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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Biosciences & Biotechnology Laboratory » Research » Publications at this Location » Publication #331967

Title: Identification of peptidoglycan hydrolase constructs with synergistic staphylolytic activity in cow's milk

item VERBREE, C - Eth Zurich
item DATWYLER, S - Eth Zurich
item EICHENSEHER, F - Eth Zurich
item MEILE, S - Eth Zurich
item Donovan, David
item LOESSNER, M - Eth Zurich
item SCHMELCHER, M - Eth Zurich

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2017
Publication Date: 3/17/2017
Citation: Verbree, C.T., Datwyler, S.M., Eichenseher, F., Meile, S., Donovan, D.M., Loessner, M.J., Schmelcher, M. 2017. Identification of peptidoglycan hydrolase constructs with synergistic staphylolytic activity in cow's milk. Applied and Environmental Microbiology.

Interpretive Summary: There is a need for novel treatments of staphylococcal bovine mastitis with antimicrobials that are refractory to resistance development. Viruses that infect bacteria (bacteriophage; phage) have co-evolved with their host and thus their cell wall degrading proteins (endolysins; peptidoglycan hydrolases) are highly refractory to resistance development. This work describes a novel screening method for the simultaneous high throughput testing of more than 170 phage endolysin constructs for activity in raw milk. Several high activity constructs were identified and tested via in vitro confirmatory assays to verify the screening method. There is still a need for in vivo testing of the constructs identified. This is the first time that a high throughput screening method has been devised for identifying enzyme antimicrobials that target a major mastitis pathogen in milk. This screen will provide a technology to rapidly identify those enzymes that could help dairy farmers world-wide.

Technical Abstract: Staphylococci are a major cause of bovine mastitis, an inflammation of the mammary gland in cows associated with high costs and posing a risk for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing drug-resistance in bacteria. Therefore, new treatment options are needed. Peptidoglycan hydrolases (PGH) rapidly and specifically kill bacteria and are refractory to resistance development, therefore holding promise as mastitis therapeutics. An important requirement for drugs administered intramammarily is that they retain activity in cow milk. In this work, a library of > 170 PGHs was screened for enzymes with staphylolytic activity in milk using a novel high-throughput protocol. Nine promising PGHs were identified and subsequently compared in time-kill assays for their efficacy against S. aureus in ultra-heat treated (UHT) milk. The strongest PGHs (lysostaphin, Ami2638A, and CHAPK_CWT-LST) eradicated S. aureus from milk at nanomolar concentrations, acted synergistically in most combinations, and were active against multiple mastitis isolates. In raw milk, lysostaphin and Ami2638A reduced bacterial numbers by > 3.5 log units within 3 h. Our results suggest high potential of the identified PGHs as mastitis therapeutics and support their further characterization in animal models.