Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/25/2016
Publication Date: N/A
Interpretive Summary: none
Technical Abstract: Background: Novel alternatives to traditional antibiotics are urgently needed for food-animal production. A goal of our laboratory is to develop and evaluate antimicrobial peptides (AMP) to control and reduce foodborne pathogens in poultry. AMP have been found in most every class of living organism where they have evolved as a host defense mechanism against invading microorganisms. Our working hypothesis is that AMP can be identified that inhibit the growth of Campylobacter jejuni and subsequently can be utilized to reduce the Campylobacter load among commercially produced chickens. Because of their modes of action, these AMP are much less likely to engender antimicrobial resistance. Methods: We chemically synthesized a set of 11 unique AMP and evaluated them for ability to inhibit growth of two strains of C. jejuni. Six of the AMP we tested produced zones of inhibition on lawns of C. jejuni. These AMP included: NRC-13, a variant of Pleurocidin isolated from the American plaice-flounder; RL-37, a 37-residue AMP of the cathelicidin family which is expressed in bone marrow of the rhesus monkey; Temporin, from the frog, Rana temporaria; a potent hybrid AMP (Cec-Mag) composed of residues 1-8 of Cecropin A (from the Cecropia moth) fused to residues 1-12 of Magainin 2 (from the African clawed frog, Xenopus Laevis); Dermaseptin from the skin of frogs of the genus Phyllomedusa; and the synthetic OAK, C12K-2b12. Three AMP were chosen for further investigation on the basis of reported reduced cytotoxicity to mammalian cells: Cec-Mag, RL-37 and C12K-2b12 were tested for ability to produce zones of inhibition in spot on lawn assays against 19 different bacteria (Table 1) including C. jejuni, C. coli and C. lari as well as two strains of Salmonella and Lactobacillus. In addition, a modification of the NCCLS M26A and Hancock assays were utilized to determine minimum inhibitory concentrations (MIC) for these AMP against three strains of C. jejuni in microtiter plates. Conclusions: • The selected AMP produced obvious zones of inhibition against growth of C. jejuni, C. coli and C. lari isolates in the classic spot-on-lawn plating assay. • MIC were approximately 3.1 ug/ml for the AMP RL-37 and C12K-2b12. • MIC were slightly higher for the Cec-Mag AMP in the range of 12.5 to 50 ug/ml. • The selected AMP also inhibited growth of pathogenic Salmonella isolates. • Additional work must be done to determine the effect of AMP on the non-pathogenic natural flora of broiler chickens. • Our next steps are to express AMP in yeast and explore encapsulation technologies to stabilize the AMP for in vivo trials in broiler chickens.