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

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.

The dual probiotic and antibiotic nature of Bdellovibrio bacteriovorus

“…One special character about Bdellovibrio that distinguishes it from other biological-based antibacterial tools, such as bacteriophages (38), is their ability to invade biofilms, penetrating them deeply and effectively destroying them, as demonstrated in the studies done by Kadouri's group (23,39). Furthermore, they reported that Bdellovibrio was even more efficient in attacking biofilms than the planktonic cells, leading to a greater loss in bacterial counts, purportedly due to the higher prey density in biofilms and hence faster and easier access to the prey.…”

Prevention and treatment of biofilms by hybrid- and nanotechnologies

“…129 It is estimated that a radial propagation of single dose of a progeny phage can treat a biofilm infection of bacterial origin, infect adjacent cells, and degrade the biofilm matrix. 130 The mechanism of action of phages is through enzyme production (depolymerisation) that hydrolyses and degrades extracellular matrix of a biofilm. [131][132][133][134] In addition to mediating direct bacterial killing, phage agents can also be incorporated into a hydrogel coating on a catheter.…”

Rapid evolution of generalized resistance mechanisms can constrain the efficacy of phage–antibiotic treatments

“…Similar to antibiotics, extracellular polysaccharides and proteins could prevent phage adsorption to its receptors while metabolically less active cells are less likely to get infected (Abedon, 2017; Labrie, Samson, & Moineau, 2010). However, certain phages have the ability to produce enzymes, which can degrade the extracellular polymeric matrix (Hanlon, Denyer, Olliff, & Ibrahim, 2001; Hughes, Sutherland, Jones, & Rutherford, 1998), while other phages are able to propagate radially through a biofilm (Doolittle, Cooney, & Caldwell, 1996). Surprisingly, little is currently known about phage–antibiotic effects in biofilms.…”