An imported case of bloody diarrhea in the Czech Republic caused by a hybrid enteroaggregative hemorrhagic Escherichia coli (EAHEC) O104:H4 strain associated with the large outbreak in Germany, May 2011
A large outbreak caused by a rare Shiga toxin-producing Escherichia coli serotype O104:H4 occurred in Germany in May to July 2011. The National Reference Laboratory for E. coli and Shigella investigated the stool sample from an American tourist with bloody diarrhea who arrived in the Czech Republic from Germany where she consumed salads with raw vegetable a week ago. Using culture of the enriched stool on extended-spectrum β-lactamase agar, we isolated E. coli strain which belonged to serotype O104:H4 as determined by conventional and molecular serotyping. The strain contained the major virulence characteristics of enterohemorrhagic E. coli (stx2 encoding Shiga toxin 2) and enteroaggregative E. coli (aggA encoding aggregative adherence fimbriae I). This unique combination of virulence traits demonstrated that this strain belongs to the hybrid enteroaggregative hemorrhagic E. coli clone which caused the German outbreak. Using advanced culture and molecular biological approaches is the prerequisite for identification of new, unusual pathogens.
Enteroaggregative Escherichia coli
Enterohemorrhagic Escherichia coli
Enteroaggregative hemorrhagic Escherichia coli
Type of β-lactamase (class A)
Type of β-lactamase (class A)
- MALDI-TOF MS
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
National Institute of Public Health
National Reference Laboratory
In May to July 2011, a large outbreak of hemolytic–uremic syndrome (HUS) and bloody diarrhea caused by Shiga toxin (Stx)-producing Escherichia coli O104:H4 occurred in Germany. As of 26 July 2011, when the Robert Koch Institute (RKI) in Berlin, Germany declared the outbreak to be officially over, 3,842 cases of E. coli O104:H4 infections have been reported in Germany, of which 855 (22.2%) were complicated by HUS. Fifty-three (1.4%) patients, including 29 HUS cases, died (RKI final report 2011). There were additional 137 cases of enterohemorrhagic E. coli (EHEC) O104:H4 infection in other 13 European countries, the USA, and Canada, most of which was associated with travel to Germany. Of these, 54 (39.4%) were HUS cases, including 2 deaths (ECDC report 2011). This was the second largest outbreak caused by Stx-producing E. coli (after the Sakai outbreak in Japan in 1996) (Michino et al. 1999; Fukushima et al. 1999), and the largest outbreak of HUS in the history of these pathogens.
France reported a local E. coli O104:H4 outbreak in Bordeaux region (Gault et al. 2011). This outbreak which involved 15 patients, including 8 cases of HUS, was not associated with travel to Germany. However, both the German and the French outbreaks had a common vehicle of transmission, in particular imported fenugreek seeds and sprouts (Gault et al. 2011; Buchholz et al. 2011).
This outbreak was caused by a previously very rare Stx-producing E. coli serotype, O104:H4. The highly virulent E. coli O104:H4 outbreak strain is a hybrid which shares the characteristics of enteroaggregative E. coli (EAEC) and EHEC, including the typical aggregative adherence to epithelial cells and production of Stx2. In contrast to typical EHEC, this strain lacks the eae gene encoding adhesin intimin and EHEC-hlyA gene encoding EHEC hemolysin. Similarly to EHEC O157:H7, the outbreak strain contains the ter gene cluster encoding tellurite resistance (Bielaszewska et al. 2011a). The whole genome sequence analyses of two German outbreak isolates revealed that this strain is most similar to EAEC (more than 90%), but, in contrast to EAEC, it harbors a prophage encoding Stx2, which is typical for EHEC (Brzuskiewicz et al. 2011; Rasko et al. 2011; Mellmann et al. 2011). On the basis of this unusual combination of its genomic features, it has been proposed that the German outbreak strain represents a new pathotype of enteroaggregative hemorrhagic E. coli (EAHEC) (Brzuskiewicz et al. 2011). The outbreak strain contains three plasmids, two larger of which are of particular interest (Rasko et al. 2011). The larger plasmid (ca. 88 kb) designated pESBL contains the genes bla CTX-M-15 [type of β-lactamase (class A)] encoding extended-spectrum β-lactamase agar (ESBL) CTX-M-15 and bla TEM-1 [type of β-lactamase (class A)] encoding TEM-1 β-lactamase. This plasmid encodes resistance of this strain to a broad spectrum of antimicrobials. The smaller plasmid (ca. 75 kb) contains virulence loci typical for EAEC, including aggR encoding master regulator of EAEC plasmid virulence genes and several genes under transcription control of AggR (Rasko et al. 2011). It also contains aggABCD cluster, encoding aggregative adherence fimbriae I (AAF/I), which mediate the typical aggregative adherence of this strain to intestinal epithelial cells (Bielaszewska et al. 2011a).
During the last 12 years, only two Stx-producing E. coli O104:H4 isolates have been reported from patients with HUS. The first strain, which was isolated in 2001 in Germany (Mellmann et al. 2008), has been later included in the HUS-associated E. coli (HUSEC) collection of the National Consulting Laboratory for HUS, University of Münster, Germany as HUSEC041 (Mellmann et al. 2008). The second strain was isolated in 2006 from a 29-year-old woman in Korea (Bae et al. 2006). However, several Stx-producing E. coli O104:H4 isolates from patients with HUS or diarrhea were identified in several laboratories during or after the O104:H4 outbreak by retrospective typing of previously uncharacterized EHEC isolates (Scavia et al. 2011; Monecke et al. 2011; Scheutz et al. 2011; EFSA report 2011).
In the Czech Republic, there is a long tradition of the detection of EHEC infections. As early as in 1988, i.e., only 5 years after E. coli O157:H7 had been reported for the first time as a cause of human diseases in the USA (Riley et al. 1983), the first HUS outbreak in the Czech Republic was identified in North Bohemia (city of Česká Lípa). This outbreak consisted of five HUS cases in children between 4 and 20 months of age, with one of these cases being fatal. EHEC O157:H7 was isolated from feces of the patients (Lhotová et al. 1990). Another HUS outbreak, including four cases of the disease in children between 10 months and 3 years of age, was reported in summer 1995 again in North Bohemia, in the districts of Teplice and Ústí nad Labem. The infection was transmitted via consumption of non-pasteurized milk from a goat that shed EHEC O157:H7 in its feces (Bielaszewska et al. 1997).
During the last 6 years, 27 cases of EHEC infection (diarrhea, bloody diarrhea, HUS) were confirmed in the National Reference Laboratory (NRL) for E. coli and Shigella of the National Institute of Public Health (NIPH), Prague. The most frequently identified EHEC serotypes in the Czech Republic have been O26:H11, O157:H7, and O111:H8/H- (Marejková et al. 2011). Over the last 2 years, three family outbreaks occurred which were caused by EHEC of serogroups O157, O26, and O145. A 2-year-old girl with HUS caused by EHEC O26:H11 died (Marejková et al. 2009).
Methods and case report
The stool sample from this patient at the stage of bloody diarrhea was sent to the NRL for E. coli and Shigella at NIPH on May 30, 2011 from the Department of Infectious Diseases, University Hospital Bulovka, Prague.
EAHEC isolation and phenotyping
The strain was isolated using the stool enrichment in GN broth Hajna (BioVendor, Czech Republic) with novobiocin supplement (Oxoid) for 5 h and subsequent plating of the enriched sample on Columbia blood agar (Oxoid) and selective media including the ESBL agar (Oxoid), glucitol (formerly “sorbitol”) MacConkey agar (SMAC; Oxoid), cefixime–tellurite glucitol MacConkey agar (CT-SMAC; Oxoid) and enterohemolysin agar (Sifin; Labmedia Servis, Czech Republic) for 18 h. The isolated strain was confirmed as E. coli [API 20E, bioMérieux; and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), Microflex, Bruker Daltonics] and serotyped using an E. coli O104 and H4 antisera from Staten Serum Institute, Denmark. β-d-Glucuronosidase activity was assessed using COLItest (Erba Lachema, Czech Republic). Production of Stx by the isolates was detected using the reverse passive latex agglutination test VTEC–RPLA (Denka Seiken, Japan).
The isolate was screened using PCR for EHEC virulence genes including stx1, stx2, eae, and EHEC-hlyA (Friedrich et al. 2002; Paton and Paton 1998), and for EAEC virulence, locus aggA which is a part of the gene cluster encoding AAF/I (Bielaszewska et al. 2011a). In addition, we used multiplex PCR targeting typical molecular features of the outbreak O104:H4 strain (rfbO104, fliCH4, stx2, and terD) which was developed in the National Consulting Laboratory for HUS in Münster, Germany (Bielaszewska et al. 2011a). The multiplex PCR was performed using PCR reaction mix (Top-Bio, Czech Republic) and primers from Generi Biotech (Czech Republic).
A 62-year-old American traveler with diarrhea arrived in the Czech Republic from northern Germany where she had consumed raw vegetables and salads a week ago. She was admitted to the Hospital Na Františku in Prague and then transferred to the Clinic of Infectious, Parasitic and Tropical Diseases of the University Hospital Bulovka on May 28 with 1 week history of abdominal pain and diarrhea, followed during 5 days hereafter by bloody diarrhea. The patient was exhausted, afebrile, with painful urination and reduced urine output. She suffered from an early stage dehydration and had mildly elevated inflammatory markers (CRP = 23.3 mg/L). There were no signs of anemia (red blood cells, 4.47 × 1012/L; hemoglobin, 141.0 g/L), thrombocytopenia (platelet count in range of 471–554 × 109/L), or hemolysis (total bilirubin, 8 μmol/L; lactate dehydrogenase was not estimated). Serum levels of creatinine and blood urea nitrogen were in normal range. Urinalysis revealed microscopic hematuria only after admission. She was without any neurological symptoms all the time.
The patient was treated symptomatically with intravenous fluids for rehydration, diosmectite (Smecta), APO-Lactobacillus, No-SPA, and vitamin K (Kanavit). Her condition improved gradually, and she was discharged after 5 days with the instruction to see her physician when she is back in the USA to be checked for her complete blood count and renal parameters.
PCR genotyping and multiplex PCR targeting the typical features of the German outbreak strain confirmed that the E. coli strain isolated from ESBL agar has all the characteristics typical for the EAHEC O104:H4, i.e., it possessed stx2, aggA, rfbO104, fliCH4, and terD. The strain did not possess stx1, eae, and EHEC-hlyA genes. The presence of somatic O104 and flagellin H4 antigens was confirmed by slide agglutination, and production of Stx2 was verified by reverse passive latex agglutination.
EAHEC O104:H4 is phenotypically classical E. coli, i.e., Gram-negative and lactose-, β-glucuronosidase-, and indol-positive rod. The strain has an enormous motility, and the colonies could be agglutinated directly from the plate (without boiling) using monovalent antiserum O104. The strain grows well on all common media used for isolation of Enterobacteriaceae. On media for isolation of EHEC, such as SMAC agar and CT-SMAC agar, EAHEC O104:H4 grows in pink, glucitol-positive colonies, similar to most normal E. coli flora, which makes its isolation from these media difficult. The ability to grow on CT-SMAC demonstrates that the tellurite resistance phenotype encoded by the ter cluster (Bielaszewska et al. 2011b) is expressed in this strain. The ESBL phenotype expressed by the outbreak strain (resistance to all penicilins, cefalosporin, and co-trimoxazole) has facilitated an easy isolation of this strain from the stool sample on the ESBL medium where the strain grew in almost pure culture.
After the EAHEC O104:H4 strain had been isolated from the American traveler, additional nine stool specimens from patients with bloody diarrhea were sent to our laboratory for screening for this organism. All the patients had a history of recent travel to Germany. However, none of these stool specimens contained EAHEC O104:H4.
This is the first and probably the only case of bloody diarrhea in the Czech Republic caused by the EAHEC O104:H4 strain that caused the large German outbreak in May–July 2011. The travel history of the patient to Germany where she consumed salads containing the sprouts epidemiologically incriminated as a vehicle of transmission of the outbreak strain (EFSA report 2011) is in accordance with the microbiological findings. Results from epidemiological studies in Germany and also from France, where a cluster of EAHEC infections in patients without travel history to Germany occurred (Gault et al. 2011), support the hypothesis that seeds used for sprouting (distributed to local producers or retail outlets) contained the EAHEC O104:H4 contamination. This ultimately led to recall of the contaminated sprouts from the market (ECDC report 2011). The EFSA technical report published on 5 July concluded that a specific lot of fenugreek seeds imported from Egypt were most probably the vehicle of the EAHEC O104:H4 infection; however, it cannot be excluded that other lots might also have been implicated. The exact point of contamination of the seeds/sprouts in the food chain has not been determined, but it is speculated that this occurred before their import (EFSA report 2011).
The largest food-borne E. coli outbreak with similar vehicle of transmission was reported in Japan in three Sakai districts in 1996, where elementary school children were infected with EHEC O157:H7 after consumption of white radish sprouts from a particular farm, which were served as a part of a school lunch (Michino et al. 1999). According to Fukushima et al. (1999), 12,680 symptomatic patients including putative secondary infections were infected in this massive outbreak; 121 cases developed HUS, and 3 girls died. However, similar to the EAHEC outbreak strain in Germany, the EHEC O157:H7 strain associated with the Sakai outbreak has never been isolated from the sprouts. The possible reason for the failure to isolate the EAHEC O104:H4 strain from the sprouts might be its ability to enter the viable but non-culturable state which might be a form in which it might survive in the environment (Aurass et al. 2011).
The recent (2011) EAHEC outbreak was the largest E. coli outbreak ever reported in Germany, with a very atypical age and sex distribution of the cases. The majority of cases involved adults, while till this time the EHEC infections were largely a pediatric problem. The number of cases affecting women outnumbered men in both HUS (68%) and EHEC infections (58%) (RKI final report 2011).
We conclude that the successful isolation of the EAHEC O104:H4 strain from the human stool in the NRL for E. coli and Shigella and subsequent prompt characterization of this strain as a close relative of the German outbreak strain were only possible by using new culture and molecular biological approaches, which is the prerequisite for identification of new, unusual pathogens.
The first author is indebted to Prof. Dr. H. Karch, the Head of the National Consulting Laboratory for Hemolytic–Uremic Syndrome at the Institute for Hygiene, University of Münster (Germany) for the possibility to get trained in this laboratory in diagnosis of EHEC infections. The authors are also thankful to Assoc. Prof. Dr. M. Bielaszewska (from the above Institute) for assistance provided to the Czech NRL for E. coli and Shigella, especially during the hectic time of the German outbreak, and for discussions during the preparation of this manuscript. The authors thank the laboratory colleagues, in particular Ms. M. Vašáková, and the collaborating microbiologists and clinicians for providing the patients’ data. The authors also thank BioVendor Ltd. (Czech Republic) for long-term access to the device Microflex for MALDI-TOF MS identification of bacteria.
- Aurass P, Prager R, Flieger A (2011) EHEC/EAEC O104:H4 strain linked with the 2011 German outbreak of hemolytic uremic syndrome enters into the viable but non-culturable state in response to various stresses and resuscitates upon stress relief. Environ Microbiol 13(12):3139–3148. doi:10.1111/j.1462-2920.2011.02604.x. PubMedCrossRefGoogle Scholar
- Bielaszewska M, Mellmann A, Zhang W, Köck R, Fruth A, Bauwens A, Peters G, Karch H (2011a) Characterization of the Escherichia coli strain associated with an outbreak of hemolytic uremic syndrome in Germany, 2011: a microbiological study. Lancet Infect Dis 11(9):671–676. doi:10.1016/S1473-3099(11)70165-7 PubMedGoogle Scholar
- Bielaszewska M, Middendorf B, Tarr PI, Zhang W, Prager R, Aldick T, Dobrindt U, Karch H, Mellmann A (2011b) Chromosomal instability in enterohemorrhagic Escherichia coli O157:H7: impact on adherence, tellurite resistance and colony phenotype. Mol Microbiol 79:1024–1044. doi:10.1111/j.1365-2958.2010.07499.x PubMedCrossRefGoogle Scholar
- Brzuskiewicz E, Thürmer A, Schuldes J et al (2011) Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: entero-aggregative-haemorrhagic Escherichia coli (EAHEC). Arch Microbiol 193(12):883–891. doi:10.1007/s00203-011-0725-6 CrossRefGoogle Scholar
- ECDC report (2011) European Centre for Disease Prevention and Control. Shiga toxin-producing E. coli (STEC): update on outbreak in the EU, 27 July 2011. Epidemiological updates; available at: http://ecdc.europa.eu/en/activities/sciadvice/Pages/Epidemiological_Updates.aspx. Accessed 30 Sept 2011
- EFSA (European Food Safety Authority) report (2011) Tracing seeds, in particular fenugreek (Trigonella foenum-graecum) seeds, in relation to the Shiga toxin-producing E. coli (STEC) O104:H4 2011 OUTbreaks in Germany and France. European Food Safety Authority, technical report; available at: http://www.efsa.europa.eu/en/supporting/doc/176e.pdf. Accessed 8 Aug 2011
- Robert Koch Institute (RKI) Final Report (2011) Final presentation and evaluation of epidemiological findings in the EHEC O104:H4 outbreak, Germany 2011. Berlin 2011; http://www.rki.de/cln_153/nn_217400/EN/Home/EHEC__final__report,templateId=raw,property=publicationFile.pdf/EHEC_final_report.pdf
- Gault G, Weill F, Mariani-Kurkdjian P, et al. (2011) Outbreak of hemolytic uremic syndrome and bloody diarrhea due to Escherichia coli O104:H4, south-west France, June 2011. Euro Surveill 16:pii = 19905; available at: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19905. Accessed 29 Jul 2011
- Lhotová H, Bielaszewska M, Kopalová E, Štolbová M (1990) Hemolytic-uremic syndrome—microbiological characterization. Čs Epidemiol Microbiol Imunol 39:13–20 (In Czech)Google Scholar
- Marejková M, Zieg J, Dušek J, Bláhová K, Petráš P (2009) Fatal case of diarrhea-positive hemolytic-uremic syndrome caused by enterohemorrhagic E. coli O26. (In Czech) Zprávy EM (SZÚ, Praha) 18:212–214; English summary available at: http://www.szu.cz/uploads/documents/CeM/Zpravy_EM/18_2009/6_cerven/212_hus.pdf
- Marejková M, Roháčová H, Reisingerová M, Petráš P (2011) A large outbreak caused by a Shiga toxigenic Escherichia coli O104:H4 and one imported case in the Czech Republic. (In Czech) Zprávy CEM (SZÚ, Praha) 20:170–173 (published on-line 7 July 2011); English summary available at: http://www.szu.cz/tema/prevence/velka-nemecka-epidemie-vyvolana-shigatoxigennim-kmenem
- Paton AW, Paton JC (1998) Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfb O111, and rfb O157. J Clin Microbiol 36:98–602Google Scholar