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Tracing the interaction of bacteriophage with bacterial biofilms using fluorescent and chromogenic probes
M M Doolittle1, J J Cooney, D E Caldwell
1Environmental Sciences Program, University of Massachusetts, Boston 02125-3393, USA.
Abstract:
Phages T4 and E79 were fluorescently-labeled with rhodamine isothiocyanate (RITC), fluoroscein isothiocyanate (FITC), and by the addition of 4'6-diamidino-2-phenylindole (DAPI) to phage-infected host cells of Escherichia coli and Pseudomonas aeruginosa. Comparisons of electron micrographs with scanning confocal laser microscope (SCLM) images indicated that single RITC-labeled phage particles could be visualized. Biofilms of each bacterium were infected by labeled phage. SCLM and epifluorescence microscopy were used to observe adsorption of phage to single-layer surface-attached bacteria and thicker biofilms. The spread of the recombinant T4 phage, YZA1 (containing an rII-LacZ fusion), within a lac E. coli biofilm could be detected in the presence of chromogenic and fluorogenic homologs of galactose. Infected cells exhibited blue pigmentation and fluorescence from the cleavage products produced by the phage-encoded beta-galactosidase activity. Fluorescent antibodies were used to detect non-labeled progeny phage. Phage T4 infected both surface-attached and surface-associated E. coli while phage E79 adsorbed to P. aeruginosa cells on the surface of the biofilm, but access to cells deep in biofilms was somewhat restricted. Temperature and nutrient concentration did not affect susceptibility to phage infection, but lower temperature and low nutrients extended the time-to-lysis and slowed the spread of infection within the biofilm.
Insights
Fluorescently labeled bacteriophages (phages) were used to visualize infection dynamics in bacterial biofilms. This method successfully tracked phage adsorption and spread within Escherichia coli and Pseudomonas aeruginosa biofilms.
Area of Science:
- Microbiology
- Bacteriology
- Virology
Background:
- Bacteriophages (phages) are viruses that infect bacteria and show promise for treating bacterial infections.
- Visualizing phage-host interactions within complex bacterial communities like biofilms remains challenging.
- Developing methods to track phage behavior in situ is crucial for understanding phage therapy efficacy.
Purpose of the Study:
- To develop and apply fluorescent labeling techniques for visualizing phage infection dynamics in bacterial biofilms.
- To investigate the adsorption and spread of bacteriophages T4 and E79 in Escherichia coli and Pseudomonas aeruginosa biofilms.
- To assess the impact of environmental factors on phage infection within biofilms.
Main Methods:
- Fluorescent labeling of bacteriophages T4 and E79 using rhodamine isothiocyanate (RITC) and fluorescein isothiocyanate (FITC).
- Infection of Escherichia coli and Pseudomonas aeruginosa biofilms with labeled phages.
- Visualization using scanning confocal laser microscopy (SCLM) and epifluorescence microscopy.
- Detection of phage-encoded enzyme activity using chromogenic and fluorogenic substrates.
Main Results:
- Single RITC-labeled phage particles were visualized using SCLM, confirming the labeling method's sensitivity.
- Phage T4 successfully infected surface-attached and surface-associated E. coli, while phage E79 adsorbed to P. aeruginosa on the biofilm surface.
- Access of phage E79 to deeper biofilm layers was restricted.
- Environmental factors like temperature and nutrient concentration influenced the rate of infection spread and lysis time but not initial susceptibility.
Conclusions:
- Fluorescently labeled phages provide a powerful tool for studying phage-biofilm interactions in real-time.
- Phage adsorption and spread within biofilms are influenced by bacterial species and biofilm architecture.
- Environmental conditions modulate the kinetics of phage infection within biofilms, impacting their therapeutic potential.