bmap-28 and Mastitis--Bovine

Bovine mastitis caused by bacterial pathogens, such as Staphylococcus (S.) aureus and Escherichia (E.) coli, is a major economic problem in dairy industry. In order to limit the presence of multi-resistant bacteria in bovine mastitis, alternatives for the treatment with antibiotics are urgently needed. Antimicrobial peptides (AMPs) have recently been discussed as a potential new strategy against bacterial infections. They are key players in the innate immune system, as they can directly act against microorganisms or modulate the immune system. The aim of our study was to test S. aureus and E. coli mastitis isolates for their susceptibility to the bovine cathelicidins, BMAP-27 and BMAP-28. Susceptibility testing was performed in analogy to the broth microdilution criteria described by the Clinical and Laboratory Standard Institute (CLSI) to determine MICs of 50 clinical S. aureus and 50 clinical E. coli isolates for BMAP-27 and BMAP-28. Based on the repetitive testing of four well-selected reference strains, the homogeneity of MIC variances for each peptide as well as the effect of temperature, oxygen level and plastic polymers on MIC testing was determined. Statistical analysis revealed not only strong peptide-specific variances, but also strain-specific variances in the technical procedure. Finally, using this technique, susceptibility testing of the field isolates revealed statistically significant peptide-specific differences in the MICs. While BMAP-27 showed lower MICs for E. coli, BMAP-28 showed lower MICs for S. aureus. However, these results clearly illustrate the need of susceptibility testing of AMPs on several unrelated strains and not only on one selected test organism.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Cathelicidins; Cattle; Escherichia coli; Female; Mastitis, Bovine; Microbial Sensitivity Tests; Proteins; Sequence Alignment; Staphylococcus aureus

The yeast-like algae of the genus Prototheca are ubiquitous saprophytes causing infections in immunocompromised patients and granulomatous mastitis in cattle. Few available therapies and the rapid spread of resistant strains worldwide support the need for novel drugs against protothecosis. Host defence antimicrobial peptides inactivate a wide array of pathogens and are a rich source of leads, with the advantage of being largely unaffected by microbial resistance mechanisms. Three structurally diverse bovine peptides [BMAP-28, Bac5 and lingual antimicrobial peptide (LAP)] have thus been tested for their capacity to inactivate Prototheca spp. In minimum inhibitory concentration (MIC) assays, they were all effective in the micromolar range against clinical mastitis isolates as well as a Prototheca wickerhamii reference strain. BMAP-28 sterilized Prototheca cultures within 30-60 min at its MIC, induced cell permeabilization with near 100% release of cellular adenosine triphosphate and resulted in extensive surface blebbing and release of intracellular material as observed by scanning electron microscopy. Bac5 and LAP inactivated Prototheca following 3-6 h incubation at fourfold their MIC and did not result in detectable surface damage despite 70-90% killing, suggesting they act via non-lytic mechanisms. In circular dichroism studies, the conformation of BMAP-28, but not that of Bac5 or LAP, was affected by interaction with liposomes mimicking algal membranes. Our results indicate that BMAP-28, Bac5 and LAP kill Prototheca with distinct potencies, killing kinetics, and modes of action and may be appropriate for protothecal mastitis treatment. In addition, the ability of Bac5 and LAP to act via non-lytic mechanisms may be exploited for the development of target-selective drugs.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; beta-Defensins; Cattle; Cell Membrane; Enterobacteriaceae; Eosinophil Granule Proteins; Female; Mastitis, Bovine; Microbial Sensitivity Tests; Molecular Sequence Data; Permeability; Protein Structure, Secondary; Proteins; Prototheca; Staphylococcus; Streptococcus