Whole genome sequence comparison of vtx2-converting phages from Enteroaggregative Haemorrhagic Escherichia coli strains
© Grande et al.; licensee BioMed Central Ltd. 2014
Received: 15 April 2014
Accepted: 3 July 2014
Published: 8 July 2014
Enteroaggregative Haemorrhagic E. coli (EAHEC) 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 (EAggEC). So far, four distinct EAHEC serotypes have been described that caused, beside the large outbreak of infection occurred in Germany in 2011, a small outbreak and six sporadic cases of HUS in the time span 1992–2012. In the present work we determined the whole genome sequence of the vtx2-phage, termed Phi-191, present in the first described EAHEC O111:H2 isolated in France in 1992 and compared it with those of the vtx-phages whose sequences were available.
The whole genome sequence of the Phi-191 phage was identical to that of the vtx2-phage P13374 present in the EAHEC O104:H4 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:H4 strains described so far. Conversely, the Phi-191 phage appeared to be different from the vtx2-phage carried by the EAHEC O111:H21 isolated in the Northern Ireland in 2012.
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.
KeywordsEnteroaggregative haemorrhagic E. coli vtx-phages Whole genome sequence Tail fibers
Diarrheagenic Escherichia coli (DEC) are a heterogeneous group of pathogenic E. coli causing a wide range of enteric diseases in humans and animals .
Enteroaggregative E. coli (EAggEC) 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 [2, 3]. 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 [4, 5].
In 2011, a large E. coli outbreak struck Germany causing more than 4,000 human infections including 50 deaths . The outbreak strain, an E. coli O104:H4, 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 (Vtx) subtype 2a (vtx2a) . In accordance with this genomic asset, the strain showed the Enteroaggregative typical “stacked brick” adhesion to cultured Hep-2 cells and was able to produce Vtx2 . 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 (VTEC), a DEC type causing haemorrhagic colitis and Haemolytic Uremic Syndrome (HUS) 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 (EAHEC) . 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 1992–2012 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:H2, O86: HNM, O104:H4 and O111:H21 [8, 10–13].
The analysis of the whole genome sequence of the EAHEC O104:H4 that caused the German outbreak in 2011 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 (aEPEC) from which the typical VTEC pathotype derives [15–17]. Moreover, the ability of vtx2-phages to infect, in the laboratory conditions, different E. coli pathogroups including ExPEC has been reported [18, 19]. This observation, together with the isolation of Enterobacteriaceae other than E. coli producing Vtx from cases of human disease [20, 21] 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:H4 and O111:H21 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.
The EAHEC O111:H2 strain ED 191 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 K12 strain LE392  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 191 has been determined. The occupancy of loci sbcB, wrbA, yehV, Z2577, and yecE has been assessed as previously described .
Infection experiments and phages propagation
The EAHEC strain ED 191 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 (LB) broth (Oxoid Limited, Basingstoke Hampshire, UK) overnight at 37°C with vigorous shaking. The culture has been diluted 1:100 in LB modified broth (LB with 0.001% thiamine V/V) and grown to 0.5 OD 600, pelleted and re-suspended in a sterile solution of CaCl2 10 mM. The culture has been exposed to UV light (130 μJoule X 100) in a crosslinker “Stratalinker® UV crosslinker” (Stratagene Cloning Systems, La Jolla, CA, USA). 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 0,22 μm pore-filters. 100 μl of phage particles suspension have been added to 100 μl of a culture of the propagator strain E. coli LE392 grown in LB modified broth at 0.5 OD 600 and maintained at 37°C for 20 minutes with static incubation. Each tube has been added with 3.5 ml of LB modified soft agar (LB modified broth with agar 7 g/L) at 42°C and immediately poured on LB modified agar plates (LB modified broth with 15 g/L agar). Plates have been incubated overnight at 37°C.
Four ml of SM buffer (100 mM NaCl, 8 mM MgSO4·7H2O, 50 mM Tris–HCl 1 M pH 7.5, Gelatin 0.002%) 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 LE392 in the conditions described above in order to increase the phage titre. Finally, the phage suspension has been concentrated by using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 tubes (Merck Millipore, Billerica, MA, USA) with a cut-off of 30 KDa. Final phage titre was 7 × 1010 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 1.5 g of CsCl, transferred to ultracentrifuge tubes, which have been filled with a CsCl solution 0.75 g/ml. The tubes have been finally sealed with mineral oil and centrifuged in a Beckman ultracentrifuge at 154,000xg, 8°C for 20 hours in a SW-41 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 8.0, 10 mM MgCl2. A final volume of 1 ml was obtained.
The suspension has been treated by adding 100 units of DNase I RNase-free (New England Biolabs, USA) 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 239.4 ng/μl.
Library preparation and whole genome sequencing of the phage DNA
Phage DNA has been sequenced with an Ion Torrent PGM semiconductor sequencer (Life Technologies, Carlsbad, USA) using the 200 bp protocol. An Ion Torrent 314 chip has been used following the manufacturer instructions (Life Technologies, Carlsbad, USA). 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 (Life Technologies, Carlsbad, USA). The sized and ligated fragments have been amplified by emulsion-PCR using the Ion OneTouch 200 Template kit and instruments (Life Technologies, Carlsbad, USA).
Assembly and further bioinformatics analyses
The reads resulting from the sequencing of the vtx2-phage DNA from the EAHEC O111:H2 strain, termed Phi-191, 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 (Applied Maths NV, Sint-Martens-Latem, BE) for analysis. To fill in the gaps between contigs, a total of 95 primers have been designed and used for sequencing by Sanger technology using a Genetic analyzer 3130 (Life Technologies, Carlsbad, USA). Mauve software  has been used to order the contigs using the sequence of the phage P13374 from the E. coli O104:H4 that caused the outbreak in Germany in 2011 [GenBank: NC_018846.1] as reference. The complete sequence of the Phi-191 phage has been annotated by Prokka tool on the online server Galaxy/CRS4  and submitted to GenBank [GenBank: KF971864]. 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 (short reads) from the EAHEC O111:H21 strain 226 were retrieved from the SRA database present on NCBI website [NCBI SRA: SRA055981] and aligned on the complete sequence of Phi-191 phage, determined in the present study and used as reference, with the Bowtie2 free software implemented in the Galaxy/CRS4 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 [29, 30]. 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 .
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:H2 strain, Phi-191, produced 320,044 reads of a mean length of 204 bp, for a total of 65.30 Mb sequenced. The assembly of the Phi-191 sequence reads using as a reference the genome of the P13374 phage [GenBank: NC_018846.1] produced 151 contigs that were further analysed using bioinformatics resources. The estimated coverage of the phage genome was 1088X.
As it has been reported for the P13374 phage, conveying the vtx2 genes in the EAHEC O104:H4 strain that caused the German outbreak in 2011 , the genome of Phi-191 (Figure 1) 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, Ea10 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-191 phage is integrated in the bacterial chromosome in the wrbA locus, as it has been described for P13374 .
Comparison of the Phi-191 whole genome sequence with other vtx-phage sequences
A BLAST search of the Phi-191 vtx2-phage whole genome sequence against those collected in the nucleotide repository held at NCBI, returned a 99% identity with that of the P13374 phage from O104:H4 strain CB13374 which caused the German outbreak in 2011 [GenBank:NC_018846.1] and a 91% identity with that of the vtx2-phage TL-2011c carried by the VTEC O103:H25 strain that caused a severe HUS outbreak in Norway in 2006 [GenBank:JQ011318] . This finding is in agreement with how reported in the literature for the vtx-phage carried by the EAHEC O104:H4 from the German outbreak and the TL-2011c phage . As expected, the Phi-191 phage sequence also showed 99% identity with the vtx2-phage from another EAHEC O104:H4 strain isolated during the 2011 German outbreak, the strain 2011C-3493 [GenBank:CP0032891.1] and was highly related (97% nucleotide sequence identity) to the sequence of two vtx2-phages from two EAHEC O104:H4 strains isolated from as many haemorrhagic colitis cases occurred in Georgia in 2009 [GenBank:CP003301.1, CP003297.1] .
List of the BLAST hits of Phi-191 DNA sequence aligned to the vtx -phages sequences from typical VTEC strains
E. coli O145:H28 str. RM13514
E. coli O103:H2 str. 12009 DNA
Phage VT2 phi_272
E. coli O157:H7 str. TW14359
E. coli O157:H7 str. EC4115
E. coli O111:H- str. 11128
E. coli O157:H7 str. Sakai
E. coli Xuzhou21
E. coli O157:H7 EDL933
Stx2 converting phage II
Stx2 converting phage I
Enterobacteria phage Min27
Stx1 converting phage
Recently, another EAHEC strain of serotype O111:H21 (strain 226), isolated from an HUS case occurred in Northern Ireland in 2012, has been described . The short reads from the whole genome-sequencing project of this strain are available in NCBI sequence reads archive [NCBI SRA: SRA055981] and have been used for comparison. Unfortunately, among the 456 contigs obtained from the de novo assembly of the reads, the one including the vtx2 genes was only 8,042 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-191 phage. The alignment was carried out with Bowtie2 software and visualized on the Integrative Genomics Viewer (IGV) free tool  (data not shown). This analysis failed to identify, in the genome of strain 226, a vtx2-phage with characteristics similar to that of Phi-191. In particular, the alignment showed that the reads from strain 226 mapped on the sequence of the Phi-191 phage only in the region comprising the Vtx2 A subunit-coding gene and extending 4.2 kb downstream the gene encoding the Vtx2 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:H21 strain 226. Nevertheless, the lack of sequencing reads aligning to the rest of the sequence of Phi-191 suggests that a different vtx2 phage intervened in the emergence of this last EAHEC strain.
Genomic comparison of Phi-191 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 (Additional file 1: Figure S1). 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 [46–48].
A deeper analysis of the Phi-191 genome was conducted by using phage sequences selected among those from typical VTEC displaying the highest similarity values (Table 1).
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, [GenBank:CP0032891.1; CP003301.1; CP003297.1] 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:H21 strain 226 from Northern Ireland.
The E. coli genome continuously changes through both small-scale variations and horizontal gene transfer of mobile genetic elements (MGE). 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 (vtx1 and vtx2) in the Verocytotoxin-producing E. coli (VTEC).
In 2011 a huge outbreak caused by an Enteroaggregative Haemorrhagic E. coli (EAHEC) O104:H4 struck Germany with more than 3,000 cases of infection, 800 HUS, and 50 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 (88% and 42 years of median age), and the heavy toll of 50 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 1992 and a case of infection in Japan in 1999 [10, 11].
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:H4 had already occurred in Europe and Asia in the time span 2001–2011 [12, 31]. Finally a HUS case occurred in Northern Ireland in 2012 was demonstrated to be associated with an EAHEC O111:H21 .
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-191, the vtx2-phage present in the EAHEC O111:H2 isolated during the French outbreak of 1992, 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-191 was almost identical to that of the vtx2-phages from the EAHEC O104:H4 strains isolated during the 2011 German outbreak about 20 years later. This is noteworthy since vtx-phages are characterized by a high degree of variability [50, 51]. 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:H21 isolated in Northern Ireland in 2012 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 1999 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-191 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:H21. 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 P13374 genome from the E. coli phage TL-2011c (O103:H25) and not exhibiting significant homology to known vtx-encoding phages .
The annotation of the Phi-191 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 (20 copies) [NCBI CDD:189968] and the Peptidase_S74 [NCBI CDD:258151]. 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 [52, 53] 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 [55, 56].
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.
Atypical enteropathogenic Escherichia coli
Diarrheagenic Escherichia coli
Enteroaggregative Escherichia coli
Enteroaggregative haemorrhagic Escherichia coli
Haemolytic uremic syndrome
Mobile genetic elements
Verocytotoxin-producing Escherichia coli
We thank Prof. Muniesa for hosting the corresponding author at University of Barcelona, Spain, where the basic techniques for working with phages have been acquired.
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