Data obtained from phenotypic characterization indicated that strain UVA1 is a Pediococcus acidilactici. The carbohydrate fermentation profile matched that described for P. acidilactici in the APILAB+ database and in the literature [8]. Furthermore, among Pediococcus species, only P. acidilactici is able to grow at 50°C [8]. It is currently accepted that the phenotypic characterization of a strain is not enough to discriminate species or even strains and a polyphasic approach for species identification is recommended [28]. The genotypic characterization using 16S rDNA sequence analysis confirmed the data from the phenotypic characterization, with strain UVA1 showing more than 99 % similarity to the 16S sequences of other P. acidilactici strains. Mora et al. 2000 [29] used restriction fragment length polymorphism (RFLP) of housekeeping genes and 16S rDNA sequence analysis to distinguish between pediocin- and non-pediocin-producing P. acidilactici strains and showed that the pediocin-producing strains represented a homogenous subpopulation. This is supported by our observation that strains UVA1 and UL5, two pediocin-producing Pediococcus acidilactici, are very closely related in regard to 16S sequence similarity.
P. acidilactici UVA1 inhibited L. ivanovii HPB28 by production of a proteinaceous compound, as demonstrated by the total loss of activity after proteolytic treatments. This compound had a molecular weight of approximately 4.5 kDa, similar to pediocin PA-1. This was confirmed by the detection in strain UVA1 of a plasmid of approximately 9.5 kb, on which we could localize the pedA-gene. We also found that the pediocin operon, which consists of the pediocin structural gene (pedA), the specific immunity gene (pedB), and genes required for processing, maturation and secretion of the bacteriocin (pedC and D), showed 99 to 100 % similarity to the published sequences for the pediocin PA-1/AcH operon [24,27]. In all pediocin-producing strains isolated so far, the genetic determinants are plasmid-encoded [5]. The comparison of genetic determinants of many pediocin-like producer strains [30] showed that all pediocin genes were carried by plasmids, but the surrounding sequences on the plasmids can differ from one strain to the other. Sequencing data for the regions upstream the regulatory region and downstream the transcription terminator of the pediocin operon in UVA1 suggested that the plasmid harboured by P. acicilactici UVA1 is probably identical to pSRQ11, a 9.4 kb plasmid from P. acidilactici PA-1 [24].
Transcription analysis by reverse-transcription PCR using primers specific for the mRNA transcript of a particular protein is a straightforward method to establish the link between the presence of the genetic determinant and the observed protein activity. With this direct method, we could show that the pedA-transcript was synthesized in exponentially growing cells of UVA1 but not in the cured, non-active derivative, bac-, and that the inhibitory activity of UVA1 was due to pediocin production. It is also interesting to notice that in P. pentosaceus DSM 20336T, the pedA-gene and the mRNA transcript were detected, but no inhibitory activity was observed against L. ivanovii HPB28 and the PCR with primers pedopF and pedopR did not yield any product (data not shown). Diep et al. [31] reported similar observations in P. pentosaceus ATCC 25745, where a truncated pen locus lead to low expression of antimicrobial activity.
Pediococci are commonly associated with various plants and their products or meat [8,32].) There are only few reports on pediococci detected in human faeces or gastrointestinal tract [12,33,34], and only Millette et al. [12], reported the presence of an antibacterial compound of proteinaceous nature, although not identified yet. Strain UVA1 is, to our knowledge, the first pediocin-producing Pediococcus to be isolated from human faecal material, which characteristic could be particularly interesting for the food industry for biopreservation as well as for possible probiotic effect. In this work, we showed, with a new real-time PCR assay, that strains containing the pedA-gene are relatively widespread in baby faecal material, but were not found in the 4 adult samples tested. Therefore, human faecal material could be a good source for isolating new pediocin-producing strains.
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