Development of a rapid cow-side bacteriophage-based diagnostic method for the detection of Staphylococcus aureus mastitis

Bovine mastitis is an inflammatory condition of the udder, typically caused by bacterial infections in dairy cattle. One of the hardest bacterial pathogens to tackle, responsible for bovine mastitis, is Staphylococcus aureus. This condition has severe economic and societal implications. Economic due to both indirect and direct costs that arise from factors including increased veterinary and labour costs and reduced milk yield and quality. Societal, due to the strains placed on public health, with the overuse of antibiotics in farms, which results to increase in the problem of antimicrobial resistance with negative effects in human health and agriculture. The current methods of diagnosis and treatment require improvement, as these are not always straightforward. In terms of the treatment and because many times the bacterial strain responsible for the infection is hard to be determined, strong broad-spectrum antibiotics are used, which in the long run increase the serious problem of antimicrobial resistance, that is the resistance of bacterial pathogens to antibiotics, posing threats on the public health.

It is therefore an urgent need to develop a rapid cow-side test for Bovine mastitis, which would provide a fundamental change in the control and treatment of S. aureus mastitis. We will focus on the detection of infection caused by Staphylococcus aureus.

For this purpose, we will use bacteriophages, specific for targeting Staphylococcus aureus, as the basis of the diagnostic method. Bacteriophages are viruses with great specificity to their bacterial host. We will genetically modify these bacteriophages to be made bioluminescent, so that in the presence of Staphylococcus aureus in the samples tested, luminescence will be produced. We will further spray dry these phages to convert those into a powder, which can be easily distributed to individual vials and form the basis of the diagnostic test. The bioluminescent bacteriophages will be optimised and will be further be tested in the field, using biological samples from cows, both in their liquid and powder format, in order to ensure their accuracy and sensitivity. Overall, we aim to develop a rapid cow-side bacteriophage-based diagnostic method for Staphylococcus aureus bovine mastitis.

A proposed solution to the rapid diagnosis of Staphylococcus aureus bovine mastitis, is the use of genetically modified bacteriophages as diagnostics. In this proposal, we will genetically modify phage K, a phage that specifically targets Staphylococcus aureus. We will design a construct with flanking regions to non-essential areas of the genome of the phage, containing a luxAB cassette, so that a stable bioluminescent K phage can be engineered.
This construct will be transformed into Staphylococcus aureus electrocompetent cells and a recombinant phage will be constructed via homologous recombination. The recombinant bioluminescent phages will be selected by plaque PCR using primers that flank the luxAB cassette, following plaque assay and a plaque that contains the correct recombinant phage will be further processed, purified and assessed for stability. In vitro experiments, combining Staph. aureus growth and then incubation with the phage, measured in different time points with luminometer, will reveal the ability of the bioluminescent phage to transduce bioluminescence and will enable us to find the optimal conditions. The bioluminescent phage will be further spray dried to be converted into powder formulation and will be distributed in vials for easy use. The liquid bioluminescent phage and the one in powder format will be tested for diagnostic accuracy and sensitivity in real conditions, using initially a selection of isolates from the large collection of bacteria that have been isolated at Cambridge. Further, these will be tested in stored mastitis samples with known microbiological diagnoses. Finally, these will be used as diagnostics directly to the dairy herd at the University Farm in Cambridge using conventional microbiology. The above will provide sensitivity and specificity values for the diagnostic method in field conditions and will form the basis for a rapid cow-side bacteriophage based diagnostic test for Staphylococcus aureus mastitis.