Development of engineered endolysins with in vitro intracellular activity against streptococcal bovine mastitis‐causing pathogens

Abstract

Bacteriophage‐derived endolysins are a novel class of antimicrobials known to rapidly kill bacteria, including antibiotic‐resistant strains. We here engineered endolysins against the bovine mastitis pathogens Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae, also targeting intracellular survival and biofilm formation. For this purpose, high‐throughput DNA assembly was used to create a library with >80,000 theoretical endolysin variants for screening of their bacteriolytic activity against Gram‐positive isolates from (sub)clinically affected cows. This lytic activity was evaluated by turbidity reduction and time‐kill assays in phosphate‐buffered saline and pasteurized whole cow's milk to allow a rank up of the most potent leading candidates. A top candidate was selected with a 4.0 log killing efficacy against S. uberis, also showing similar activity against S. agalactiae and S. dysgalactiae. This top candidate eradicated S. uberis biofilm and showed intracellular activity in two bovine mammary epithelial cell lines as was confirmed by confocal microscopy. A potentiating effect on cloxacillin, a beta‐lactam penicillin used to intramammarily treat bovine Gram‐positive mastitis, was observed for this top candidate endolysin in raw cow's milk from (sub)clinically infected udders. Our in vitro results indicate that engineered endolysins may have a future role as add‐on in the treatment of bovine streptococcal mastitis.

Endolysin NC5 improves early cloxacillin treatment in a mouse model of Streptococcus uberis mastitis

“…An explanation for this unexpected division into slow- vs. fast-responding animals might lie in the rational choice for a short (4 h) duration of the treatment and the deliberate use of outbred mice (Woodward and Whittem 2019 ). Cloxacillin, being an isoxazolyl penicillin, needs to be incorporated into the cell wall of multiplying Gram-positive bacteria to effectively eliminate them (Kim et al 2023 ).…”

“…As a result, the CFU levels in the cloxacillin treatment groups, with or without addition of endolysin NC5, were as expected not completely reduced to the limit of detection after only 4 h. Nevertheless, in our previous in vitro study, an accelerated reduction in S. uberis CFUs was observed in raw milk between the 4 and 8 h time points for the supplemental strategy (Vander Elst et al 2023b ). Therefore, in this preclinical in vivo study, we deliberately choose to evaluate the combination treatment after only 4 h to specifically capture the small discrepancies between the experimental groups mid-treatment (Woodward and Whittem 2019 ). However, this limits the current study to one early endpoint and does not provide a comprehensive kinetic profile to allow a more holistic interpretation of the obtained results.…”

Bacteriophage-derived endolysins as innovative antimicrobials against bovine mastitis-causing streptococci and staphylococci: a state-of-the-art review

“…The spot-on-plate assay is regarded more stringent than the spot-on-lawn assay. Of note, the diameter of the halo does not correlate with the lytic activity of the endolysin [4,9,30,31]. It is rather dependent on the size of the endolysin that diffuses through the agar.…”

“…The turbidity reduction assay (TRA) is a method to evaluate the biochemical activity of an enzyme (Fig. 3B) [16,[31][32][33][34][35][36][37]. It measures a decrease of the optical density (OD) over time using a spectrophotometer, typically monitored at a wavelength of 600 nm (OD 600nm ).…”

Emerging Approaches for Mitigating Biofilm-Formation-Associated Infections in Farm, Wild, and Companion Animals

“…In 2023, two studies were developed in the context of bovine mastitis, however, for two different pathogens, Streptococcus uberis and E. faecalis . Elst et al described an endolysin with activity against S. uberis in raw cow’s milk [ 239 ]. After exposure of the S. uberis biofilm to a concentration of 1.5 uM NC5 for 2 h 30 min, a significant reduction of around 70% of the biofilm mass was observed.…”