Characterization of a virulent bacteriophage LK1 specific for Citrobacter freundii isolated from sewage water

Abstract

Citrobacter freundii is a worldwide emerging nosocomial pathogen with escalating incidence of multidrug resistance. Citrobacter freundii exists in natural environment, especially in health care settings and is difficult to eradicate. Phage therapy is considered as an alternative way of controlling bacterial infections and contaminations. In this study, we have described isolation and characterization of a virulent bacteriophage LK1 capable of specifically infecting Citrobacter freundii. A virulent bacteriophage LK1, specific for Citrobacter freundii was isolated from sewage water sample. TEM showed that phage Lk1 has an icosahedral head 70 nm in diameter and short tail of 17 nm, and can be classified as a member of the Podoviridae family. Restriction analysis indicated that phage LK1 was a dsDNA virus with an approximate genome size of 20-23 kb. Proteomic pattern generated by SDS PAGE using purified LK1 phage particles, revealed three major and six minor protein bands with molecular weight ranging from 25 to 80 kDa. Adsorption rate of LK1 relative to the host bacterium was also determined which showed significant improvement in adsorption with the addition of CaCl2 . In a single step growth experiment, LK1 exhibited a latent period of 24 min and burst size of 801 particle/cell. Moreover, pH and thermal stability of phage LK1 demonstrated a pH range of 5.0-6.0 and phage viability decreased to 0% at 65 °C. When LK1 was used to infect six other clinically isolated pathogenic strains, it showed relatively narrow host range. LK1 was capable of eliciting efficient lysis of Citrobacter freundii, revealing its potential as a non-toxic sanitizer for controlling Citrobacter freundii infection and contamination in both hospital and other public environments.

Keywords: Bacteriophage; Citrobacter freundii; LK1 phage; Phage therapy.