Molecular Characterisation of Bacteriophage K Towards Applications for the Biocontrol of Pathogenic Staphylococci.

End of Project ReportThe aim of this work was to characterise staphylococcal bacteriophage (a bacterial virus) and to assess their potential as therapeutic agents against pathogenic strains of Staphylococcus aureus, particularly mastitis-causing strains. The project included the use of two newly isolated phage CS1 and DW2, and an existing polyvalent phage. The new phage were isolated from the farmyard and characterised by electron microscopy and restriction analysis. Both phage were shown to belong to the Siphoviridae family and were lytic for representatives of all three clonal groups of Irish mastitis-associated staphylococci. A cocktail of three phage (CS1, DW2 and K) at 108 (plaque forming units) PFU/ml was infused into cows teats in animal trials. The lack of an increase in somatic cell counts in milks indicated strongly that the phage did not irritate the animal. In addition, the most potent phage used in this study, phage K, was further studied by genome sequencing, which revealed a linear DNA genome of 127,395 base pairs, which encodes 118 putative ORFs (open reading frames). Interesting features of the genome include; 1) a region exhibiting high homology to the structural module from Listeria phage A511, 2) genes which potentially encodes proteins necessary for its own replisome, 3) an absence of GATC sites and 4) three introns encoding putative endonucleases were located in the genome, (two in the putative DNA polymerase gene and one in the lysin gene). Unlike both CS1 and DW2, the polyvalent phage K, exhibited a broad host range within the genus Staphylococcus. In in vitro inhibitory assays, phage K lysed all staphylococcal strains tested including nine different species. In preliminary application-type studies, anti-staphylococcal activity was also evident in a hand wash Project 4942 2 and phage cream. An unexpected result was the observation that phage K was unable to replicate in raw milk, which could limit its applications in mastitis treatments. This may have been due to clumping of the bacteria caused by immunoglobulins. However, inhibition activity was lost after milk was heat-treated. The overall results in this study provide new insights into the biology of the broad host range phage K and indicate that phage K has potential for treatment and prevention of infections caused by pathogenic staphylococci