Experimental reproducibility in flow-chamber biofilms

Heydorn, Arne; Ersbøll, Bjarne Kjær; Hentzer, Morten; Parsek, Matthew R.; Givskov, Michael; Molin, Søren

doi:10.1099/00221287-146-10-2409

Cited by 240 publications

(186 citation statements)

References 34 publications

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“…Digital images were acquired with the OpenLab 4.0.3 software package (Improvision) and volumes were deconvolved by iterative restoration using the Volocity 3.5.1 software (Improvision). Quantitative analysis of 3D biofilm structures was achieved with the COMSTAT image analysis software package 7,8 .…”

Section: Representative Resultsmentioning

confidence: 99%

Co-culture Models of <em>Pseudomonas aeruginosa</em> Biofilms Grown on Live Human Airway Cells

Moreau‐Marquis

Redelman

Anderson

2010

JoVE

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Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm. . CFBE cells should be seeded at a concentration of 10 6 cells/well in a 6-well tissue culture plate or 2 X 10 5 in a 24-well tissue culture plate in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 2mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin. We use 1.5 mL medium per well in 6-well plates and 0.5 mL medium per well in 24-well plates. 2. Cells should be grown at 37°C and 5% CO2-95% air for 7-10 days to form a confluent monolayer before inoculation with bacteria. The medium must be changed every 2-3 days. These conditions have been shown to lead to formation of a confluent monolayer and tight junctions. 3. Grow P. aeruginosa in 5 mL LB for 18 hours at 37°C on an incubator shaker at 200 rpm. Under these conditions, P. aeruginosa cultures will typically reach a density of 5x10 9 CFU/ mL. 4. For bacterial inoculation, remove the medium from CFBE cells and add an equal volume of MEM without phenol red, supplemented with 2 mM L-glutamine (Microscopy medium). Confluent CFBE monolayers are inoculated with P. aeruginosa at a multiplicity of infection of approximately 30:1 relative to the number of CFBE cells originally seeded. This equates to 2 X 10 7 CFU/mL in 1.5 mL MEM/well for 6-well plates and 1.2 X 10 7 CFU/mL in 0.5 mL MEM/well for 24-well plates. 5. Incubate plates for 1 hour at 37°C and 5% CO2-95% air. 6. Following the 1 hour incubation, the supernatant should be removed and replaced with fresh Microscopy medium supplement...

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Section: Representative Resultsmentioning

confidence: 99%

Co-culture Models of <em>Pseudomonas aeruginosa</em> Biofilms Grown on Live Human Airway Cells

Moreau‐Marquis

Redelman

Anderson

2010

JoVE

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“…The emission wavelengths for eCFP, eYFP and DsRedExpress were 476, 527 and 584 nm, respectively. For each flow cell channel, five image stacks were acquired from the centre of the channel (2 mm from the walls), starting at a distance of 5-10 mm from the inlet (unless otherwise stated) and spaced approximately 5 mm apart (Heydorn et al, 2000). The five image stacks covered a total area of approximately 9 Â 10 5 mm 2 , which was more than the minimum of 1 Â 10 5 mm 2 required for representative data (Korber et al, 1993).…”

Section: Development and Resilience Of A Mixed-species Biofilm Kwk Lementioning

confidence: 99%

Biofilm development and enhanced stress resistance of a model, mixed-species community biofilm

et al. 2013

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Most studies of biofilm biology have taken a reductionist approach, where single-species biofilms have been extensively investigated. However, biofilms in nature mostly comprise multiple species, where interspecies interactions can shape the development, structure and function of these communities differently from biofilm populations. Hence, a reproducible mixed-species biofilm comprising Pseudomonas aeruginosa, Pseudomonas protegens and Klebsiella pneumoniae was adapted to study how interspecies interactions affect biofilm development, structure and stress responses. Each species was fluorescently tagged to determine its abundance and spatial localization within the biofilm. The mixed-species biofilm exhibited distinct structures that were not observed in comparable single-species biofilms. In addition, development of the mixed-species biofilm was delayed 1-2 days compared with the single-species biofilms. Composition and spatial organization of the mixed-species biofilm also changed along the flow cell channel, where nutrient conditions and growth rate of each species could have a part in community assembly. Intriguingly, the mixed-species biofilm was more resistant to the antimicrobials sodium dodecyl sulfate and tobramycin than the single-species biofilms. Crucially, such community level resilience was found to be a protection offered by the resistant species to the whole community rather than selection for the resistant species. In contrast, community-level resilience was not observed for mixed-species planktonic cultures. These findings suggest that community-level interactions, such as sharing of public goods, are unique to the structured biofilm community, where the members are closely associated with each other.

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“…Besides the many bacterial factors that have been implicated, the biophysical properties of the substrate and surrounding medium can also greatly influence bacterial biofilm formation (Heydorn et al 2000;Prouty and Gunn 2003). For example, C. elegans mutants with an altered surface cuticle do not accumulate Yersinia biofilm (Höflich et al 2004;Joshua et al 2003).…”

Section: Environmental Factorsmentioning

confidence: 99%

Yersinia pestis Biofilm in the Flea Vector and Its Role in the Transmission of Plague

Hinnebusch¹,

Erickson²

2008

Current Topics in Microbiology and Immunology

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Transmission by fleabite is a relatively recent evolutionary adaptation of Yersinia pestis, the bacterial agent of bubonic plague. To produce a transmissible infection, Y. pestis grows as an attached biofilm in the foregut of the flea vector. Biofilm formation both in the flea foregut and in vitro is dependent on an extracellular matrix (ECM) synthesized by the Yersinia hms gene products. The hms genes are similar to the pga and ica genes of Escherichia coli and Staphylococcus epidermidis, respectively, that act to synthesize a poly-β-1,6-N-acetyl-dglucosamine ECM required for biofilm formation. As with extracellular polysaccharide production in many other bacteria, synthesis of the Hms-dependent ECM is controlled by intracellular levels of cyclic-di-GMP. Yersinia pseudotuberculosis, the food-and water-borne enteric pathogen from which Y. pestis evolved recently, possesses identical hms genes and can form biofilm in vitro but not in the flea. The genetic changes in Y. pestis that resulted in adapting biofilm-forming capability to the flea gut environment, a critical step in the evolution of vector-borne transmission, have yet to be identified. During a flea bite, Y. pestis is regurgitated into the dermis in a unique biofilm phenotype, and this has implications for the initial interaction with the mammalian innate immune response.

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Experimental reproducibility in flow-chamber biofilms

Cited by 240 publications

References 34 publications

Co-culture Models of <em>Pseudomonas aeruginosa</em> Biofilms Grown on Live Human Airway Cells

Co-culture Models of <em>Pseudomonas aeruginosa</em> Biofilms Grown on Live Human Airway Cells

Biofilm development and enhanced stress resistance of a model, mixed-species community biofilm

Yersinia pestis Biofilm in the Flea Vector and Its Role in the Transmission of Plague

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