In vitro evaluation of a novel bacteriophage cocktail as a preventative for bovine coliform mastitis

Abstract

The objective of this study was to investigate the potential use of bacteriophage in preventing Escherichia coli mastitis on dairies. A cocktail consisting of 4 distinct bacteriophages was generated by screening against 36 E. coli isolates from dairy cows in Washington State with clinical mastitis. The bacteriophage significantly inhibited growth of 58% of the Washington State isolates and 54% of E. coli mastitis isolates from New York State, suggesting that the cocktail of phages had a relatively broad spectrum of action against relevant strains from 2 distinct geographies. The ability to suppress bacterial growth of these isolates in a liquid growth medium was not affected by the ratio of bacteriophage particles to bacterial cells (multiplicity of infection, MOI). For those E. coli that were completely inhibited by the phage cocktail, an MOI as low as 10 had the same effect as 10 µg/mL of ceftiofur on the growth rate of E. coli over a 12-h period using optical density measurements. A 3.3- to 5.6-log reduction of growth was achieved when E. coli was co-incubated with our phage cocktail in raw milk over a 12-h period at physiologic temperature. A modified gentamicin protection assay using bovine mammary epithelial cells provided a model to test whether bacteriophage could prevent cell attachment and invasion by chronic coliform mastitis strains. Pretreatment of cell cultures with the phage cocktail significantly reduced adhesion and intracellular survival of E. coli compared with controls. When combined with a bismuth-based teat sealant, the phage cocktail was able to inhibit bacterial growth when challenged with 1.6 × 10(3) cfu/mL of a clinical mastitis E. coli strain. In vitro results show bactericidal activity by our phage in raw milk and mammary tissue culture systems. Before a bacteriophage-based dry-cow treatment becomes a potential option for dairies, in vivo studies must be able to demonstrate that a specific dose of bacteriophage can protect cows from experimentally induced E. coli mastitis without inducing an inflammatory reaction.

Identification of Key Candidate Genes in Dairy Cow in Response to Escherichia coli Mastitis by Bioinformatical Analysis

“…Genes coding for proteins such as haptoglobin, cathelicidin antimicrobial peptide, and lingual antimicrobial peptide have been identified as potential biomarkers for mastitis detection (Sharifi et al, 2018). At present, antibiotics are mainly used as systemic treatment of mastitis (Porter et al, 2016). Antibiotic should be chosen based on specific mastitis pathogen.…”

Potentiële therapeutische toepassing van bacteriofagen en faag-afgeleide endolysinen als alternatieve behandeling van boviene mastitis

“…It may therefore be useful to develop cocktails consisting of different bacteriophages or to supplement the phage therapy with endolysins. The expansion of the working spectrum has already been tested and confirmed by two recent studies (Porter et al, 2016;Breyne et al, 2017). The main disadvantage of the classical endolysins remains their Gram-positive spectrum.…”

“…Recently, a cocktail consisting of four different bacteriophages has been proposed against E. coli through several in vitro tests (Porter et al, 2016). A 3.3 to 5.6 log reduction of growth in raw milk was observed when E. coli was co-incubated with this phage cocktail for twelve hours.…”

“…Recently, several researchers have claimed that promising candidate bacteriophages and endolysins should now be tested in vivo to evaluate the effects of this novel treatment strategy in mammary gland of rodents, but preferably in the target species i.e. the cow (Schmelcher et al, 2015;Porter et al, 2016). How-ever, even if the envisaged phage therapy confirms to be promising in these follow-up studies, several practical hurdles will raise during its development.…”

Bacteriophages’ action against mastitis-causing bactéria

“…Bacteriophage therapy combining several phages (cocktail) also proved to be a potential alternative in the prevention of E. coli mastitis by reducing their adhesion and intracellular survival in breast tissue. In the strains where there was total inhibition by phage Research, Society and Development, v. 9, n. 10, e1849108541, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8541 cocktail, there was no statistically significant difference compared to 10 µg/mL of ceftiofur over a period of 12 hours (Porter et al, 2016).…”