TRACING THE INTERACTION OF BACTERIOPHAGE WITH BACTERIAL BIOFILMS USING FLUORESCENT AND CHROMOGENIC PROBES

Phages T4 and E79 were fluorescently-labeled with rhodamine isothiocya nate (RITC), fluoroscein isothiocyanate (FITC), and by the addition of 4'6-diamidino-2-phenylindole (DAPI) to phage-infected host cells of E scherichia coli and Pseudomonas aeruginosa, Comparisons of electron mi crographs with scanning confocal laser microscope (SCLM) images indica ted that single RITC-labeled phage particles could be visualized, Biof ilms of each bacterium were infected by labeled phage, SCLM and epiflu orescence microscopy were used to observe adsorption of phage to singl e-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 chromogeni c 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 nonlabeled progeny phage. Phage T4 infected both s urface-attached and surface-associated E. coli while phage E79 adsorbe d to P. aeruginosa cells on the surface of the biofilm, but access to cells deep in biofilms was somewhat restricted, Temperature and nutrie nt concentration did not affect susceptibility to phage infection, but lower temperature and low nutrients extended the time-to-lysis and sl owed the spread of infection within the biofilm.