shiga toxin ( stx ) producing escherichia coli ( stec ) were first described as vero toxin producing e. coli ( vtec ) leading to bloody diarrhea ( hemorrhagic colitis ) and the hemolytic uremic syndrome ( hus ) about 30  years ago [ 1 , 2 ] . hus is characterized by hemolytic anemia , thrombocytopenia , acute kidney injury ( aki ) , and , in severe cases , neurologic complications . numerous outbreaks , as well as sporadic cases of stec infections and hus , have been documented worldwide . this , however , does not necessarily mirror an increased frequency of stec infections in developed countries but , rather , results from a reporting bias due to diagnostic resources , nationwide reporting systems , and publication activities . alternatively , in contrast to locally restricted farming in developing countries , modern industrialized large - scale food production might serve as a widespread vector in cases of food contamination . stec strains carry phages that encode shigatoxins 1 and/or 2 , also known as vero toxins or verocytotoxins [ 2 , 4 ] . for both stx 1 and stx 2 , enterohemorrhagic e. coli ( ehec ) , the  classical  subset of stec , possess the eae gene of enteropathogenic e. coli as an additional virulence factor conferring adherence to the intestinal mucosa . stec not harboring eae were long believed to be less virulent but have also been shown to be the causative agent of stec outbreaks [ 6 , 7 ] . the pathogenesis of hus development is incompletely understood . in brief , stec are incorporated by macrophages and m cells of the colon mucosa and produce toxin , which is released when the host cell undergoes apoptosis . after local and systemic distribution , stx causes damage to vascular endothelial cells of the kidney and brain by direct cytotoxicity , as well as indirectly via cytokine secretion , complement activation , and subsequent generalized disease . however , in some geographic regions , including germany , non - o157 serotypes have been reported to account for up to half of hus cases [ 10 , 11 ] . in germany , stec were commonly recognized as pathogens causing rare but severe disease almost exclusively in younger children . before 2011 , about 1,000 infections per year and fewer than 100 cases of hus were registered throughout germany . this review focuses on the 2011 outbreak of a stec of serotype o104:h4 , with unusual genetic properties leading to atypical epidemiologic and clinical presentation . new developments in the diagnosis and treatment of stec and hus are highlighted , as well as questions remaining open . however , most information about stec disease and its therapeutic implications was drawn from larger food - borne outbreaks , because the responsible strains were usually well characterized and epidemiological and clinical data were sufficiently available to gain statistically solid insight . the 2011 outbreak of a stec o104:h4 in northern germany started at the beginning of may , reaching its peak on may 22 . until july 4 , when the end of the outbreak was officially declared , 855 cases of hus , 2,987 cases of acute gastroenteritis , and 53 deaths were registered in germany [ 13 , 14 ] . additionally , 83 ehec cases and 54 cases of hus were recorded outside germany , most of them linked to travel to northern germany in may 2011 [ 14 ] . fenugreek sprouts were identified as the most likely vehicle of infection and were traced back to a single producer . despite all efforts , the strain could not be isolated from the implicated seeds . shortly after the discovery of sprouts as the source of infection , a smaller outbreak by stec o104:h4 was reported from western france , including 24 patients with 7 cases progressing to hus . here the outbreak proved a major challenge to clinicians , microbiologists , and epidemiologists , not only because of its severity , but also due to its unusual presentation . in contrast to earlier outbreaks , the majority of patients were adults , predominantly middle - aged and otherwise healthy women . the incidence rate of hus ( 22  % ) was higher than reported in previous outbreaks ( 1  % 15  % ) [ 17 ] and was accompanied by an unusually high number of cases with severe neurological symptoms ( aphasia , seizures , and delirium ) . the incubation time was approximately 8  days , which is longer than the incubation period reported for stec o157 during an outbreak in washington in 1994 . early during the outbreak , the national hus reference center identified the strain as being of serotype o104:h4 and as belonging to a clone ( husec041 ) originally isolated from an hus patient in germany in 2001 [ 20 ] . previously , there were reports of six sporadic cases of hus caused by a stec o104:h4 in germany , france , korea , and georgia ; however , molecular fingerprinting of the available strains revealed them to be not identical . the 2011 outbreak strain combined virulence factors of both enteroaggregative e. coli ( eaec ) and stec , harboring the paa plasmid of eaec , as well as a stx 2 producing phage , combined with an extended spectrum betalactamase encoding plasmid [ 20 ] , and was therefore called an  enteroaggregative stec . whole genome sequencing was undertaken , and its results were also available early on during the outbreak [ 2224 ] . moreover , a multiplex polymerase chain reaction pcr assay for the parallel detection of several characteristic virulence factors ( stx2 , terd , rfbo104 , flic h4 ) was rapidly developed and made publicly available [ 20 ] . despite the observation that the constitutive stx production of stec o104:h4 was lower , as compared with o157:h7 strains , the proportion of patients developing hus was greater than in most outbreaks before . this led to speculation about a possible hypervirulence of stec o104:h4 , potentially mediated by a facilitated stx uptake due to the very efficient adherence of eaec to the intestinal mucosa . while the core genome of stec o104:h4 is highly similar to other eaec of the same serotype , the stx - phage was shown to be most closely related to the phage of an o111:h- strain   and has probably been acquired quite recently in the phylogenetic history of the outbreak strain . during the outbreak , the mean shedding period was reported to be more than 34  days for stec o104:h4 in patients not receiving antibiotics [ 27 ] , with some of them still shedding the pathogen more than 1  year after the outbreak ( unpublished data ) . these data indicate that the outbreak strain might be carried for longer periods , as compared with stec o157 , for which a maximal shedding period of 124  days was reported . there are numerous reports on stec o157:h7 as the most common serotype associated with hus , especially in children . the historical outbreak most comparable to the recent german epidemic was likewise linked to sprouts and took place in japan in 1996 ( sakai city outbreak ) . about 6,000 persons were affected after white radish sprouts had been served at school canteens , exposing 47,000 children to the contaminated food [ 29 , 30 ] . as compared with the german outbreak in 2011 , the incidence of hus in the sakai city outbreak was considerably lower ( 106 cases out of 6,000 infections ) . also in 1996 , another outbreak of a stec o157:h7 in scotland involved 512 persons , of whom 279 cases were microbiologically confirmed . in this outbreak , the majority of patients developing hus were adults ( 28 of the 34 ) . another massive outbreak of o157:h7 that gained particular attention happened in the western u.s . in 1993 , comprising 501 cases , 151 hospitalizations , and 45 cases of hus . this outbreak was linked to consumption of undercooked hamburger meat at a fast food chain   and promoted the ( misleading ) opinion that ground beef is the primary vehicle for stec infections . several reports on non - o157 stec underline their potential to cause sporadic disease as well as epidemics . one of the largest non - o157 stec outbreaks was caused by stec o111:nm in oklahoma in 2008 . out of 341 cases with gastroenteritis , 71 patients required hospitalization . the hus rate was 17  % , and more than 50  % of hus patients were adults . in 1992 , stec o111:h2 was isolated from the stools of 5 out of 10 children hospitalized with hus and of 3 healthy contact persons in south picardy , france . later on , this strain was shown to adhere to epithelial cells in an aggregative pattern and to possess the eaec virulence plasmid , while also producing stx 2 . however , this eaec was consistently shown to have the potential of causing severe gastroenteritis in the absence of shiga toxins . the largest eaec outbreak occurred in japan in 1993 , when 2,967 children developed severe gastroenteritis after having consumed the same school lunch . stools from 30 patients were studied , and 27 strains of an e. coli ont : h10 were isolated . the strain adhered to epithelial cells in vitro in the typical aggregative pattern of eaec but was negative for all virulence factors of diarrheagenic e. coli , including shiga toxins except for the presence of the enteroaggregative heat - stable toxin . these and other reports show the high diversity of stec strains and the potential of pathogenic e. coli to acquire virulence factors by horizontal gene transfer , resulting in new hybrid phenotypes and  atypical  clinical presentations . this challenges the detection of new stec strains and the prediction of their pathogenicity , underlining the need for further investigation . the 2011 outbreak of a stec o104:h4 in northern germany started at the beginning of may , reaching its peak on may 22 . until july 4 , when the end of the outbreak was officially declared , 855 cases of hus , 2,987 cases of acute gastroenteritis , and 53 deaths were registered in germany [ 13 , 14 ] . additionally , 83 ehec cases and 54 cases of hus were recorded outside germany , most of them linked to travel to northern germany in may 2011 [ 14 ] . fenugreek sprouts were identified as the most likely vehicle of infection and were traced back to a single producer . despite all efforts , the strain could not be isolated from the implicated seeds . shortly after the discovery of sprouts as the source of infection , a smaller outbreak by stec o104:h4 was reported from western france , including 24 patients with 7 cases progressing to hus . here again , consumption of fenugreek sprouts was associated with infections . the outbreak proved a major challenge to clinicians , microbiologists , and epidemiologists , not only because of its severity , but also due to its unusual presentation . in contrast to earlier outbreaks , the majority of patients were adults , predominantly middle - aged and otherwise healthy women . the incidence rate of hus ( 22  % ) was higher than reported in previous outbreaks ( 1  % 15  % ) [ 17 ] and was accompanied by an unusually high number of cases with severe neurological symptoms ( aphasia , seizures , and delirium ) . the incubation time was approximately 8  days , which is longer than the incubation period reported for stec o157 during an outbreak in washington in 1994 . early during the outbreak , the national hus reference center identified the strain as being of serotype o104:h4 and as belonging to a clone ( husec041 ) originally isolated from an hus patient in germany in 2001 [ 20 ] . previously , there were reports of six sporadic cases of hus caused by a stec o104:h4 in germany , france , korea , and georgia ; however , molecular fingerprinting of the available strains revealed them to be not identical . the 2011 outbreak strain combined virulence factors of both enteroaggregative e. coli ( eaec ) and stec , harboring the paa plasmid of eaec , as well as a stx 2 producing phage , combined with an extended spectrum betalactamase encoding plasmid [ 20 ] , and was therefore called an  enteroaggregative stec . whole genome sequencing was undertaken , and its results were also available early on during the outbreak [ 2224 ] . moreover , a multiplex polymerase chain reaction pcr assay for the parallel detection of several characteristic virulence factors ( stx2 , terd , rfbo104 , flic h4 ) was rapidly developed and made publicly available [ 20 ] . despite the observation that the constitutive stx production of stec o104:h4 was lower , as compared with o157:h7 strains , the proportion of patients developing hus was greater than in most outbreaks before . this led to speculation about a possible hypervirulence of stec o104:h4 , potentially mediated by a facilitated stx uptake due to the very efficient adherence of eaec to the intestinal mucosa . while the core genome of stec o104:h4 is highly similar to other eaec of the same serotype , the stx - phage was shown to be most closely related to the phage of an o111:h- strain   and has probably been acquired quite recently in the phylogenetic history of the outbreak strain . during the outbreak , the mean shedding period was reported to be more than 34  days for stec o104:h4 in patients not receiving antibiotics [ 27 ] , with some of them still shedding the pathogen more than 1  year after the outbreak ( unpublished data ) . these data indicate that the outbreak strain might be carried for longer periods , as compared with stec o157 , for which a maximal shedding period of 124  days was reported there are numerous reports on stec o157:h7 as the most common serotype associated with hus , especially in children . the historical outbreak most comparable to the recent german epidemic was likewise linked to sprouts and took place in japan in 1996 ( sakai city outbreak ) . about 6,000 persons were affected after white radish sprouts had been served at school canteens , exposing 47,000 children to the contaminated food [ 29 , 30 ] . as compared with the german outbreak in 2011 , the incidence of hus in the sakai city outbreak was considerably lower ( 106 cases out of 6,000 infections ) . also in 1996 , another outbreak of a stec o157:h7 in scotland involved 512 persons , of whom 279 cases were microbiologically confirmed . in this outbreak , the majority of patients developing hus were adults ( 28 of the 34 ) . contaminated meat products were identified as the outbreak source [ 31 , 32 ] . another massive outbreak of o157:h7 that gained particular attention happened in the western u.s . in 1993 , comprising 501 cases , 151 hospitalizations , and 45 cases of hus . this outbreak was linked to consumption of undercooked hamburger meat at a fast food chain   and promoted the ( misleading ) opinion that ground beef is the primary vehicle for stec infections . several reports on non - o157 stec underline their potential to cause sporadic disease as well as epidemics . one of the largest non - o157 stec outbreaks was caused by stec o111:nm in oklahoma in 2008 . the hus rate was 17  % , and more than 50  % of hus patients were adults . in 1992 , stec o111:h2 was isolated from the stools of 5 out of 10 children hospitalized with hus and of 3 healthy contact persons in south picardy , france . later on , this strain was shown to adhere to epithelial cells in an aggregative pattern and to possess the eaec virulence plasmid , while also producing stx 2 . however , this eaec was consistently shown to have the potential of causing severe gastroenteritis in the absence of shiga toxins . the largest eaec outbreak occurred in japan in 1993 , when 2,967 children developed severe gastroenteritis after having consumed the same school lunch . stools from 30 patients were studied , and 27 strains of an e. coli ont : h10 were isolated . the strain adhered to epithelial cells in vitro in the typical aggregative pattern of eaec but was negative for all virulence factors of diarrheagenic e. coli , including shiga toxins except for the presence of the enteroaggregative heat - stable toxin . these and other reports show the high diversity of stec strains and the potential of pathogenic e. coli to acquire virulence factors by horizontal gene transfer , resulting in new hybrid phenotypes and  atypical  clinical presentations . this challenges the detection of new stec strains and the prediction of their pathogenicity , underlining the need for further investigation . during the 2011 outbreak in northern germany , clinicians were confronted with a large number of mainly adult patients with hus associated with severe hemolysis and neurological complications [ 18 , 36 ] . a large proportion of patients required renal replacement therapy . however , we also observed some stec - infected patients with severe neurological disorders but lacking renal or hematologic signs of hus ( unpublished data ) . prior to the outbreak , no standardized causative treatment existed for stec - hus , and randomized clinical trials approving any therapeutic concept to be beneficial beyond best supportive therapy were missing . therefore , different therapeutic concepts were rapidly proposed   on the basis of theoretical considerations and preceding observations , but without evidence for the effectiveness of such  best guess  strategies . moreover , these ad hoc strategies were adjusted to new observations   made during the outbreak . thus , individual medical centers used varying therapeutic regimens [ 17 ] , including plasmapheresis , glucocorticoids , and the anti - c5 monoclonal antibody eculizumab . the use of plasmapheresis was based on observations from the scotland outbreak in 1996 , with 24 adult patients suffering from hus . in a multicenter case control analysis of the 2011 german outbreak with stec o104:h4 , no short term benefit for plasmapheresis with or without additional application of glucocorticoids was found in 251 patients receiving plasmapheresis , as compared with 47 patients without plasma exchange [ 17 ] . in an additional single - center analysis of 130 hus patients , again no benefit of plasmaphesesis was observed , whereas the risk of severe aki and neurological complication was increased . likewise , analysis of 90 children with hus caused by stec o104:h4 did not justify recommending plasma exchange in pediatric hus . this is in accordance with a recent prospective follow - up study of 274 children showing that plasma exchange therapy was associated with a poor long - term outcome for children with stec - hus . unfortunately , most of these studies are limited by their retrospective character and biased by indication , since sicker patients were more often treated with plasmapheresis . taken together , the relevance for plasma exchange therapy in stec - hus is highly questionable and might even be adverse . a small group of 12 patients with severe neurological complications were enrolled in a non - controlled trial with immunoadsorption therapy   after several other interventions were assumed to be ineffective . hence , the authors stated that antibodies could play a role in the pathogenesis of neurological disorders and that immunoadsorption might amend the neurological symptoms . this study is limited by very heterogeneous treatment procedures and a very small sample size . at the end of may 2011 , a case series of three children suffering from stec - hus was published reporting rapid clinical improvement under therapy with eculizumab . therefore , starting from the end of may , over 300 patients of the german outbreak were treated with eculizumab off - label , initially as a compassionate use trial that was transformed to a nonrandomized trial including 198 patients . a short - term outcome analysis of 67 patients treated with eculizumab outside the above - mentioned trial showed no significant therapeutic benefit , as compared with a matched control group not having received eculizumab [ 17 ] . however , in many medical centers , eculizumab was administered only to patients who had no clinical improvement during plasmapheresis and/or were suffering from severe neurological complications , which might imply a selection bias . therefore , no definite conclusions can be drawn concerning the effect of eculizumab on the course of hus . however , the treatment with eculizumab can be considered as a save intervention , and therefore , its prognostic impact , as well as optimal time - point and dosage , should be evaluated in future prospective randomized trials . a major issue discussed during the outbreak was the question of whether or not antibiotics should be used . in previous retrospective analyses of stec outbreaks and sporadic infections , patients treated with antibiotics were found to have an increased risk of hus development [ 32 , 44 ] . this dogma was recently confirmed by a large multicenter trial investigating risk factors for the development of hus in ehec o157:h7 infected children . therefore , the use of antibiotics was strongly discouraged unless secondary complications made antibiotic treatment urgent . in our center , we strictly abstained from using antibiotics unless it was unavoidable . however , since the c5a antibody eculizumab disrupts the complement cascade and , thereby , increases the risk for bacterial meningitis , antibiotic meningitis prophylaxis was mandatory in patients receiving eculizumab . because several in vitro studies did not show any induction but , rather , suppression of stx expression by azithromycin , this antibiotic was selected for recommendation by the german society of nephrology in the ad hoc guidelines for meningitis prophylaxis to be given orally for 14  days . the close monitoring of stec - shedding in stool samples revealed that all patients receiving azithromycin were rapidly decolonized from the outbreak strain , while untreated patients remained positive for stec significantly longer [ 27 ] . in detail , among antibiotic - treated patients , long - term stec carriage ( > 28  days ) was observed in 1 of 22 patients ( 4.5  % ; 95  % ci , 0  % 13.3  % ) , as compared with 35 of 43 patients ( 81.4  % ; 95  % ci , 69.8  % 93.0  % ) who were not treated with antibiotics ( p  <  .001 ) . all 22 patients receiving azithromycin had at least 3 stec - negative stool specimens after the completion of treatment , and no recurrence of stec was observed . therefore , as a proof of principle , a 3-day course of oral azithromycin was offered as decolonization therapy to long - term carriers ( > 28  days ) of stec o104:h4 who were initially not treated with antibiotics , if they were restricted in their social or working life ( e.g. , ban from work ) . all 15 patients treated with azithromycin for stec decolonization had negative stool specimens after the 3-day course , without any deterioration of renal function or development of other hus - related symptoms [ 27 ] . therefore , successful decolonization treatment was extended to more than 40 patients , without any adverse effects until now ( unpublished data ) . such decolonization regimen has always to be weighed cautiously against the risk of other potential , pathogen - independent adverse drug side effects . the in vitro finding that azithromycin does not induce stx expression was confirmed recently for stec o104:h4 by bielaszewska et al . [ 48 ] . comparing the effect of subinhibitory concentrations of several antibiotics on the induction of stx production of stec o104:h4 , they found that ciprofloxacin increased stx production , while meropenem , rifaximin , tigecycline , and azithromycin did not [ 48 ] . interestingly , stec o104:h4 appears to respond differently to antibiotics , as compared with the  classical  stec strains o157:h7 . in the situation of acute stec disease , the differential response of various stec strains to antibiotics requires an early evaluation of these interactions in order to enable precise warnings or recommendations concerning antibiotic treatment . despite the in vitro induction of shiga toxin expression by quinolones , preemptive therapy of stec - hus patients with a combination therapy of meropenem and ciprofloxacin in one other medical center resulted in statistically significant reduction of death , seizures , and stec shedding [ 17 ] . however , it has to be taken into account that all promising results concerning the use of antibiotics for the treatment of stec during the german stec o104:h4 outbreak were retrieved either in patients suffering already from hus or in clinically recovered , now asymptomatic long - term carriers with a shedding time of at least 28  days . therefore , at present , no definite conclusions can be drawn for the use of antibiotics in acute stec diarrhea . any antibiotics should be handled cautiously in patients with acute bloody diarrhea caused by stec until their benefit is approved in controlled trials . during the northern german outbreak of stec o104:h4 in 2011 , new aspects regarding the therapy of stec infections and hus were investigated retrospectively , raising new questions that still need to be answered for stec disease in general . from a clinical point of view the use of plasma exchange therapy in stec - hus can be regarded as obsolete . the previous dogma that antibiotics are absolutely contraindicated in stec disease needs to be revised . especially in patients with already established hus , as well as in long - term stec carriers who have recovered from their acute gastrointestinal manifestation , treatment with azithromycin however , there is no approved indication for antibiotic treatment during the acute stec - related bloody diarrhea . new diagnostic methods for immediate serotyping , as well as rapid analysis of phage induction and modulation of shiga toxin expression by different antibiotics , are essential for clinical decision making ( e.g. , antibiotic therapy ) in future stec infections .
in 2011 , a large outbreak caused by a shiga toxin producing e. coli ( stec ) occurred in northern germany , with a satellite outbreak in western france , including the highest number of hemolytic uremic syndrome ( hus ) cases ever encountered during a stec outbreak . 
 the outbreak strain was characterized as an enteroaggregative e. coli of serotype o104:h4 expressing a phage - encoded shiga toxin 2 . 
 the majority of stec infections and hus cases were observed in adults , with a preponderance of the female gender . 
 the outbreak imposed huge challenges on clinicians , microbiologists , and epidemiologists but also provided important new insight for the understanding of stec infection . 
 thus , novel therapeutic strategies in the treatment of hus in adults and for decolonization of long - term stec carriers were evaluated . 
 this review highlights the unusual features of the recent o104:h4 outbreak and focuses on emerging new strategies in diagnostics and treatment of acute stec - related disease , as well as stec long - term carriage .