escherichia coli of serotype o157:h7 was first recognized in 1982 as a human pathogen associated with outbreaks of bloody diarrhea in the united states and is now considered a major cause of foodborne infections ( 1 , 2 ) . the virulence of e. coli o157:h7 depends on the presence of a number of mobile genetic elements ( mge ) , such as shiga toxin ( stx)-converting bacteriophages carrying different genes encoding stx1 and stx2 , the virulence plasmid po157 , the locus of enterocyte effacement ( lee ) , o islands , an arginine translocation system , and various adhesion factors ( 3 ) . in stx - producing e. coli ( stec ) , stx is thought to be responsible for the most severe form of the infection causing the life - threatening hemorrhagic colitis ( hc ) and hemolytic uremic syndrome ( hus ) ( 4 ) . strains causing these clinical symptoms are also known as enterohemorrhagic e. coli ( ehec ) strains ( 5 ) . the genes encoding stxs ( stx1 and stx2 ) are found in lysogenic lambdoid bacteriophages ( 6 ) , which can integrate into the host chromosome via specific insertion sites . integration sites for stx2-converting phages in stec o157:h7 include wrba , argw , sbcb , and yece , whereas stx1-converting phages integrate in the yehv region ( 711 ) . stec o157:h7 strains generally do not ferment sorbitol ( non - sorbitol - fermenting [ nsf ] strains ) , and this feature is widely used to identify these pathogenic strains . nevertheless , sorbitol - fermenting ( sf ) stec o157:nm ( nonmotile ) strains , as an emerging and important pathogen in europe , have been isolated from patients with hus and diarrhea ( 12 ) . both sf and nsf o157:h7/nm strains are thought to have evolved from a common nonpathogenic ancestor of serotype o55:h7 following the modification of the o - antigen gene cluster and the acquisition of a number of virulence - associated mge via horizontal gene transfers ( 13 ) . the most current and accepted evolutionary model proposes that e. coli o157:h7 lost the o55 rfb - gnd gene cluster and acquired the stx2 bacteriophage and the o157 rfb - gnd gene cluster . after the diversification of the two lineages , e. coli o157:h7 acquired stx1 via acquisition of the bacteriophage containing the stx1 gene and lost the ability to ferment sorbitol , while the sorbitol - fermenting stx2-producing e. coli o157 lost its motility and evolved into nonmotile e. coli o157:nm ( 14 , 15 ) . most e. coli o157 isolates produce a large outer membrane protein , intimin ( encoded by the eae gene ) , which is the genetic determinant of the formation of attaching and effacing ( a / e ) lesions , a central mechanism in the pathogenesis of enteropathogenic e. coli ( epec ) ( 16 ) . as described in previous studies , stx - negative e. coli o157:h7/nm isolates were obtained from patients with hus and diarrhea . it was assumed , especially for the hus cases , that these patients were originally infected with an ehec strain and that excision of the stx bacteriophage occurred during the infection . those stx - negative o157:h7/nm isolates appeared to be closely related to stx - positive o157:h7 isolates as determined by conventional molecular typing methods ( 1821 ) . in this study , whole - genome sequencing ( wgs ) was used to obtain a more detailed molecular characterization of stx - negative e. coli o157:h7/nm isolates and to reveal their genetic relationship with stx - positive o157:h7 isolates obtained from patients with gastrointestinal complaints . fecal samples were collected from patients with gastrointestinal complaints in the regions of groningen and rotterdam during the period april 2013 to march 2014 as part of a large multicenter study ( stec - id - net , unpublished data ) . samples were screened for the presence of stx1 , stx2 , and escv ( used as an alternative marker for the lee instead of the eae gene ) and for o - serogroup determination ( o26 , o103 , o104 , o111 , o121 , o145 , and o157 ) by real - time pcr as described previously ( 22 ) . this resulted in the collection of 34 e. coli o157 isolates ( with or without stx ) from 34 different patients . pcr for the detection of the flich7 gene was performed as described before ( 23 ) , and only the isolates positive for flich7 were then subjected to wgs . the publically available genomes of e. coli o157:h7 strains sakai , edl933 , and ss52 , e. coli o157:h45 strain c639_08 , e. coli o127:h6 strain e2348/69 , and e. coli o55:h7 cb9615 were also included in the comparative analysis . moreover , as no complete genomes of sf stec o157:nm and stx - negative o157:h7/nm were available in the ncbi database when the study was performed , we sequenced the genomes of one sf stec o157:nm ( e09/10 ) strain and one stx - negative o157:nm ( e09/224 ) strain and included them as control strains in all the analyses . the information on the isolates used in this study is presented in table s1 in the supplemental material . sorbitol fermentation was determined using ct - smac ( sorbitol macconkey agar with cefixime and tellurite ) plates , and motility was tested using motility test medium with triphenyltetrazolium chloride ( mediaproducts bv , groningen , the netherlands ) . the production of beta - glucuronidase and urease was checked using macconkey ii agar with 4-methylumbelliferryl--d - glucuronide ( mug ) ( bd diagnostics , breda , the netherlands ) and urea - triple sugar iron ( tsi ) agar ( mediaproducts bv , groningen , the netherlands ) , respectively . the o and h serotypes of the isolates were determined by seroagglutination performed at the national institute for public health and the environment ( rivm , bilthoven , the netherlands ) . dna was extracted using the ultraclean microbial dna isolation kit ( mo bio laboratories , carlsbad , ca , usa ) according to the manufacturer 's protocol . a dna library was prepared using the nextera xt kit ( illumina , san diego , ca , usa ) according to the manufacturer 's instructions and then run on a miseq instrument ( illumina ) for generating paired - end 250-bp reads , aiming at a coverage of at least 60-fold . de novo assembly was performed using clc genomics workbench v7.0.3 ( clc bio a / s , aarhus , denmark ) after quality trimming ( qs , 28 ) with optimal word sizes based on the maximum n50 value . annotation was performed by uploading the assembled genome on the rast server version 2.0 ( 24 ) . the sequence type ( st ) was identified by uploading the assembled genomes to the multilocus sequence type ( mlst ) server ( version 1.7 ) ( 25 ) , and the virulence genes and stx subtypes were determined with virulence finder 1.2 ( 26 ) . the serogenotype of the isolates was determined using the cge serotypefinder tool ( 27 ) . to analyze the sequence homology of the lee pathogenicity island in the isolates , the contigs of each sample were subjected to a blast search against the lee region of e. coli o157:h7 strain 71074 ( accession no . gq338312 ) as a reference and plotted by blast ring image generator ( brig ) ( 28 ) . to identify plasmids , po157 ( accession no . nc_009602 ) of the e. coli o157:nm strain 3072/96 were used as references for blast analyses . for identifying proposed marker genes for differentiating nsf stec o157:h7/nm from sf stec o157:nm , including the complete cdt cluster , the complete sequence of efa1 , the tellurite resistance- and adherence - conferring pathogenicity island ( tai ) , and the urease gene cluster ( 29 ) , contigs were either subjected to a blast search using blastn ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) or mapped by clc genomics workbench v7.0.3 using default settings against a reference sequence artificially generated by concatenating sequences of the marker genes . to determine the phylogenetic relationship of the isolates , a gene - by - gene typing approach was performed using seqsphere v1.0 ( ridom gmbh , mnster , germany ) . briefly , an in - house defined mlst+ ( also referred to as core genome [ cg ] mlst ) scheme was developed using the genome of e. coli o157:h7 strain sakai as reference genome to extract open reading frames ( orfs ) from the genome of each isolate by seqsphere . only the orfs without a premature stop codon and ambiguous nucleotides from contigs of assembled genomes were included . the genes shared by the genomes of all isolates analyzed were defined as the core genome for phylogenetic analysis ( 30 , 31 ) . a neighbor - joining ( nj ) tree was constructed based on a distance matrix among the isolates depending on the core genomes of all isolates . the most common and well described stx phage integration sites for stec o157:h7/nm were analyzed in our stx - negative isolates to reveal if any phage was occupying these sites or that they were available for future integration of a phage , including one that contains stx genes . five integration sites were studied : yehv ( for the stx1 phage ) , wrba and argw ( for the stx2a phage ) , sbcb ( for the stx2c phage ) , and yece ( for the stx2a phage of sf stec ) ( 11 ) . these five loci were identified in the contigs , the adjacent regions were extracted , and the presence of phage integrases was detected using the blastn algorithm . the genbank accession numbers of the isolates analyzed in this study are ldoz00000000 , lfua00000000 , lfub00000000 , lgaz00000000 , lfuh00000000 , lgba00000000 , lgbb00000000 , lgbc00000000 , lgbd00000000 , lgbe00000000 , lgbf00000000 , lgbg00000000 , lgbh00000000 , lgbq00000000 , lgbi00000000 
 , lgbj00000000 
 , lgbk00000000 
 , lgbl00000000 
 , lgbm00000000 
 , lgbn00000000 
 , lgbo00000000 
 , and lgbp00000000 
 . among 34 e. coli o157 isolates initially obtained in this study , 16 were stec ( all were flich7 positive ) and 18 were categorized as epec ( as they were stx negative but eae positive ) . among the 18 epec isolates , four o157:h7 , for the subsequent comparative study , 20 isolates positive for flich7 ( 16 stec o157:h7 and 4 epec o157:h7/nm ) were used . the phenotypic characteristics of the 20 o157:h7 isolates of this study and the two control strains are shown in table 1 . the 16 stec o157:h7 isolates did not ferment sorbitol ( nsf ) and were beta - glucuronidase negative , whereas among the four epec isolates , one ( epec 287 ) was nsf and beta - glucuronidase negative . the remaining three isolates ( epec 393 , epec 1572 , and epec 1669 ) and the control strain ( e09/224 ) were sorbitol fermenting ( sf ) and beta - glucuronidase positive , as was the sf stec control strain e09/10 ( see table s1 in the supplemental material ) . among the nsf stec isolates , phenotypic and molecular characteristics of the isolates nsf , non - sorbitol fermenting ; sf , sorbitol fermenting ; nm , nonmotile . the type of the eae gene was determined from wgs data using blastn . this isolate was obtained from germany and used as a control strain for sf stec o157:nm isolates . one of these four isolates was obtained from germany and used as a control strain for the stx - negative o157:nm isolates . stx subtyping of stec isolates revealed eight ( 50% ) isolates with stx1a and stx2c subtypes , two ( 12.5% ) with stx1a and stx2a subtypes , and six ( 37.5% ) with the stx2c subtype only . all isolates contained the o - serotyping gene wzxo157 and the flagellar genes flich7 and eae ( type gamma ) and were assigned to st11 ( table 1 ) . all nsf stec isolates had identical virulence profiles , with exception of isolate stec 1109 , which contained the cdt - v gene , encoding cytolethal distending toxin . some of the virulence genes , such as serine protease autotransporter - encoding gene espp , toxin - encoding gene toxb , catalase peroxidase - encoding gene katp , the tellurite resistance- and adherence - conferring island ( tai ) , and the urease gene cluster , were present only in nsf stec and in the only nsf epec isolate but not in any sf isolates . on the other hand , sfpa , the complete efa1 , and the cdt gene cluster were present only in sf isolates . the sf epec isolates carried almost all the virulence genes carried by the german sf stec o157:nm isolate , with the exception of the tccp gene ( encoding tir cytoskeletal coupling protein ) , which was present only in sf epec isolates . distribution of virulence and other genes among stx - positive and stx - negative o157:h7/nm isolates encoding cytolethal distending toxin a , b , and c subunits . tellurite resistance- and adherence - conferring island carrying adhesin gene iha and putative tellurite resistance genes tlra , tlrb , tlrc , and tlrd . ure gene cluster containing urea , ureb , urec , ured , uree , uref , and ureg . all the stec isolates contained an lee region highly similar to the lee of stec o157:h7 strain 71074 , used as a reference . however , some nsf stec isolates lacked two genes encoding the mobile element proteins orfa and orfb located in the insertion sequence is911 . three stec isolates ( stec 2257 , stec 2820 , and stec 2821 ) did not possess the intl gene , which is known to encode an integrase of the putative prophage 933l carried in the lees of other stec o157:h7 isolates . the sequences of the lees of our nsf and sf epec isolates were more similar to those of nsf and sf stec , respectively , than to the lee of epec reference genomes e2348/69 and c639_08 ( fig . 1 ) . comparison of lee pathogenicity islands , showing a blast comparison of stec and epec isolates , depicted by each ring , against the reference lee sequence ( core black circle ) . the colors of different groups as well as the order of the rings for each isolate ( from inner to outer ) with the color gradient for sequence identity are shown at the right . no sequence variation was observed in regions carrying putative virulence genes such as , e.g. , genes involved in the type ii secretion system , hemolysins , toxins , and catalase peroxidase in po157 of nsf stec . however , with the exception of the two stx2a - positive isolates ( stec 2112 and stec 2868 ) , all stec isolates lacked the po157p35 gene , encoding a reverse transcriptase . only three stec isolates ( stec 605 , stec 989 , and stec 1109 ) harbored the plasmid posak1 . among the epec isolates , the nsf one ( epec 287 ) had almost the intact po157 plasmid , lacking only an intact espp gene . the sf epec isolates contained an almost identical copy of plasmid psfo157 of sf stec o157:nm ( fig . 2 ) . comparison of plasmids , showing a blast comparison of stec and epec isolates , depicted by each ring , against the reference plasmid composed of three plasmids shown in the outermost ring by three different colors ( black , blue , and orange represent plasmids po157 , poska1 , and psfo157 , respectively ) . the color of the rings represents sequence identity on a sliding scale ; the more gray the ring is , the lower the percent identity . the colors of different groups as well as the order of the rings for each isolate ( from inner to outer ) with the color gradient for sequence identity are shown at the right . core genome phylogenetic analysis was performed to evaluate the evolutionary relationship between the stx - positive ( stec ) and stx - negative ( epec ) o157:h7/nm isolates . in total , 3,005 orfs were shared by all isolates analyzed in this study , and these were defined as the core genome for phylogenetic analysis . this analysis separated epec c639_08 and epec e2348/69 from the o157:h7/nm isolates in this study ( fig . the latter isolates formed two separated clusters : sf isolates ( cluster 1 ) and nsf isolates ( cluster 2 ) . remarkably , in cluster 1 , four sf epec isolates ( epec 393 , epec 1572 , epec 1669 , and e09/224 ) clustered together with the sf stec isolate . cluster 2 ( nsf o157:h7 isolates ) could be divided into three subclusters : cluster 2a , containing stec o157:nm ( nonmotile ) isolates together with one nsf epec isolate ( epec 287 ) ; cluster 2b , containing six of the motile stec o157:h7 isolates ; and cluster 2c , containing two stx2a - positive isolates ( stec 2112 and stec 2868 ) clustered closely with two previously described stec outbreak isolates , sakai and edl933 ( 1 , 32 ) . the last subcluster also included two other motile isolates ( stec 1109 and stec 989 ) and stec o157:h7 strain ss52 ( stx2a and stx2c positive ) , isolated from super shedder cattle ( 33 ) . taken together , the data indicate that the epec o157:nm isolates clustered with stec isolates but not with epec isolates ( fig . the nj tree was constructed based on a distance matrix among the isolates depending on their core genomes . in the sf epec strains ( epec 393 , epec 1572 , epec 1669 , and epec e09/224 ) , the phage insertion sites analyzed were intact , with the exception of argw , which was occupied in isolates epec 393 and epec 1572 . in epec 287 , although yehv and argw were occupied by phages , the wrba and sbcb loci ( integration sites for stx2a and stx2c phage , respectively ) were unoccupied . a red arrow indicates the presence of a phage integrase adjacent to the integration site . ( a ) sf stec isolate e09/10 ; the yece region is occupied by phage . ( e ) nsf epec isolate epec 287 ; the yehv region occupied by phage . e. coli o157:h7 is one of the major causes of foodborne illness and represents a considerable public health concern worldwide ( 34 ) . this study aimed at determining the phylogenetic relationships and comparing the virulence factors of stx - negative e. coli o157:h7/nm with those of stx - positive e. coli o157:h7 with the highest resolution and greatest possible detail using a wgs approach . the e. coli o157:h7 isolates used in this study were obtained from patients with diarrhea and other gastrointestinal complaints from two different regions in the netherlands ( groningen and rotterdam ) . isolates e09/10 and e09/224 were sequenced and used as control strains for sf stec o157:nm and stx - negative o157:nm isolates , respectively . as the stx - negative isolates were found to be positive for eae but negative for the bfpa gene , they were considered atypical epec . notably , the epec isolates belonged to st11 and contained type gamma intimin , which are not typical features of epec but are frequently associated with stec o157:h7 ( 35 ) . remarkably , all the typical genes ( e.g. , ehxa , asta , lpfa , katp , etpd , espp , the pathogenicity island tai , and the urease gene cluster ) of nsf stec o157:h7 described previously were present in epec 287 . in contrast , sf epec isolates possessed the sfp gene cluster , the cdt gene cluster , and the complete efa1 gene but lacked the genes toxb , katp , espp carried on plasmid po157 , the urease gene cluster , and the pathogenicity island tai , which are typical features of sf stec o157:nm ( 18 , 29 ) . subsequent analyses showed that epec 287 harbored a po157 plasmid almost identical to that of the stec isolates , with sequence variability mostly in mobile genetic elements . the sf epec isolates contained a plasmid similar to the psfo157 present in the sf stec o157:nm isolate strain 3072/96 . additionally , our nsf and sf epec isolates shared an almost identical lee pathogenicity island with the stec isolates containing additional orfs encoding a putative prophage normally not present in the lee of epec ( 36 ) . all these results together suggest that the epec o157:h7/nm isolates investigated shared a virulence profile similar to that of stec isolates rather than to the virulence profile of epec . the genetic relationship of the o157:h7/nm isolates of this study was confirmed by the phylogenetic analysis using a gene - by - gene comparison by core genome mlst . the nsf epec isolate clustered together with the nonmotile nsf stec isolates , and the three sf epec isolates clustered together with the control strain of sf stec o157:nm . it has already been described that the nonmotile characteristic of sf stec o157 is due to a 12-bp deletion in the master regulator of flagellar biosynthesis , the flhc gene ( 37 ) . we also observed this deletion in the same gene in our nonmotile sf epec isolates but not in our nonmotile nsf stec isolates , further strengthening the hypothesis of their derivation from stx - positive sf o157:nm . taken together , the results of our analyses provide further evidence that the nsf and sf epec isolates were mostly related to the stec group , but lacking the stx - converting phages , and might be referred to as ehec strains that have lost the shiga toxin ( ehec - lst ) , as has also been proposed previously ( 19 , 20 ) . studying in more detail the typical stx phage insertion sites ( yece for sf epec and wrba and sbcb for nsf epec ) revealed that they were unoccupied in the stx - negative isolates . such unoccupied regions could be an indication of loss of the stx bacteriophage , e.g. , in the course of infection or during isolation or subculture ( 38 ) . conversely , stx - negative strains could be progenitors of stec prepared to acquire one or more stx - converting phages ( 18 , 39 ) . in our case , the stx genes were not lost in the laboratory during isolation or subculture , as they were not detected in the feces of the patients . therefore , the patients may have been infected with an stx - negative variant , which could explain the mild symptoms they displayed . in routine diagnostic testing , these stx - negative isolates may be missed , as most microbiological laboratories depend on the molecular screening of stec by the detection of the stx genes only . screening for several additional genes ( including eae , saa , and sfpa ) in routine diagnostics to identify these pathogens has already been proposed ( 18 ) . moreover , our findings bring into question whether classifying pathogenic e. coli into stec and epec based on detection of the stx and eae genes , respectively , is reliable enough . due to integrating and interchanging mobile genetic elements , e.g. , the stx - converting bacteriophages , which could integrate into several e. coli pathogroups , it is sometimes complicated to precisely define the classification of pathogenic e. coli ( 40 , 41 ) . obviously , screening a number of accessory virulence genes of stec would be laborious for routine diagnostics . in our opinion , screening of at least the flich7 gene , together with the o157-encoding gene , may help to identify ehec - lst of the most virulent clone of ehec ( o157:h7 ) , as all the flich7-positive isolates were genetically related to stec o157:h7 in this study . our results are consistent with the finding that the proportion of stx - negative variants among sf o157:nm isolates is generally higher than that among nsf o157:h7 isolates ( 20 , 42 ) . nevertheless , the number of isolates studied was too low to draw firm conclusions . however , to our best knowledge , this is the first report where the genetic relationship of stx - negative variants of e. coli o157:h7/nm with stx - positive o157:h7/nm has been confirmed using wgs . in conclusion , stx - negative e. coli o157:h7/nm strains are a cause of gastrointestinal disease in the netherlands , and because of the presence of the complete set of accessory virulence genes , these isolates should be considered of public health concern similar to that for their stx - positive variants . additional diagnostic approaches should be implemented to identify these ehec - lst isolates for surveillance purposes and to allow appropriate treatment measures and for preventing their transmission .
the ability of escherichia coli o157:h7 to induce cellular damage leading to disease in humans is related to numerous virulence factors , most notably the stx gene , encoding shiga toxin ( stx ) and carried by a bacteriophage . 
 loss of the stx - encoding bacteriophage may occur during infection or culturing of the strain . here , we collected stx - positive and stx - negative variants of e. coli o157:h7/nm ( nonmotile ) isolates from patients with gastrointestinal complaints . 
 isolates were characterized by whole - genome sequencing ( wgs ) , and their virulence properties and phylogenetic relationship were determined . because of the presence of the eae gene but lack of the bfpa gene , the stx - negative isolates were considered atypical enteropathogenic e. coli ( aepec ) . 
 however , they had phenotypic characteristics similar to those of the shiga toxin - producing e. coli ( stec ) isolates and belonged to the same sequence type , st11 . 
 furthermore , epec and stec isolates shared similar virulence genes , the locus of enterocyte effacement region , and plasmids . core genome phylogenetic analysis using 
 a gene - by - gene typing approach showed that the sorbitol - fermenting ( sf ) stx - negative isolates clustered together with an sf stec isolate and that one non - sorbitol - fermenting ( nsf ) stx - negative isolate clustered together with nsf stec isolates . 
 therefore , these stx - negative isolates were thought either to have lost the stx phage or to be a progenitor of stec o157:h7/nm . as detection of stec infections is often based solely on the identification of the presence of stx genes , these may be misdiagnosed in routine laboratories . 
 therefore , an improved diagnostic approach is required to manage identification , strategies for treatment , and prevention of transmission of these potentially pathogenic strains .