Flow cytometric and microscopic analysis of GFP-tagged Pseudomonas fluorescens bacteria

Tombolini, Riccardo

doi:10.1016/s0168-6496(96)00068-2

Cited by 79 publications

(122 citation statements)

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“…4 (7 to 17.5 h). These observations suggest that the loss of fluorescence might result from the detachment of cells from the biofilm rather than degradation of GFP, which is normally a stable protein in eubacteria (1,47).…”

Section: Resultsmentioning

confidence: 99%

Quorum Sensing in Staphylococcus aureus Biofilms

et al. 2004

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Several serious diseases are caused by biofilm-associated Staphylococcus aureus, infections in which the accessory gene regulator (agr) quorum-sensing system is thought to play an important role. We studied the contribution of agr to biofilm development, and we examined agr-dependent transcription in biofilms. Under some conditions, disruption of agr expression had no discernible influence on biofilm formation, while under others it either inhibited or enhanced biofilm formation. Under those conditions where agr expression enhanced biofilm formation, biofilms of an agr signaling mutant were particularly sensitive to rifampin but not to oxacillin. Time lapse confocal scanning laser microscopy showed that, similar to the expression of an agr-independent fluorescent reporter, biofilm expression of an agr-dependent reporter was in patches within cell clusters and oscillated with time. In some cases, loss of fluorescence appeared to coincide with detachment of cells from the biofilm. Our studies indicate that the role of agr expression in biofilm development and behavior depends on environmental conditions. We also suggest that detachment of cells expressing agr from biofilms may have important clinical implications.Staphylococcus aureus, a leading cause of nosocomial infections worldwide, is the etiologic agent of a wide range of diseases, from relatively benign skin infections to potentially fatal systemic disorders. Many of these diseases, including endocarditis, osteomyelitis, and foreign-body related infections, appear to be caused by biofilm-associated S. aureus (12, 18, 30, and 44). Biofilms are sessile microbial communities embedded in a self-produced extracellular polymeric matrix (12, 44). There is increasing awareness that biofilms have a special clinical relevance. Biofilm-associated bacteria show an innate resistance to antibiotics (5), disinfectants (36), and clearance by host defenses (reference 43; also reviewed in reference 12). These properties likely contribute to the persistence and recalcitrance to treatment of staphylococcal biofilm infections.Two stages of staphylococcal biofilm formation have been described (reviewed in reference 18). The first stage involves attachment of cells to a surface. This stage of biofilm formation is likely to be mediated in part by cell wall-associated adhesins, including the microbial surface components recognizing adhesive matrix molecules. The second stage of biofilm development includes cell multiplication and formation of a mature structure consisting of many cell layers. This stage is associated with the production of extracellular factors, including the polysaccharide intercellular adhesin component of the extracellular matrix.Intercellular signaling, often referred to as quorum sensing, has been shown to be involved in biofilm development by several bacteria, including Pseudomonas aeruginosa (11), Burkholderia cepacia (22, 23), Streptococcus mutans (26, 31), and others (27,46,49). For example, a quorum-sensing-defective mutant of P. aeruginosa is unable to ...

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Section: Resultsmentioning

confidence: 99%

Quorum Sensing in Staphylococcus aureus Biofilms

et al. 2004

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“…Therefore, the inducible IPTGbased expression system, as compared to constitutive expression systems, allows for a control of GFP expression and guarantees a balance between fluorescent and healthy bacteria (Figure 4c,d). Since eGFP is a very stable protein [41,42] new GFP variants with reduced half-lives, e.g. GFP (LVA), have been suggested to study transient gene expression in bacteria [43].…”

Section: Discussionmentioning

confidence: 99%

“…For live cell imaging, GFP expression in bacteria is used extensively and expression systems are available for many different bacterial species, including clinically relevant strains of Salmonella, Streptococcus, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli O157:H7 [41,[44][45][46][47]. Plasmid based gene expression is a well-established and long-used method in microbiology.…”

Section: Discussionmentioning

confidence: 99%

Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinant bacteria

et al. 2013

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Bacterial adhesion and biofilm growth can cause severe biomaterial-related infections and failure of medical implants. To assess the antifouling properties of engineered coatings, advanced approaches are needed for in situ monitoring of bacterial viability and growth kinetics as the bacteria colonize a surface. Here, we present an optimized protocol for optical real-time quantification of bacterial viability. To stain living bacteria, we replaced the commonly used fluorescent dye SYTO ® 9 with endogenously expressed eGFP, as SYTO ® 9 inhibited bacterial growth. With the addition of nontoxic concentrations of propidium iodide (PI) to the culture medium, the fraction of live and dead bacteria could be continuously monitored by fluorescence microscopy as demonstrated here using GFP expressing Escherichia coli as model organism. The viability of bacteria was thereby monitored on untreated and bioactive dimethyloctadecyl [3-(trimethoxysilyl)propyl]ammonium chloride (DMOAC)-coated glass substrates over several hours. Pre-adsorption of the antimicrobial surfaces with serum proteins, which mimics typical protein adsorption to biomaterial surfaces upon contact with host body fluids, completely blocked the antimicrobial activity of the DMOAC surfaces as we observed the recovery of bacterial growth. Hence, this optimized eGFP/PI viability assay provides a protocol for unperturbed in situ monitoring of bacterial viability and colonization on engineered biomaterial surfaces with single-bacteria sensitivity under physiologically relevant conditions.

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“…The flow cytometer can also detect cells which are specifically stained by in situ hybridization probes (FISH) or which have a marker gene inserted into the genome such as gfp. Since the excitation peak of natural gfp does not match the standard 488nm laser of the flow cytometer, Tombolini et al (1997) used a red shifted mutant gene which displayed a high and stable fluorescence signal. The gfp gene was expressed by a constitutive psbA promoter from Amaranthus hybridus which resulted in a stable and evenly distributed signal in all Pseudomonas fluorescens cells.…”

Section: Requirements For Marker Traitsmentioning

confidence: 99%

“…During the growth phase fluorescent intensity varied. The fluorescence intensity decreased to 30% in exponential phase while it increased again in stationary until the intensity per cell was the same as that of the inoculum (Tombolini et al, 1997) The choice of a specific detection method in combination with a certain marker gene and promoter sequence depends on the aim of the experiment. If researchers want to study the survival of a microorganism, it is best to use a constitutively expressed, stable gfp marker gene which is indicative for the presence of a bacterium and suited to determine cell number by FacsSort analysis.…”

Section: Requirements For Marker Traitsmentioning

confidence: 99%

Safety Assessment of Transgenic Organisms, Volume 4

2010

Harmonisation of Regulatory Oversight in Biotechnology

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io s a fe t y nv ir on me nt ag ri cu lt ur e bi os af et y g r ic u lt u r e e n vi r o n m e n t gr ic ul tu re bi os af et y en vi ro nm en t n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o io sa fe ty en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en v io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t nv ir on me nt ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm e g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a f gr ic ul tu re bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t a n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u io sa fe ty en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m nv ir on me nt ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a f gr ic ul tu re bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t a n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u io sa fe ty en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e nv ir on me nt ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y e g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a fe t y a g r ic u lt u r e e n vi r o n m e n t b io s a f ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et y en vi ro nm en t ag ri cu lt ur e bi os af et...

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Flow cytometric and microscopic analysis of GFP-tagged Pseudomonas fluorescens bacteria

Cited by 79 publications

References 0 publications

Quorum Sensing in Staphylococcus aureus Biofilms

Quorum Sensing in Staphylococcus aureus Biofilms

Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinant bacteria

Safety Assessment of Transgenic Organisms, Volume 4

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