• Development and Application of Strategies to Generate Bacteriophage Resistant Strains for Use in Milk Fermentation Processes

      Ross, R Paul; Fitzgerald, Gerald F; Coffey, Aidan; Coakley, M.; O'Sullivan, Daniel (Teagasc, 1999-02-01)
      The objectives of this project were firstly, the identification of natural phage resistance systems for exploitation, secondly, the development of methodologies to utilise these systems to improve the bacteriophage resistance of starter strains for use in milk fermentation processes, and thirdly, the actual application of these methodologies to improving starter strains. The main conclusions were as follows: Three new natural plasmid (DNA)-associated bacteriophage resistance systems were identified at Moorepark. The detailed genetic makeup of the phage resistance plasmid (pMRC01) was elucidated. Bacteriophages currently evolving in the industrial cheese-making environment were monitored to facilitate the judicious choice of phage resistance systems for use in commercial starter cultures which can more effectively target the documented problematic phage types. Two highly virulent phages targeting important cheese starters were identified in the industrial cheese-making environment. A reliable food-grade method to facilitate the transfer of phage resistance systems to cheese-making starter strains was developed. This is based on bacteriocin immunity-linked phage resistance. Phage resistant cheese starter cultures were developed through natural selection and by molecular manipulation using phage resistance plasmids. The phage resistance plasmid pMRC01 was introduced to 31 cheese starter strains.
    • Molecular Characterisation of Bacteriophage K Towards Applications for the Biocontrol of Pathogenic Staphylococci.

      O'Flaherty, Sarah; Flynn, James; Coffey, Aidan; Fitzgerald, Gerald F; Meaney, William J; Ross, R Paul (Teagasc, 2006-01-01)
      The 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.