BACKGROUND
diarrheagenic escherichia coli  are a heterogeneous group of pathogenic e. coli causing a wide range of enteric diseases in humans and animals  <cit> .
enteroaggregative e. coli  are a dec pathotype inducing a gastrointestinal illness characterized by long-lasting watery, mucoid, secretory diarrhoea with low-grade fever and little or no vomiting  <cit> .
eaggec infections are a common cause of acute diarrheal illness among children in low-income countries, but sporadic cases and outbreaks are recorded in industrialized countries as well  <cit> .
in  <dig>  a large e. coli outbreak struck germany causing more than  <dig>  human infections including  <dig> deaths  <cit> .
the outbreak strain, an e. coli o104:h <dig>  showed the presence of the typical virulence genes of eaggec such as aggr, aaic, sepa, aata and, at the same time, it carried a bacteriophage conveying the genes encoding the verocytotoxin  subtype 2a   <cit> .
in accordance with this genomic asset, the strain showed the enteroaggregative typical “stacked brick” adhesion to cultured hep- <dig> cells and was able to produce vtx <dig>  <cit> .
the infecting strain thus displayed an unusual combination of virulence features comprising the colonization repertoire from eaggec coupled with the production of a toxin typically produced by vtx-producing e. coli , a dec type causing haemorrhagic colitis and haemolytic uremic syndrome  worldwide  <cit> .
the impact of the german outbreak was so huge that the epidemic strain became iconic of a new dec type: the enteroaggregative haemorrhagic e. coli   <cit> .
the occurrence of the german outbreak also caused the scientific community to look retrospectively at the reported hus cases linked to infections with atypical vtec types or to browse the scientific literature in order to assess if other eahec cases of infection could be retrieved.
it turned out that in the time period 1992– <dig> a small outbreak and at least six sporadic cases of hus had been described as being associated with eahec strains belonging to four different serotypes: o111:h <dig>  o86: hnm, o104:h <dig> and o111:h <dig> .
the analysis of the whole genome sequence of the eahec o104:h <dig> that caused the german outbreak in  <dig> showed that the vtx2-phage is inserted in a bacterial genomic backbone typical of eaggec  <cit> , therefore the eahec pathotype seems to have arisen from the acquisition of vtx2-phages by classical eaggec strains.
the appearance of the eahec group has shown that the stable acquisition of vtx-phages seems to have occurred at least twice by two different dec groups, the eaggec and the atypical epec  from which the typical vtec pathotype derives .
moreover, the ability of vtx2-phages to infect, in the laboratory conditions, different e. coli pathogroups including expec has been reported  <cit> .
this observation, together with the isolation of enterobacteriaceae other than e. coli producing vtx from cases of human disease  <cit>  suggests that vtx-phages can infect a range of bacterial hosts wider than expected, confirming the pivotal role of phages in the evolution of bacterial pathogens.
in the present work we determined the whole genome sequence of the vtx2-phage present in the first eahec ever described and compared it with that of the vtx2-phages present in the eahec o104:h <dig> and o111:h <dig> available in the public repositories and with those of other vtx-phages, with the aim of investigating the mechanisms underlying the evolution of the eahec pathotype.
methods
bacterial strains
the eahec o111:h <dig> strain ed  <dig> has been used to obtain the vtx2-phage subjected to whole genome sequencing and is part of the collections held at istituto superiore di sanità.
the strain’s characteristics have been described in a previous publication  <cit> .
e. coli k <dig> strain le <dig>  <cit>  has been used as a propagator strain in infection experiments for the vtx2-phage amplification prior to sequencing.
determination of the vtx2-phages integration sites in the e. coli genome
the vtx2-phage integration site in the e. coli strain ed  <dig> has been determined.
the occupancy of loci sbcb, wrba, yehv, z <dig>  and yece has been assessed as previously described  <cit> .
infection experiments and phages propagation
the eahec strain ed  <dig> has been exposed to uv light in order to induce the excision of phage genome from the bacterial chromosome  <cit> .
in detail, the bacterial strain has been grown in luria-bertrani  broth  overnight at 37°c with vigorous shaking.
the culture has been diluted 1: <dig> in lb modified broth  and grown to  <dig>  od  <dig>  pelleted and re-suspended in a sterile solution of cacl <dig> 10 mm.
the culture has been exposed to uv light  in a crosslinker “stratalinker® uv crosslinker” .
after induction, the culture has been diluted in lb modified broth and incubated at 37°c for 5 hours with vigorous shaking.
the culture has been centrifuged and the supernatant containing phages particles filtered with  <dig>  μm pore-filters.
100 μl of phage particles suspension have been added to 100 μl of a culture of the propagator strain e. coli le <dig> grown in lb modified broth at  <dig>  od  <dig> and maintained at 37°c for 20 minutes with static incubation.
each tube has been added with  <dig>  ml of lb modified soft agar  at 42°c and immediately poured on lb modified agar plates .
plates have been incubated overnight at 37°c.
four ml of sm buffer  have been dispensed to each plate in order to recover phages particles from the lytic plaques and kept overnight at 4°c.
the phage suspension in sm has been recovered and chloroform has been added at 5% final concentration.
the phage suspension has been centrifuged at 500xg 10 minutes twice for removing agar debris and used to re-infect the propagator e. coli strain le <dig> in the conditions described above in order to increase the phage titre.
finally, the phage suspension has been concentrated by using amicon ultra- <dig> centrifugal filter unit with ultracel- <dig> tubes  with a cut-off of  <dig> kda.
final phage titre was  <dig> ×  <dig> pfu/ml.
cscl gradient and viral dna extraction
the suspension has been purified by isopycnic centrifugation through cscl equilibrium gradient as described by sambrook and russell  <cit> .
briefly, 2 ml of the phage suspension have been added with  <dig>  g of cscl, transferred to ultracentrifuge tubes, which have been filled with a cscl solution  <dig>  g/ml.
the tubes have been finally sealed with mineral oil and centrifuged in a beckman ultracentrifuge at  <dig> xg, 8°c for 20 hours in a sw- <dig> rotor.
the band containing the phage particles has been collected with a syringe by puncturing the tube.
the recovered solution containing the purified phage particles has been dialyzed in against 10 mm nacl, 50 mm tris–hcl ph  <dig> , 10 mm mgcl <dig>  a final volume of 1 ml was obtained.
the suspension has been treated by adding  <dig> units of dnase i rnase-free  at 37°c for one hour to eliminate free dna contaminating the phage suspension.
finally a treatment with proteinase k 50 μg/ml at 56°c for one hour has been carried out to disrupt the phage capsid followed by dna extraction with phenol-chloroform-isoamyl alcohol  <cit> .
phage dna concentration after the purification step was estimated to be  <dig>  ng/μl.
library preparation and whole genome sequencing of the phage dna
phage dna has been sequenced with an ion torrent pgm semiconductor sequencer  using the 200 bp protocol.
an ion torrent  <dig> chip has been used following the manufacturer instructions .
genomic library has been obtained by shearing 1 μg of dna in blunt-ended fragments followed by linking the ion adapters using the protocol included in the ion xpress™ plus fragment library kit .
the sized and ligated fragments have been amplified by emulsion-pcr using the ion onetouch  <dig> template kit and instruments .
assembly and further bioinformatics analyses
the reads resulting from the sequencing of the vtx2-phage dna from the eahec o111:h <dig> strain, termed phi- <dig>  have been assembled in contigs by using the open source mira software integrated in the ion torrent server.
contigs have been imported in kodon software  for analysis.
to fill in the gaps between contigs, a total of  <dig> primers have been designed and used for sequencing by sanger technology using a genetic analyzer  <dig> .
mauve software  <cit>  has been used to order the contigs using the sequence of the phage p <dig> from the e. coli o104:h <dig> that caused the outbreak in germany in  <dig>  as reference.
the complete sequence of the phi- <dig> phage has been annotated by prokka tool on the online server galaxy/crs <dig>  <cit>  and submitted to genbank .
the g + c content has been analysed by the gc calculator free online tool  <cit> .
identification of putative trna genes has been performed using trnascan-se  <cit> .
the raw sequence data  from the eahec o111:h <dig> strain  <dig> were retrieved from the sra database present on ncbi website  and aligned on the complete sequence of phi- <dig> phage, determined in the present study and used as reference, with the bowtie <dig> free software implemented in the galaxy/crs <dig> server  <cit> .
genomic comparisons between the available vtx-phages sequences have been performed by using the blast algorithm available at ncbi  <cit>  and the mauve free alignment software  <cit> .
comparison map between vtx-phages has been generated by circoletto online tool  <cit> .
for the pictogram construction, bit-score values have been used to describe the quality of the alignment at a given point.
the bit-score is a normalized version of the score value returned by the blast searches, expressed in bits  <cit> .
ethics
this work does not include animal testing and does not report human data.
all the information regarding outbreaks and cases of infection are all from already published papers properly referenced in the text.
RESULTS
sequencing of the vtx2-phage from the eahec o111:h2
the whole genome sequencing of the vtx2-phage from the eahec o111:h <dig> strain, phi- <dig>  produced  <dig>  reads of a mean length of 204 bp, for a total of  <dig>  mb sequenced.
the assembly of the phi- <dig> sequence reads using as a reference the genome of the p <dig> phage  produced  <dig> contigs that were further analysed using bioinformatics resources.
the estimated coverage of the phage genome was 1088x.
the length of the whole sequence of phi- <dig> resulted to be about  <dig> kb   with a mean g + c content of  <dig> %.sequence annotation revealed the presence of  <dig> predicted coding sequences including the genes encoding the subtype 2a of the verocytotoxin and three transfer rnas  .figure  <dig>
genomic organization of phi- <dig> phage.
coding sequences are represented as blue bars in the outer circle.
putative genes are labelled according to the predicted functions of their products, if known.
the whole gc content and gc skew of leading and lagging strand is shown in the black and coloured inner circles, respectively.
as it has been reported for the p <dig> phage, conveying the vtx <dig> genes in the eahec o104:h <dig> strain that caused the german outbreak in  <dig>  <cit> , the genome of phi- <dig>  only included the lambda genes ci and cro while the other genes typically composing the regulatory repertoire of lambda phages such as cii, ciii, n, ea <dig> and gam seemed not to be present  <cit> .
most of these genes are involved in the regulation of the switch between lysogeny and lytic cycle in lambda-like phages.
therefore, their absence suggests the existence of an alternative mechanism used to regulate the choice of entry into lysogenic state.
the phi- <dig> phage is integrated in the bacterial chromosome in the wrba locus, as it has been described for p <dig>  <cit> .
comparison of the phi- <dig> whole genome sequence with other vtx-phage sequences
a blast search of the phi- <dig> vtx2-phage whole genome sequence against those collected in the nucleotide repository held at ncbi, returned a 99% identity with that of the p <dig> phage from o104:h <dig> strain cb <dig> which caused the german outbreak in  <dig>  and a 91% identity with that of the vtx2-phage tl-2011c carried by the vtec o103:h <dig> strain that caused a severe hus outbreak in norway in  <dig>   <cit> .
this finding is in agreement with how reported in the literature for the vtx-phage carried by the eahec o104:h <dig> from the german outbreak and the tl-2011c phage  <cit> .
as expected, the phi- <dig> phage sequence also showed 99% identity with the vtx2-phage from another eahec o104:h <dig> strain isolated during the  <dig> german outbreak, the strain 2011c- <dig>  and was highly related  to the sequence of two vtx2-phages from two eahec o104:h <dig> strains isolated from as many haemorrhagic colitis cases occurred in georgia in  <dig>   <cit> .
the other scores included hits with query coverage values ranging from 87% down to 60% and having 98%-99% sequence identity, with a number of other vtx-phages identified in different vtec strains .
the alignment of all the phage sequences comprised in the 100%-60% similarity range is shown in a pictogram generated with the circoletto online tool  <cit>   and in another one produced by mauve software .table  <dig>
list of the blast hits of phi- <dig> dna sequence aligned to the
vtx
-phages sequences from typical vtec strains
the similarity score indicates the query coverage values with 98%-99% sequence identity.
the hits with similarity values down to 60% are shown.
sequence similarities between phi- <dig> and other
vtx
-phages.
the picture shows the results of the blast local alignments using phi- <dig> as a query against the vtx-phage sequences with 99% to 60% similarity listed in table  <dig>  the colours codes blue, green, orange and red represent the overall quality of the aligned segments along the phage sequences, evaluated on the basis of the bit-score values in the worst-to-the-best order .
the bit-score is a normalized version of the score value returned by the blast searches, expressed in bits  <cit> .
the height of the coloured bars in the histogram on the top of the phi- <dig> ideogram shows how many times each colour hits a specific fragment of the other phage sequences.
a twist in a ribbon indicates that the local alignment is inverted .
recently, another eahec strain of serotype o111:h <dig> , isolated from an hus case occurred in northern ireland in  <dig>  has been described  <cit> .
the short reads from the whole genome-sequencing project of this strain are available in ncbi sequence reads archive  and have been used for comparison.
unfortunately, among the  <dig> contigs obtained from the de novo assembly of the reads, the one including the vtx <dig> genes was only  <dig>  bp long, hindering the analysis of the entire sequence of the vtx2-encoding phage.
nevertheless, its whole-genome sequencing reads were aligned to the complete genomic sequence from the phi- <dig> phage.
the alignment was carried out with bowtie <dig> software and visualized on the integrative genomics viewer  free tool  <cit>  .
this analysis failed to identify, in the genome of strain  <dig>  a vtx2-phage with characteristics similar to that of phi- <dig>  in particular, the alignment showed that the reads from strain  <dig> mapped on the sequence of the phi- <dig> phage only in the region comprising the vtx <dig> a subunit-coding gene and extending  <dig>  kb downstream the gene encoding the vtx <dig> b subunit.
it should be noted that alignments conducted using the whole short reads set could have generated a too high background noise deriving from the presence of sequences from other bacteriophages integrated in different regions of the chromosome of the o111:h <dig> strain  <dig>  nevertheless, the lack of sequencing reads aligning to the rest of the sequence of phi- <dig> suggests that a different vtx <dig> phage intervened in the emergence of this last eahec strain.
genomic comparison of phi- <dig> with other vtx2-phages
in order to delve into the genomic organization of the different vtx2-phages we performed a progressive mauve alignment between all the phages sequences used in the nucleotide comparison .
this analysis onfirmed the presence of similarities between the sequences but also highlighted that an extensive re-arrangement between the sequence-blocks must have occurred at some points during the phages’ evolution.
this observation is in agreement with how reported in literature .
a deeper analysis of the phi- <dig> genome was conducted by using phage sequences selected among those from typical vtec displaying the highest similarity values .
the alignment of the phi- <dig> sequence with those of phage tl-2011c , and the phages from the vtec strains rm <dig>  and  <dig> , the causative agent of a romaine lettuce-associated outbreak occurred in the us in  <dig>  <cit>  and isolated in japan from a sporadic case of diarrhoea  <cit> , respectively, showed the presence of two sequence blocks that seemed to be peculiar to the eahec vtx2-phage and divergent from the same regions in the three classical vtec associated-phages .
one of the two regions was  <dig>  bp long and comprised 140 bp of the 5’-terminus of a gene encoding a lysozyme , two complete genes encoding the lysis protein s and a hypothetical protein, and 440 bp of the 3’-terminus of a gene annotated as a hypothetical protein.
the  <dig>  bp region corresponded to the nucleotidic positions  <dig> - <dig>  in the p <dig> genome .
the other fragment was 900 bp long and spanned the region comprised between nucleotides  <dig>  and  <dig>  in the genome of the p <dig> phage.
the 900 bp fragment shared 100% homology with a region of 730 bp at the 3’-terminus of the orf <dig> of p <dig> phage encoding a phage tail fiber and a 72 bp fragment of the orf <dig> coding for a tail fiber adesine.figure  <dig>
mauve progressive alignment of
vtx2
-phage genomes from eahec and vtec strains showing the eahec-specific regions.
blocks with the same colours indicate the vtx2-phages regions with identical dna sequence.
white blocks in a phage sequence indicate regions lacking of correspondence in the other sequences.
fragments of the phage genomes that are peculiar to eahec vtx2-phages are marked with circles: a red circle indicates the sequence encoding a hypothetical protein; the sequence encoding the phage fiber tail is encircled in blue.
distribution and analysis of the putative eahec-associated sequence regions amongst the vtx-phages
a blast comparison was conducted with the aim of investigating the distribution of the two putative eahec-associated regions among all the vtx-phage sequences available at ncbi.
such an analysis showed that only the 900 bp region encoding phage fiber tail was present in all the fully sequenced vtx2-phages from eahec strains,  and divergent or absent in all the other phage sequences investigated.
accordingly, the presence of the sole sequence of 900 bp was assessed in the contigs derived from the de novo assembly of the short reads from the genome sequence of the o111:h <dig> strain  <dig> from northern ireland.
discussion
the e. coli genome continuously changes through both small-scale variations and horizontal gene transfer of mobile genetic elements .
among mges, bacteriophages play a pivotal role in the evolution of e. coli pathogenic clones  <cit>  by providing a mean for genomic remodelling and conveying important virulence genes such as those encoding the verocytotoxins  in the verocytotoxin-producing e. coli .
in  <dig> a huge outbreak caused by an enteroaggregative haemorrhagic e. coli  o104:h <dig> struck germany with more than  <dig>  cases of infection,  <dig> hus, and  <dig> deaths  <cit> .
the causative agent was a mosaic strain deriving from the lysogenization of an enteroaggregative e. coli strain with a vtx2-phage  <cit> .
such a virulence combination was indeed associated with elevate pathogenicity, as demonstrated by the high rate of human infections evolving to hus, even among adults , and the heavy toll of  <dig> deaths  <cit> .
this arrangement of virulence factors in e. coli strains from human disease had been occasionally reported before during the investigation of a small outbreak of hus occurred in france in  <dig> and a case of infection in japan in  <dig>  <cit> .
the occurrence of the german outbreak caused the scientific community to look back retrospectively at the repositories of vtec infections records and culture collections and it turned out that some other sporadic cases of infections with vtx2-producing enteroaggregative e. coli o104:h <dig> had already occurred in europe and asia in the time span 2001– <dig>  <cit> .
finally a hus case occurred in northern ireland in  <dig> was demonstrated to be associated with an eahec o111:h <dig>  <cit> .
the observation of sporadic cases and outbreaks occurring throughout a 20-years time span, all caused by vtx2-producing eaggec and belonging to different serotypes, strengthens the hypothesis that these pathogenic e. coli represent a new pathogroup, as it has been recently proposed  <cit> .
to better understand the events underlying the emergence of eahec we determined the whole genome sequence of phi- <dig>  the vtx2-phage present in the eahec o111:h <dig> isolated during the french outbreak of  <dig>  and compared it with the sequences of the vtx2-phage present in the eahec strains described in the following years and available in genbank.
interestingly, the genomic sequence of phi- <dig> was almost identical to that of the vtx2-phages from the eahec o104:h <dig> strains isolated during the  <dig> german outbreak about 20 years later.
this is noteworthy since vtx-phages are characterized by a high degree of variability  <cit> .
it is conceivable that the same vtx2-phage has been acquired in two different events and that the selective pressure impeded the accumulation of changes in the phage sequence before the phage infection events occurred.
however, the eahec o111:h <dig> isolated in northern ireland in  <dig> seems to host a different type of vtx2-phage, suggesting that at least two different vtx2-phage types have been successfully transferred to eaggec.
unfortunately, the sequence of the phage of the eahec o86: hnm isolated in japan in  <dig> was not available for comparison  <cit> .
it has been hypothesized that the infection with a lambdoid phage can be mediated by the cross-talking between the bacterium and the phage resulting in host specificity  <cit> .
an extended comparison of the eahec vtx2-phages with the whole genome sequences of vtx-phages from vtec strains available at ncbi returned a wide range of similarities between sequences, going from 87% to 60% and lower.
this picture is in line with how reported for the general variability of vtx-phages sequences  <cit> .
interestingly, one region of 900 bp, identified in the phi- <dig> and encoding a tail fiber, was present in all the vtx2-phages from eahec and was also present in the short reads dataset from the eahec o111:h <dig>  at the same time this dna sequence was absent in all the vtx-phage sequences identified in vtec strains and stored at ncbi.
this is in agreement with previously reported data which pointed at a larger fragment including this region as one of those differentiating the p <dig> genome from the e. coli phage tl-2011c  and not exhibiting significant homology to known vtx-encoding phages  <cit> .
the annotation of the phi- <dig> genome showed that this sequence peculiar to eahec vtx2-phages contains part of a gene encoding a type of phage tail fiber displaying some conserved aminoacidic motifs such as a collagen triple helix repeat   and the peptidase_s <dig> .
the latter is the c-terminal domain of the bacteriophage protein endosialidase, which forms homotrimeric molecules and releases itself from the end-tail-spike of the bacteriophages  <cit> .
the 900 bp-long sequence could potentially encode part of the mechanism defining the specificity of the vtx2-phages for eaggec strains, being directly involved in the phage-bacterium interaction.
as a matter of fact, several authors reported that the interactions between phage tail fibers and host proteins, such as lamb and ompc  <cit>  contribute to the success of the infection, as demonstrated by the finding that lamb gene mutations block phage adsorption  <cit> .
it is therefore conceivable that differences in phage tail fibers may contribute to define vtx2-phages tropism for e. coli recipients, although this hypothesis together with the mechanisms underlying this process still need to be verified.
for a successful infection to occur, suitable vtx-phages and e. coli acceptors need to meet in the same environment.
in the case of the emergence of typical vtec, the events of vtx-phage acquisition probably occurred at the level of the gastrointestinal tract of ruminants  <cit>  where both vtx-phages and aepec are abundant  <cit> .
conversely, the eahec emergence is probably not directly connected to an animal reservoir since eaggec are human pathogens with an inter-human transmission of the infection  <cit> .
the environment, in turn, plays a role in the pathogen’s amplification cycles, particularly in geographic areas characterised by poor hygienic conditions, where the lack of effective human sewage treatments make the infections with enteric pathogens, including eaggec, endemic  <cit> .
in such a scenario, an environment contaminated with ruminant’s excreta might have been the source of the vtx2-phages found in eahec as it has been recently proposed  <cit> .
such a picture may account for the existence of a favourable setting for the eaggec and the vtx-phages to come in contact and for the following selection process resulting in the occasional emergence of an e. coli strain matching the eahec definition.
CONCLUSIONS
the vtx2-phages characterising eahec seem to belong to a sub-population of vtx-phages kept under selective pressure and characterised by the presence of a gene encoding a tail fiber, which could be involved in the mechanism used to recognize the eaggec.
the new eahec pathogroup may have emerged following multiple vtx2-phage acquisition events favoured by an overlapping of a human reservoir of pathogenic e. coli, the eaggec, with the known animal reservoir of vtx-phages.
the emergence of this new e. coli pathogroup further witnesses the great adaptability and plasticity of this bacterial species and underlines the need to rethink the global asset of hygienic practices to mitigate enteric infections worldwide; particularly in the presence of a global market of foodstuffs that is extending its boundaries towards low-income countries in the quest of new sources to meet the always increasing demand of cheap and exotic food commodities.
electronic supplementary material
additional file 1: figure s1: mauve progressive alignment of phi- <dig> genome with vtx2-phage genomes from eahec and vtec strains showing the highest score of similarity.
blocks with the same colours indicate the vtx2-phages regions with identical dna sequence.
white fragments in a phage sequence indicate regions lacking of correspondence in the other sequences.
connecting lines link the same genomic block in different genomes and help to pinpoint the re-arrangement between vtx-phage genomes.
abbreviations
aepecatypical enteropathogenic escherichia coli
decdiarrheagenic escherichia coli
eaggecenteroaggregative escherichia coli
eahecenteroaggregative haemorrhagic escherichia coli
hchaemorrhagic colitis
hushaemolytic uremic syndrome
mgemobile genetic elements
vtecverocytotoxin-producing escherichia coli
vtxverocytotoxin genes.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
gl carried out the infection experiments, contributed to the sequencing and drafted the manuscript; mv performed the whole genome sequencing and bioinformatics analysis; tr contributed to all the steps of the whole genome sequencing and revised critically the manuscript; rp carried out part of infection experiments and cscl gradient; ma helped in the revision of the draft manuscript; ca contributed to the revision of the draft manuscript; ms conceived the study and strongly contributed to revise the manuscript.
finally, all the authors approved the manuscript to be published.

 diarrheagenic escherichia coli are a heterogeneous group of pathogenic e. coli causing a wide range of enteric diseases in humans and animals. enteroaggregative e. coli are a dec pathotype inducing a gastrointestinal illness characterized by long-lasting watery, mucoid, secretory diarrhoea with low-grade fever and little or no vomiting. eaggec infections are a common cause of acute diarrheal illness among children in low-income countries, but sporadic cases and outbreaks are recorded in industrialized countries as well. in a large e. coli outbreak struck germany causing more than human infections including deaths. the outbreak strain, an e. coli o104:h showed the presence of the typical virulence genes of eaggec such as aggr, aaic, sepa, aata and, at the same time, it carried a bacteriophage conveying the genes encoding the verocytotoxin subtype 2a. in accordance with this genomic asset, the strain showed the enteroaggregative typical “stacked brick” adhesion to cultured hep- cells and was able to produce vtx. the infecting strain thus displayed an unusual combination of virulence features comprising the colonization repertoire from eaggec coupled with the production of a toxin typically produced by vtx-producing e. coli, a dec type causing haemorrhagic colitis and haemolytic uremic syndrome worldwide. the impact of the german outbreak was so huge that the epidemic strain became iconic of a new dec type: the enteroaggregative haemorrhagic e. coli. the occurrence of the german outbreak also caused the scientific community to look retrospectively at the reported hus cases linked to infections with atypical vtec types or to browse the scientific literature in order to assess if other eahec cases of infection could be retrieved. it turned out that in the time period 1992– a small outbreak and at least six sporadic cases of hus had been described as being associated with eahec strains belonging to four different serotypes: o111:h o86: hnm, o104:h and o111:h. the analysis of the whole genome sequence of the eahec o104:h that caused the german outbreak in showed that the vtx2-phage is inserted in a bacterial genomic backbone typical of eaggec, therefore the eahec pathotype seems to have arisen from the acquisition of vtx2-phages by classical eaggec strains. the appearance of the eahec group has shown that the stable acquisition of vtx-phages seems to have occurred at least twice by two different dec groups, the eaggec and the atypical epec from which the typical vtec pathotype derives. moreover, the ability of vtx2-phages to infect, in the laboratory conditions, different e. coli pathogroups including expec has been reported. this observation, together with the isolation of enterobacteriaceae other than e. coli producing vtx from cases of human disease suggests that vtx-phages can infect a range of bacterial hosts wider than expected, confirming the pivotal role of phages in the evolution of bacterial pathogens. in the present work we determined the whole genome sequence of the vtx2-phage present in the first eahec ever described and compared it with that of the vtx2-phages present in the eahec o104:h and o111:h available in the public repositories and with those of other vtx-phages, with the aim of investigating the mechanisms underlying the evolution of the eahec pathotype. methods bacterial strains the eahec o111:h strain ed has been used to obtain the vtx2-phage subjected to whole genome sequencing and is part of the collections held at istituto superiore di sanità. the strain’s characteristics have been described in a previous publication. e. coli k strain le has been used as a propagator strain in infection experiments for the vtx2-phage amplification prior to sequencing. determination of the vtx2-phages integration sites in the e. coli genome the vtx2-phage integration site in the e. coli strain ed has been determined. the occupancy of loci sbcb, wrba, yehv, z and yece has been assessed as previously described. infection experiments and phages propagation the eahec strain ed has been exposed to uv light in order to induce the excision of phage genome from the bacterial chromosome. in detail, the bacterial strain has been grown in luria-bertrani broth overnight at 37°c with vigorous shaking. the culture has been diluted 1: in lb modified broth and grown to od pelleted and re-suspended in a sterile solution of cacl 10 mm. the culture has been exposed to uv light in a crosslinker “stratalinker® uv crosslinker”. after induction, the culture has been diluted in lb modified broth and incubated at 37°c for 5 hours with vigorous shaking. the culture has been centrifuged and the supernatant containing phages particles filtered with  μm pore-filters. 100 μl of phage particles suspension have been added to 100 μl of a culture of the propagator strain e. coli le grown in lb modified broth at od and maintained at 37°c for 20 minutes with static incubation. each tube has been added with  ml of lb modified soft agar at 42°c and immediately poured on lb modified agar plates. plates have been incubated overnight at 37°c. four ml of sm buffer have been dispensed to each plate in order to recover phages particles from the lytic plaques and kept overnight at 4°c. the phage suspension in sm has been recovered and chloroform has been added at 5% final concentration. the phage suspension has been centrifuged at 500xg 10 minutes twice for removing agar debris and used to re-infect the propagator e. coli strain le in the conditions described above in order to increase the phage titre. finally, the phage suspension has been concentrated by using amicon ultra- centrifugal filter unit with ultracel- tubes with a cut-off of kda. final phage titre was × pfu/ml. cscl gradient and viral dna extraction the suspension has been purified by isopycnic centrifugation through cscl equilibrium gradient as described by sambrook and russell. briefly, 2 ml of the phage suspension have been added with  g of cscl, transferred to ultracentrifuge tubes, which have been filled with a cscl solution  g/ml. the tubes have been finally sealed with mineral oil and centrifuged in a beckman ultracentrifuge at xg, 8°c for 20 hours in a sw- rotor. the band containing the phage particles has been collected with a syringe by puncturing the tube. the recovered solution containing the purified phage particles has been dialyzed in against 10 mm nacl, 50 mm tris–hcl ph , 10 mm mgcl a final volume of 1 ml was obtained. the suspension has been treated by adding units of dnase i rnase-free at 37°c for one hour to eliminate free dna contaminating the phage suspension. finally a treatment with proteinase k 50 μg/ml at 56°c for one hour has been carried out to disrupt the phage capsid followed by dna extraction with phenol-chloroform-isoamyl alcohol. phage dna concentration after the purification step was estimated to be  ng/μl. library preparation and whole genome sequencing of the phage dna phage dna has been sequenced with an ion torrent pgm semiconductor sequencer using the 200 bp protocol. an ion torrent chip has been used following the manufacturer instructions. genomic library has been obtained by shearing 1 μg of dna in blunt-ended fragments followed by linking the ion adapters using the protocol included in the ion xpress™ plus fragment library kit. the sized and ligated fragments have been amplified by emulsion-pcr using the ion onetouch template kit and instruments. assembly and further bioinformatics analyses the reads resulting from the sequencing of the vtx2-phage dna from the eahec o111:h strain, termed phi- have been assembled in contigs by using the open source mira software integrated in the ion torrent server. contigs have been imported in kodon software for analysis. to fill in the gaps between contigs, a total of primers have been designed and used for sequencing by sanger technology using a genetic analyzer. mauve software has been used to order the contigs using the sequence of the phage p from the e. coli o104:h that caused the outbreak in germany in as reference. the complete sequence of the phi- phage has been annotated by prokka tool on the online server galaxy/crs and submitted to genbank. the g + c content has been analysed by the gc calculator free online tool. identification of putative trna genes has been performed using trnascan-se. the raw sequence data from the eahec o111:h strain were retrieved from the sra database present on ncbi website and aligned on the complete sequence of phi- phage, determined in the present study and used as reference, with the bowtie free software implemented in the galaxy/crs server. genomic comparisons between the available vtx-phages sequences have been performed by using the blast algorithm available at ncbi and the mauve free alignment software. comparison map between vtx-phages has been generated by circoletto online tool. for the pictogram construction, bit-score values have been used to describe the quality of the alignment at a given point. the bit-score is a normalized version of the score value returned by the blast searches, expressed in bits. ethics this work does not include animal testing and does not report human data. all the information regarding outbreaks and cases of infection are all from already published papers properly referenced in the text.  sequencing of the vtx2-phage from the eahec o111:h2 the whole genome sequencing of the vtx2-phage from the eahec o111:h strain, phi- produced reads of a mean length of 204 bp, for a total of  mb sequenced. the assembly of the phi- sequence reads using as a reference the genome of the p phage produced contigs that were further analysed using bioinformatics resources. the estimated coverage of the phage genome was 1088x. the length of the whole sequence of phi- resulted to be about kb with a mean g + c content of %.sequence annotation revealed the presence of predicted coding sequences including the genes encoding the subtype 2a of the verocytotoxin and three transfer rnas.figure genomic organization of phi- phage. coding sequences are represented as blue bars in the outer circle. putative genes are labelled according to the predicted functions of their products, if known. the whole gc content and gc skew of leading and lagging strand is shown in the black and coloured inner circles, respectively. as it has been reported for the p phage, conveying the vtx genes in the eahec o104:h strain that caused the german outbreak in, the genome of phi- only included the lambda genes ci and cro while the other genes typically composing the regulatory repertoire of lambda phages such as cii, ciii, n, ea and gam seemed not to be present. most of these genes are involved in the regulation of the switch between lysogeny and lytic cycle in lambda-like phages. therefore, their absence suggests the existence of an alternative mechanism used to regulate the choice of entry into lysogenic state. the phi- phage is integrated in the bacterial chromosome in the wrba locus, as it has been described for p. comparison of the phi- whole genome sequence with other vtx-phage sequences a blast search of the phi- vtx2-phage whole genome sequence against those collected in the nucleotide repository held at ncbi, returned a 99% identity with that of the p phage from o104:h strain cb which caused the german outbreak in and a 91% identity with that of the vtx2-phage tl-2011c carried by the vtec o103:h strain that caused a severe hus outbreak in norway in. this finding is in agreement with how reported in the literature for the vtx-phage carried by the eahec o104:h from the german outbreak and the tl-2011c phage. as expected, the phi- phage sequence also showed 99% identity with the vtx2-phage from another eahec o104:h strain isolated during the german outbreak, the strain 2011c- and was highly related to the sequence of two vtx2-phages from two eahec o104:h strains isolated from as many haemorrhagic colitis cases occurred in georgia in. the other scores included hits with query coverage values ranging from 87% down to 60% and having 98%-99% sequence identity, with a number of other vtx-phages identified in different vtec strains. the alignment of all the phage sequences comprised in the 100%-60% similarity range is shown in a pictogram generated with the circoletto online tool and in another one produced by mauve software.table list of the blast hits of phi- dna sequence aligned to the vtx -phages sequences from typical vtec strains the similarity score indicates the query coverage values with 98%-99% sequence identity. the hits with similarity values down to 60% are shown. sequence similarities between phi- and other vtx -phages. the picture shows the results of the blast local alignments using phi- as a query against the vtx-phage sequences with 99% to 60% similarity listed in table  the colours codes blue, green, orange and red represent the overall quality of the aligned segments along the phage sequences, evaluated on the basis of the bit-score values in the worst-to-the-best order. the bit-score is a normalized version of the score value returned by the blast searches, expressed in bits. the height of the coloured bars in the histogram on the top of the phi- ideogram shows how many times each colour hits a specific fragment of the other phage sequences. a twist in a ribbon indicates that the local alignment is inverted. recently, another eahec strain of serotype o111:h, isolated from an hus case occurred in northern ireland in has been described. the short reads from the whole genome-sequencing project of this strain are available in ncbi sequence reads archive and have been used for comparison. unfortunately, among the contigs obtained from the de novo assembly of the reads, the one including the vtx genes was only  bp long, hindering the analysis of the entire sequence of the vtx2-encoding phage. nevertheless, its whole-genome sequencing reads were aligned to the complete genomic sequence from the phi- phage. the alignment was carried out with bowtie software and visualized on the integrative genomics viewer free tool. this analysis failed to identify, in the genome of strain a vtx2-phage with characteristics similar to that of phi- in particular, the alignment showed that the reads from strain mapped on the sequence of the phi- phage only in the region comprising the vtx a subunit-coding gene and extending  kb downstream the gene encoding the vtx b subunit. it should be noted that alignments conducted using the whole short reads set could have generated a too high background noise deriving from the presence of sequences from other bacteriophages integrated in different regions of the chromosome of the o111:h strain nevertheless, the lack of sequencing reads aligning to the rest of the sequence of phi- suggests that a different vtx phage intervened in the emergence of this last eahec strain. genomic comparison of phi- with other vtx2-phages in order to delve into the genomic organization of the different vtx2-phages we performed a progressive mauve alignment between all the phages sequences used in the nucleotide comparison. this analysis onfirmed the presence of similarities between the sequences but also highlighted that an extensive re-arrangement between the sequence-blocks must have occurred at some points during the phages’ evolution. this observation is in agreement with how reported in literature. a deeper analysis of the phi- genome was conducted by using phage sequences selected among those from typical vtec displaying the highest similarity values. the alignment of the phi- sequence with those of phage tl-2011c, and the phages from the vtec strains rm and, the causative agent of a romaine lettuce-associated outbreak occurred in the us in and isolated in japan from a sporadic case of diarrhoea, respectively, showed the presence of two sequence blocks that seemed to be peculiar to the eahec vtx2-phage and divergent from the same regions in the three classical vtec associated-phages. one of the two regions was  bp long and comprised 140 bp of the 5’-terminus of a gene encoding a lysozyme, two complete genes encoding the lysis protein s and a hypothetical protein, and 440 bp of the 3’-terminus of a gene annotated as a hypothetical protein. the  bp region corresponded to the nucleotidic positions - in the p genome. the other fragment was 900 bp long and spanned the region comprised between nucleotides and in the genome of the p phage. the 900 bp fragment shared 100% homology with a region of 730 bp at the 3’-terminus of the orf of p phage encoding a phage tail fiber and a 72 bp fragment of the orf coding for a tail fiber adesine.figure mauve progressive alignment of vtx2 -phage genomes from eahec and vtec strains showing the eahec-specific regions. blocks with the same colours indicate the vtx2-phages regions with identical dna sequence. white blocks in a phage sequence indicate regions lacking of correspondence in the other sequences. fragments of the phage genomes that are peculiar to eahec vtx2-phages are marked with circles: a red circle indicates the sequence encoding a hypothetical protein; the sequence encoding the phage fiber tail is encircled in blue. distribution and analysis of the putative eahec-associated sequence regions amongst the vtx-phages a blast comparison was conducted with the aim of investigating the distribution of the two putative eahec-associated regions among all the vtx-phage sequences available at ncbi. such an analysis showed that only the 900 bp region encoding phage fiber tail was present in all the fully sequenced vtx2-phages from eahec strains, and divergent or absent in all the other phage sequences investigated. accordingly, the presence of the sole sequence of 900 bp was assessed in the contigs derived from the de novo assembly of the short reads from the genome sequence of the o111:h strain from northern ireland. discussion the e. coli genome continuously changes through both small-scale variations and horizontal gene transfer of mobile genetic elements. among mges, bacteriophages play a pivotal role in the evolution of e. coli pathogenic clones by providing a mean for genomic remodelling and conveying important virulence genes such as those encoding the verocytotoxins in the verocytotoxin-producing e. coli. in a huge outbreak caused by an enteroaggregative haemorrhagic e. coli o104:h struck germany with more than cases of infection, hus, and deaths. the causative agent was a mosaic strain deriving from the lysogenization of an enteroaggregative e. coli strain with a vtx2-phage. such a virulence combination was indeed associated with elevate pathogenicity, as demonstrated by the high rate of human infections evolving to hus, even among adults, and the heavy toll of deaths. this arrangement of virulence factors in e. coli strains from human disease had been occasionally reported before during the investigation of a small outbreak of hus occurred in france in and a case of infection in japan in. the occurrence of the german outbreak caused the scientific community to look back retrospectively at the repositories of vtec infections records and culture collections and it turned out that some other sporadic cases of infections with vtx2-producing enteroaggregative e. coli o104:h had already occurred in europe and asia in the time span 2001–. finally a hus case occurred in northern ireland in was demonstrated to be associated with an eahec o111:h. the observation of sporadic cases and outbreaks occurring throughout a 20-years time span, all caused by vtx2-producing eaggec and belonging to different serotypes, strengthens the hypothesis that these pathogenic e. coli represent a new pathogroup, as it has been recently proposed. to better understand the events underlying the emergence of eahec we determined the whole genome sequence of phi- the vtx2-phage present in the eahec o111:h isolated during the french outbreak of and compared it with the sequences of the vtx2-phage present in the eahec strains described in the following years and available in genbank. interestingly, the genomic sequence of phi- was almost identical to that of the vtx2-phages from the eahec o104:h strains isolated during the german outbreak about 20 years later. this is noteworthy since vtx-phages are characterized by a high degree of variability. it is conceivable that the same vtx2-phage has been acquired in two different events and that the selective pressure impeded the accumulation of changes in the phage sequence before the phage infection events occurred. however, the eahec o111:h isolated in northern ireland in seems to host a different type of vtx2-phage, suggesting that at least two different vtx2-phage types have been successfully transferred to eaggec. unfortunately, the sequence of the phage of the eahec o86: hnm isolated in japan in was not available for comparison. it has been hypothesized that the infection with a lambdoid phage can be mediated by the cross-talking between the bacterium and the phage resulting in host specificity. an extended comparison of the eahec vtx2-phages with the whole genome sequences of vtx-phages from vtec strains available at ncbi returned a wide range of similarities between sequences, going from 87% to 60% and lower. this picture is in line with how reported for the general variability of vtx-phages sequences. interestingly, one region of 900 bp, identified in the phi- and encoding a tail fiber, was present in all the vtx2-phages from eahec and was also present in the short reads dataset from the eahec o111:h at the same time this dna sequence was absent in all the vtx-phage sequences identified in vtec strains and stored at ncbi. this is in agreement with previously reported data which pointed at a larger fragment including this region as one of those differentiating the p genome from the e. coli phage tl-2011c and not exhibiting significant homology to known vtx-encoding phages. the annotation of the phi- genome showed that this sequence peculiar to eahec vtx2-phages contains part of a gene encoding a type of phage tail fiber displaying some conserved aminoacidic motifs such as a collagen triple helix repeat and the peptidase_s. the latter is the c-terminal domain of the bacteriophage protein endosialidase, which forms homotrimeric molecules and releases itself from the end-tail-spike of the bacteriophages. the 900 bp-long sequence could potentially encode part of the mechanism defining the specificity of the vtx2-phages for eaggec strains, being directly involved in the phage-bacterium interaction. as a matter of fact, several authors reported that the interactions between phage tail fibers and host proteins, such as lamb and ompc contribute to the success of the infection, as demonstrated by the finding that lamb gene mutations block phage adsorption. it is therefore conceivable that differences in phage tail fibers may contribute to define vtx2-phages tropism for e. coli recipients, although this hypothesis together with the mechanisms underlying this process still need to be verified. for a successful infection to occur, suitable vtx-phages and e. coli acceptors need to meet in the same environment. in the case of the emergence of typical vtec, the events of vtx-phage acquisition probably occurred at the level of the gastrointestinal tract of ruminants where both vtx-phages and aepec are abundant. conversely, the eahec emergence is probably not directly connected to an animal reservoir since eaggec are human pathogens with an inter-human transmission of the infection. the environment, in turn, plays a role in the pathogen’s amplification cycles, particularly in geographic areas characterised by poor hygienic conditions, where the lack of effective human sewage treatments make the infections with enteric pathogens, including eaggec, endemic. in such a scenario, an environment contaminated with ruminant’s excreta might have been the source of the vtx2-phages found in eahec as it has been recently proposed. such a picture may account for the existence of a favourable setting for the eaggec and the vtx-phages to come in contact and for the following selection process resulting in the occasional emergence of an e. coli strain matching the eahec definition.  the vtx2-phages characterising eahec seem to belong to a sub-population of vtx-phages kept under selective pressure and characterised by the presence of a gene encoding a tail fiber, which could be involved in the mechanism used to recognize the eaggec. the new eahec pathogroup may have emerged following multiple vtx2-phage acquisition events favoured by an overlapping of a human reservoir of pathogenic e. coli, the eaggec, with the known animal reservoir of vtx-phages. the emergence of this new e. coli pathogroup further witnesses the great adaptability and plasticity of this bacterial species and underlines the need to rethink the global asset of hygienic practices to mitigate enteric infections worldwide; particularly in the presence of a global market of foodstuffs that is extending its boundaries towards low-income countries in the quest of new sources to meet the always increasing demand of cheap and exotic food commodities. electronic supplementary material additional file 1: figure s1: mauve progressive alignment of phi- genome with vtx2-phage genomes from eahec and vtec strains showing the highest score of similarity. blocks with the same colours indicate the vtx2-phages regions with identical dna sequence. white fragments in a phage sequence indicate regions lacking of correspondence in the other sequences. connecting lines link the same genomic block in different genomes and help to pinpoint the re-arrangement between vtx-phage genomes. abbreviations aepecatypical enteropathogenic escherichia coli decdiarrheagenic escherichia coli eaggecenteroaggregative escherichia coli eahecenteroaggregative haemorrhagic escherichia coli hchaemorrhagic colitis hushaemolytic uremic syndrome mgemobile genetic elements vtecverocytotoxin-producing escherichia coli vtxverocytotoxin genes. competing interests the authors declare that they have no competing interests. authors’ contributions gl carried out the infection experiments, contributed to the sequencing and drafted the manuscript; mv performed the whole genome sequencing and bioinformatics analysis; tr contributed to all the steps of the whole genome sequencing and revised critically the manuscript; rp carried out part of infection experiments and cscl gradient; ma helped in the revision of the draft manuscript; ca contributed to the revision of the draft manuscript; ms conceived the study and strongly contributed to revise the manuscript. finally, all the authors approved the manuscript to be published. 
at least two different vtx2-phages, both characterized by the presence of a peculiar tail fiber-coding gene, intervened in the emergence of eahec. the comparison of the vtx2-phages sequences from eahec strains with those from the vtx-phages of typical verocytotoxin-producing e. coli strains showed the presence of a 900 bp sequence uniquely associated with eahec phages and encoding a tail fiber. so far, four distinct eahec serotypes have been described that caused, beside the large outbreak of infection occurred in germany in a small outbreak and six sporadic cases of hus in the time span 1992– in the present work we determined the whole genome sequence of the vtx2-phage, termed phi- present in the first described eahec o111:h isolated in france in and compared it with those of the vtx-phages whose sequences were available. the whole genome sequence of the phi- phage was identical to that of the vtx2-phage p present in the eahec o104:h strain isolated during the german outbreak 20 years later. enteroaggregative haemorrhagic e. colivtx-phageswhole genome sequencetail fibersissue-copyright-statement© the author 2014 enteroaggregative haemorrhagic e. coli is a new pathogenic group of e. coli characterized by the presence of a vtx2-phage integrated in the genomic backbone of enteroaggregative e. coli. the observation that different eahec infections have been traced back to countries where eaggec infections are endemic and the treatment of human sewage is often ineffective suggests that such countries may represent the cradle for the emergence of the eahec pathotype. moreover, it was also almost identical to those of the other vtx2-phages of eahec o104:h strains described so far. conversely, the phi- phage appeared to be different from the vtx2-phage carried by the eahec o111:h isolated in the northern ireland in the finding of an identical vtx2-phage in two eaggec strains isolated after 20 years in spite of the high variability described for vtx-phages is unexpected and suggests that such vtx2-phages are kept under a strong selective pressure.
 enteroaggregative haemorrhagic e. coli is a new pathogenic group of e. coli characterized by the presence of a vtx2-phage integrated in the genomic backbone of enteroaggregative e. coli. so far, four distinct eahec serotypes have been described that caused, beside the large outbreak of infection occurred in germany in a small outbreak and six sporadic cases of hus in the time span 1992– in the present work we determined the whole genome sequence of the vtx2-phage, termed phi- present in the first described eahec o111:h isolated in france in and compared it with those of the vtx-phages whose sequences were available.  the whole genome sequence of the phi- phage was identical to that of the vtx2-phage p present in the eahec o104:h strain isolated during the german outbreak 20 years later. moreover, it was also almost identical to those of the other vtx2-phages of eahec o104:h strains described so far. conversely, the phi- phage appeared to be different from the vtx2-phage carried by the eahec o111:h isolated in the northern ireland in the comparison of the vtx2-phages sequences from eahec strains with those from the vtx-phages of typical verocytotoxin-producing e. coli strains showed the presence of a 900 bp sequence uniquely associated with eahec phages and encoding a tail fiber.  at least two different vtx2-phages, both characterized by the presence of a peculiar tail fiber-coding gene, intervened in the emergence of eahec. the finding of an identical vtx2-phage in two eaggec strains isolated after 20 years in spite of the high variability described for vtx-phages is unexpected and suggests that such vtx2-phages are kept under a strong selective pressure. the observation that different eahec infections have been traced back to countries where eaggec infections are endemic and the treatment of human sewage is often ineffective suggests that such countries may represent the cradle for the emergence of the eahec pathotype. in these regions, eaggec of human origin can extensively contaminate the environment where they can meet free vtx-phages likely spread by ruminants excreta. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords enteroaggregative haemorrhagic e. colivtx-phageswhole genome sequencetail fibersissue-copyright-statement© the author 2014 