Detection of the pediocin gene pedA in strains from human faeces by real-time PCR and characterization of Pediococcus acidilactici UVA1

Abstract

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.

Conclusion: 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.

Figure 1

Molecular weight determination of the bacteriocin produced by strain UVA1. (a) Coomassie stained SDS-PAGE gel. (b) SDS gel overlaid with the indicator strain Listeria ivanovii HPB28. Std: polypeptide molecular weight standard in kDa, F3c: active FPLC-fraction 10-fold concentrated. CFSc: cell-free supernatant of a UVA1 culture, 10-fold concentrated. CFS: cell-free supernatant not concentrated.

Figure 2

Localization of the pedA gene on the plasmid by curing and Southern blotting. (a) Agarose gel electrophoresis of plasmid DNA. UVA1: P. acidilactici UVA1. UL5: P. acidilactici UL5. bac-: cured derivative of UVA1. M. Supercoiled DNA ladder in kb. ch: chromosomal DNA band. (b) Southern blot DNA hybridization of the DIG labeled pedA-probe with plasmidic DNA from P. acidilactici bac-, UVA1 and UL5. (c) and (d) Agar-well diffusion assay with CFS of cultures of P. acidilactici UVA1 (c) and its cured derivative P. acidilactici bac- (d).

Figure 3

Transcription analysis of pedA. pedA-reverse transcription-PCR on cDNA from P. acidilactici bac- (1, 2, 3) or UVA1 (7, 8, 9) after 1 h 30, 2 h 30 and 3 h 30 of growth, respectively, and from P. acidilactici DSM 20284^T(4) or UL5 (6) and P. pentosaceus DSM 20336^T (5) after 2 h 30 of growth. 10: water instead of DNA. lm: low molecular weight DNA ladder (in bp). h: Tridye 100-bp DNA ladder (in bp). Expected product size: 100 bp.

Figure 4

Real-time PCR on faecal DNA samples. (a) Ct values of spiked samples plotted against spiked cell concentration in faecal DNA sample. (b) Ct values obtained for children (C1-C13) and adult (A1-A4) faecal samples. F0: faecal sample free of DNA. w: water instead of DNA. Values represented are means and standard deviations for 3 repetitions.