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

Bacteriophage-encoded endolysins, peptidoglycan hydrolases breaking down the Gram-positive bacterial cell wall, represent a groundbreaking class of novel antimicrobials to revolutionize the veterinary medicine field. Wild-type endolysins exhibit a modular structure, consisting of enzymatically active and cell wall-binding domains, that enable genetic engineering strategies for the creation of chimeric fusion proteins or so-called 'engineered endolysins'. This biotechnological approach has yielded variants with modified lytic spectrums, introducing new possibilities in antimicrobial development. However, the discovery of highly similar endolysins by different groups has occasionally resulted in the assignment of different names that complicate a straightforward comparison. The aim of this review was to perform a homology-based comparison of the wild-type and engineered endolysins that have been characterized in the context of bovine mastitis-causing streptococci and staphylococci, grouping homologous endolysins with ≥ 95.0% protein sequence similarity. Literature is explored by homologous groups for the wild-type endolysins, followed by a chronological examination of engineered endolysins according to their year of publication. This review concludes that the wild-type endolysins encountered persistent challenges in raw milk and in vivo settings, causing a notable shift in the field towards the engineering of endolysins. Lead candidates that display robust lytic activity are nowadays selected from screening assays that are performed under these challenging conditions, often utilizing advanced high-throughput protein engineering methods. Overall, these recent advancements suggest that endolysins will integrate into the antibiotic arsenal over the next decade, thereby innovating antimicrobial treatment against bovine mastitis-causing streptococci and staphylococci.

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

Streptococcus uberis frequently causes bovine mastitis, an infectious udder disease with significant economic implications for dairy cows. Conventional antibiotics, such as cloxacillin, sometimes have limited success in eliminating S. uberis as a stand-alone therapy. To address this challenge, the study objective was to investigate the VersaTile engineered endolysin NC5 as a supplemental therapy to cloxacillin in a mouse model of bovine S. uberis mastitis. NC5 was previously selected based on its intracellular killing and biofilm eradicating activity. To deliver preclinical proof-of-concept of this supplemental strategy, lactating mice were intramammarily infected with a bovine S. uberis field isolate and subsequently treated with cloxacillin (30.0 µg) combined with either a low (23.5 µg) or high (235.0 µg) dose of NC5. An antibiotic monotherapy group, as well as placebo treatment, was included as controls. Two types of responders were identified: fast (n = 17), showing response after 4-h treatment, and slow (n = 10), exhibiting no clear response at 4 h post-treatment across all groups. The high-dose combination therapy in comparison with placebo treatment impacted the hallmarks of mastitis in the fast responders by reducing (i) the bacterial load 13,000-fold (4.11 ± 0.78 Δlog10; p < 0.001), (ii) neutrophil infiltration 5.7-fold (p > 0.05), and (iii) the key pro-inflammatory chemokine IL-8 13-fold (p < 0.01). These mastitis hallmarks typically followed a dose response dependent on the amount of endolysin added. The current in vivo study complements our in vitro data and provides preclinical proof-of-concept of NC5 as an adjunct to intramammary cloxacillin treatment. KEY POINTS: • Engineered endolysin NC5 was preclinically evaluated as add-on to cloxacillin treatment. • Two types of mice (slow and fast responding) were observed. • The add-on treatment decreased bacterial load, neutrophil influx, and pro-inflammatory mediators.