Rapid culture-based identification of Shiga toxin-producing Escherichia coli and Shigella spp./Enteroinvasive E. coli using the eazyplex® EHEC complete assay

  • Anika Penzel
  • Katrin Schützler
  • Jana Dröge
  • Alexander Mellmann
  • Ralf Ehricht
  • Ines Engelmann
  • Sascha D. Braun
  • Benjamin T. Schleenvoigt
  • Bettina Löffler
  • Jürgen RödelEmail author
Original Article


Shiga toxin–producing Escherichia coli (STEC) and Shigella spp./enteroinvasive E. coli (EIEC) are common diarrheagenic bacteria that cause sporadic diseases and outbreaks. Clinical manifestations vary from mild symptoms to severe complications. For microbiological diagnosis, culture confirmation of a positive stool screening PCR test is challenging because of time-consuming methods for isolation of strains, wide variety of STEC pathotypes, and increased emergence of non-classical strains with unusual serotypes. Therefore, molecular assays for the rapid identification of suspect colonies growing on selective media are very useful. In this study, the performance of the newly introduced eazyplex® EHEC assay based on loop-mediated isothermal amplification (LAMP) was evaluated using 18 representative STEC and Shigella strains and 31 isolates or positive-enrichment broths that were collected from clinical stool samples following screening by BD MAX™ EBP PCR. Results were compared to real-time PCR as a reference standard. Overall, sensitivities and specificities of the eazyplex® EHEC were as follows: 94.7% and 100% for Shiga toxin 1 (stx1), 100% and 100% for stx2, 93.3% and 97.1% for intimin (eae), 100% and 100% for enterohemolysin A (ehlyA), and 100% and 100% for invasion-associated plasmid antigen H (ipaH) as Shigella spp./EIEC target, respectively. Sample preparation for LAMP took only some minutes, and the time to result of the assay ranged from 8.5 to 13 min. This study shows that eazyplex® EHEC is a very fast and easy to perform molecular assay that provides reliable results as a culture confirmation assay for the diagnosis of STEC and Shigella spp./EIEC infections.


Shiga toxin E. coli Shigella LAMP Stool culture 


Funding information

This work was supported by a grant from the German Federal Ministry of Education and Research (BMBF, 13N13890).

Compliance with ethical standards

Ethical approval

The study protocol for the evaluation of the eazyplex® EHEC complete assay for clinical samples was reviewed and approved by the ethics committee of the Jena University Hospital (5548-05/18).

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Knabl L, Grutsch I, Orth-Höller D (2016) Comparison of the BD MAX® enteric bacterial panel assay with conventional diagnostic procedures in diarrheal stool samples. Eur J Clin Microbiol Infect Dis 35:131–136. CrossRefPubMedGoogle Scholar
  2. 2.
    DeRauw K, Jacobs S, Piérard D (2018) Twenty-seven years of screening for Shiga toxin-producing Escherichia coli in a university hospital. Brussels, Belgium, 1987-2014. PLoS One 13:e0199968. CrossRefGoogle Scholar
  3. 3.
    Jenssen GR, Veneti L, Lange H, Vold L, Naseer U, Brandal LT (2019) Implementation of multiplex PCR diagnostics for gastrointestinal pathogens linked to increase of notified Shiga toxin-producing Escherichia coli cases in Norway, 2007-2017. Eur J Clin Microbiol Infect Dis 38:801–809. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Nüesch-Inderbinen M, Morach M, Cernela N, Althaus D, Jost M, Mäusezahl M, Bloomberg G, Stephan R (2018) Serotypes and virulence profiles of Shiga toxin-producing Escherichia coli strains isolated during 2017 from human infections in Switzerland. Int J Med Microbiol 308:933–939. CrossRefPubMedGoogle Scholar
  5. 5.
    Valilis E, Ramsey A, Sidiq S, DuPont HL (2018) Non-O157 Shiga toxin-producing Escherichia coli-a poorly appreciated enteric pathogen: systematic review. Int J Infect Dis 76:82–87. CrossRefPubMedGoogle Scholar
  6. 6.
    Karch H, Tarr PI, Bielaszewska M (2005) Enterohaemorrhagic Escherichia coli in human medicine. Int J Med Microbiol 295:405–418. CrossRefPubMedGoogle Scholar
  7. 7.
    Newell DG, La Ragione RM (2018) Enterohemorrhagic and other Shiga toxin-producing Escherichia coli (STEC): where are we now regarding diagnostics and control strategies? Transbound Emerg Dis 65(S1):49–71. CrossRefPubMedGoogle Scholar
  8. 8.
    Karmali MA (2017) Emerging public health challenges of Shiga toxin-producing Escherichia coli related to changes in the pathogen, the population, and the environment. Clin Infect Dis 64:371–376. CrossRefPubMedGoogle Scholar
  9. 9.
    Saupe A, Edel B, Pfister W, Löffler B, Ehricht R, Rödel J (2017) Acute diarrhea due to a Shiga toxin 2e-producing Escherichia coli O8:H19. JMM Case Rep 4:e005099. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Kotloff KL, Riddle MS, Platts-Mills JA, Pavlinac P, Zaidi AKM (2018) Shigellosis. Lancet 391:801–812. CrossRefPubMedGoogle Scholar
  11. 11.
    van den Beld MJC, Reubsaet FAG (2012) Differentiation between Shigella, enteroinvasive Escherichia coli (EIEC) and noninvasive Escherichia coli. Eur J Clin Microbiol Infect Dis 31:899–904. CrossRefPubMedGoogle Scholar
  12. 12.
    Ud-Din A, Wahid S (2014) Relationship among Shigella spp. and enteroinvasive Escherichia coli (EIEC) and their differentiation. Braz J Microbiol 45:1131–1138. CrossRefPubMedGoogle Scholar
  13. 13.
    Eigner U, Hiergeist A, Veldenzer A, Rohlfs M, Schwarz R, Holfelder M (2017) Evaluation of a new real-time PCR assay for the direct detection of diarrheagenic Escherichia coli in stool specimens. Diagn Microbiol Infect Dis 88:12–16. CrossRefPubMedGoogle Scholar
  14. 14.
    Zhi S, Szelewicki J, Ziebell K, Parsons B, Chui L (2019) General detection of Shiga toxin 2 and subtyping of Shiga toxin 1 and 2 in Escherichia coli using qPCR. J Microbiol Methods 159:51–55 https://10.1016/j.mimet.2019.02.008 CrossRefGoogle Scholar
  15. 15.
    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Rödel J, Bohnert JA, Stoll S, Wassill L, Edel B, Karrasch M, Löffler B, Pfister W (2017) Evaluation of loop-mediated isothermal amplification for the rapid identification of bacteria and resistance determinants in positive blood cultures. Eur J Clin Microbiol Infect Dis 36:1033–1040. CrossRefPubMedGoogle Scholar
  17. 17.
    Geue L, Stieber B, Monecke S, Engelmann I, Gunzer F, Slickers P, Braun SD, Ehricht R (2014) Development of a rapid microarray-based DNA subtyping assay for the alleles of Shiga toxins 1 and 2 of Escherichia coli. J Clin Microbiol 52:2898–2904. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Mellmann A, Harmsen D, Cummings CA, Zentz EB, Leopold SR, Rico A, Prior K, Szczepanowski R, Ji Y, Zhang W, McLaughlin SF, Henkhaus JK, Leopold B, Bielaszewska M, Prager R, Brzoska PM, Moore RL, Guenther S, Rothberg JM, Karch H (2011) Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology. PLoS One 6:e22751. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Morin N, Santiago AE, Ernst RK, Guillot SJ, Nataro JP (2013) Characterization of the AggR regulon in enteroaggregative Escherichia coli. Infect Immun 81:122–132. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Golshani M, Oloomi M, Bouzari S (2017) In silico analysis of Shiga toxins (Stxs) to identify new potential vaccine targets for Shiga toxin-producing Escherichia coli. In Silico Pharmacol 5:2. CrossRefPubMedCentralGoogle Scholar
  21. 21.
    Friesema I, van der Zwaluw K, Schuurman T, Kooistra-Smid M, Franz E, van Duynhoven Y, van Pelt W (2014) Emergence of Escherichia coli endocing Shiga toxin 2f in human Shiga toxin-producing E. coli (STEC) infections in the Netherlands, January 2008 to December 2011. Euro Surveill 19:26–32PubMedGoogle Scholar
  22. 22.
    Bielaszewska M, Aldick T, Bauwens A, Karch H (2014) Hemolysin of enterohemorrhagic Escherichia coli: structure, transport, biologicalactivity and putative role in virulence. In J Med Microbiol 304:521–529. CrossRefGoogle Scholar
  23. 23.
    Gaudio PA, Sethabutr O, Echeverria P, Hoge CW (1997) Utility of a polymerase chain reaction diagnostic system in a study of the epidemiology of shigellosis among dysentery patients, family contacts, and well controls living in a shigellosis-endemic area. J Infect Dis 176:1013–1018. CrossRefPubMedGoogle Scholar
  24. 24.
    Anderson NW, Buchan BW, Ledeboer NA (2014) Comparsion of the BD MAX enteric bacterial panel to routine culture methods for detection of Campylobacter, enterohemorrhagic Escherichia coli (O157), Salmonella, and Shigella isolates in preserved stool specimens. J Clin Microbiol 52:1222–1224. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Martinez-Castillo A, Quirós P, Navarro F, Miró E, Muniesa M (2013) Shiga toxin 2-encoding bacteriophages in human fecal samples from healthy individuals. Appl Environ Microbiol 79:4862–4868. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Bielaszewska M, Prager R, Kock R, Mellmann A, Zhang W, Tschäpe H, Tarr PI, Karch H (2007) Shiga toxin gene loss and transfer in vitro and in vivo during enterohemorrhagic Escherichia coli O26 infection in humans. Appl Environ Microbiol 73:3144–3150. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Senthakumaran T, Brandal LT, Lindstedt BA, Jørgensen SB, Charnock C, Tunsjø HS (2018) Implications of stx loss for clinical diagnostics of Shiga toxin-producing Escherichia coli. Eur J Clin Microbiol Infect Dis 37:2361–2370. CrossRefPubMedGoogle Scholar
  28. 28.
    Pedersen RM, Nielsen MTK, Möller S, Ethelberg S, Skov MN, Kolmos HJ, Scheutz F, Holt HM, Rosenvinge FS (2018) Shiga toxin-producing Escherichia coli: incidence and clinical features in a setting with complete screening of patients with suspected infective diarrhea. Clin Microbiol Infect 24:635–639. CrossRefPubMedGoogle Scholar
  29. 29.
    Bielaszewska M, Stoewe F, Fruth A, Zhang W, Prager R, Brockmeyer J, Mellmann A, Karch H, Friedrich AW (2009) Shiga toxin, cytolethal distending toxin, and hemolysin repertoires in clinical Escherichia coli O91 isolates. J Clin Microbiol 47:2061–2066. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Fruth A, Prager R, Tietze E, Rabsch W, Flieger A (2015) Molecular epidemiological view on Shiga toxin-producing Escherichia coli causing human disease in Germany: diversity, prevalence, and outbreaks. Int J Med Microbiol 305:697–704. CrossRefPubMedGoogle Scholar
  31. 31.
    Escher M, Scavia G, Morabito S, Tozzoli R, Maugliani A, Cantoni S, Fracchia S, Bettati A, Casa R, Gesu GP, Torresani E, Caprioli A (2014) A severe foodborne outbreak of diarrhoea linked to a canteen in Italy caused by enteroinvasive Escherichia coli, an uncommon agent. Epidemiol Infect 142:2559–2566. CrossRefPubMedGoogle Scholar
  32. 32.
    Newitt S, MacGregor V, Robbins V, Bayliss L, Chattaway MA, Dallman T, Ready D, Aird H, Puleston R, Hawker J (2016) Two linked enteroinvasive Escherichia coli outbreaks, Nottingham, UK, June 2014. Emerg Infect Dis 22:1178–1184. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Pettengill EA, Hoffmann M, Binet R, Roberts RJ, Payne J, Allard M, Michelacci V, Minelli F, Morabito S (2015) Complete genome sequence of enteroinvasive Escherichia coli O96:H19 associated with a severe foodborne outbreak. Genome Announc 3:e00883–e00815. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Parsons BD, Zelyas N, Berenger BM, Chui L (2016) Detection, characterization, and typing of Shiga toxin-producing Escherichia coli. Front Microbiol 7:478. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Okeke IN, Aboderin AO, Opintan JA (2016) Enteroinvasive Escherichia coli may account for uncultured Shigella. Am J Trop Med Hyg 94:480–481. CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Quinn E, Najjar Z, Huhtinen E, Jegasothy E, Gupta L (2019) Culture-positive shigellosis cases are epidemiologically different to culture-negative/PCR-positive cases. Aust N Z J Public Health 43:41–45. CrossRefPubMedGoogle Scholar
  37. 37.
    Gould LH, Bopp C, Strockbine N, Atkinson R, Baselski V, Body B, Carey R, Crandall C, Hurd S, Kaplan R, Neill M, Shea S, Somsel P, Tobin-D’Angelo M, Griffin PM, Gerner-Smidt P, Centers for Disease Control and Prevention (CDC) (2009) MMWR Recomm Rep 58:1–14PubMedGoogle Scholar
  38. 38.
    De Rauw K, Breynaert J, Piérard D (2016) Evaluation of the Alere SHIGA TOXIN QUICK CHEK™ in comparison to multiplex Shiga toxin PCR. Diagn Microbiol Infect Dis 86:35–39. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Anika Penzel
    • 1
  • Katrin Schützler
    • 1
  • Jana Dröge
    • 1
  • Alexander Mellmann
    • 2
  • Ralf Ehricht
    • 3
  • Ines Engelmann
    • 4
  • Sascha D. Braun
    • 3
  • Benjamin T. Schleenvoigt
    • 5
  • Bettina Löffler
    • 1
  • Jürgen Rödel
    • 1
    Email author
  1. 1.Institute of Medical MicrobiologyJena University HospitalJenaGermany
  2. 2.Institute of Hygiene and National Consulting Laboratory for Hemolytic Uremic Syndrome (HUS)University Hospital MünsterMünsterGermany
  3. 3.Leibniz Institute of Photonic Technology (IPHT)JenaGermany
  4. 4.Blink AGJenaGermany
  5. 5.Institute of Infectious Diseases and Infection ControlJena University HospitalJenaGermany

Personalised recommendations