteria isolated from sputa was measured and correlated with the rRNA contents of the same bacteria growing in vitro at defined rates. The results showed that most cells were actively growing with doubling times of between 100 and 200 min, with some growing even faster. Only a small stationary-phase subpopulation seemed to be present in sputa. This was found for both mucoid and nonmucoid variants despite their different organizations in sputum. The results suggest that the bacterial population may be confronted with selection forces that favor optimized growth activities. This scenario constitutes a new perspective on the adaptation and evolution of P. aeruginosa during chronic infections in CF patients in particular and on long-term infections in general.

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Use of In-Biofilm Expression Technology To Identify Genes Involved in Pseudomonas aeruginosa Biofilm Development

Journal of Bacteriology ◽

10.1128/jb.185.9.2700-2710.2003 ◽

2003 ◽

Vol 185 (9) ◽

pp. 2700-2710 ◽

Cited By ~ 47

Author(s):

Antonio Finelli ◽

Claude V. Gallant ◽

Keith Jarvi ◽

Lori L. Burrows

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Three Dimensional ◽

Streptomyces Griseus ◽

Form Complex ◽

Technology System ◽

Planktonic Growth ◽

Biofilm Development

ABSTRACT Mature Pseudomonas aeruginosa biofilms form complex three-dimensional architecture and are tolerant of antibiotics and other antimicrobial compounds. In this work, an in vivo expression technology system, originally designed to study virulence-associated genes in complex mammalian environments, was used to identify genes up-regulated in P. aeruginosa grown to a mature (5-day) biofilm. Five unique cloned promoters unable to promote in vitro growth in the absence of purines after recovery from the biofilm environment were identified. The open reading frames downstream of the cloned promoter regions were identified, and knockout mutants were generated. Insertional mutation of PA5065, a homologue of Escherichia coli ubiB, was lethal, while inactivation of PA0240 (a porin homologue), PA3710 (a putative alcohol dehydrogenase), and PA3782 (a homologue of the Streptomyces griseus developmental regulator adpA) had no effect on planktonic growth but caused defects in biofilm formation in static and flowing systems. In competition experiments, mutants demonstrated reduced fitness compared with the parent strain, comprising less than 0.0001% of total biofilm cells after 5 days. Therefore, using in-biofilm expression technology, we have identified novel genes that do not affect planktonic growth but are important for biofilm formation, development, and fitness.

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A Survival Strategy for Pseudomonas aeruginosa That Uses Exopolysaccharides To Sequester and Store Iron To Stimulate Psl-Dependent Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.01307-16 ◽

2016 ◽

Vol 82 (21) ◽

pp. 6403-6413 ◽

Cited By ~ 20

Author(s):

Shan Yu ◽

Qing Wei ◽

Tianhu Zhao ◽

Yuan Guo ◽

Luyan Z. Ma

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Iron Concentration ◽

Survival Strategy ◽

Iron Storage ◽

Content Type ◽

High Level ◽

Biofilm Development ◽

Biofilm Matrix

ABSTRACTExopolysaccharide Psl is a critical biofilm matrix component inPseudomonas aeruginosa, which forms a fiber-like matrix to enmesh bacterial communities. Iron is important forP. aeruginosabiofilm development, yet it is not clearly understood how iron contributes to biofilm development. Here, we showed that iron promoted biofilm formation via elevating Psl production inP. aeruginosa. The high level of iron stimulated the synthesis of Psl by reducing rhamnolipid biosynthesis and inhibiting the expression of AmrZ, a repressor ofpslgenes. Iron-stimulated Psl biosynthesis and biofilm formation held true in mucoidP. aeruginosastrains. Subsequent experiments indicated that iron bound with Pslin vitroand in biofilms, which suggested that Psl fibers functioned as an iron storage channel inP. aeruginosabiofilms. Moreover, among three matrix exopolysaccharides ofP. aeruginosa, Psl is the only exopolysaccharide that can bind with both ferrous and ferric ion, yet with higher affinity for ferrous iron. Our data suggest a survival strategy ofP. aeruginosathat uses exopolysaccharide to sequester and store iron to stimulate Psl-dependent biofilm formation.IMPORTANCEPseudomonas aeruginosais an environmental microorganism which is also an opportunistic pathogen that can cause severe infections in immunocompromised individuals. It is the predominant airway pathogen causing morbidity and mortality in individuals affected by the genetic disease cystic fibrosis (CF). Increased airway iron and biofilm formation have been proposed to be the potential factors involved in the persistence ofP. aeruginosain CF patients. Here, we showed that a high level of iron enhanced the production of the key biofilm matrix exopolysaccharide Psl to stimulate Psl-dependent biofilm formation. Our results not only make the link between biofilm formation and iron concentration in CF, but also could guide the administration or use of iron chelators to interfere with biofilm formation inP. aeruginosain CF patients. Furthermore, our data also imply a survival strategy ofP. aeruginosaunder high-iron environmental conditions.

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Recombinant R2-pyocin cream is effective in treating Pseudomonas aeruginosa-infected wounds

Canadian Journal of Microbiology ◽

10.1139/cjm-2021-0207 ◽

2021 ◽

Author(s):

Abdulaziz Alqahtani ◽

London Mena ◽

Dean Scholl ◽

Cassandra Kruczek ◽

Jane A. Colmer-Hamood ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Chronic Wounds ◽

Opportunistic Pathogen ◽

Biofilm Model ◽

Major Species ◽

Infected Wounds ◽

New Antibiotics ◽

Biofilm Development ◽

Antibiofilm Agents

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is one of the major species isolated from infected chronic wounds. The multidrug resistance exhibited by P. aeruginosa plus its ability to form biofilms that are difficult to eradicate, along with rising cost of producing new antibiotics, has necessitated the search for alternatives to standard antibiotics. Pyocins are antimicrobial compounds produced by P. aeruginosa to protect itself from competitors. We synthesized and purified recombinant P. aeruginosa R2 pyocin and used it in aqueous solution (rR2P) or formulated in polyethylene glycol (rR2PC) to treat P. aeruginosa-infected wounds. Clinical strains of P. aeruginosa were found to be sensitive (completely), partially sensitive, or resistant to rR2P. In the in vitro biofilm model, rR2P inhibited biofilm development by rR2P-sensitive isolates; while rR2PC eliminated partial biofilms formed by these strains in the in vitro wound biofilm model. In the murine model of excision wound, and at 24 h post infection, rR2PC application significantly reduced the bioburden of clinical isolate BPI86. Application of rR2PC containing two glycoside hydrolase antibiofilm agents eliminated BPI86 from the infected wound. These results suggest that the topical application of rR2PC is an effective therapy to treat wounds infected with R2P-senstive P. aeruginosa strains.

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Computationally designed pyocyanin demethylase acts synergistically with tobramycin to kill recalcitrant Pseudomonas aeruginosa biofilms

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2022012118 ◽

2021 ◽

Vol 118 (12) ◽

pp. e2022012118

Author(s):

Chelsey M. VanDrisse ◽

Rosalie Lipsh-Sokolik ◽

Olga Khersonsky ◽

Sarel J. Fleishman ◽

Dianne K. Newman

Keyword(s):

Pseudomonas Aeruginosa ◽

Protein Design ◽

Chronic Wounds ◽

Fold Increase ◽

Survival Strategies ◽

Design Algorithm ◽

Hypoxic Conditions ◽

Opportunistic Human Pathogen ◽

Biofilm Development

Pseudomonas aeruginosa is an opportunistic human pathogen that develops difficult-to-treat biofilms in immunocompromised individuals, cystic fibrosis patients, and in chronic wounds. P. aeruginosa has an arsenal of physiological attributes that enable it to evade standard antibiotic treatments, particularly in the context of biofilms where it grows slowly and becomes tolerant to many drugs. One of its survival strategies involves the production of the redox-active phenazine, pyocyanin, which promotes biofilm development. We previously identified an enzyme, PodA, that demethylated pyocyanin and disrupted P. aeruginosa biofilm development in vitro. Here, we asked if this protein could be used as a potential therapeutic for P. aeruginosa infections together with tobramycin, an antibiotic typically used in the clinic. A major roadblock to answering this question was the poor yield and stability of wild-type PodA purified from standard Escherichia coli overexpression systems. We hypothesized that the insufficient yields were due to poor packing within PodA’s obligatory homotrimeric interfaces. We therefore applied the protein design algorithm, AffiLib, to optimize the symmetric core of this interface, resulting in a design that incorporated five mutations leading to a 20-fold increase in protein yield from heterologous expression and purification and a substantial increase in stability to environmental conditions. The addition of the designed PodA with tobramycin led to increased killing of P. aeruginosa cultures under oxic and hypoxic conditions in both the planktonic and biofilm states. This study highlights the potential for targeting extracellular metabolites to assist the control of P. aeruginosa biofilms that tolerate conventional antibiotic treatment.

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