BACKGROUND
escherichia coli clone o45:k1:h <dig>  belonging to virulence sequence type  <dig>  is a major cause of neonatal meningitis and of urosepsis in young infants in france  <cit> .
the recently sequenced o45:k1:h <dig> strain s <dig>  isolated from cerebrospinal fluid of a neonate, harbors a plasmid of 134 kb, named ps <dig>  involved in meningeal virulence and bacteremia  <cit> .
epidemiological studies have shown that major genetic determinants of this plasmid are not restricted to e. coli clone o45:k1:h <dig> but are widely distributed among e. coli neonatal meningitis  clones, uropathogenic e. coli strains , and avian pathogenic e. coli strains   <cit> .
sequencing of ps <dig> revealed  <dig> orfs, including genes involved in the plasmid machinery , is-like genes, two colicins , and several virulence genes of known or putative functions, such iron-uptake system.
these iron-uptake systems include aerobactin , salmochelin  and the sitabcd transport system  <cit> .
the s <dig> plasmid also contains the serum survival gene iss <cit> , the etsabc genes, encoding a putative type  <dig> secretion system  <cit> , omptp, encoding a putative outer-membrane protease differing from the e. coli chromosomal ompt gene  <cit>  and hlyf, encoding a hemolysin  <cit> .
finally,  <dig> orfs have unknown functions and may represent new virulence genes.
few studies have analyzed the transcriptional profile of human extraintestinal e. coli  strains responsible for urinary tract infection  <cit> .
to further unravel the role of ps <dig> in the virulence of clone o45:k1:h <dig>  we analyzed the transcriptional response of plasmid ps <dig> to growth in urine and serum, representing two steps required for meningeal invasion  <cit> .
we also analyzed the transcriptome of a ps88-like plasmid recovered from a neonate with urinary tract infection .
RESULTS
validation of transcriptional analysis
the transcriptional analysis was validated first by qrt-pcr amplification of transcripts of  <dig> genes  in serial dilutions of rna extracted from s <dig> grown in lb broth.
the ct values showed a linear relation with the template dilution .
similar results were obtained after growth in lb broth containing the iron chelator  <dig> ’-dipyridyl .
we also conduced three independent biological replicates of ps <dig> after growth in lb broth, named experiments  <dig>   <dig> and  <dig>  to compare the ct values which each other.
as expected, most of the fold changes were close to  <dig>  and 98% of values were between  <dig>  and  <dig> .
therefore, we considered that an orf was upregulated or downregulated if the change in expression was smaller or larger than  <dig> -fold and 4-fold, respectively, with p values ≤ <dig> .
these thresholds are in line with those selected by mobley et al.
<cit> .
global analysis of the ps <dig> transcriptome ex vivo and the pamm transcriptome in vivo
table  <dig> shows the transcriptome patterns for ps <dig> grown in iron-depleted lb, in human urine and serum, as well as that of pamm .
a transcript was detected for all  <dig> orfs tested, except for orf  <dig>  overall,  <dig> orfs ,  <dig> of which corresponded to  <dig> operons, were upregulated in at least one of the three ex vivo conditions.
the only down-regulated genes were traa in urine, and ydfa and orf  <dig> in iron-depleted lb broth.
the transcriptome pattern of pamm largely matched the ex vivo patterns, indicating that growth in human urine ex vivo was a relevant model.
interestingly, the fold changes observed in vivo were far higher than those observed ex vivo and in vitro.
a fold changes of transcription levels relative to reference condition .
fold change >  <dig> are in bold print.
b p value in student’s t test for the comparison of the three biological replicates for each experiment in different growth conditions and the reference condition.
p <  <dig>  are in bold print.
c orfs present in plasmid ps <dig> but absent from plasmid pamm.
d housekeeping genes.
expression of iron uptake systems
the concentration of free iron in human urine and serum is low, because iron is sequestered by host molecules  <cit> .
e. coli has developed several strategies to acquire iron in such environments.
ten orfs were upregulated after growth in urine, in serum, and in iron-depleted lb, suggesting they were induced by the low iron concentrations in these media.
five of these  <dig> orfs corresponded to iron-uptake and iron-assimilation systems, namely iuta and iuca , irob  and sita and sitb .
these iron-uptake systems have previously been linked to the virulence of expec and apec  <cit> .
mobley et al.
also observed upregulation of upec iron-acquisition systems such as aerobactin, salmochelin and the sitabcd system in urinary isolates from experimentally infected mice and from women with uti  <cit> .
likewise, li et al.
found that genes involved in iron acquisition were among the most significantly upregulated genes during growth in chicken serum of the apec strain o <dig>  <cit> , which harbours a plasmid  closely related to ps <dig>  <cit> .
our study represents the first transcriptional analysis of an e. coli plasmid after growth in human serum.
surprisingly, we found that the salmochelin receptor iron was not upregulated in our ex vivo experiments, and that the transcript level of the aerobactin receptor iuta was markedly lower than that of the siderophore iuca.
in contrast the salmochelin receptor iron was upregulated 28-fold in the isolate from a neonate with uti.
such discrepancies have been previously described.
in the murine uti model used by mobley et al.
<cit> , iron was upregulated but its transcript level was also lower than that of irob.
moreover, in their transcriptome analysis of e. coli isolates from eight women with urinary tract infection, iron and iuta were only upregulated in two isolates  <cit> .
colicin expression
another group of genes upregulated in iron-deficient conditions were the genes encoding the microcin v  and colicin ia, which were also upregulated in human serum and urine.
previous reports have shown the influence of bacterial intracellular iron levels on colicin expression, but the reason of such induction is still poorly understood  <cit> .
of note, transcription of immunity protein for both colicins was not upregulated in any of the conditions studied except for colicin ia in human serum.
expression of orfs of unknown function in iron-deficient environments
two orfs with unknown functions, shif and orf  <dig>  were upregulated in iron-deficient conditions, with large fold changes in vivo and ex vivo.
orf  <dig> was the most strongly upregulated  in the  <dig> test conditions, and was expressed  <dig> to  <dig> times more strongly than the iron acquisition systems.
a nucleotide homology search using the blast program  <cit>  showed that orf  <dig> is highly homologous  to an orf present in e. coli plasmids possessing a cvp region  or located on the chromosome of upec strains such as cft <dig>  and  <dig> .
no homologous gene is found in the commensal e. coli strain mg <dig>  transcriptome analysis by mobley et al.
<cit>  showed over-expression of c <dig> transcripts in e. coli cft <dig> in a mouse model of uti.
the putative protein encoded by orf  <dig> showed 45-50% identity to three phospho-2-dehydro-3-deoxyheptonate aldolases that catalyze the first reaction of the shikimate pathway and are present on the chromosome of e. coli k <dig>  this pathway involves seven enzymatic reactions that generate chorismate, a factor involved in the synthesis of three aromatic amino acids   <cit> .
however, this pathway is also involved in other reactions, such as biosynthesis of siderophore group nonribosomal peptides such as yersiniabactin and enterobactin.
in plasmid ps <dig>  as in other cvp-containing plasmids, orf  <dig> lies just upstream of iron and is preceded by a sequence resembling the fur box consensus sequence   <cit> .
blast analysis of complete genomes available on publicly available database showed that orf  <dig> is only found when the salmochelin operon is present but the reciprocity is not true, as for example in strain uti <dig>  which harbors only an iro locus.
on the chromosome of e. coli strains cft <dig> and  <dig>  this orf  is located in a pathogenicity island containing an iro locus but is 20–30 kb distant from the iro locus.
because of its putative function, its high inducibility in iron-depleted conditions, and its physical proximity to the iro locus, we suspected that this orf might be an auxiliary gene that boosts the synthesis of iron acquisition systems such as salmochelin by enhancing the production of chorismate and, consequently, enterobactin, the precursor of salmochelin  <cit> .
the shif orf was also upregulated in iron-deficient environments.
shif was first described in the pathogenicity island shi- <dig> in shigella flexneri <cit>  and encodes a putative protein belonging to the major facilitator superfamily.
the latter is one of the two largest families of membrane transporters capable of transporting small solutes in response to chemiosmotic ion gradients.
transcriptome analysis of apec o <dig> grown in chicken serum showed that shif was also upregulated  <cit> .
blast analysis revealed that shif is present in many upec and apec strains, but only when the locus encoding aerobactin is present, although the two do not always colocalize.
of interest, in ps <dig>  as in shi- <dig>  shif is located just upstream of the aerobactin operon, on the opposite strand, and shares the same fur box.
these results suggest that shif induction is at least partly regulated by iron deficiency and that, like orf  <dig>  shif may be an auxiliary gene that promotes the transport of lysine, the precursor of aerobactin.
specific orf expression in serum and urine
a minority of orfs were upregulated in serum and/or urine but not in iron-depleted lb broth.
two of these orfs were upregulated only in urine , while  <dig> orfs were upregulated in both serum and urine .
meanwhile the putative role of orf  <dig>  orf  <dig> and psia in the steps studied could not be predicted, the most strongly upregulated orf in urine, orf  <dig>  could play a role in the infection process.
this orf codes for a putative enolase, an enzyme involved in the penultimate step of glycolysis and that catalyses 2-phosphoglycerate conversion to phosphoenolpyruvate.
intriguingly this latter molecule is the substrate of the phospho-2-dehydro-3-deoxyheptonate aldolase involved in the shikimate pathway.
orf  <dig> might therefore help to optimize the synthesis of iron-uptake systems in urine.
other putative virulence genes
other putative virulence factors like omptp, etsciss and hlyf <cit>  were not upregulated in any of the conditions studied here.
nolan et al.
has reported upregulation of the etsabc genes  in apec o <dig> strains, including papec-o1-colbm, grown in chicken serum at 37°c  <cit> .
in contrast, in their transcriptional analysis of  <dig> genes in papec-o2-colv grown in chicken serum and human urine, they found that iss, but not etsc, was upregulated in chicken serum  <cit> .
moreover, hlyf was also upregulated in chicken serum but not in human urine.
variability between commercial chicken serum could explain the observed differences in the previously mentioned studies.
alternatively, these putative virulence genes may be induced in highly specific conditions that remain to be determined.
CONCLUSIONS
while several studies have examined e. coli virulence gene expression in animal models, little is known about their expression during human infection  <cit> .
here we identified several genes that may play a key role during e. coli growth in human serum and urine.
further studies are necessary to determine the roles of these candidate virulence genes and to understand the contribution of plasmid ps <dig> to the virulence of e. coli strain s <dig>  in particular its aptitude to cross the human blood–brain barrier.

 escherichia coli clone o45:k1:h belonging to virulence sequence type is a major cause of neonatal meningitis and of urosepsis in young infants in france. the recently sequenced o45:k1:h strain s isolated from cerebrospinal fluid of a neonate, harbors a plasmid of 134 kb, named ps involved in meningeal virulence and bacteremia. epidemiological studies have shown that major genetic determinants of this plasmid are not restricted to e. coli clone o45:k1:h but are widely distributed among e. coli neonatal meningitis clones, uropathogenic e. coli strains, and avian pathogenic e. coli strains. sequencing of ps revealed orfs, including genes involved in the plasmid machinery, is-like genes, two colicins, and several virulence genes of known or putative functions, such iron-uptake system. these iron-uptake systems include aerobactin, salmochelin and the sitabcd transport system. the s plasmid also contains the serum survival gene iss, the etsabc genes, encoding a putative type secretion system, omptp, encoding a putative outer-membrane protease differing from the e. coli chromosomal ompt gene and hlyf, encoding a hemolysin. finally, orfs have unknown functions and may represent new virulence genes. few studies have analyzed the transcriptional profile of human extraintestinal e. coli strains responsible for urinary tract infection. to further unravel the role of ps in the virulence of clone o45:k1:h we analyzed the transcriptional response of plasmid ps to growth in urine and serum, representing two steps required for meningeal invasion. we also analyzed the transcriptome of a ps88-like plasmid recovered from a neonate with urinary tract infection.  validation of transcriptional analysis the transcriptional analysis was validated first by qrt-pcr amplification of transcripts of genes in serial dilutions of rna extracted from s grown in lb broth. the ct values showed a linear relation with the template dilution. similar results were obtained after growth in lb broth containing the iron chelator ’-dipyridyl. we also conduced three independent biological replicates of ps after growth in lb broth, named experiments and to compare the ct values which each other. as expected, most of the fold changes were close to and 98% of values were between and. therefore, we considered that an orf was upregulated or downregulated if the change in expression was smaller or larger than -fold and 4-fold, respectively, with p values ≤. these thresholds are in line with those selected by mobley et al.. global analysis of the ps transcriptome ex vivo and the pamm transcriptome in vivo table  shows the transcriptome patterns for ps grown in iron-depleted lb, in human urine and serum, as well as that of pamm. a transcript was detected for all orfs tested, except for orf overall, orfs, of which corresponded to operons, were upregulated in at least one of the three ex vivo conditions. the only down-regulated genes were traa in urine, and ydfa and orf in iron-depleted lb broth. the transcriptome pattern of pamm largely matched the ex vivo patterns, indicating that growth in human urine ex vivo was a relevant model. interestingly, the fold changes observed in vivo were far higher than those observed ex vivo and in vitro. a fold changes of transcription levels relative to reference condition. fold change >  are in bold print. b p value in student’s t test for the comparison of the three biological replicates for each experiment in different growth conditions and the reference condition. p <  are in bold print. c orfs present in plasmid ps but absent from plasmid pamm. d housekeeping genes. expression of iron uptake systems the concentration of free iron in human urine and serum is low, because iron is sequestered by host molecules. e. coli has developed several strategies to acquire iron in such environments. ten orfs were upregulated after growth in urine, in serum, and in iron-depleted lb, suggesting they were induced by the low iron concentrations in these media. five of these orfs corresponded to iron-uptake and iron-assimilation systems, namely iuta and iuca, irob and sita and sitb. these iron-uptake systems have previously been linked to the virulence of expec and apec. mobley et al. also observed upregulation of upec iron-acquisition systems such as aerobactin, salmochelin and the sitabcd system in urinary isolates from experimentally infected mice and from women with uti. likewise, li et al. found that genes involved in iron acquisition were among the most significantly upregulated genes during growth in chicken serum of the apec strain o, which harbours a plasmid closely related to ps. our study represents the first transcriptional analysis of an e. coli plasmid after growth in human serum. surprisingly, we found that the salmochelin receptor iron was not upregulated in our ex vivo experiments, and that the transcript level of the aerobactin receptor iuta was markedly lower than that of the siderophore iuca. in contrast the salmochelin receptor iron was upregulated 28-fold in the isolate from a neonate with uti. such discrepancies have been previously described. in the murine uti model used by mobley et al., iron was upregulated but its transcript level was also lower than that of irob. moreover, in their transcriptome analysis of e. coli isolates from eight women with urinary tract infection, iron and iuta were only upregulated in two isolates. colicin expression another group of genes upregulated in iron-deficient conditions were the genes encoding the microcin v and colicin ia, which were also upregulated in human serum and urine. previous reports have shown the influence of bacterial intracellular iron levels on colicin expression, but the reason of such induction is still poorly understood. of note, transcription of immunity protein for both colicins was not upregulated in any of the conditions studied except for colicin ia in human serum. expression of orfs of unknown function in iron-deficient environments two orfs with unknown functions, shif and orf were upregulated in iron-deficient conditions, with large fold changes in vivo and ex vivo. orf was the most strongly upregulated in the test conditions, and was expressed to times more strongly than the iron acquisition systems. a nucleotide homology search using the blast program showed that orf is highly homologous to an orf present in e. coli plasmids possessing a cvp region or located on the chromosome of upec strains such as cft and. no homologous gene is found in the commensal e. coli strain mg transcriptome analysis by mobley et al. showed over-expression of c transcripts in e. coli cft in a mouse model of uti. the putative protein encoded by orf showed 45-50% identity to three phospho-2-dehydro-3-deoxyheptonate aldolases that catalyze the first reaction of the shikimate pathway and are present on the chromosome of e. coli k this pathway involves seven enzymatic reactions that generate chorismate, a factor involved in the synthesis of three aromatic amino acids. however, this pathway is also involved in other reactions, such as biosynthesis of siderophore group nonribosomal peptides such as yersiniabactin and enterobactin. in plasmid ps as in other cvp-containing plasmids, orf lies just upstream of iron and is preceded by a sequence resembling the fur box consensus sequence. blast analysis of complete genomes available on publicly available database showed that orf is only found when the salmochelin operon is present but the reciprocity is not true, as for example in strain uti which harbors only an iro locus. on the chromosome of e. coli strains cft and this orf is located in a pathogenicity island containing an iro locus but is 20–30 kb distant from the iro locus. because of its putative function, its high inducibility in iron-depleted conditions, and its physical proximity to the iro locus, we suspected that this orf might be an auxiliary gene that boosts the synthesis of iron acquisition systems such as salmochelin by enhancing the production of chorismate and, consequently, enterobactin, the precursor of salmochelin. the shif orf was also upregulated in iron-deficient environments. shif was first described in the pathogenicity island shi- in shigella flexneri and encodes a putative protein belonging to the major facilitator superfamily. the latter is one of the two largest families of membrane transporters capable of transporting small solutes in response to chemiosmotic ion gradients. transcriptome analysis of apec o grown in chicken serum showed that shif was also upregulated. blast analysis revealed that shif is present in many upec and apec strains, but only when the locus encoding aerobactin is present, although the two do not always colocalize. of interest, in ps as in shi- shif is located just upstream of the aerobactin operon, on the opposite strand, and shares the same fur box. these results suggest that shif induction is at least partly regulated by iron deficiency and that, like orf shif may be an auxiliary gene that promotes the transport of lysine, the precursor of aerobactin. specific orf expression in serum and urine a minority of orfs were upregulated in serum and/or urine but not in iron-depleted lb broth. two of these orfs were upregulated only in urine, while orfs were upregulated in both serum and urine. meanwhile the putative role of orf orf and psia in the steps studied could not be predicted, the most strongly upregulated orf in urine, orf could play a role in the infection process. this orf codes for a putative enolase, an enzyme involved in the penultimate step of glycolysis and that catalyses 2-phosphoglycerate conversion to phosphoenolpyruvate. intriguingly this latter molecule is the substrate of the phospho-2-dehydro-3-deoxyheptonate aldolase involved in the shikimate pathway. orf might therefore help to optimize the synthesis of iron-uptake systems in urine. other putative virulence genes other putative virulence factors like omptp, etsciss and hlyf were not upregulated in any of the conditions studied here. nolan et al. has reported upregulation of the etsabc genes in apec o strains, including papec-o1-colbm, grown in chicken serum at 37°c. in contrast, in their transcriptional analysis of genes in papec-o2-colv grown in chicken serum and human urine, they found that iss, but not etsc, was upregulated in chicken serum. moreover, hlyf was also upregulated in chicken serum but not in human urine. variability between commercial chicken serum could explain the observed differences in the previously mentioned studies. alternatively, these putative virulence genes may be induced in highly specific conditions that remain to be determined.  while several studies have examined e. coli virulence gene expression in animal models, little is known about their expression during human infection. here we identified several genes that may play a key role during e. coli growth in human serum and urine. further studies are necessary to determine the roles of these candidate virulence genes and to understand the contribution of plasmid ps to the virulence of e. coli strain s in particular its aptitude to cross the human blood–brain barrier. 
to identify possible genetic determinants of ps virulence, we examined the transcriptomes of plasmidic orfs corresponding to known and putative virulence genes, and orfs of unknown function. we also analyzed the transcriptome of a ps88-like plasmid recovered from a neonate with urinary tract infection. the transcriptome obtained after ex vivo growth in serum and urine was very similar to those obtained in iron-depleted lb broth. transcriptome analysis of the ps88-like plasmid recovered in vivo showed a similar pattern of induction but at much higher levels. genes encoding iron acquisition systems were strongly upregulated. the transcriptome of e. coli strain s grown in human serum and urine ex vivo were compared to that obtained during growth in luria bertani broth, with and without iron depletion. we identify new ps genes potentially involved in the growth of e. coli meningitis strain s in human serum and urine. four orfs were induced ex vivo, independently of the iron concentration. other putative virulence genes such as iss, etsc, omptp and hlyf were not upregulated in any of the conditions studied.
 the sequenced o45:k1:h escherichia coli meningitis strain s harbors a large virulence plasmid. to identify possible genetic determinants of ps virulence, we examined the transcriptomes of plasmidic orfs corresponding to known and putative virulence genes, and orfs of unknown function.  quantification of plasmidic transcripts was obtained by quantitative real-time reverse transcription of extracted rna, normalized on three housekeeping genes. the transcriptome of e. coli strain s grown in human serum and urine ex vivo were compared to that obtained during growth in luria bertani broth, with and without iron depletion. we also analyzed the transcriptome of a ps88-like plasmid recovered from a neonate with urinary tract infection. the transcriptome obtained after ex vivo growth in serum and urine was very similar to those obtained in iron-depleted lb broth. genes encoding iron acquisition systems were strongly upregulated. shif and orf two orfs encoding protein with hypothetical function and physically linked to aerobactin and salmochelin loci, respectively, were also highly expressed in iron-depleted conditions and may correspond to ancillary iron acquisition genes. four orfs were induced ex vivo, independently of the iron concentration. other putative virulence genes such as iss, etsc, omptp and hlyf were not upregulated in any of the conditions studied. transcriptome analysis of the ps88-like plasmid recovered in vivo showed a similar pattern of induction but at much higher levels.  we identify new ps genes potentially involved in the growth of e. coli meningitis strain s in human serum and urine. escherichia coliplasmidtranscriptomernavirulenceironsiderophore 