The O26 verocytotoxin-producing Escherichia coli (VTEC)-associated outbreak of hemolytic uremic syndrome (HUS) cases in Romania during 2016 showed the need to improve the current methodology of non-O157 VTEC detection and surveillance. An in-house assay based on xTAG Luminex technology was optimized to identify seven of the most relevant diarrheagenic E.coli serogroups (O-specific wzx genes), two convenient VTEC virulence markers (eaeA and ehxA genes), and a species-specific control gene (uidA). Twenty-nine strains previously characterized in terms of serogroup and virulence genes were tested with the optimized protocol and the results were as expected. The ratio of sample signal to background varied from 66.7 (ehxA) to 7.6 (uidA) for positive samples, with a cut-off of 3. Sensitivity varied depending on the target to be amplified from approximately 102 genomic copies to approximately 104 genomic copies per reaction, respectively. The current approach seems an affordable alternative to commercially available assays that can be further exploited to improve existing autochthonous strategies to prevent future VTEC outbreaks.
4. Scheutz F, Strockbine NA. Escherichia. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology. New York: Springer; 2005. P. 607-624.
5. Fratamico PM, DebRoy C, Liu Y, Needleman DS, Baranzoni GM, Feng P. Advances in molecular serotyping and subtyping of Escherichia coli. Front Microbiol. 2016;7:644. DOI: 10.3389/fmicb.2016.00644
6. Iguchi A, Iyoda S, Kikuchi T, Ogura Y, Katsura K, Ohnishi M et al. A complete view of the genetic diversity of the Escherichia coli O-antigen biosynthesis gene cluster. DNA Res 2015;22(1):101-7. DOI: 10.1093/dnares/dsu043
7. Johnson KE, Thorpe CM, Sears CL. The emergingclinical importance of non-O157 Shiga toxin-producing Escherichia coli. Clin Infect Dis 2006;43:1587-95.DOI: 10.1086/509573
8. Muniesa M, Hammerl JA, Hertwig S, Appel B, Brüssow H. Shiga toxin-producing Escherichia coli O104:H4: a new challenge for microbiology. Appl Environ Microbiol 2012;78(12):4065-73. DOI: 10.1128/AEM.00217-12
9. Peron E, Zaharia A, Zota LC, Severi E, Mårdh O, Usein C, et al. Early findings in outbreak of haemolytic uraemic yndrome among young children caused by Shiga toxin-producing Escherichia coli, Romania, January to February 2016. Euro Surveill 2016;21(11):30170. DOI: 10.2807/1560-7917.ES.2016.21.11.30170
10. Usein CR, Ciontea AS, Militaru CM, Condei M, Dinu S, Oprea M, et al. Molecular characterisation of human Shiga toxin-producing Escherichia coli O26 strains: results of an outbreak investigation, Romania, February to August 2016. Euro Surveill 2017;22(47):17-00148. DOI: 10.2807/1560-7917.ES.2017.22.47.17-00148
11. Luminex xMAP® Cookbook. A collection of methods and protocols for developing multiplex assays with xMAP Technology. 2rd Edition. Available at http://info.luminexcorp.com/en-us/download-the-xmap-cookbook Accessed November 14, 2016.
12. DebRoy C, Roberts E, Valadez AM, Dudley EG, Cutter CN. Detection of Shiga toxin-producing Escherichia coli O26, O45, O103, O111, O113, O121, O145, and O157 serogroups by multiplex polymerase chain reaction of the wzx gene of the O-antigen gene cluster. Foodborne Pathog Dis 2011;8(5):651-2. DOI: 10.1089/fpd.2010.0769
13. Leszczyńska B, Ziółkowska H, Podsiadły E, Szych J, Rastawicki W, Demkow U, et al. Diagnostic value of serological tests against verotoxigenic Escherichia coli in hemolytic uremic syndrome in children. Adv Clin Exp Med. 2015;24(6):1031-6. DOI: 10.17219/acem/41931
14. Rice T, Quinn N, Sleator RD, Lucey B. Changing diagnostic methods and increased detection of verotoxigenic Escherichia coli, Ireland. Emerg Infect Dis. 2016;22(9):1656-7. DOI: 10.3201/eid2209.160477
15. European Centre for Disease Prevention and Control. Annual Epidemiological Report 2016 - Shigatoxin/ verocytotoxin-producing Escherichia coli infection. Available at http://ecdc.europa.eu/en/healthtopics/stec/Pages/Annual-epidemiological-report-2016.aspx Accessed December 20, 2017.
16. Wessels E, Rusman LG, van Bussel MJ, Claas EC. Added value of multiplex Luminex Gastrointestinal Pathogen Panel (xTAG® GPP) testing in the diagnosis of infectious gastroenteritis. Clin Microbiol Infect 2014;20(3):O182-O187. DOI: 10.1111/1469-0691.12364
17. Carter JM1 Lin A, Clotilde L, Lesho M. Rapid, multiplexed characterization of Shiga toxin-producing Escherichia coli (STEC) Isolates Using Suspension Array Technology. Front Microbiol 2016;7:439.
18. Jiang L, Ren H, Zhou H, Qin T, Chen Y. Simultaneous detection of nine key bacterial respiratory pathogens using Luminex xTAG(®) technology. Int J Environ Res Public Health 2017;14(3):E223. DOI: 10.3390/ijerph14030223
19. Lin A, Nguyen L, Lee T, Clotilde LM, Kase JA, Son I, Carter JM, Lauzon CR. Rapid O serogroup identification of the ten most clinically relevant STECs by Luminex microbead-based suspension array. J Microbiol Methods 2011;87(1):105-10. DOI: 10.1016/j.mimet.2011.07.019
20. Clotilde LM, Bernard C 4th, Hartman GL, Lau DK, Carter JM. Microbead-based immunoassay for simultaneous detection of Shiga toxins and isolation of Escherichia coli O157 in foods. J Food Prot 2011;74(3):373-9. DOI: 10.4315/0362-028X.JFP-10-344