Detection of the pediocin gene pedA in strains from human faeces by real-time PCR and characterization of Pediococcus acidilactici UVA1
Sophie Mathys*, Ueli von Ah*, Christophe Lacroix, Ernö Staub, Raffaella Mini, Tania Cereghetti and Leo Meile
Laboratory of Food Biotechnology, Institute of Food Science and Nutrition, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland
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°C and 16S rDNA sequencing. The partially purified bacteriocin was heat resistant up to 100°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.
Background
The lactic acid bacteria are an inhomogeneous group which includes among others the genera Pediococcus, Enterococcus, Lactococcus, Lactobacillus and Streptococcus. Among the Pediococcus group, P. acidilactici and P. pentosaceus are widely used for fermentation of food products like vegetable and meat products. By production of organic acids, resulting in pH decrease, they contribute to control the microbial succession during fermentation. They also act as protective cultures preventing the growth of food-borne pathogens such as Listeria monocytogenes or Staphylococcus aureus [1] and by doing this, they extend storage life and enhance safety of food products [2,3]. Beside production of classical antimicrobial compounds such as organic acids or hydrogen peroxide, the protective effect also results from the production of bacteriocins [4]. Bacteriocins are ribosomally synthesized, small, heat-stable antimicrobial peptides produced by bacteria. Pediocin AcH/PA-1 is the most studied class IIa bacteriocin (non modified, nonlantibiotic peptides) and has potential for use as food preservative, due to its strong antilisterial activity [5]. Since amino-acid sequence determination of the pediocin AcH/PA-1 in 1992 [6,7], several pediocin-producing P. acidilactici and P. pentosaceus strains have been screened from a large variety of plants and fruits [8], but also from the gastrointestinal tract of poultry, ducks and other animals [9-11]. Millette et al. [12] recently isolated a strain of Pediococcus acidilactici from human faeces which produces an unidentified antimicrobial proteinaceous compound. To our knowledge, no pediocin-producing Pediococcus has been isolated so far from human faeces and it is therefore unknown to which extent these strains are widespread in the human intestinal microbiota and contribute to the microbial balance of the complex gut ecosystem.
Real-time PCR is a very sensitive and rapid molecular method for the detection of specific genes in complex samples. It is particularly suitable for measuring non cultivable bacteria, because detection is independent of growth conditions of the target organism [13]. It has for example recently been used successfully on faecal samples to assess the survival of Lactococcus lactis subsp. cremoris FC after transit through the gastrointestinal tract [14] or to detect the presence of noroviruses in clinical stool samples [15].
In this work, we report the identification of a new bacteriocin-producing strain of Pediococcus acidilactici (UVA1), which was co-isolated from baby faeces with the bacteriocinogenic strain Bifidobacterium thermophilum RBL67 [16]. The biochemical and genetic characterization of the antimicrobial compound was performed. The distribution of pedA-containing strains in human faeces was also investigated using a newly designed real-time PCR assay targeting the pediocin structural gene.
BMC Biotechnology 2007, 7:55. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0).
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