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Purification and identification of the pediocin produced by Pediococcus acidilactici MM33, a new human intestinal
M Millette1, C Dupont, F Shareck
1INRS-Institut Armand-Frappier, Research Laboratories in Sciences Applied to Food, Canadian Irradiation Centre, Laval, QC, Canada.
Aims:
The aim of this study was to purify and identify the bacteriocin produced by Pediococcus acidilactici MM33, a strain previously isolated from human gut.
Methods And Results:
Purification of the bacteriocin was performed by cationic exchange chromatography followed by a reverse phase step. Biochemical and mass spectrometry analysis showed homology with pediocin PA-1. To verify if P. acidilactici MM33 carried the pediocin PA-1 gene, total DNA was used to amplify the pediocin gene. The PCR product obtained was then sequenced and the nucleotide sequence revealed to be identical to that of pediocin PA-1. Treatment of P. acidilactici MM33 with novobiocin resulted in a plasmid-cured strain without bacteriocin-producing capacity. Antimicrobial assay and molecular analysis demonstrated that this strain was ped(-) suggesting that the ped cluster is plasmid encoded. Antimicrobial assay revealed that pediocin was bactericidal against Listeria monocytogenes, showing a minimal inhibitory concentration (MIC) of 200 AU ml(-1).
Conclusions:
A two-step purification procedure was elaborated in this study. The bacteriocin secreted by the human strain P. acidilactici MM33 is carried on a plasmid and the amino acid sequence is identical to pediocin PA-1.
Significance And Impact Of The Study:
Pediococcus acidilactici MM33 is the first human pediocin-producing strain reported and could be used as probiotic to prevent enteric pathogen colonization.
Insights
Researchers purified and identified a bacteriocin from human gut bacteria Pediococcus acidilactici MM33. This bacteriocin, identical to pediocin PA-1, is plasmid-encoded and effective against Listeria monocytogenes.
Area of Science:
- Microbiology
- Bacteriocins
- Probiotics
Background:
- Pediococcus acidilactici is a lactic acid bacterium found in fermented foods and the human gut.
- Bacteriocins are ribosomally synthesized antimicrobial peptides with a narrow spectrum of activity.
- Pediocin PA-1 is a well-characterized bacteriocin produced by Pediococcus acidilactici.
Purpose of the Study:
- To purify and identify the bacteriocin produced by Pediococcus acidilactici MM33, a human gut isolate.
- To determine the genetic basis of bacteriocin production in this strain.
- To evaluate the antimicrobial activity of the purified bacteriocin.
Main Methods:
- Bacteriocin purification using cationic exchange chromatography and reverse-phase chromatography.
- Biochemical and mass spectrometry analysis for identification.
- Polymerase chain reaction (PCR) and DNA sequencing to identify the bacteriocin gene.
- Plasmid curing experiments using novobiocin.
- Antimicrobial assays to determine the minimal inhibitory concentration (MIC).
Main Results:
- The purified bacteriocin showed homology to pediocin PA-1.
- DNA sequencing confirmed the presence of the pediocin PA-1 gene.
- Plasmid curing eliminated bacteriocin production, indicating plasmid-encoded bacteriocin synthesis.
- The bacteriocin was bactericidal against Listeria monocytogenes with an MIC of 200 AU/ml.
Conclusions:
- Pediococcus acidilactici MM33 produces pediocin PA-1, encoded on a plasmid.
- This is the first reported human-derived pediocin-producing Pediococcus acidilactici strain.
- The strain and its bacteriocin hold potential as probiotics for preventing enteric pathogen colonization.
