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  <cit>  and by doing this, they extend storage life and enhance safety of food products  <cit> .
beside production of classical antimicrobial compounds such as organic acids or hydrogen peroxide, the protective effect also results from the production of bacteriocins  <cit> .
bacteriocins are ribosomally synthesized, small, heat-stable antimicrobial peptides produced by bacteria.
pediocin ach/pa- <dig> is the most studied class iia bacteriocin  and has potential for use as food preservative, due to its strong antilisterial activity  <cit> .
since amino-acid sequence determination of the pediocin ach/pa- <dig> in  <dig>  <cit> , several pediocin-producing p. acidilactici and p. pentosaceus strains have been screened from a large variety of plants and fruits  <cit> , but also from the gastrointestinal tract of poultry, ducks and other animals  <cit> .
millette et al.
<cit>  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  <cit> .
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  <cit>  or to detect the presence of noroviruses in clinical stool samples  <cit> .
in this work, we report the identification of a new bacteriocin-producing strain of pediococcus acidilactici , which was co-isolated from baby faeces with the bacteriocinogenic strain bifidobacterium thermophilum rbl <dig>  <cit> .
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.
methods
bacterial strains, and growth conditions and plasmid-curing
lactobacillus paracasei subsp.
paracasei dsm5622t, pediococcus acidilactici dsm 20284t and pediococcus pentosaceus dsm 20336t  were used as reference strains for analysis of the carbohydrate fermentation profile.
listeria ivanovii hpb <dig>  was used as indicator strain for the detection of pediocin activity  <cit> .
p. acidilactici ul <dig>  was used as pediocin producer control  <cit> .
pediococcus acidilactici uva <dig> was previously isolated from human baby faeces as a stable consortium with the bifidobacterium strain rbl <dig>  <cit> , which was recently identified as bifidobacterium thermophilum.
strain uva <dig> was purified by subsequent selective plating and analysis of single colonies by genus-specific probes .
a non-bacteriocin producing derivative of p. acidilactici uva <dig>  named bac-, was obtained after curing by novobiocin treatment  as already described  <cit> .
sixty colonies were screened by the overlay method for absence of antimicrobial activity against l. ivanovii hpb <dig> and four mutants producing no inhibition halo were detected.
one of them was purified on mrsc agar, consisting of mrs  supplemented with  <dig>  % l- cysteine hydrochloride and  <dig>  % agar, and propagated in mrsc broth.
the cell-free supernatant was checked for the absence of antilisterial activity and absence of the peda-gene was confirmed by pcr.
all lactic acid bacteria were routinely grown overnight in mrsc medium, with incubation at 37°c in anaerobic jars with an atmosphere generation system .
l. ivanovii hpb <dig> was propagated in tsy medium consisting of tryptic soy broth  containing  <dig>  %  yeast extract  overnight at 30°c.
for agar plates and soft-agar, the media were supplemented with  <dig>  % and  <dig>  %  agar, respectively.
bacterial stocks were stored at -80°c in appropriate media supplemented with  <dig> %  glycerol and subcultured three times at one day intervals on fresh agar plates before use.
cultures were routinely checked under a light microscope for contamination.
carbohydrate fermentation profile
the carbohydrate fermentation profile of strain uva <dig> was determined using api  <dig> fermentation strips .
a 2-ml volume of an overnight culture  was centrifuged at 14' <dig> × g for  <dig> min at 4°c.
the pellet was washed once and resuspended in  <dig> ml of sterile, double distilled water.
the suspension was then added to  <dig> ml of chl  <dig> medium .
each tube of the api chl <dig> strips was then inoculated with  <dig> μl of the bacterial suspension in chl <dig> medium and sealed with sterile paraffin.
the strips were incubated at 37°c under anaerobic conditions, the results were visually assessed after  <dig>   <dig> and  <dig> h and analysed with the apilab+ software version  <dig> .
<dig> according to the manufacturer's instructions.
p. acidilactici ul <dig>  p. acidilactici dsm20284t, p. pentosaceus dsm20336t, and l. paracasei subsp.
paracasei dsm5622t were used as reference strains.
all tests were repeated three times.
determination of growth at 50°c
strain uva <dig> was incubated at 50°c in  <dig> ml mrsc medium inoculated with  <dig> % of an overnight culture.
p. acidilactici ul <dig> and dsm20284t and p. pentosaceus dsm20336t were used as controls.
the optical density at  <dig> nm was measured with an uvikon 810p photometer  after  <dig>   <dig> and  <dig> h. the ability to grow at 50°c was positive if od <dig> exceeded  <dig>  after  <dig> h. od <dig> of  <dig>  was set as the limit of growth and corresponded to twice the value of the od of freshly inoculated medium.
all tests were performed twice.
inhibition assay
antibacterial activity was assessed by the agar-well diffusion method.
briefly,  <dig> ml of soft-agar  was inoculated with  <dig>  % of an overnight culture of the indicator strain l. ivanovii hbp <dig>  poured into a petri dish and allowed to set for  <dig> min at room temperature.
holes  were then punched in the agar and filled with  <dig> μl of sample.
the plates were incubated at 4°c for  <dig> min to allow bacteriocin diffusion and overnight at 30°c for growth of the indicator strain.
the diameter of the inhibition zone was measured.
effect of temperature, ph, enzymes and other agents on bacteriocin activity
cell-free supernatant  was obtained after centrifugation at 13' <dig> g for  <dig> min at 4°c of a 16-h culture in mrsc at ph  <dig> and 37°c.
the supernatant was heated  <dig> min at 95°c.
the effect of temperature on the antibacterial activity was tested after heating at 121°c for  <dig> min in an autoclave and at 100°c for  <dig> and  <dig> min using a water-bath.
the effect of ph was tested by adjusting the ph of the cfs to values in a range from  <dig> to  <dig> using either  <dig> m hcl or  <dig> m naoh.
residual activity was measured by the agar-well diffusion method, after one day, one week and one month storage at 4°c.
to test the sensitivity to proteases and other agents, the cfs was incubated for  <dig> h at 37°c in the presence of  <dig> mg ml- <dig> chymotrypsin, pepsin, protease, proteinase k, trypsin or lysozyme or  <dig> % sds, urea, catalase, rnase a, tween  <dig>  tween  <dig>  triton-x or  <dig>   <dig> or  <dig> mm edta.
for enzyme denaturation, the samples were finally heated at 95°c for  <dig> min and residual activity was measured by the agar-well diffusion method.
all enzymes and other chemicals were purchased from sigma-aldrich chemie gmbh , except proteinase k and trypsin, which were obtained from applichem .
residual activity was defined as the ratio of the diameter of the halo produced by the treated sample compared to the untreated control and expressed in percentage.
all assays were performed twice
molecular weight determination
the bacteriocin produced by p. acidilactici uva <dig> was partially purified by injecting  <dig> ml cfs at a rate of  <dig> ml min- <dig> in a 60-ml sp sepharose column connected to a fplc chromatography system .
the column was first equilibrated with  <dig> column volumes of  <dig> mm ammonium acetate buffer , 5-ml fractions were collected in a fraction collector  and the bacteriocin-like activity was eluted with  <dig>  m nacl in the same buffer.
the cfs and active fractions after fplc were 10-fold concentrated by ultrafiltration  and  <dig> μl of the samples were loaded on two parallel sds gels, along with  <dig> μl of polypeptide sds-page molecular weight standard .
the gels were prepared according to schägger and jagow  <cit>  and consisted of a  <dig> % acrylamide-bisacrylamide stacking gel and a  <dig>  % separating gel.
separation was done with constant voltage  for  <dig> h  <dig> using a vertical slab gel apparatus .
one of the gels was stained with coomassie brilliant blue r <dig>  and the other was used for activity detection: the gel was first soaked for  <dig> h in fixation solution  and rinsed overnight in hplc-grade water before being overlaid with  <dig> ml soft tsy agar inoculated with  <dig>  % of an overnight culture of l. ivanovii hbp <dig>  the molecular weight was estimated by comparison of the mobility of the inhibition zone to that of the molecular weight marker run simultaneously.
the whole procedure was repeated twice.
dna sequencing and pcr conditions
sequencing of dna was performed by microsynth  and similarity searches were conducted with the blast program from ncbi .
primers and probe used in this study are listed in table  <dig>  they were designed with the program primer <dig>  <cit>  and synthesised by microsynth.
the pcr reactions were set up in a total volume of  <dig> μl containing  <dig>  u eurotaq-dna-polymerase ,  <dig>  mm magnesium chloride ,  <dig>  mm dntp's ,  <dig>  μm of each primer and either  <dig> μl of dna or  <dig> μl of cell suspension .
the 16s rdna of p. acidilactici uva <dig> and ul <dig> were amplified using a slightly modified protocol from schürch  <cit> .
the annealing temperature for the bak11w/bak <dig> primers was increased to 62°c.
the 16s rdna sequences of uva <dig> and ul <dig> are deposited at genbank under accession numbers  and , respectively.
for the amplification and sequencing of the first  <dig> bp of the pediocin pa- <dig> operon, primers p <dig> and p <dig> and conditions described by rodriguez et al.
<cit>  were used.
the second part of the operon  was amplified with primers pedopf and pedopr, designed on the basis of the reported sequence for psrq <dig>  <cit> .
amplification conditions were as follow:  <dig> min at 95°c,  <dig> cycles of  <dig> min at 94°c,  <dig> s at 45°c and  <dig> min at 72°c and final elongation step  <dig> min at 72°c.
oligonucleotides pedopf and pedopr as well as pedseq a, b, c and d, designed every  <dig> bp along the pcr product, were used as sequencing primers.
additionally, a 1009-bp sequence directly upstream and a 1417-bp sequence directly downstream of the operon were amplified and sequenced with primer pairs pedseq l and h and pedseq m and n, respectively.
amplification conditions were:  <dig> min at 95°c,  <dig> cycles of  <dig> min at 95°c,  <dig> s at 55°c and  <dig> min at 72°c and final elongation step  <dig> min at 72°c.
plasmid isolation and southern blotting
extrachromosomal dna elements were extracted from p. acidilactici uva <dig>  ul <dig> and the bac- mutant using a modified method after anderson and mckay for small scale plasmid isolation  <cit> .
shortly, for cell lysis,  <dig>  μl mutanolysin  was added to the lysis solution  and plasmid dna was resuspended in  <dig> × te buffer.
finally, the rna was digested with  <dig> μg rnase a .
the dna was visualized after electrophoresis on a  <dig>  % agarose gel in  <dig> × tbe at  <dig> v for  <dig> h  <dig>  the supercoiled dna ladder  was used as size standard.
dig-labelling of the peda-probe , blotting on nylon membrane, hybridisation  and chemiluminescent detection were conducted with the dig-high prime dna labeling and detection starter kit ii  according to supplier's instructions.
plasmid preparation, blotting and hybridization were performed twice.
rna isolation and reverse transcription
the rna was isolated during the exponential growth-phase of p. acidilactici uva <dig>  bac-, ul <dig> and dsm 20284t, and p. pentosaceus dsm 20336t using the rneasy mini kit .
the protocol was slightly modified by addition of  <dig> u mutanolysin in the lysis solution.
the samples were finally treated with rnase-free dnase i  for  <dig> min at 37°c.
first strand cdna synthesis was performed with the omniscript reverse transcription kit  and  <dig> μl of the product were used for pcr amplification of a  <dig> bp-fragment with primers peda2rtf and peda2rtr.
pcr products were separated on a  <dig> % agarose gel in  <dig> × tae buffer by electrophoresis at  <dig> v for  <dig> h  <dig>  the low molecular weight dna ladder and tridye  <dig> bp dna-ladder  were used as size standards.
preparation of dna from faecal samples
twenty-one human faecal samples were collected in collaboration with the department of gastroenterology .
thirteen faecal samples were collected from children donors aged one month to  <dig> years and  <dig> from adults.
faecal samples were collected within  <dig> h after defecation, placed in anaerobic jars and rapidly transported to our laboratory.
they were immediately frozen at -20°c upon arrival, i.e.
no more than  <dig> h after defecation.
total dna was isolated from  <dig> mg of each sample using the qiaamp dna stool mini kit  according to the manufacturer's instructions.
before dna extraction, one faecal sample was autoclaved twice  to obtain a sample free of dna.
ten aliquots of this sample were spiked with a 10-fold serial dilution of p. acidilactici uva <dig>  at concentrations ranging from  <dig> to  <dig> bacteria cells per g faeces.
the extracted dna was stored at -20°c.
real-time pcr
primers and taqman probe listed in table  <dig> were designed based on the peda sequence with the software primerexpress  <dig>   and synthesized by microsynth.
the taqman probe was labeled with 5'-fam as a fluorescent reporter dye and 3'-tamra as a quencher.
their specificity was tested using the blast program from ncbi.
reactions were set in a total volume of  <dig> μl, containing  <dig>  μl of faecal dna extract,  <dig>  μl of qpcr mastermix from eurogentec ,  <dig>  μm of each primer and  <dig>  μm of the taqman probe.
reactions were run on an abi prism  <dig> sequence detector .
the amplification conditions were  <dig> min at 50°c,  <dig> min denaturation at 95°c, followed by  <dig> cycles of  <dig> sec at 95°c and  <dig> min at 60°c.
the cycle threshold , corresponding to the number of cycle after which the target-dna concentration increase become exponential, was monitored.
results were analysed using the sds  <dig>  software .
all reactions were done in triplicate and repeated three times.
RESULTS
phenotypic identification of strain uva <dig> as pediococcus acidilactici
a carbohydrate fermentation profile was performed using api  <dig> chl strips after  <dig> h anaerobic incubation at 37°c.
using the apilab+ software and p. acidilactici dsm20284t, p. acidilactici ul <dig>  and p. pentosaceus dsm20336t as control strains, uva <dig> was identified as pediococcus acidilactici.
strains uva <dig>  ul <dig> and dsm20284t differed from each other.
mannose was fermented by ul <dig> and dsm20284t but not by uva <dig>  saccharose was only fermented by ul <dig> and dsm20336t whereas d-trehalose was only fermented by dsm20284t and dsm20336t.
using optical density measurement at  <dig> nm after  <dig> h incubation, strain uva <dig>  as well as p. acidilactici dsm20284tand ul <dig>  were shown to grow at 50°c, whereas p. pentosaceus dsm20336t didn't.
genotypic confirmation of the identity of p. acidilactici uva1
the 16s rdna sequences of strain uva <dig> and p. acidilactici ul <dig> were analysed using the software bioedit  <cit> .
the assembly of sequence fragments produced a  <dig> and  <dig> bp sequences for strains uva <dig> and ul <dig>  respectively.
the sequences, compared with 16s rdna sequences in the genbank, showed  <dig> % similarity with the type strain pediococcus acidilactici dsm20284t.
the results of 16s rdna sequencing identified strain uva <dig> as a new pediococcus acidilactici strain.
furthermore, the 16s sequences of strains p. acidilactici uva <dig> and ul <dig> were also 99% similar, showing a close relationship between these two strains.
biochemical characterization of the antimicrobial compound produced by p. acidilactici uva1
heating the cell-free supernatant  at 121°c for  <dig> min destroyed the inhibitory activity against listeria ivanovii hpb <dig>  whereas heat treatments at 100°c for  <dig> and  <dig> min only yielded a slight reduction of the activity with  <dig> and  <dig> % residual activity, respectively.
activity remained unaffected after one month storage at 4°c at ph values from  <dig> to  <dig>  at ph  <dig> and  <dig>  total loss of activity was already observed after  <dig> week storage and only 60% of the initial activity was still measurable after  <dig> month at ph  <dig>  treatment with proteolytic enzymes such as chymotrypsin, pepsin, protease, proteinase k or trypsin resulted in a total loss of activity while lysozyme, catalase and other agents tested had no effect.
determination of the molecular weight
as shown in figure  <dig>  sds-page of the partially purified bacteriocin  showed a diffuse band on the coomassie-stained gel while a clear inhibition halo was visible on the activity-gel for the concentrated fplc-fraction as well as for the cfs.
the relative mobility of the inhibition zone on the gel overlaid with listeria ivanovii hbp <dig> was compared to that of the standards stained with coomassie blue.
the apparent molecular weight of the bacteriocin was estimated in the range of  <dig>  to  <dig> kda.
the biochemical properties and the molecular weight described above, as well as the fact that this proteinaceous compound is produced by a pediococcus strain suggest that the anti-listeria compound is a pediocin-like bacteriocin.
a genetic approach was used to confirm this assumption.
genetic characterization of the bacteriocin
primers p <dig> and p <dig> were used to amplify and sequence a 711-bp fragment  including the ribosome binding site as well as the -35/- <dig> region.
the remaining sequence of the operon  was amplified with primers pedopf and pedopr  and the generated dna fragment was sequenced.
this resulted in a 3473-bp sequence, comprising the four genes constituting the operon including the upstream region of peda , presumably containing the regulatory elements of the operon.
the whole nucleotide sequence showed more than  <dig>  % similarity to the published sequences for p. acidilactici k <dig> , p. acidilactici h , p. acidilactici pac <dig>  , p. pentosaceus , lactobacillus plantarum , p. parvulus , and bacillus coagulans .
two sequences of  <dig> and  <dig> bp, upstream and downstream of the operon, respectively, were also pcr-amplified and sequenced.
the resulting 5368-bp sequence was  <dig> % identical to the pediocin-encoding plasmid psrq <dig> described for p. acidilactici pa- <dig>  <cit> .
the antimicrobial activity is linked to the presence of the plasmid and the expression of peda
agarose gel electrophoresis of plasmid dna preparation from strain uva <dig> showed three bands: two extrachromosomal dna elements at  <dig>  kb and at > <dig> kb and one corresponding to chromosomal dna, respectively ).
for strain bac-, a non-inhibitory derivative of p. acidilactici uva <dig> obtained after plasmid curing, in contrast, only chromosomal dna was visible.
plasmid dna prepared from the control pediocin producing strain ul <dig> exhibited the same bands as detected for uva <dig>  in addition to two larger extrachromosomal dna elements.
southern hybridization with the peda-probe ) yielded positive signals for the  <dig> -kb and the >10-kb extrachromosomal elements present in ul <dig> and uva <dig>  but no hybridization occurred for bac-, confirming the presence of the peda-gene on the  <dig> -kb plasmid, the band at > <dig> kb probably being the relaxed circular form of the same plasmid.
the presence of the peda-gene on the plasmid was confirmed by an agar-well diffusion assay, where the supernatant of a culture of the bac- strain failed to exhibit inhibition of the indicator strain listeria ivanovii hpb <dig>  and 2).
furthermore, the peda-transcript was detected by reverse-transcription-pcr on cdna from p. acidilactici uva <dig>  but not from the cured derivative bac- , providing the ultimate link between presence of the plasmid-localized genetic determinant and expression of the bacteriocin.
the presence of a slight band at  <dig> bp for the non-plasmid containing strains  could not be explained, but the very weak intensity compared to lanes  <dig> to  <dig> does not represent a positive signal.
a second pcr, performed with primers p <dig> and p <dig> , which encompass the regulatory region upstream of the transcriptional start, resulted in no amplification in samples using cdna as template, allowing us to exclude genomic dna contamination in the rna preparation of uva <dig>  ul <dig> and p. pentosaceus dsm 20336t .
distribution of peda-containing strains in human faecal samples
the designed primers and probe allowed specific amplification of a 100-bp fragment located within the peda-gene.
as determined with spiked samples, the reaction was linear for concentrations ranging from  <dig> to  <dig> of p. acidilactici uva <dig> cells per g of spiked faeces, corresponding to ct values comprised between  <dig> and  <dig> ).
a ct value of  <dig> was thus fixed as the upper limit for detection.
samples with a higher value were considered not to contain any peda-gene.
with this assay, the pediocin gene was detected in dna isolated from  <dig> out of  <dig> children faecal samples tested, but in none of the  <dig> adult samples ).
discussion
data obtained from phenotypic characterization indicated that strain uva <dig> is a pediococcus acidilactici.
the carbohydrate fermentation profile matched that described for p. acidilactici in the apilab+ database and in the literature  <cit> .
furthermore, among pediococcus species, only p. acidilactici is able to grow at 50°c  <cit> .
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  <cit> .
the genotypic characterization using 16s rdna sequence analysis confirmed the data from the phenotypic characterization, with strain uva <dig> showing more than  <dig> % similarity to the 16s sequences of other p. acidilactici strains.
mora et al.
<dig>  <cit>  used restriction fragment length polymorphism  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 uva <dig> and ul <dig>  two pediocin-producing pediococcus acidilactici, are very closely related in regard to 16s sequence similarity.
p. acidilactici uva <dig> inhibited l. ivanovii hpb <dig> 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  <dig>  kda, similar to pediocin pa- <dig>  this was confirmed by the detection in strain uva <dig> of a plasmid of approximately  <dig>  kb, on which we could localize the peda-gene.
we also found that the pediocin operon, which consists of the pediocin structural gene , the specific immunity gene , and genes required for processing, maturation and secretion of the bacteriocin , showed  <dig> to  <dig> % similarity to the published sequences for the pediocin pa-1/ach operon  <cit> .
in all pediocin-producing strains isolated so far, the genetic determinants are plasmid-encoded  <cit> .
the comparison of genetic determinants of many pediocin-like producer strains  <cit>  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 uva <dig> suggested that the plasmid harboured by p. acicilactici uva <dig> is probably identical to psrq <dig>  a  <dig>  kb plasmid from p. acidilactici pa- <dig>  <cit> .
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 uva <dig> but not in the cured, non-active derivative, bac-, and that the inhibitory activity of uva <dig> 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 hpb <dig> and the pcr with primers pedopf and pedopr did not yield any product .
diep et al.
<cit>  reported similar observations in p. pentosaceus atcc  <dig>  where a truncated pen locus lead to low expression of antimicrobial activity.
pediococci are commonly associated with various plants and their products or meat  <cit> .)
there are only few reports on pediococci detected in human faeces or gastrointestinal tract  <cit> , and only millette et al.
<cit> , reported the presence of an antibacterial compound of proteinaceous nature, although not identified yet.
strain uva <dig> 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  <dig> adult samples tested.
therefore, human faecal material could be a good source for isolating new pediocin-producing strains.
CONCLUSIONS
strain uva <dig>  isolated from new born faeces, was clearly identified as a pediococcus acidilactici using a polyphasic approach.
data for the molecular size, sensitivity to protease and sequence of the plasmid-borne genetic determinant indicated that the antibacterial compound produced by uva <dig> is pediocin pa-1/ach.
to our knowledge, pediococcus acidilactici uva <dig> is the first pediocin-producing pediococcus strain isolated from human faeces.
real-time pcr was an efficient method for detection of specific genes in faecal samples harbouring a complex microbiota.
we showed a large distribution of peda-containing strains in baby faecal samples.
authors' contributions
sm participated in the study design and carried out the identification of the bacteriocin and the genetic study.
uva contributed to the study design, isolated and identified the strain uva <dig> and participated in the writing of the manuscript.
es contributed to the characterisation of the strain.
rm performed the real-time pcr screening of faecal samples.
tc participated in the identification of the genetic determinant.
lm and cl provided guidance during all parts of the work.
all authors read and approved the final manuscript.

 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 and by doing this, they extend storage life and enhance safety of food products. beside production of classical antimicrobial compounds such as organic acids or hydrogen peroxide, the protective effect also results from the production of bacteriocins. bacteriocins are ribosomally synthesized, small, heat-stable antimicrobial peptides produced by bacteria. pediocin ach/pa- is the most studied class iia bacteriocin and has potential for use as food preservative, due to its strong antilisterial activity. since amino-acid sequence determination of the pediocin ach/pa- in, several pediocin-producing p. acidilactici and p. pentosaceus strains have been screened from a large variety of plants and fruits, but also from the gastrointestinal tract of poultry, ducks and other animals. millette et al. 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. 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 or to detect the presence of noroviruses in clinical stool samples. in this work, we report the identification of a new bacteriocin-producing strain of pediococcus acidilactici, which was co-isolated from baby faeces with the bacteriocinogenic strain bifidobacterium thermophilum rbl. 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. methods bacterial strains, and growth conditions and plasmid-curing lactobacillus paracasei subsp. paracasei dsm5622t, pediococcus acidilactici dsm 20284t and pediococcus pentosaceus dsm 20336t were used as reference strains for analysis of the carbohydrate fermentation profile. listeria ivanovii hpb was used as indicator strain for the detection of pediocin activity. p. acidilactici ul was used as pediocin producer control. pediococcus acidilactici uva was previously isolated from human baby faeces as a stable consortium with the bifidobacterium strain rbl, which was recently identified as bifidobacterium thermophilum. strain uva was purified by subsequent selective plating and analysis of single colonies by genus-specific probes. a non-bacteriocin producing derivative of p. acidilactici uva named bac-, was obtained after curing by novobiocin treatment as already described. sixty colonies were screened by the overlay method for absence of antimicrobial activity against l. ivanovii hpb and four mutants producing no inhibition halo were detected. one of them was purified on mrsc agar, consisting of mrs supplemented with % l- cysteine hydrochloride and % agar, and propagated in mrsc broth. the cell-free supernatant was checked for the absence of antilisterial activity and absence of the peda-gene was confirmed by pcr. all lactic acid bacteria were routinely grown overnight in mrsc medium, with incubation at 37°c in anaerobic jars with an atmosphere generation system. l. ivanovii hpb was propagated in tsy medium consisting of tryptic soy broth containing % yeast extract overnight at 30°c. for agar plates and soft-agar, the media were supplemented with % and % agar, respectively. bacterial stocks were stored at -80°c in appropriate media supplemented with % glycerol and subcultured three times at one day intervals on fresh agar plates before use. cultures were routinely checked under a light microscope for contamination. carbohydrate fermentation profile the carbohydrate fermentation profile of strain uva was determined using api fermentation strips. a 2-ml volume of an overnight culture was centrifuged at 14' × g for min at 4°c. the pellet was washed once and resuspended in ml of sterile, double distilled water. the suspension was then added to ml of chl medium. each tube of the api chl strips was then inoculated with μl of the bacterial suspension in chl medium and sealed with sterile paraffin. the strips were incubated at 37°c under anaerobic conditions, the results were visually assessed after and h and analysed with the apilab+ software version. according to the manufacturer's instructions. p. acidilactici ul p. acidilactici dsm20284t, p. pentosaceus dsm20336t, and l. paracasei subsp. paracasei dsm5622t were used as reference strains. all tests were repeated three times. determination of growth at 50°c strain uva was incubated at 50°c in ml mrsc medium inoculated with % of an overnight culture. p. acidilactici ul and dsm20284t and p. pentosaceus dsm20336t were used as controls. the optical density at nm was measured with an uvikon 810p photometer after and h. the ability to grow at 50°c was positive if od exceeded after h. od of was set as the limit of growth and corresponded to twice the value of the od of freshly inoculated medium. all tests were performed twice. inhibition assay antibacterial activity was assessed by the agar-well diffusion method. briefly, ml of soft-agar was inoculated with % of an overnight culture of the indicator strain l. ivanovii hbp poured into a petri dish and allowed to set for min at room temperature. holes were then punched in the agar and filled with μl of sample. the plates were incubated at 4°c for min to allow bacteriocin diffusion and overnight at 30°c for growth of the indicator strain. the diameter of the inhibition zone was measured. effect of temperature, ph, enzymes and other agents on bacteriocin activity cell-free supernatant was obtained after centrifugation at 13' g for min at 4°c of a 16-h culture in mrsc at ph and 37°c. the supernatant was heated min at 95°c. the effect of temperature on the antibacterial activity was tested after heating at 121°c for min in an autoclave and at 100°c for and min using a water-bath. the effect of ph was tested by adjusting the ph of the cfs to values in a range from to using either m hcl or m naoh. residual activity was measured by the agar-well diffusion method, after one day, one week and one month storage at 4°c. to test the sensitivity to proteases and other agents, the cfs was incubated for h at 37°c in the presence of mg ml- chymotrypsin, pepsin, protease, proteinase k, trypsin or lysozyme or % sds, urea, catalase, rnase a, tween tween triton-x or or mm edta. for enzyme denaturation, the samples were finally heated at 95°c for min and residual activity was measured by the agar-well diffusion method. all enzymes and other chemicals were purchased from sigma-aldrich chemie gmbh, except proteinase k and trypsin, which were obtained from applichem. residual activity was defined as the ratio of the diameter of the halo produced by the treated sample compared to the untreated control and expressed in percentage. all assays were performed twice molecular weight determination the bacteriocin produced by p. acidilactici uva was partially purified by injecting ml cfs at a rate of ml min- in a 60-ml sp sepharose column connected to a fplc chromatography system. the column was first equilibrated with column volumes of mm ammonium acetate buffer, 5-ml fractions were collected in a fraction collector and the bacteriocin-like activity was eluted with m nacl in the same buffer. the cfs and active fractions after fplc were 10-fold concentrated by ultrafiltration and μl of the samples were loaded on two parallel sds gels, along with μl of polypeptide sds-page molecular weight standard. the gels were prepared according to schägger and jagow and consisted of a % acrylamide-bisacrylamide stacking gel and a % separating gel. separation was done with constant voltage for h using a vertical slab gel apparatus. one of the gels was stained with coomassie brilliant blue r and the other was used for activity detection: the gel was first soaked for h in fixation solution and rinsed overnight in hplc-grade water before being overlaid with ml soft tsy agar inoculated with % of an overnight culture of l. ivanovii hbp the molecular weight was estimated by comparison of the mobility of the inhibition zone to that of the molecular weight marker run simultaneously. the whole procedure was repeated twice. dna sequencing and pcr conditions sequencing of dna was performed by microsynth and similarity searches were conducted with the blast program from ncbi. primers and probe used in this study are listed in table they were designed with the program primer and synthesised by microsynth. the pcr reactions were set up in a total volume of μl containing u eurotaq-dna-polymerase, mm magnesium chloride, mm dntp's, μm of each primer and either μl of dna or μl of cell suspension. the 16s rdna of p. acidilactici uva and ul were amplified using a slightly modified protocol from schürch. the annealing temperature for the bak11w/bak primers was increased to 62°c. the 16s rdna sequences of uva and ul are deposited at genbank under accession numbers and, respectively. for the amplification and sequencing of the first bp of the pediocin pa- operon, primers p and p and conditions described by rodriguez et al. were used. the second part of the operon was amplified with primers pedopf and pedopr, designed on the basis of the reported sequence for psrq. amplification conditions were as follow: min at 95°c, cycles of min at 94°c, s at 45°c and min at 72°c and final elongation step min at 72°c. oligonucleotides pedopf and pedopr as well as pedseq a, b, c and d, designed every bp along the pcr product, were used as sequencing primers. additionally, a 1009-bp sequence directly upstream and a 1417-bp sequence directly downstream of the operon were amplified and sequenced with primer pairs pedseq l and h and pedseq m and n, respectively. amplification conditions were: min at 95°c, cycles of min at 95°c, s at 55°c and min at 72°c and final elongation step min at 72°c. plasmid isolation and southern blotting extrachromosomal dna elements were extracted from p. acidilactici uva ul and the bac- mutant using a modified method after anderson and mckay for small scale plasmid isolation. shortly, for cell lysis, μl mutanolysin was added to the lysis solution and plasmid dna was resuspended in × te buffer. finally, the rna was digested with μg rnase a. the dna was visualized after electrophoresis on a % agarose gel in × tbe at v for h the supercoiled dna ladder was used as size standard. dig-labelling of the peda-probe, blotting on nylon membrane, hybridisation and chemiluminescent detection were conducted with the dig-high prime dna labeling and detection starter kit ii according to supplier's instructions. plasmid preparation, blotting and hybridization were performed twice. rna isolation and reverse transcription the rna was isolated during the exponential growth-phase of p. acidilactici uva bac-, ul and dsm 20284t, and p. pentosaceus dsm 20336t using the rneasy mini kit. the protocol was slightly modified by addition of u mutanolysin in the lysis solution. the samples were finally treated with rnase-free dnase i for min at 37°c. first strand cdna synthesis was performed with the omniscript reverse transcription kit and μl of the product were used for pcr amplification of a bp-fragment with primers peda2rtf and peda2rtr. pcr products were separated on a % agarose gel in × tae buffer by electrophoresis at v for h the low molecular weight dna ladder and tridye bp dna-ladder were used as size standards. preparation of dna from faecal samples twenty-one human faecal samples were collected in collaboration with the department of gastroenterology. thirteen faecal samples were collected from children donors aged one month to years and from adults. faecal samples were collected within h after defecation, placed in anaerobic jars and rapidly transported to our laboratory. they were immediately frozen at -20°c upon arrival, i.e. no more than h after defecation. total dna was isolated from mg of each sample using the qiaamp dna stool mini kit according to the manufacturer's instructions. before dna extraction, one faecal sample was autoclaved twice to obtain a sample free of dna. ten aliquots of this sample were spiked with a 10-fold serial dilution of p. acidilactici uva at concentrations ranging from to bacteria cells per g faeces. the extracted dna was stored at -20°c. real-time pcr primers and taqman probe listed in table were designed based on the peda sequence with the software primerexpress and synthesized by microsynth. the taqman probe was labeled with 5'-fam as a fluorescent reporter dye and 3'-tamra as a quencher. their specificity was tested using the blast program from ncbi. reactions were set in a total volume of μl, containing μl of faecal dna extract, μl of qpcr mastermix from eurogentec, μm of each primer and μm of the taqman probe. reactions were run on an abi prism sequence detector. the amplification conditions were min at 50°c, min denaturation at 95°c, followed by cycles of sec at 95°c and min at 60°c. the cycle threshold, corresponding to the number of cycle after which the target-dna concentration increase become exponential, was monitored. results were analysed using the sds software. all reactions were done in triplicate and repeated three times.  phenotypic identification of strain uva as pediococcus acidilactici a carbohydrate fermentation profile was performed using api chl strips after h anaerobic incubation at 37°c. using the apilab+ software and p. acidilactici dsm20284t, p. acidilactici ul and p. pentosaceus dsm20336t as control strains, uva was identified as pediococcus acidilactici. strains uva ul and dsm20284t differed from each other. mannose was fermented by ul and dsm20284t but not by uva saccharose was only fermented by ul and dsm20336t whereas d-trehalose was only fermented by dsm20284t and dsm20336t. using optical density measurement at nm after h incubation, strain uva as well as p. acidilactici dsm20284tand ul were shown to grow at 50°c, whereas p. pentosaceus dsm20336t didn't. genotypic confirmation of the identity of p. acidilactici uva1 the 16s rdna sequences of strain uva and p. acidilactici ul were analysed using the software bioedit. the assembly of sequence fragments produced a and bp sequences for strains uva and ul respectively. the sequences, compared with 16s rdna sequences in the genbank, showed % similarity with the type strain pediococcus acidilactici dsm20284t. the results of 16s rdna sequencing identified strain uva as a new pediococcus acidilactici strain. furthermore, the 16s sequences of strains p. acidilactici uva and ul were also 99% similar, showing a close relationship between these two strains. biochemical characterization of the antimicrobial compound produced by p. acidilactici uva1 heating the cell-free supernatant at 121°c for min destroyed the inhibitory activity against listeria ivanovii hpb whereas heat treatments at 100°c for and min only yielded a slight reduction of the activity with and % residual activity, respectively. activity remained unaffected after one month storage at 4°c at ph values from to at ph and total loss of activity was already observed after week storage and only 60% of the initial activity was still measurable after month at ph treatment with proteolytic enzymes such as chymotrypsin, pepsin, protease, proteinase k or trypsin resulted in a total loss of activity while lysozyme, catalase and other agents tested had no effect. determination of the molecular weight as shown in figure sds-page of the partially purified bacteriocin showed a diffuse band on the coomassie-stained gel while a clear inhibition halo was visible on the activity-gel for the concentrated fplc-fraction as well as for the cfs. the relative mobility of the inhibition zone on the gel overlaid with listeria ivanovii hbp was compared to that of the standards stained with coomassie blue. the apparent molecular weight of the bacteriocin was estimated in the range of to kda. the biochemical properties and the molecular weight described above, as well as the fact that this proteinaceous compound is produced by a pediococcus strain suggest that the anti-listeria compound is a pediocin-like bacteriocin. a genetic approach was used to confirm this assumption. genetic characterization of the bacteriocin primers p and p were used to amplify and sequence a 711-bp fragment including the ribosome binding site as well as the -35/- region. the remaining sequence of the operon was amplified with primers pedopf and pedopr and the generated dna fragment was sequenced. this resulted in a 3473-bp sequence, comprising the four genes constituting the operon including the upstream region of peda, presumably containing the regulatory elements of the operon. the whole nucleotide sequence showed more than % similarity to the published sequences for p. acidilactici k, p. acidilactici h, p. acidilactici pac, p. pentosaceus, lactobacillus plantarum, p. parvulus, and bacillus coagulans. two sequences of and bp, upstream and downstream of the operon, respectively, were also pcr-amplified and sequenced. the resulting 5368-bp sequence was % identical to the pediocin-encoding plasmid psrq described for p. acidilactici pa-. the antimicrobial activity is linked to the presence of the plasmid and the expression of peda agarose gel electrophoresis of plasmid dna preparation from strain uva showed three bands: two extrachromosomal dna elements at kb and at > kb and one corresponding to chromosomal dna, respectively ). for strain bac-, a non-inhibitory derivative of p. acidilactici uva obtained after plasmid curing, in contrast, only chromosomal dna was visible. plasmid dna prepared from the control pediocin producing strain ul exhibited the same bands as detected for uva in addition to two larger extrachromosomal dna elements. southern hybridization with the peda-probe ) yielded positive signals for the -kb and the >10-kb extrachromosomal elements present in ul and uva but no hybridization occurred for bac-, confirming the presence of the peda-gene on the -kb plasmid, the band at > kb probably being the relaxed circular form of the same plasmid. the presence of the peda-gene on the plasmid was confirmed by an agar-well diffusion assay, where the supernatant of a culture of the bac- strain failed to exhibit inhibition of the indicator strain listeria ivanovii hpb and 2). furthermore, the peda-transcript was detected by reverse-transcription-pcr on cdna from p. acidilactici uva but not from the cured derivative bac-, providing the ultimate link between presence of the plasmid-localized genetic determinant and expression of the bacteriocin. the presence of a slight band at bp for the non-plasmid containing strains could not be explained, but the very weak intensity compared to lanes to does not represent a positive signal. a second pcr, performed with primers p and p, which encompass the regulatory region upstream of the transcriptional start, resulted in no amplification in samples using cdna as template, allowing us to exclude genomic dna contamination in the rna preparation of uva ul and p. pentosaceus dsm 20336t. distribution of peda-containing strains in human faecal samples the designed primers and probe allowed specific amplification of a 100-bp fragment located within the peda-gene. as determined with spiked samples, the reaction was linear for concentrations ranging from to of p. acidilactici uva cells per g of spiked faeces, corresponding to ct values comprised between and ). a ct value of was thus fixed as the upper limit for detection. samples with a higher value were considered not to contain any peda-gene. with this assay, the pediocin gene was detected in dna isolated from out of children faecal samples tested, but in none of the adult samples ). discussion data obtained from phenotypic characterization indicated that strain uva is a pediococcus acidilactici. the carbohydrate fermentation profile matched that described for p. acidilactici in the apilab+ database and in the literature. furthermore, among pediococcus species, only p. acidilactici is able to grow at 50°c. 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. the genotypic characterization using 16s rdna sequence analysis confirmed the data from the phenotypic characterization, with strain uva showing more than % similarity to the 16s sequences of other p. acidilactici strains. mora et al. used restriction fragment length polymorphism 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 uva and ul two pediocin-producing pediococcus acidilactici, are very closely related in regard to 16s sequence similarity. p. acidilactici uva inhibited l. ivanovii hpb 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 kda, similar to pediocin pa- this was confirmed by the detection in strain uva of a plasmid of approximately kb, on which we could localize the peda-gene. we also found that the pediocin operon, which consists of the pediocin structural gene, the specific immunity gene, and genes required for processing, maturation and secretion of the bacteriocin, showed to % similarity to the published sequences for the pediocin pa-1/ach operon. in all pediocin-producing strains isolated so far, the genetic determinants are plasmid-encoded. the comparison of genetic determinants of many pediocin-like producer strains 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 uva suggested that the plasmid harboured by p. acicilactici uva is probably identical to psrq a kb plasmid from p. acidilactici pa-. 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 uva but not in the cured, non-active derivative, bac-, and that the inhibitory activity of uva 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 hpb and the pcr with primers pedopf and pedopr did not yield any product. diep et al. reported similar observations in p. pentosaceus atcc where a truncated pen locus lead to low expression of antimicrobial activity. pediococci are commonly associated with various plants and their products or meat.) there are only few reports on pediococci detected in human faeces or gastrointestinal tract, and only millette et al., reported the presence of an antibacterial compound of proteinaceous nature, although not identified yet. strain uva 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 adult samples tested. therefore, human faecal material could be a good source for isolating new pediocin-producing strains.  strain uva isolated from new born faeces, was clearly identified as a pediococcus acidilactici using a polyphasic approach. data for the molecular size, sensitivity to protease and sequence of the plasmid-borne genetic determinant indicated that the antibacterial compound produced by uva is pediocin pa-1/ach. to our knowledge, pediococcus acidilactici uva is the first pediocin-producing pediococcus strain isolated from human faeces. real-time pcr was an efficient method for detection of specific genes in faecal samples harbouring a complex microbiota. we showed a large distribution of peda-containing strains in baby faecal samples. authors' contributions sm participated in the study design and carried out the identification of the bacteriocin and the genetic study. uva contributed to the study design, isolated and identified the strain uva and participated in the writing of the manuscript. es contributed to the characterisation of the strain. rm performed the real-time pcr screening of faecal samples. tc participated in the identification of the genetic determinant. lm and cl provided guidance during all parts of the work. all authors read and approved the final manuscript. 
the newly isolated strain uva was identified as a pediococcus acidilactici by carbohydrate fermentation profile, growth at 50°c and 16s rdna sequencing. 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. 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. the mrna transcript for peda could be detected in p. acidilactici uva but not in the cured derivative, confirming the expression of the peda-gene in uva using a new real-time pcr assay, eleven out of seventeen human faecal samples tested were found to contain peda-dna. bacteriocin-producing lactic acid bacteria are commonly used as natural protective cultures. the partially purified bacteriocin was heat resistant up to 100°c, active over a wide range of ph and susceptible to proteolytic enzymes. southern hybridization using the dig-labelled peda-probe established that the bacteriocin gene was plasmid-borne as for all pediocin described so far. p. acidilactici uva harboured a -kb plasmid that could be cured easily, which resulted in the loss of the antimicrobial activity. the molecular weight, estimated by sds-page, was similar to that of pediocin ach/pa-.
 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.  the newly isolated strain uva 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 and susceptible to proteolytic enzymes. the molecular weight, estimated by sds-page, was similar to that of pediocin ach/pa-. p. acidilactici uva harboured a -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 showed almost % similarity to the pediocin ach/pa- operon. the mrna transcript for peda could be detected in p. acidilactici uva but not in the cured derivative, confirming the expression of the peda-gene in uva using a new real-time pcr assay, eleven out of seventeen human faecal samples tested were found to contain peda-dna.  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. 