Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk.
Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have screened a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity againstStaphylococcus aureusin milk, using a microtiter plate-based protocol. Nine suitable PGH constructs were identified by this approach and further compared in time-kill assays for their efficacy againstS. aureusin heat-treated milk. The three most active enzymes (lysostaphin, Ami2638A, and CHAPK_CWT-LST) reducedS. aureusin milk to undetectable numbers within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity in most combinations, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The most effective PGH combination completely eradicatedS. aureusfrom milk, with no more bacteria being detected within 24 h after addition of the enzymes, corresponding to a reduction of >9 log units compared to the control. Efficacy was also retained at different inoculum levels (3 versus 6 log CFU/ml) and whenS. aureuswas grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas both Ami2638A and lysostaphin retained their activity, reducing bacterial numbers by >3.5 log units within 3 h.IMPORTANCEStaphylococci andS. aureusin particular are a major cause of bovine mastitis, an inflammation of the mammary gland in cows associated with high costs and risks for consumers of milk products.S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell wall and are refractory to resistance development, therefore holding promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk within a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and support their further characterization in animal models.
Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland.
Carolin T
CT
Verbree
Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland.
Steven M
SM
Dätwyler
Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland.
Susanne
S
Meile
Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland.
Fritz
F
Eichenseher
Animal Biosciences and Biotechnology Laboratory, ANRI, NEA, ARS, U.S. Department of Agriculture, Beltsville, Maryland, USA.
David M
DM
Donovan
Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland.
Martin J
MJ
Loessner
Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland mathias.schmelcher@hest.ethz.ch.
Mathias
M
Schmelcher
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002417:Cattle; D002418:Cattle Diseases / Q000188:drug therapy / Q000382:microbiology; D055808:Drug Discovery; D004357:Drug Synergism; D005260:Female; D015723:Gene Library; D006358:Hot Temperature; D008321:Mammary Glands, Animal / Q000382:microbiology; D008414:Mastitis, Bovine / Q000188:drug therapy / Q000382:microbiology; D008826:Microbial Sensitivity Tests; D008892:Milk / Q000382:microbiology*; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D010457:Peptidoglycan / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013203:Staphylococcal Infections / Q000188:drug therapy / Q000382:microbiology / Q000662:veterinary; D013211:Staphylococcus aureus / Q000187:drug effects*
D000900:Anti-Bacterial Agents; D010457:Peptidoglycan; D011994:Recombinant Proteins; D009238:N-Acetylmuramoyl-L-alanine Amidase
antibiotic resistance; antimicrobial agents; bovine mastitis; peptidoglycan hydrolases