Tracing the interaction of bacteriophage with bacterial biofilms using fluorescent and chromogenic probes.

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.