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United States Department of Agriculture

Agricultural Research Service

Research Project: DEVELOPING GENETIC BIOTECHNOLOGIES FOR INCREASED FOOD ANIMAL PRODUCTION, INCLUDING NOVEL ANTIMICROBIALS FOR IMPROVED HEALTH & PRODUCT SAFETY

Location: Animal Biosciences and Biotechnology Laboratory

Title: Identification and characterization of peptidoglycan hydrolase constructs with activity in cow milk as potential antimicrobials for treatment of staphylococcal bovine mastitis

Authors
item Verbree, Caroline -
item Schmelcher, Mathias -
item Donovan, David
item Loessner, Martin -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: May 21, 2014
Publication Date: N/A

Technical Abstract: Mastitis in dairy cows is a widespread infection of the mammary glands that leads to high losses in dairy production. Most members of the Gram-positive genus Staphylococcus can cause mastitis, with Staphylococcus S. aureus being one of the major pathogens. Intramammary application of antibiotics is a commonly used treatment, but due to increasing drug resistance and low efficacy, there is a need for alternative treatment methods. The use of bacteriophage endolysins (peptidoglycan hydrolases; PGH), which exhibit lytic activity against Gram-positive bacteria, constitutes a promising approach. However, a crucial requirement for novel anti-mastitis drugs is high activity in cow milk. In this work, a PGH collection consisting of over 180 PGH constructs (parental enzyme, truncations and fusions) from two different labs was screened for proteins with high activity in milk using a novel 96-well plate-based screening protocol. Nine promising protein candidates were identified and subsequently analyzed by viable count assays with S. aureus-inoculated milk in an effort to further narrow down the selection of enzymes. The constructs Ami2638 and CHAPK_CWT-LST were identified as the most effective proteins, with activities similar to that of the reportedly strong PGH antimicrobial Lysostaphin. Since the two selected enzymes and Lysostaphin together represent three different types of catalytic specificity (MurNAc-L-Ala amidase; D-Ala-Gly endopeptidase; Gly-Gly endopeptidase), they were further analyzed for potential synergistic effects. Classical checkerboard assays in growth media revealed strong synergy between CHAPK_CWT-LST and Lysostaphin, moderate synergy between CHAPK_CWT-LST and Ami2638, and weak synergy between Ami2638 and Lysostaphin. A modified checkerboard assay using milk instead of broth, as well as time-kill experiments demonstrated synergy in milk against S. aureus for CHAPK_CWT-LST and Lysostaphin, CHAPK_CWT-LST and Ami2638, and the combination of all three proteins. The most effective combination (CHAPK_CWT-LST and Lysostaphin) was shown to be active against all strains within a collection of S. aureus and coagulase-negative Staphylococcus mastitis isolates. The results of this work together with previously published data suggest high potential of the identified proteins as antimicrobials for treatment of mastitis and support their further characterization in animal models.

Last Modified: 10/20/2014
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