Application of Microcin N and Tridecaptin A1 to control bacterial pathogens

• Author / Creator

  Satchwell, Katherine L

• Bacterial pathogens are responsible for a number of infectious diseases affecting humans and animals. Peptide based antimicrobial interventions may be an effective means to control pathogens. Enterohaemorrhagic Escherichia coli and Salmonella enterica are foodborne pathogens that are commonly implicated in outbreaks stemming from contaminated meat products. Listonella anguillarum is the etiological agent responsible for vibriosis in farmed salmon. The antimicrobial peptides, Microcin N (McnN) and Tridecaptin A1 (TriA1), were investigated for their ability to control these pathogens in foodstuff, designated for human and aquatic consumption, respectively. McnN is a bacteriocin produced by a non-pathogenic strain of E. coli that displays activity against E. coli and Salmonella species. TriA1 is a cationic linear lipopeptide produced by Paenibacillus terrae that displays potent antimicrobial activity against gram-negative microorganisms. Synthesis of an analogue of TriA1, Octyl-Tridecaptin A1, produced via acylation of octanoic acid to the N-terminus of the peptide was performed by collaborators and was found to exhibit antimicrobial activity similar to the native TriA1. Hydrophobic interaction chromatography was used to partially purify Microcin N from the supernatant of E. coli MC4100 pGOB18. A compound similar in molecular mass to of McnN was identified in the partially purified McnN fraction using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. High performance liquid chromatography and ion chromatography failed to yield pure McnN. The partially pure preparation of McnN obtained from hydrophobic interactions chromatography failed to reduce counts of 5-strain cocktails of E. coli O157:H7 when applied to raw or cooked ground beef and incubated for 72 h at 8°C, respectively. Additionally, 5-strain cocktails of S. enterica were not inhibited by the partially pure McnN preparation under the same storage conditions in raw or cooked ground beef. While the results with McnN were not as expected, future work to isolate pure bacteriocin could provide an antimicrobial effective against gram-negative foodborne pathogens. The application of antimicrobials to foodstuffs for the control of bacterial pathogens is a viable means to reduce the burden associated with contamination of these organisms. TriA1 was purified from the supernatant of P. terrae B-NRRL 30644 using hydrophobic interactions chromatography and confirmed using high performance liquid chromatography. Both the antimicrobial preparation (AMP) and Oct-TriA1 were active against L. anguillarum in vitro in spot-on-lawn assays. The minimum inhibitory concentration of Oct-TriA1 was determined against a collection of foodborne and aquatic organisms, and MIC values ranged from 0.6 to 38 μg/mL. Lyophilization of the AMP provided a water-soluble powdered preparation that could be concentrated for application. Both the concentrated AMP and Oct-TriA1 were active against L. anguillarum when assessed directly using spot-on-lawn assays and when treated commercial feed was over-layered with agar containing the indicator strain. Encapsulation of antimicrobials using alginate was successfully performed, though Oct-TriA1 was superior in terms of consistent inhibition of L. anguillarum. Oct-TriA1 did not interact with constituents in salmon feed whereas Polymyxin B and Polymyxin E were inactive against L. anguillarum after exposure to commercial feed. The concentration of TriA1 in the concentrated AMP is likely the limiting factor in the variability of activity of encapsulated products against indicators. Sufficient quantities are needed to elicit an antimicrobial effect when working with medicated feed. Based on these findings TriA1 is an effective antimicrobial for use against pathogens that afflict Atlantic salmon. Oct-TriA1 has great potential for industrial application as an antimicrobial for medicated feed formulation for use in challenge studies with Atlantic salmon.  

• Subjects / Keywords

  • Microcin N
  • Tridecaptin A1

• Graduation date

  Fall 2016

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3CN6Z46C

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.