BACKGROUND
enterobacter sakazakii, previously known as yellow-pigmented e. cloacae  <cit> , has been identified as the causative agent of rare but often severe invasive infections in infants.
neonates and infants less than  <dig> months of age appear to be groups at particular risk  <cit> .
meningitis is the most frequently reported clinical symptom in neonatal e. sakazakii infections  <cit> .
infants in whom meningitis developed were generally <  <dig> week of age at the onset of infection and had near-term gestational age and birthweight  <cit> .
they frequently develop complications including seizures and brain abscesses, resulting in high mortality  and morbidity.
septicaemia or necrotizing enterocolitis are also associated with e. sakazakii-related infections  <cit> .
compared with infants with meningitis, infants in whom bacteremia alone developed tended to be born very prematurely and have extremely low birthweight .
they had generally surpassed the neonatal period at the onset of their disease  <cit> .
mortality among these cases is lower   <cit> .
e. sakazakii is emerging as a hazard in powdered infant formulae .
in several e. sakazakii-related outbreaks and sporadic cases, pif was epidemiologically or microbiologically established as the source of infection  <cit> .
this organism was however isolated at very low levels from commercial pif and dry environmental samples collected from infant formula factories.
the contamination was in most cases not exceeding  <dig> cfu per  <dig> g  <cit> .
prompted by the health risks associated with e. sakazakii, the international standard organisation  and the international dairy federation  have recently jointly adopted a technical specification , quoted as a reviewed method for idf , defining a method for the detection of e. sakazakii in pif.
the method is based on the selective enrichment procedure developed by guillaume-gentil et al.
<cit> .
isolation of the organism involves  a preenrichment in buffered peptone water ,  an enrichment in modified lauryl sulfate tryptose broth , and  a plating on the chromogenic enterobacter sakazakii isolation agar .
identification include  observation of yellow pigmentation for suspected e. sakazakii colonies on tryptone soy agar  at 25°c and  confirmation of the species via biochemical characteristics.
the isolation and confirmation procedures usually require  <dig> to  <dig> days to be completed.
although at present, the gold standard for the detection of microorganisms in food is in general conventional culture, molecular methodologies based on nucleic acid detection have been successfully developed these last years.
they offer an interesting alternative to rapidly and specifically identify organisms from a wide variety of sources.
with regards to e. sakazakii detection, several pcr-based approaches have already been published  <cit> .
however, some of these methods do not include an internal amplification control  that allows a simultaneous assessment of pcr inhibition.
moreover, none of them have been evaluated with naturally contaminated food samples, thus not addressing the ecological diversity of naturally contaminated food matrices.
the present study evaluated a real-time pcr-based assay including an iac and an automated nucleic acid extraction method that can be used in combination with the iso-idf enrichment steps for the routine examination of naturally contaminated pif.
RESULTS
specificity of the real-time pcr assay
the dna region located between the trna-glu and 23s rrna genes was selected as target for detecting the e. sakazakii species.
primers esfor and esrevb were demonstrated to amplify a 158-bp fragment in all  <dig> strains of e. sakazakii tested with no cross-reaction with other non-e. sakazakii bacterial strains .
real-time pcrs were next conducted with a first set of hybridisation probes in the presence of  <dig> molecules of iac.
all but one e. sakazakii strains gave a positive signal in real-time pcr assay .
for strain es  <dig>  we repeatedly failed to detect any fluorescent signal and this lack of e. sakazakii specific signal was curiously also accompanied by a lack of the iac signal.
to attempt to improve the assays, the esfor/esrevb amplified products specific to this strain were sequenced, as well as those of the strain atcc  <dig>  one of the more phylogenetically distinct e. sakazakii strain which displayed one of the higher ct value in this assay .
sequencing of both 158-bp target fragments did reveal some mismatches between the lc1es hybridisation probes and its binding sequences.
a new lc1es probe incorporating three degenerated bases/positions compared to the initial one was therefore tested.
the new designed real-time pcr was able to correctly detect all e sakazakii strains tested, including the es  <dig> isolates.
the use of a more degenerated lc1es probe did not significantly affect the ct value obtained for each strain but increased the intensity of fluorescence measured for some of them, including strain atcc  <dig>  this new probe was used in the rest of the study.
for the exclusivity test, a total of  <dig> non-e. sakazakii enterobacteriaceae and  <dig> non-enterobacteriaceae strains were chosen .
all  <dig> non-e. sakazakii strains tested were negative by either pcr or real-time pcr while a positive iac signal was always detected .
the new e. sakazakii-iac real-time pcr developed could therefore distinguish e. sakazakii isolates from non-target bacteria.
sensitivity of the real-time pcr assay
two reference strains belonging to distinct lineages  <cit>  were selected to determine the sensitivity of the assay.
the type strain atcc  <dig> is representative of a majority of e. sakazakii strains.
the previously mentioned strain atcc  <dig>  the preceptrol™  strain, is more closely related to taxa including e. pyrinus, e. hormaechei and c. koseri  <cit> .
the sensitivity of the real-time pcr assay in the presence of  <dig> molecules of iac was experimentally determined by using purified dna.
tenfold dilutions of e. sakazakii genomic dna in the concentration range of  <dig> to  <dig> equivalent genome per reaction were amplified in triplicate.
table  <dig> summarises the mean ct values obtained for both strains.
a detection limit of 5- and 25-genome copies was assessed for the strains atcc  <dig> and atcc  <dig>  respectively.
a positive reaction could however be detected in some cases with, respectively, as few as  <dig> and  <dig> dna molecules per tube .
to assess the detection limit of the assay combined with the dna extraction step, tenfold serial cell dilutions of calibrated cultures containing  <dig>   <dig> cfu/ml were performed and dna extracted using the magna pure lc system.
results showed that at least  <dig> and  <dig> cfu/ml  need to be present to get a positive reaction with the strains atcc  <dig> and atcc  <dig>  respectively .
100% of positive reactions were recorded in samples containing, respectively,  <dig>   <dig> cfu/ml and  <dig>   <dig> cfu/ml.
results showed a linear log correlation over the 10-fold dilution series , indicating that real-time pcr combined with magna pure dna extraction system could be used to quantify e. sakazaki cell suspensions.
evaluation of the real-time pcr assay coupled with the iso-idf enrichment procedure with artificially contaminated infant formula samples
infant formula powders from three different commercial brands were inoculated with the strains atcc  <dig> or atcc  <dig> at four levels of contamination  plus negative control.
briefly, twenty five grams of pif were dissolved in  <dig> ml of buffered peptone water and then inoculated with diluted e. sakazakii culture.
the contaminations were carried out in triplicate, except the blank which was twice repeated.
samples were analysed in parallel by the conventional iso-idf  method and by real-time pcr after a common cultural enrichment.
both methods gave the same results for all samples and replicates .
non-inoculated samples  were negative.
all but one artificially contaminated samples tested positive for e. sakazakii, demonstrating that both methods allow the detection of less than  <dig> e. sakazakii cells in  <dig> g of dried infant formulae.
mean ct values obtained by real-time pcr ranged from  <dig> to  <dig> cycles.
as observed for the sensitivity analysis, ct values were systematically higher for the preceptrol strain than for the e. sakazakii type strain atcc  <dig>  a slight matrix-dependent effect was also noticed, with ct values for one of the infant formulae's brand higher than those of the others brands.
in contrast, no significant difference was observed according to the contamination level, indicating that e. sakazakii has likely reached a similar growth density at the end of the enrichment procedure for all samples.
the negative result found for one replicate by both methods suggested that the artificial contamination failed.
heterogeneity in artificial contamination at low inoculation levels  may explain the likely absence of e. sakazakii in this sample.
given the reported presence of e. sakazakii at low level in pif, a contamination level of  <dig> cfu/ <dig> g was also attempted using the phylogenetically more distinct strain atcc  <dig>  <cit> .
out of the  <dig> replicates performed,  <dig> were positive for e. sakazakii.
as previously mentioned, the  <dig> negative ones were likely not contaminated.
the method is therefore able to detect as few as  <dig>  cells per gram of dried infant formulae in as little as  <dig> h of total analytical time.
in an attempt to further shorten this duration, we also tested whether the assay could be carried out after a single pre-enrichment of samples in bpw for  <dig> h. equivalent results, with mean ct values in the same range as previously found, were obtained .
the real-time pcr analytical time could therefore be shortened to about  <dig> h without reducing the method's sensitivity.
detection of es in naturally contaminated samples: comparison of real-time pcr detection and conventional iso-idf culture method
the presence of e. sakazakii in a total of forty-one samples suspected to be naturally contaminated, including infant formulae and samples from the production environment of infant formulae factories, were investigated using the iso cultural method and real-time pcr in parallel .
twenty-five grams test portions were analysed in duplicate for each sample after bpw-mlst enrichments as recommended by the iso-idf procedure.
twenty-two samples were positive for e. sakazakii by the iso-idf method and  <dig> were tested positive by real-time pcr, giving more than  <dig> % concordance between both methods.
the sample  that tested positive by pcr and negative by the culture method had a mean ct of  <dig> .
this value, largely higher than those found for the other positive samples , indicated a lower e. sakazakii cell density in the enriched sample.
extrapolation of this value based on standard curves previously calculated  suggested that the cell load achieved at the end of the two-step enrichment might not exceed 103– <dig> cfu/ml.
moreover, when tested after a single  <dig> h-bpw enrichment, e. sakazakii was not detected by real-time pcr in this sample in contrast to the other  <dig> positive ones , indicating that growth was actually occurring during the second enrichment and that a detectable level could not be reached without a two-days incubation.
all together, these data suggested that the additional pcr-positive sample was not a false positive result but an indication of the higher sensitivity of the real-time pcr assay compared to the conventional culture method.
discussion
given the diversity and the limited sequence information available concerning the e. sakazaki genome, we selected the 16s–23s rdna internal transcribed spacer  sequence to design a fluorescence resonance energy transfer  real-time pcr assay.
this region has the advantages to be highly conserved throughout eubacteriae, to somewhat differ in length and primary sequence between genus and species, and to be well characterised in numerous species and strains for phylogenetic purposes.
the set of primers esfor and esrevb was shown to be specific enough to distinguish e. sakazakii from other isolates of bacteria by conventional pcr amplification.
they amplified a 158-pb dna fragment located between the trna-glu and 23s rrna genes.
taking account of the sequence variations encountered in this region among our collection of e. sakazakii isolates, the upstream internal probe lc1es designed was degenerated in several positions to match the requirement for 100% homology between sequences of probes and templates.
no false-positive or false-negative result was indeed obtained, indicating that the fret real-time pcr assay developed was highly specific to e. sakazakii.
the diversity existing between e. sakazakii strains was found to affect the sensitivity of the real-time pcr results.
ct values varied to some extent for same cell concentration, using different strains.
while the detection limit was approximately  <dig> to  <dig> equivalent genome per reaction for the strain atcc  <dig> , it was approximately  <dig> copies  for the phylogenetically more distinct strain atcc  <dig>  the use of a very sensitive detection method is essential given the presence at low levels of e. sakazakii in pif.
an enrichment step was therefore included in the real-time pcr method to increase the bacterial population largely above the detection limit.
the enrichment procedures recommended by the iso-idf  method was evaluated for this purpose.
this enrichment allowed to detect an initial contamination level of  <dig> cell per  <dig> g of pif.
the pcr sensitivity depends on the efficiency of the method used to recover bacterial dna from food.
sufficient nucleic acid extraction, with the removal of substances inhibitory to amplification, is important for an optimal detection of microbial pathogens by pcr, especially with a complex and rich food matrix such as pif.
the fret real-time pcr was therefore combined with a robust nucleic acid extraction procedure, the magna pure lc automated dna extraction system.
in this study, it was observed that by using the magna pure system in combination with a lightcycler, reproducible results could be obtained with extraction of genomic dna from pif suspensions to real-time-pcr in approximately  <dig> to  <dig> h. another interesting aspect of this system is the high level of purity of the resulting dna.
pcr detection in pif is often hampered by pcr-inhibition problems .
in this respect, the method offers a good dna purification step prior to amplification and was shown to be satisfactory for analysis of pif.
moreover, to assess pcr inhibition, a competitive iac template was incorporated in the design of the assay.
all real-time pcr reactions were performed in the presence of a given number of its copies.
this allowed to distinguish negative responses due to the absence of target sequence in the sample from negative responses due to an amplification failure.
all negative results in this study were then systematically validated by the iac amplification.
despite the use of an automated dna extraction system, matrix-dependent effects affecting real-time pcr results were noticed.
mean ct values obtained from one of the three brands of artificially contaminated pif used were found to differ from the others, being systematically slightly higher.
the pif composition is suspected to directly and significantly affect the pcr assay sensitivity.
compared to the  <dig> other brands, the involved pif contained higher fat and skimmed milk content and was enriched with starch and bifidobacterium.
in addition, this powder was more difficult to handle .
the shift in ct values for this matrix might also reflect less favourable growth conditions which would not support the same cell density as the other pif, linked perhaps to the presence of competitive microflora.
however, the methodology developed, combining real-time pcr detection, automated dna extraction and enrichment, was able to correctly identify all samples containing e. sakazakii irrespective of pif brand, contamination level and strains analysed.
reliable detection of pathogens in a timely manner is a major challenge for product quality control in the food industry, for official controls and for outbreak investigation.
in this respect, an interesting strategy has been recently reported by mullane et al.
to quickly detect e. sakazakii in pif  <cit> .
the developed method requires a short enrichment period , followed by capture of the bacteria using charged paramagnetic beads and subsequent identification after plating onto selective agar.
real-time pcr technology coupled to automated nucleic acid extraction also meets this requirement.
however, the method developed in this work includes a significant enrichment time that does not enable a same-day analysis.
the enrichment procedure recommended by the iso-idf method that we used involves a pre-enrichment in bpw for  <dig> h, followed by an enrichment in mlst for  <dig> h. to shorten the total analytical time to less than  <dig> h, the enrichment step was limited to the single pre-enrichment in bpw.
a clear real-time pcr positive signal was obtained with this one-step enrichment for all artificially or naturally contaminated samples, previously found positive using the two-step iso-idf enrichment, with one exception.
significant difference in term of ct, with a shift to more elevated values, were however observed with very low level of contamination, i.e.,  <dig> cfu/ <dig> g of pif, and naturally contaminated samples, indicating a lower cell density at the time of the analysis for those samples.
for one naturally contaminated sample , e. sakazakii was only detectable by the assay with a two-step enrichment, due to probable late and laborious cell recovery.
the presence in pif and the environment of infant formulae factories of injured or stressed cells due to the type of process is plausible.
in this case, an increased variability of individual lag times and of the detection times  is expected  <cit> .
the second enrichment in mlst might therefore be useful for samples containing a low number of injured or stress cells.
in this study,  <dig> of  <dig> samples was concerned.
the analysis of more naturally contaminated samples need to be carried out to assess the frequency of such event and to assess whether the second enrichment might be omitted without the risk of false-negative results.
CONCLUSIONS
compared to the iso-idf cultural method, a high degree of agreement with the new developed real-time pcr method was found.
agreement  was obtained with artificially contaminated pif samples and  <dig> % with naturally contaminated samples.
the pcr detection system gave one additional positive.
this result might suggest that, besides being significantly faster, molecular methods could also be more sensitive than the cultural method.
in conclusion, the method developed can be considered as a fast alternative for identification of e. sakazakii in pif.

 enterobacter sakazakii, previously known as yellow-pigmented e. cloacae, has been identified as the causative agent of rare but often severe invasive infections in infants. neonates and infants less than months of age appear to be groups at particular risk. meningitis is the most frequently reported clinical symptom in neonatal e. sakazakii infections. infants in whom meningitis developed were generally < week of age at the onset of infection and had near-term gestational age and birthweight. they frequently develop complications including seizures and brain abscesses, resulting in high mortality and morbidity. septicaemia or necrotizing enterocolitis are also associated with e. sakazakii-related infections. compared with infants with meningitis, infants in whom bacteremia alone developed tended to be born very prematurely and have extremely low birthweight. they had generally surpassed the neonatal period at the onset of their disease. mortality among these cases is lower. e. sakazakii is emerging as a hazard in powdered infant formulae. in several e. sakazakii-related outbreaks and sporadic cases, pif was epidemiologically or microbiologically established as the source of infection. this organism was however isolated at very low levels from commercial pif and dry environmental samples collected from infant formula factories. the contamination was in most cases not exceeding cfu per g. prompted by the health risks associated with e. sakazakii, the international standard organisation and the international dairy federation have recently jointly adopted a technical specification, quoted as a reviewed method for idf, defining a method for the detection of e. sakazakii in pif. the method is based on the selective enrichment procedure developed by guillaume-gentil et al.. isolation of the organism involves a preenrichment in buffered peptone water, an enrichment in modified lauryl sulfate tryptose broth, and a plating on the chromogenic enterobacter sakazakii isolation agar. identification include observation of yellow pigmentation for suspected e. sakazakii colonies on tryptone soy agar at 25°c and confirmation of the species via biochemical characteristics. the isolation and confirmation procedures usually require to days to be completed. although at present, the gold standard for the detection of microorganisms in food is in general conventional culture, molecular methodologies based on nucleic acid detection have been successfully developed these last years. they offer an interesting alternative to rapidly and specifically identify organisms from a wide variety of sources. with regards to e. sakazakii detection, several pcr-based approaches have already been published. however, some of these methods do not include an internal amplification control that allows a simultaneous assessment of pcr inhibition. moreover, none of them have been evaluated with naturally contaminated food samples, thus not addressing the ecological diversity of naturally contaminated food matrices. the present study evaluated a real-time pcr-based assay including an iac and an automated nucleic acid extraction method that can be used in combination with the iso-idf enrichment steps for the routine examination of naturally contaminated pif.  specificity of the real-time pcr assay the dna region located between the trna-glu and 23s rrna genes was selected as target for detecting the e. sakazakii species. primers esfor and esrevb were demonstrated to amplify a 158-bp fragment in all strains of e. sakazakii tested with no cross-reaction with other non-e. sakazakii bacterial strains. real-time pcrs were next conducted with a first set of hybridisation probes in the presence of molecules of iac. all but one e. sakazakii strains gave a positive signal in real-time pcr assay. for strain es we repeatedly failed to detect any fluorescent signal and this lack of e. sakazakii specific signal was curiously also accompanied by a lack of the iac signal. to attempt to improve the assays, the esfor/esrevb amplified products specific to this strain were sequenced, as well as those of the strain atcc one of the more phylogenetically distinct e. sakazakii strain which displayed one of the higher ct value in this assay. sequencing of both 158-bp target fragments did reveal some mismatches between the lc1es hybridisation probes and its binding sequences. a new lc1es probe incorporating three degenerated bases/positions compared to the initial one was therefore tested. the new designed real-time pcr was able to correctly detect all e sakazakii strains tested, including the es isolates. the use of a more degenerated lc1es probe did not significantly affect the ct value obtained for each strain but increased the intensity of fluorescence measured for some of them, including strain atcc this new probe was used in the rest of the study. for the exclusivity test, a total of non-e. sakazakii enterobacteriaceae and non-enterobacteriaceae strains were chosen. all non-e. sakazakii strains tested were negative by either pcr or real-time pcr while a positive iac signal was always detected. the new e. sakazakii-iac real-time pcr developed could therefore distinguish e. sakazakii isolates from non-target bacteria. sensitivity of the real-time pcr assay two reference strains belonging to distinct lineages were selected to determine the sensitivity of the assay. the type strain atcc is representative of a majority of e. sakazakii strains. the previously mentioned strain atcc the preceptrol™ strain, is more closely related to taxa including e. pyrinus, e. hormaechei and c. koseri. the sensitivity of the real-time pcr assay in the presence of molecules of iac was experimentally determined by using purified dna. tenfold dilutions of e. sakazakii genomic dna in the concentration range of to equivalent genome per reaction were amplified in triplicate. table summarises the mean ct values obtained for both strains. a detection limit of 5- and 25-genome copies was assessed for the strains atcc and atcc respectively. a positive reaction could however be detected in some cases with, respectively, as few as and dna molecules per tube. to assess the detection limit of the assay combined with the dna extraction step, tenfold serial cell dilutions of calibrated cultures containing cfu/ml were performed and dna extracted using the magna pure lc system. results showed that at least and cfu/ml need to be present to get a positive reaction with the strains atcc and atcc respectively. 100% of positive reactions were recorded in samples containing, respectively, cfu/ml and cfu/ml. results showed a linear log correlation over the 10-fold dilution series, indicating that real-time pcr combined with magna pure dna extraction system could be used to quantify e. sakazaki cell suspensions. evaluation of the real-time pcr assay coupled with the iso-idf enrichment procedure with artificially contaminated infant formula samples infant formula powders from three different commercial brands were inoculated with the strains atcc or atcc at four levels of contamination plus negative control. briefly, twenty five grams of pif were dissolved in ml of buffered peptone water and then inoculated with diluted e. sakazakii culture. the contaminations were carried out in triplicate, except the blank which was twice repeated. samples were analysed in parallel by the conventional iso-idf method and by real-time pcr after a common cultural enrichment. both methods gave the same results for all samples and replicates. non-inoculated samples were negative. all but one artificially contaminated samples tested positive for e. sakazakii, demonstrating that both methods allow the detection of less than e. sakazakii cells in g of dried infant formulae. mean ct values obtained by real-time pcr ranged from to cycles. as observed for the sensitivity analysis, ct values were systematically higher for the preceptrol strain than for the e. sakazakii type strain atcc a slight matrix-dependent effect was also noticed, with ct values for one of the infant formulae's brand higher than those of the others brands. in contrast, no significant difference was observed according to the contamination level, indicating that e. sakazakii has likely reached a similar growth density at the end of the enrichment procedure for all samples. the negative result found for one replicate by both methods suggested that the artificial contamination failed. heterogeneity in artificial contamination at low inoculation levels may explain the likely absence of e. sakazakii in this sample. given the reported presence of e. sakazakii at low level in pif, a contamination level of cfu/ g was also attempted using the phylogenetically more distinct strain atcc. out of the replicates performed, were positive for e. sakazakii. as previously mentioned, the negative ones were likely not contaminated. the method is therefore able to detect as few as cells per gram of dried infant formulae in as little as h of total analytical time. in an attempt to further shorten this duration, we also tested whether the assay could be carried out after a single pre-enrichment of samples in bpw for h. equivalent results, with mean ct values in the same range as previously found, were obtained. the real-time pcr analytical time could therefore be shortened to about h without reducing the method's sensitivity. detection of es in naturally contaminated samples: comparison of real-time pcr detection and conventional iso-idf culture method the presence of e. sakazakii in a total of forty-one samples suspected to be naturally contaminated, including infant formulae and samples from the production environment of infant formulae factories, were investigated using the iso cultural method and real-time pcr in parallel. twenty-five grams test portions were analysed in duplicate for each sample after bpw-mlst enrichments as recommended by the iso-idf procedure. twenty-two samples were positive for e. sakazakii by the iso-idf method and were tested positive by real-time pcr, giving more than % concordance between both methods. the sample that tested positive by pcr and negative by the culture method had a mean ct of. this value, largely higher than those found for the other positive samples, indicated a lower e. sakazakii cell density in the enriched sample. extrapolation of this value based on standard curves previously calculated suggested that the cell load achieved at the end of the two-step enrichment might not exceed 103– cfu/ml. moreover, when tested after a single h-bpw enrichment, e. sakazakii was not detected by real-time pcr in this sample in contrast to the other positive ones, indicating that growth was actually occurring during the second enrichment and that a detectable level could not be reached without a two-days incubation. all together, these data suggested that the additional pcr-positive sample was not a false positive result but an indication of the higher sensitivity of the real-time pcr assay compared to the conventional culture method. discussion given the diversity and the limited sequence information available concerning the e. sakazaki genome, we selected the 16s–23s rdna internal transcribed spacer sequence to design a fluorescence resonance energy transfer real-time pcr assay. this region has the advantages to be highly conserved throughout eubacteriae, to somewhat differ in length and primary sequence between genus and species, and to be well characterised in numerous species and strains for phylogenetic purposes. the set of primers esfor and esrevb was shown to be specific enough to distinguish e. sakazakii from other isolates of bacteria by conventional pcr amplification. they amplified a 158-pb dna fragment located between the trna-glu and 23s rrna genes. taking account of the sequence variations encountered in this region among our collection of e. sakazakii isolates, the upstream internal probe lc1es designed was degenerated in several positions to match the requirement for 100% homology between sequences of probes and templates. no false-positive or false-negative result was indeed obtained, indicating that the fret real-time pcr assay developed was highly specific to e. sakazakii. the diversity existing between e. sakazakii strains was found to affect the sensitivity of the real-time pcr results. ct values varied to some extent for same cell concentration, using different strains. while the detection limit was approximately to equivalent genome per reaction for the strain atcc, it was approximately copies for the phylogenetically more distinct strain atcc the use of a very sensitive detection method is essential given the presence at low levels of e. sakazakii in pif. an enrichment step was therefore included in the real-time pcr method to increase the bacterial population largely above the detection limit. the enrichment procedures recommended by the iso-idf method was evaluated for this purpose. this enrichment allowed to detect an initial contamination level of cell per g of pif. the pcr sensitivity depends on the efficiency of the method used to recover bacterial dna from food. sufficient nucleic acid extraction, with the removal of substances inhibitory to amplification, is important for an optimal detection of microbial pathogens by pcr, especially with a complex and rich food matrix such as pif. the fret real-time pcr was therefore combined with a robust nucleic acid extraction procedure, the magna pure lc automated dna extraction system. in this study, it was observed that by using the magna pure system in combination with a lightcycler, reproducible results could be obtained with extraction of genomic dna from pif suspensions to real-time-pcr in approximately to h. another interesting aspect of this system is the high level of purity of the resulting dna. pcr detection in pif is often hampered by pcr-inhibition problems. in this respect, the method offers a good dna purification step prior to amplification and was shown to be satisfactory for analysis of pif. moreover, to assess pcr inhibition, a competitive iac template was incorporated in the design of the assay. all real-time pcr reactions were performed in the presence of a given number of its copies. this allowed to distinguish negative responses due to the absence of target sequence in the sample from negative responses due to an amplification failure. all negative results in this study were then systematically validated by the iac amplification. despite the use of an automated dna extraction system, matrix-dependent effects affecting real-time pcr results were noticed. mean ct values obtained from one of the three brands of artificially contaminated pif used were found to differ from the others, being systematically slightly higher. the pif composition is suspected to directly and significantly affect the pcr assay sensitivity. compared to the other brands, the involved pif contained higher fat and skimmed milk content and was enriched with starch and bifidobacterium. in addition, this powder was more difficult to handle. the shift in ct values for this matrix might also reflect less favourable growth conditions which would not support the same cell density as the other pif, linked perhaps to the presence of competitive microflora. however, the methodology developed, combining real-time pcr detection, automated dna extraction and enrichment, was able to correctly identify all samples containing e. sakazakii irrespective of pif brand, contamination level and strains analysed. reliable detection of pathogens in a timely manner is a major challenge for product quality control in the food industry, for official controls and for outbreak investigation. in this respect, an interesting strategy has been recently reported by mullane et al. to quickly detect e. sakazakii in pif. the developed method requires a short enrichment period, followed by capture of the bacteria using charged paramagnetic beads and subsequent identification after plating onto selective agar. real-time pcr technology coupled to automated nucleic acid extraction also meets this requirement. however, the method developed in this work includes a significant enrichment time that does not enable a same-day analysis. the enrichment procedure recommended by the iso-idf method that we used involves a pre-enrichment in bpw for h, followed by an enrichment in mlst for h. to shorten the total analytical time to less than h, the enrichment step was limited to the single pre-enrichment in bpw. a clear real-time pcr positive signal was obtained with this one-step enrichment for all artificially or naturally contaminated samples, previously found positive using the two-step iso-idf enrichment, with one exception. significant difference in term of ct, with a shift to more elevated values, were however observed with very low level of contamination, i.e., cfu/ g of pif, and naturally contaminated samples, indicating a lower cell density at the time of the analysis for those samples. for one naturally contaminated sample, e. sakazakii was only detectable by the assay with a two-step enrichment, due to probable late and laborious cell recovery. the presence in pif and the environment of infant formulae factories of injured or stressed cells due to the type of process is plausible. in this case, an increased variability of individual lag times and of the detection times is expected. the second enrichment in mlst might therefore be useful for samples containing a low number of injured or stress cells. in this study, of samples was concerned. the analysis of more naturally contaminated samples need to be carried out to assess the frequency of such event and to assess whether the second enrichment might be omitted without the risk of false-negative results.  compared to the iso-idf cultural method, a high degree of agreement with the new developed real-time pcr method was found. agreement was obtained with artificially contaminated pif samples and % with naturally contaminated samples. the pcr detection system gave one additional positive. this result might suggest that, besides being significantly faster, molecular methods could also be more sensitive than the cultural method. in conclusion, the method developed can be considered as a fast alternative for identification of e. sakazakii in pif. 
enterobacter sakazakii is the causative agent of rare but severe food-borne infections associated with meningitis, necrotizing enterocolitis and sepsis in infants. performance of the assay, coupled to an automated dna extraction system and the e. sakazakii iso-idf enrichment procedure, was evaluated on infant formulae and samples from production environment. this method, combining specific real-time pcr, automated dna extraction and iso-idf standard enrichments, provides a useful tool for rapid screening of e. sakazakii in food and environmental matrices. the real-time pcr assay had 100% specificity as assessed using e. sakazakii and non-e. sakazakii strains. according to the e. sakazakii strains tested, the detection limits ranged from to genomic copies. rehydrated powdered infant formulae have been implicated as the source of infection in several outbreaks and sporadic cases. in this work, a real time fluorescence resonance energy transfer pcr assay incorporating an internal amplification control was developed for the specific detection of e. sakazakii in foods. out of naturally contaminated infant formulae and environmental samples analysed for the presence of e. sakazakii, were positive by real-time pcr and by the conventional culture method, giving % concordance with the iso-idf reference method.
 enterobacter sakazakii is the causative agent of rare but severe food-borne infections associated with meningitis, necrotizing enterocolitis and sepsis in infants. rehydrated powdered infant formulae have been implicated as the source of infection in several outbreaks and sporadic cases. in this work, a real time fluorescence resonance energy transfer pcr assay incorporating an internal amplification control was developed for the specific detection of e. sakazakii in foods. performance of the assay, coupled to an automated dna extraction system and the e. sakazakii iso-idf enrichment procedure, was evaluated on infant formulae and samples from production environment.  the real-time pcr assay had 100% specificity as assessed using e. sakazakii and non-e. sakazakii strains. according to the e. sakazakii strains tested, the detection limits ranged from to genomic copies. assays on pure cultures gave a sensitivity of about to cfu/ml. out of naturally contaminated infant formulae and environmental samples analysed for the presence of e. sakazakii, were positive by real-time pcr and by the conventional culture method, giving % concordance with the iso-idf reference method.  this method, combining specific real-time pcr, automated dna extraction and iso-idf standard enrichments, provides a useful tool for rapid screening of e. sakazakii in food and environmental matrices. 