Novel Bacteriophages Lyse Clinical Isolates of Serratia marcescens

Abstract

Purpose: : To isolate novel bacteriophages that could be used for molecular biological analysis of bacterial physiology, biochemistry, and pathogenesis, and for a second potential use as a bactericidal agent for ocular pathogens.

Methods: : Soil, manure and compost samples were suspended in PBS and inoculated separately with four different strains of S. marcescens. After 2-3 months of incubation at room temperature, aqueous fraction was passed through a 0.22 micron filter and were screened for the presence of bacteriophages using laboratory and six different human keratitis isolates of S. marcescens. The host range for phage from purified plaques was determined for 17 different human keratitis isolates, one environmental isolate and two laboratory strains of S. marcescens, by spotting high-titer phage preparations on bacterial samples. Transmission electron microscopy was performed on high-titer phage preparations that were negative stained with uranyl acetate. To find host-receptors for phage attachment, we used isogenic fimbriae (fimC) and LPS (waaG) mutants of S. marcescens and determined phage infectivity. All experiments were repeated at least twice on different days.

Results: : Three different bacteriophages were isolated from three different enrichment cultures. Each phage exhibited a differential host range, infecting up to 40% of clinical and non-clinical S. marcescens strains (n=20). The extent of lysis ranged from complete lysis to cloudy or incomplete lysis according both the individual phage and the host-strain of bacteria. Electron microscopy revealed the presence of phage with typical head and tail structures. Phage number two was capable of infecting both fimC and waaG strains, whereas phages 1 and 3 only poorly infected the strain in which the mutants were made.

Conclusions: : We have isolated novel bacteriophages able to kill S. marcescens clinical and laboratory isolates. Phage two uses a receptor other than type I fimbriae or LPS. These phage could potentially be used for epidemiological purposes, as well as molecular biology tools. Individual phage and multi-phage mixtures will be tested for disruption of bacterial biofilms in the future.

Keywords: bacterial disease • antibiotics/antifungals/antiparasitics • microbial pathogenesis: experimental studies