Purification of Microcin N – A Bacteriocin Effective against Salmonella enterica and Escherichia coli

Introduction: Salmonella enterica and Escherichia coli O157:H7 are foodborne pathogens commonly associated with disease outbreaks from the consumption of meat. Microcin N (McnN), a bacteriocin produced by a non-pathogenic strain of E. coli MC4100 pGOB18, has potential to control the growth and survival of these enteric pathogens. Purification of MccN for use in food systems has not been previously done.

Purpose: The objective of this study was to purify stable peptides for use as an antimicrobial in meat.

Methods: E. coli MC4100 pGOB18 was grown in minimal media and the production of McnN was confirmed with a deferred inhibition assay using S. enterica and E. coli as the indicator organisms.  The supernatant of an overnight culture was passed through an Amberlite XAD16N column and the peptide eluted with 80% isopropanol containing 0.1% trifluoroacetic acid.  Antimicrobial activity was confirmed by spot-on-lawn assay using S. enterica as the indicator organism and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to determine the presence of McnN.  High performance liquid chromatography with a C18 column was used to further purify the active compounds. Fractions were concentrated and tested for antimicrobial activity using a spot-on-lawn assay. Active fractions were analyzed using MALDI-TOF MS to confirm the presence of peptides. 

Results: After column purification, antimicrobial activity against S. enterica and E. coli was detected.  MALDI-TOF MS revealed the presence of a peptide with a mass similar to that of McnN (7.3 kDa).  Further purification with HPLC resulted in activity in spot-on-lawn assays but MALDI-TOF MS did not reveal the presence of a peptide with a mass consistent with McnN.  However, smaller peptides were present. 

Significance: Partial purification of McnN with column chromatography may provide a crude extract that could be used to control target pathogens in meat. Further research is required to obtain pure McnN.