Methods for STEC Identification and Serotyping

White Paper

Methods for STEC Identification and Serotyping Overview of STEC First reported in 1982, Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that has been linked to outbreaks of human illness from contaminated food such as dairy, meat, and fresh (uncooked) produce.1 Other terms that are used to describe STEC are enterohemorrhagic E. coli (EHEC), which refers specifically to pathogenic STEC, and verotoxic E. coli (VTEC).

Identified as an important global public health concern, STEC causes approximately 2.8 million acute illnesses annually that result in 3,890 cases of hemolytic uremic syndrome, 270 cases of end-stage renal disease, and 230 deaths.2 The estimated cost for acute care of STEC patients in the United States alone is $2 billion annually.3 STEC can cause mild-to-bloody diarrhea, abdominal cramps, vomiting, strokes, hemorrhagic colitis, hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura.4 Between 5% and 10% of STEC infections result in severe illness and HUS, mostly in older adults and children under 5 years of age.2 Severe cases can end with chronic renal failure, chronic nervous system deficiencies, and death.5 The main toxins produced by STEC are Shiga toxins type 1 (stx1) and type 2 (stx2), which are responsible for most clinical symptoms of STEC illness.1,6 The serotype stx2 is much more powerful than stx1. Shiga toxin is a member of the AB5 toxin family, which is composed of a B-pentamer that binds to and internalizes an A subunit, thereby inhibiting protein synthesis by a mechanism similar to that of ricin.7

Identification of STEC O-Serogroups During Foodborne Outbreaks The most common type of STEC in the United States is E. coli O157:H7 (often shortened to E. coli O157 or even just O157).2 Over 100 other STEC serogroups, which are called non-O157, are responsible for more than 60% of STEC infections.8,9 The U.S. Department of Agriculture’s zero-tolerance policy standard includes the top 6 non-O157 STEC serogroups (O26, O45, O103, O111, O121, and O145) in raw, non-intact beef products. These non-O157 STEC serogroups are referred to as the “Big Six” and

account for 75% to 80% of non-O157 STEC isolations in clinical samples in the United States.10 STEC O91, O113, and O128 have also been previously reported to cause hemorrhagic colitis and HUS.3 A STEC O104-caused outbreak in Germany in 2011 sickened 3,816 individuals, making it one of the largest HUS outbreaks ever reported.3 Identification and serotyping of STEC is important for prevention, identification of sources, control of outbreaks, and removal of sources from commerce.11 Identifying O-serogroups of STECs can help to differentiate pathogenic STECs from STECs that are not associated with human illness.12 During foodborne outbreaks, this can aid in matching clinical, food, and environmental isolates when trying to identify the sources of illness and ultimately food contamination.3,12 Rapid detection and identification of STEC O-serogroups directly benefits regulatory agencies by minimizing analysis time, and protects consumer health by allowing early diagnosis of STEC infection for determining appropriate treatment.1,3 Another factor for foodborne outbreaks is the presence of latent virulence factors including Shiga toxins (stx1 and stx2), intimin (eaeA) and hemolysin (hlyA) which are responsible for adhesion, colonization, and invasion of bacterial cells into the intestinal walls.5

Methods of STEC Detection and Serotyping Current assays test for the presence of Shiga toxin via enzyme immunoassay (EIA)/lateral flow (LF) tests, and polymerase chain reaction (PCR) tests to detect the stx1 and stx2 encoding genes. Many identification methods for pathogenic E. coli are time-consuming and provide limited information for clinical diagnosis and food safety analysis.13 Effective detection and isolation of non-O157 STEC strains from contamination sources remain challenging.11 Due to the need for faster and less labor-intensive methods, there has been a great interest in the development of rapid and reliable detection methods for STEC (targeting common virulence factors) and specific STEC serogroups (targeting serogroup-specific traits).3,11,13

Current approaches used for detecting non-O157 STEC in food include culture, immunological, and molecular methods, as well as several novel technologies.11 Culture-based methods consist of multiple incubation steps (pre-enrichment, selective enrichment, selective and differential plating) for bacterial isolation followed by additional biological, serological, or molecular tests for confirmation. These methods are labor intensive and time consuming.14 Immunological-based methods use antibodies that recognize Shiga toxins or specific O-antigens allowing for the direct detection of STEC serogroups (either common virulence genes or O-serogroup– specific genes). These methods are used to detect STEC serogroups in food samples, or aid in their isolation through immunomagnetic separation (IMS) or colony immunoblot. FDA-cleared enzyme immunoassays for in vitro diagnosis of STEC infections require 20 minutes to 4 hours to run, although overnight enrichment prior to the analysis is strongly recommended.15 Molecular-based methods include nucleic acid amplification tests such as PCR (conventional single analyte and multiplex PCR), real-time quantitative PCR (qPCR), and isothermal amplification techniques that have been adopted to detect non-O157 STEC.3,11 Detection of virulence genes is not a guarantee of gene expression, and in the case of multiple STEC strains contaminating a single food sample, further confirmation is needed to determine whether the gene profiles detected are from a single strain or multiple different strains.16,17 Examples of advanced technologies are microbead-based suspension arrays (Luminex’s xMAP® Technology), which are used in an immunoassay to detect Shiga toxins1 and in a probe-based assay to detect 10 clinically relevant STEC serogroups (top 6, O91, O113, O128, and O157).18 Low-density DNA oligonucleotide microarrays have been designed to combine the detection of multiple O-serogroups and H types, as well as virulence genes; these assays have not been directly applied in food testing but are confirmation tools after pure cultures have been isolated (DNA purification is necessary).19 The use of clustered regularly interspaced short palindromic repeat sequence polymorphisms has been explored for detection of serotypes by real-time PCR; further studies are needed to evaluate their applicability in food testing.20 Culture-Independent Diagnostic Testing (CIDT) for enteric pathogen detection, including both antigen detection and multiplex nucleic acid amplification techniques, is becoming more widespread.21 CIDTs can test for an array of clinically important infections, such as respiratory, bloodstream, and enteric infections, more quickly and effectively than other methods can.21 From 2012 to 2015, there has been a significant increase in the percentage of enteric infections, including STEC, diagnosed only by a CIDT. Although routine submission of culture isolates is less likely to occur with CIDTs, access to isolates of enteric pathogens continues to be essential for public health surveillance, detection, and tracking of outbreaks.21

xMAP® Technology is Widely Used for Identification and Serotyping of STEC The U.S. FDA developed a microbead-based multiplex suspension array to screen colonies for 11 clinically relevant STEC serogroups (O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157), which is available in Section R of the Bacteriological Analytical Manual.22 Kase et al. (2016) evaluated the usefulness of this method to identify STEC-positive enrichment samples before agar plating compared with RT-PCR.23 As the STEC microbeadbased suspension array accurately screened food enrichments for the eleven (11) O serogroups, Kase et. al. recommend the expanded use of this assay as a rapid first screen of undiluted E. coli food enrichment cultures to provide an early indication of contamination with potentially pathogenic STEC. A PCR-based xMAP® suspension array was also developed to rapidly identify the presence of potentially virulent O157:H7 and non-O157.6 The assay detects five bacterial targets consisting of the genes coding for four virulence factors (stx1, stx2, eae, and ehxA), plus the O157:H7-specific +93 uidA single nucleotide polymorphism. Internal amplification control (IAC) was added to the assay to avoid false-negative results due to the presence of PCR inhibitors. Excellent accuracy with this assay has been demonstrated for all the strains tested.

Two multiplex PCR-based and antibody-based xMAP assays were compared with conventional serotyping methods using antisera for the serotyping of 161 STEC strains isolated from fecal samples of California cattle.4 The xMAP-based PCR assay was able to serotype 22 additional strains over conventional serotyping methods using antisera and an additional ten strains over antibody-based xMAP assay. The xMAP assays also provide simultaneous testing for multiple serogroups using a single sample and thus require less sample material.

A multiplex suspension array assay was developed for molecular serotyping of the seven most prevalent STEC serogroups (O26, O45, O103, O111, O121, O145, and O157).24 Fluorescence values of 59 STEC-positive samples were 30 to more than 270 times greater than the signals of negative controls, demonstrating the effectiveness of STEC molecular serotyping.

A 7-plex microbead-based immunoassay was developed for STEC serotyping (O26, O45, O103, O111, O121, O145, and O157).25 The specificity of the immunoassay was tested against a collection of 79 PCR typed E. coli strains belonging to the top 7 E. coli. Nearly all strains (98.7%; 78/79) were correctly identified on the Bio-Plex® 100 instrument in less than 4 hours.

A multiplex immunoassay was developed to simultaneously serotype E. coli O157 and detect stx1 and/or stx2 and used to test 34 E. coli isolates belonging to various O serogroups phenotypically different for Stx.1 The xMAP format, which has a high number of replicates, was shown to provide greater precision compared with the microplate sandwich format using enzyme-linked

2

immunosorbent assay (ELISA). In addition, the xMAP format has faster analysis time and increased sensitivity of up to 1,000 times compared with the microplate format.

The Luminex xMAP multiplexing platform has the ability to determine 11 top STEC serogroups and two virulence factors including: 12

The xMAP® STEC Molecular Serotyping Assay

•• O157—the most common STEC serogroup in the United

Currently, there are no other commercially available kits capable of simultaneously detecting STEC O157 and its toxins in food such as ground beef, lettuce, and milk. Based on this unmet need, Luminex developed a test that provides results in less than 24 hours. The xMAP® STEC Molecular Serotyping Assay works with magnetic fluorescent microbeads capable of detecting both STEC O157 and its toxins, while simplifying recovery of the bacteria from food.3

About the Product The xMAP® STEC Molecular Serotyping Assay is a multiplex, nucleic acid-based assay for research and epidemiological use in identifying the serogroup of a STEC isolate. The PCR-based identification test was developed by the U.S. FDA by Lin et al.

(2011) and validated in multisite studies.18,26 Adapted from previous TaqMan™ assays that targeted the O-group specific regions of the wzx (flippase) and wzy (polymerase) genes, the test is an xMAP microbead-based suspension array that identifies the O-serogroup of the ten most clinically relevant STECs (O26, O45, O91, O103, O111, O113, O121, O128, O145, and O157).18 The test consists of uniquely colored fluorescent microspheres conjugated to specific DNA probe sequences for each of the STEC target O-serogroups.

Test performance was evaluated in multiple laboratories with a panel of eleven (11) different STEC serogroups (one of each O-serogroup and one unidentified O-serogroup [ATCC 25922]) on the Bio-Plex® 200 and MAGPIX® instruments. All 114 STEC isolates tested were correctly identified with 100% accuracy and no false positives among 46 negative control isolates (26 other STEC strains and 20 non–E. coli bacteria).18 Lin et al. (2013) further developed the multiplex assay with the addition of the STEC serogroup O104. The accuracy, reproducibility, and practicality of the xMAP assay were evaluated in a blinded multilaboratory collaborative study involving 10 participants from 9 laboratories.25 Out of 55 STEC strains tested, 44 (4 of each of eleven (11) O serogroups) were identifiable with the assay; 11 were included as negative controls (these were STECs isolated from similar sources [i.e., human, bovine, and produce] but were not of the target eleven (11) O serogroups). Five of 10 participants correctly identified all 55 strains correctly; 2 participants each had one false-positive result; 1 participant had one false-negative result, and 1 participant had two false-negative results. Out of 495 analyses, there were two false-positive results and three falsenegative results, yielding an accuracy between 96.4 % and 100% among nine laboratories. The participants ranged in their experience using the Luminex technology from none to moderate experience, indicating that the xMAP-based assay produces accurate and consistent results in various laboratory settings among technicians with differing skill sets.

States, accounting for the majority of cases

•• O26, O45, O103, O111, O121, and O145—the “Big Six”

non-O157 serogroups, causing more than 70% of infections in the United States

•• O91, O113, and O128—less common serogroups, but

accounting for a sizeable number, and are associated with HUS; may also lack eae gene but possess ehxA

•• O104—responsible for recent outbreak in Germany with 850

cases of HUS and 32 deaths; this H4 subtype carried the EAEC genome as well

•• eae and aggR are two virulence markers that have been added to the assay

•• eae—important for attachment and effacement of microvilli; present in many different serogroups, the eae gene seems to be important in causing disease27 •• aggR—a transcriptional regulator in EAEC that has been proposed as the defining factor for “typical EAEC” and regulates a large number of virulence factors27 The Luminex xMAP® STEC Molecular Serotyping Assay allows for the simultaneous determination of STEC serogroups and important virulence markers while avoiding the need to develop a custom assay. The identification test is a qualitative multiplex PCR assay that can be run in conjunction with immunoassay-based STEC testing on the same instrument. As a high throughput molecular test, the xMAP assay offers better reproducibility and less hands-on time compared with traditional agglutination methods.1 See Figure 1 for the complete workflow.

Developed and validated by the U.S. FDA for epidemiological use, the xMAP STEC Molecular Serotyping Assay has been shown to be rapid, accurate, robust, and adaptable. The performance of the xMAP assay has been well established in multisite studies.

Providing a much faster turnaround time (hours vs. days) and much less hands-on time with a total assay time of approximately 3 hours, the multiplex assay provides a high-throughput solution to conventional E. coli identification methods that are very labor intensive.3,25

About xMAP® Technology The xMAP® multiplexing platform uses fluorescent dyes to create sets of microbeads with unique spectral identities. Unique capture molecules are coupled to each set of microbeads, which bind to different analytes of interest. After binding to a detection molecule that is coupled to a fluorescent reporter, the microbeads are read in a flow cytometer or in a bead imager to determine both the presence and quantity of analyte(s) in the sample.

3

Figure 1: xMAP®Molecular STEC Serotyping Assay Workflow

xMAP Technology provides a high throughput and high-plex open architecture assay platform that detects up to 500 different analytes from one sample enabling a significant reduction of sample input. The flexible throughput of the xMAP platform allows testing of a few samples to a full 96-well or 384-well plate. The reduced time to results of xMAP compared with traditional single-plex testing offers valuable time-saving benefits.

200

Step 1

Step 2

Step 3

Step 4

Step 5

Culture to Generate Pure DNA Isolate

PCR

Hybridization

Add Reporter/ Incubation

Detection

~100 minutes

~30 minutes

~15 minutes

<1 minute/sample

~15 minutes

Learn more about xMAP® Technology at https://www.luminexcorp.com/xmap.

In addition to STEC serogroup testing, Luminex also offers numerous other assays, including the xMAP® Salmonella Serotyping Assay (SSA) and the xMAP® Biothreat Toxin Panel (BTP), which may be of interest to public health laboratories.

Request assistance or pricing from a Luminex Business Manager at http://info.luminexcorp.com/sales-contact-request.

References 1.

Clotilde LM, Bernard Ct, Hartman GL, et. al. Microbead-based immunoassay for simultaneous detection of Shiga toxins and isolation of Escherichia coli O157 in foods. J Food Prot 2011;74:373-9.

2.

Majowicz SE, Scallan E, Jones-Bitton A, et. al. Global incidence of human Shiga toxin-producing Escherichia coli infections and deaths: a systematic review and knowledge synthesis. Foodborne Pathog Dis. 2014;11(6):447-55.

3.

Carter JM, 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.Horner P, Blee K, Adams E. Time to manage Mycoplasma genitalium as an STI: but not with azithromycin 1 g! Curr Opin Infect Dis 2014 Feb;27(1):68-74.

4.

Clotilde LM, Salvador A, Bernard Ct, et. al. Comparison of multiplex immunochemical and molecular serotyping methods for Shiga toxin-producing Escherichia coli. Foodborne Pathog Dis 2015;12:118-21.

5.

Ranjbar R, Masoudimanesh M, Dehkordi FS, Jonaidi-Jafari N, Rahimi E. Shiga (Vero)-toxin producing Escherichia coli isolated from the hospital foods; virulence factors, o-serogroups and antimicrobial resistance properties. Antimicrob Resist Infect Control 2017;6:4.

6.

Son I, Binet R, Lin A, Hammack TS, Kase JA. Identification of five Shiga toxin-producing Escherichia coli genes by Luminex microbead-based suspension array. J Microbiol Methods 2015;111:108-10.

7.

Hall G, Kurosawa S, Stearns-Kurosawa DJ. Shiga Toxin Therapeutics: Beyond Neutralization. Toxins (Basel) 2017;9.

8.

Grant M, Hedberg C, Johnson R, et al. The significance of non-O157 Shiga toxin producing Escherichia coli in food. Food Prot Trends 2011;31:33–45.

9.

Scallan E, Hoekstra RM, Angulo FJ, et al. Foodborne illness acquired in the United States—major pathogens. Emerg Infect Dis 2011;17:7-15.

10. Gould LH, Mody RK, Ong KL, et al. Increased recognition of non-O157 Shiga toxin-producing Escherichia coli infections in the United States during 2000-2010: epidemiologic features and comparison with E. coli O157 infections. Foodborne Pathog Dis 2013;10:453-60.

11.

Wang F, Yang Q, Kase JA, et al. Current trends in detecting non-O157 Shiga toxin-producing Escherichia coli in food. Foodborne Pathog Dis 2013;10:665-77.

12. STEC molecular serotyping and virulence profiling protocol. Luminex-based suspension array to identify STEC O serogroups O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, O157, eae, and aggR. FDA (Internet). Cited 2017 November.

Available from: https://www.fda.gov/downloads/Food/FoodScienceResearch/RFE/UCM470888.pdf.

13. Patel IR, Gangiredla J, Lacher DW, et al. FDA Escherichia coli Identification (FDA-ECID) Microarray: a Pangenome Molecular Toolbox for Serotyping, Virulence Profiling, Molecular Epidemiology, and Phylogeny. Appl Environ Microbiol 2016;82:3384-94.

4

14. Wang Y, Salazar JK. Culture-independent rapid detection methods for bacterial pathogens and toxins in food matrices. Comprehensive Reviews in Food Science and Food Safety 2016;15:183-205.

15. Recommendations for Diagnosis of Shiga Toxin — Producing Escherichia coli Infections by Clinical Laboratories. CDC (Internet). Cited 2018 May. Available from: https://www.cdc.gov/mmwr/preview/mmwrhtml/rr5812a1.htm.

16. Feng PC, Jinneman K, Scheutz F, Monday SR. Specificity of PCR and serological assays in the detection of Escherichia coli Shiga toxin subtypes. Appl Environ Microbiol 2011;77:6699-702.

17. Gould LH, Bopp C, Strockbine N, et al. Recommendations for diagnosis of shiga toxin—producing Escherichia coli infections by clinical laboratories.

MMWR Recomm Rep 2009;58:1-14.

18. Lin A, Nguyen L, Lee T, et al. Rapid O serogroup identification of the ten most clinically relevant STECs by Luminex microbead-based suspension array.

J Microbiol Methods 2011;87:105-10.

19. Bugarel M, Beutin L, Martin A, Gill A, Fach P. Micro-array for the identification of Shiga toxin-producing Escherichia coli (STEC) seropathotypes associated with Hemorrhagic Colitis and Hemolytic Uremic Syndrome in humans. Int J Food Microbiol 2010;142:318-29.

20. Delannoy S, Beutin L, Burgos Y, Fach P. Specific detection of enteroaggregative hemorrhagic Escherichia coli O104:H4 strains by use of the CRISPR locus as a target for a diagnostic real-time PCR. J Clin Microbiol 2012;50:3485-92.

21. Shea S, Kubota KA, Maguire H, et al. Clinical microbiology laboratories’ adoption of culture-independent diagnostic tests is a threat to foodborne-disease surveillance in the United States. J Clin Microbiol 2017;55:10-9.

22. . Bacteriological Analytical Manual. Chapter 4A. FDA (Internet). Diarrheagenic Escherichia coli. Cited 2017 October.

Available from: https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070080.htm).

23. Kase JA, Maounounen-Laasri A, Lin A. Rapid identification of Shiga toxin-producing Escherichia coli O Serogroups from fresh produce and raw milk enrichment cultures by Luminex bead-based suspension array. J Food Prot 2016;79:1623-9.

24. Toro M, Najjar MB, Ju W, Brown E, et al. Molecular serogrouping of Shiga toxin-producing Escherichia coli using suspension array. Foodborne Pathog Dis 2013;10:478-80.

25. Clotilde LM, Bernard Ct, Salvador A, et al. A 7-plex microbead-based immunoassay for serotyping Shiga toxin-producing Escherichia coli. J Microbiol Methods 2013;92:226-30.

26. Lin A, Kase JA, Moore MM, et al. Multilaboratory validation of a Luminex microbead-based suspension array for the identification of the 11 most clinically relevant Shiga toxin-producing Escherichia coli O serogroups. J Food Prot 2013;76:867-70.

27. Navarro-Garcia F. Escherichia coli O104:H4 Pathogenesis: an enteroaggregative E. coli/Shiga toxin-producing E. coli explosive cocktail of high virulence.

Microbiol Spectr 2014;2.

To learn more, please visit: www.luminexcorp.com/STEC For Research Use Only. Not for use in diagnostic procedures. This assay method has not been independently evaluated by a third party certifier of government agency. Please contact support@luminexcorp.com to obtain the appropriate product information for your country of residence. ©2018 Luminex Corporation. Luminex, xMAP, and MAGPIX are trademarks of Luminex Corporation, registered in the U.S. and other Countries. Bio-Plex is a registered trademark of Bio-Rad Laboratories, Inc. TaqMan is a trademark of Roche Diagnostics, Inc.

www.luminexcorp.com HEADQUARTERS UNITED STATES

EUROPE

CANADA

CHINA

JAPAN

+1.512.219.8020 info@luminexcorp.com

+31.73.800.1900 europe@luminexcorp.com

+1.416.593.4323 info@luminexcorp.com

+86.21.8036.9888 infocn@luminexcorp.com

+81.3.5545.7440 infojp@luminexcorp.com

WP84381