Detection of the pediocin gene pedA in strains from human faeces by real-time PCR and characterization of Pediococcus acidilactici UVA1.
BACKGROUND
Bacteriocin-producing lactic acid bacteria are commonly used as natural protective cultures. Among them, strains of the genus Pediococcus are particularly interesting for their ability to produce pediocin, a broad spectrum antimicrobial peptide with a strong antagonistic activity against the food-borne pathogen Listeria monocytogenes. Furthermore, there is increasing interest in isolating new bacteriocin-producing strains of human intestinal origin that could be developed for probiotic effects and inhibition of pathogenic bacteria in the gut. In this work, we typed a new strain, co-isolated from baby faeces together with a Bifidobacterium thermophilum strain, and characterized its proteinaceous compound with strong antilisterial activity.


RESULTS
The newly isolated strain UVA1 was identified as a Pediococcus acidilactici by carbohydrate fermentation profile, growth at 50 degrees C and 16S rDNA sequencing. The partially purified bacteriocin was heat resistant up to 100 degrees C, active over a wide range of pH (2 to 9) and susceptible to proteolytic enzymes. The molecular weight, estimated by SDS-PAGE, was similar to that of pediocin AcH/PA-1 (4.5 kDa). P. acidilactici UVA1 harboured a 9.5-kb plasmid that could be cured easily, which resulted in the loss of the antimicrobial activity. Southern hybridization using the DIG-labelled pedA-probe established that the bacteriocin gene was plasmid-borne as for all pediocin described so far. Nucleotide sequence of the whole operon (3.5 kb) showed almost 100 % similarity to the pediocin AcH/PA-1 operon. The mRNA transcript for pedA could be detected in P. acidilactici UVA1 but not in the cured derivative, confirming the expression of the pedA-gene in UVA1. Using a new real-time PCR assay, eleven out of seventeen human faecal samples tested were found to contain pedA-DNA.


CONCLUSIONS
We identified and characterised the first pediocin produced by a human intestinal Pediococcus acidilactici isolate and successfully developed a new real-time PCR assay to show the large distribution of pedA-containing strains in baby faecal samples.
Mathys
Sophie
S
Laboratory of Food Biotechnology, Institute of Food Science and Nutrition, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland. sophie.mathys@ilw.agrl.ethz.ch
von Ah
Ueli
U
Lacroix
Christophe
C
Staub
Ernö
E
Mini
Raffaella
R
Cereghetti
Tania
T
Meile
Leo
L
D001430:Bacteriocins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D015139:Blotting, Southern; D004591:Electrophoresis, Polyacrylamide Gel; D005243:Feces / Q000382:microbiology*; D005285:Fermentation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008087:Listeria / Q000187:drug effects; D008970:Molecular Weight; D010376:Pediococcus / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D013696:Temperature
D001430:Bacteriocins; D012336:RNA, Ribosomal, 16S