FOODBORNE PATHOGENS AND DISEASE Volume 10, Number 8, 2013 Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2012.

1448

Review

Current Trends in Detecting Non-O157 Shiga ToxinProducing Escherichia coli in Food

1,2 1 Fei Wang, Qianru Yang, Julie A. Kase,3 Jianghong Meng,2 Laurie M. Clotilde,4 Andrew Lin,4 and Beilei Ge1,5

Abstract

Non-O157 Shiga toxinproducing Escherichia coli (non-O157 STEC) strains are increasingly recognized as important foodborne pathogens worldwide. Together with E. coli O157:H7, six additional STEC serogroups (O26, O45, O103, O111, O121, and O145) are now regulated as adulterants in certain raw beef products in the United States. However, effective detection and isolation of non-O157 STEC strains from food matrices remain challenging. In the past decade, great attention has been paid to developing rapid and reliable detection methods for STEC in general (targeting common virulence factors) and specic STEC serogroups in particular (targeting serogroup-specic traits). This review summarizes current trends in detecting non-O157 STEC in food, including culture, immunological, and molecular methods, as well as several novel technologies.

Introduction mong well over 100 Shiga toxinproducing Escherichia coli (STEC) serotypes associated with sporadic and epidemic human infections, E. coli O157:H7 has to date received the most attention in terms of research and regulatory framework (Grant et al., 2011). First recognized as a foodborne pathogen in 1982 (Riley et al., 1983), it remains the single most common STEC serotype today, causing more than one third of the total estimated STEC illnesses and all of the estimated STEC deaths in the United States (Scallan et al., 2011). On September 28, 1994, the U.S. Department of Agriculture (USDA)s Food Safety and Inspection Service (FSIS) declared E. coli O157:H7 an adulterant in raw ground beef (Taylor, 1994). Since then, sampling and testing at federally inspected plants and retail stores have resulted in 234 beef recalls, which may have partly contributed to the overall decline in the incidence of E. coli O157 infections (CDC, 2011). On the other hand, non-O157 STEC strains are increasingly recognized as important foodborne pathogens worldwide, with reported outbreaks linking to produce, milk, juice, and beef, among others (Eblen, 2007; Mathusa et al., 2010; Grant et al., 2011; CDC, 2012b; USDA, 2012b). Although non-O157 STEC strains appear to cause watery diarrhea more often than bloody diarrhea, in certain geographic regions (e.g., Latin
1 2 3

American, Australia, and Europe), the frequency of non-O157 STEC implicated in hemolyticuremic syndrome cases actually rivaled that of E. coli O157:H7 ( Karmali et al., 2003; Johnson et al., 2006). Reporting of non-O157 STEC in the United States has increased every year since it was designated a nationally notiable infection in 2000 (Atkinson et al., 2006). Recent FoodNet data suggest that non-O157 STEC infections have started to gain predominance over O157 cases in the United States (CDC, 2011, 2012a, 2013). Six STEC serogroupsO26, O45, O103, O111, O121, and O145 (referred to as top 6 hereafter) accounted for approximately 75% of total non-O157 STEC illnesses in the United States annually (Brooks et al., 2005; USDA, 2012b), while other highly pathogenic serogroups (e.g., O91, O104, O113, and O128) appear more prevalent in countries outside of the United States (Karmali et al., 2003; Johnson et al., 2006; Bettelheim, 2007; EFSA/ECDC, 2012). Considering the rising public health concern about nonO157 STEC, effective June 4, 2012, FSIS expanded the zerotolerance policy for E. coli O157:H7 to include the top 6 non-O157 STEC serogroups in raw, nonintact beef products (USDA, 2012c). With this regulation, it is imperative that rapid, accurate, and reliable detection methods be available to test for non-O157 STEC in beef and other high-risk foods. Strategic Consulting Inc. (www.strategic-consult.com)

Department of Food Science, Louisiana State University Agricultural Center, Baton Rouge, Louisiana. Department of Nutrition and Food Science, University of Maryland, College Park, Maryland. Division of Microbiology, Ofce of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland. 4 San Francisco District Laboratory, Ofce of Regulatory Affairs, U.S. Food and Drug Administration, Alameda, California. 5 Division of Animal and Food Microbiology, Ofce of Research, Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, Maryland.

2 estimated that in 2010, testing for E. coli O157:H7 in the United States totaled 4.9 million tests, with red meat accounting for 80% of the test volume. This group projected that STEC screening test volume in 2012 will eventually equal to or exceed 2010 E. coli O157:H7 test volume, a major shift in the U.S. testing market (Byron, 2012). All of these have fueled the development of detection methods for STEC as a group and specic STEC serogroups in particular. In this review, we summarize the challenges and current trends in detecting non-O157 STEC in food. Challenges There are many inherent challenges associated with pathogen detection in food, such as low levels of injured/stressed target cells, high levels of background ora, nonhomogeneous distribution of target pathogens, and complex food matrices involved (Ge and Meng, 2009). As a result, effective sampling and sample preparation prior to the actual analyses are deemed critical (Stevens and Jaykus, 2004; Brehm-Stecher et al., 2009; Dwivedi and Jaykus, 2011). Enrichment is often used to overcome these challenges and primarily serves to (1) resuscitate injured/stressed target cells; (2) increase the target cell numbers as much as a million-fold; and (3) dilute the effect

WANG ET AL. of food inhibitors and background ora on the assay. However, with enrichment, the sample-to-result time is inevitably extended to days rather than hours (Ge and Meng, 2009). Additional challenges unique to non-O157 STEC detection in food are evident. First, unlike E. coli O157:H7, phenotypic characteristics (e.g., sorbitol fermentation) that distinguish non-O157 STEC from generic E. coli are lacking. Therefore, non-O157 STEC is neither readily nor routinely cultured in clinical diagnostic and food-testing laboratories (Gould et al., 2009). Second, non-O157 STEC encompasses a diverse group of organisms with a total of 174 E. coli O-serogroups identied to date, although not all are capable of producing Shiga toxins (DebRoy et al., 2011a). Even highly pathogenic non-O157 STEC still comprises 410 serogroups (Karmali et al., 2003; Johnson et al., 2006; EFSA, 2009; USDA, 2012a), requiring a suite of assays for detection and conrmation. Third, a stepwise approach is commonly used for non-O157 STEC, rst screening for virulence genes (e.g., Shiga toxin genes [stx] and the E. coli attaching and effacing gene [eae]), then testing for O serogroupspecic genes (USDA, 2012a). Due to the lack of unequivocal virulence markers, nonpathogenic STEC strains may also give signals for the target genes at the rst-step screening. It is also challenging to conrm that the signals for stx, eae, and serogroup-specic genes in the enrichment

FIG. 1. Schematic diagrams of current procedures employed by the U.S. Food and Drug Administration (FDA) (A) and the U.S. Department of Agriculture (USDA) (B) for detecting non-O157 Shiga toxinproducing Escherichia coli (STEC) in food. Diagram A was drawn based on the text in FDA bacteriological analytical manual chapter 4A (FDA, 2012a), while diagram B was modied based on USDA microbiology laboratory guidebook 5B Appendix 4.01 (USDA, 2012a). mBPWp, modied buffered peptone water with pyruvate; qPCR, real-time quantitative polymerase chain reaction; L-EMB, Levines eosinmethylene blue agar; SHIBAM, STEC heart infusion washed blood agar with mitomycin C; TSAYE, trypticase soy agar with yeast extract; CC, ColiComplete disc; mTSB + n, modied trypticase soy broth with 8 mg/L novobiocin plus casamino acids; IMS, immunomagnetic separation; mRBA, modied rainbow agar; SBA, sheep blood agar.

DETECTION METHODS FOR NON-O157 STEC

FIG. 1.

(Continued)

samples are originated from individual cell or from a mixture of different cells; therefore, further conrmation steps are essential. Overview of Method Developments In the United States, food safety regulatory agencies such as the U.S. Food and Drug Administration (FDA) and USDA have each developed and validated a set of detection methods for foodborne pathogens of interest in various foods. Methods adopted for non-O157 STEC are available online at FDAs Bacteriological Analytical Manual (BAM) chapter 4A (FDA, 2012a) and FSISs Microbiology Laboratory Guidebook (MLG) method 5B (USDA, 2012a). Schematic diagrams of the two methods are shown in Figure 1; the methods combine culture, immunological, and molecular techniques as described below and take about 45 days. Many test kit manufacturing companies are also geared toward developing rapid detection methods for emerging pathogens such as non-O157 STEC. Table 1 includes a select list of commercially available non-O157 STEC test methods, several of which have recently received no-objection letters issued by FSIS (USDA, 2012d). Validation and certication of such methods are conducted by independent organizations such as the AOAC International, the International Organization for Standardization (ISO), and the Association French Normalization Organization Regulation. Very recently, ISO approved a real-time PCR-based method (ISO/TS 13136) for the detection of STEC O157, O111, O26, O103 and

O145 in food and animal feed (ISO, 2012). Furthermore, many academic and government research laboratories are actively involved in method development for foodborne pathogens, including non-O157 STEC, driving the innovation to an even faster pace. Below current trends in non-O157 STEC detection, isolation, and conrmation in food are reviewed. Culture-Based Methods Generally regarded as the criterion standard for pathogen detection in food, 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 conrmation (Feng, 2007; Ge and Meng, 2009). The hallmark of such methods is the use of suitable selective and differential culture media. Many of the media currently in use for nonO157 STEC are modied from those originally developed for E. coli O157:H7. For example, novobiocin is added to nonO157 enrichment broth at a lower concentration (e.g., 16 lg/mL) than the usual 20 lg/mL for E. coli O157:H7 due to less tolerance of non-O157 STEC to novobiocin (Vimont et al., 2007). Replacing sorbitol used in sorbitol MacConkey (SMAC) for culturing E. coli O157:H7 with rhamnose has led to the development of rhamnose-MacConkey (RMAC) for differential isolation of STEC O26 due to their inability to ferment rhamnose (Hiramatsu et al., 2002). Similar to E. coli O157:H7, tellurite-containing media have been used widely for selective culturing of STEC O26, O111, and O145 because of

Table 1. Partial List of Commercially Available Products for Detecting Non-O157 Shiga ToxinProducing Escherichia coli (STEC) Product name CHROMagar STEC Rainbow Agar O157 SHIBAM agar Target non-O157 STECa Manufacturer CHROMagar Microbiology, Paris, France Biolog, Inc., Hayward, CA Hardy Diagnostics, Santa Maria, CA KPL, Gaithersburg, MD Lab M Ltd., Lancashire, UK Life Technologies, Foster City, CA Denka Seiken Co., Ltd., Tokyo, Japan SDIX, Newark, DE Merck, Darmstadt, Germany Meridian BioScience, Inc., Cincinnati, OH Oxoid Ltd., Hampshire, UK

Assay type

Assay technology

Culture

Media

Immunological O26, O103, O111, O145 O26, O103, O111, O145 O26, O111 Top 6 All STEC

Enzyme-linked immunosorbent assay Immunomagnetic separation (IMS)

O26, O103, O104, O111, O118, O121, O145, and others Top 6 and others All STEC producing enterohemolysin Top 6

Lateral ow EIA

Latex agglutination Premier EHECb ProSpecT STECb BioStar OIA SHIGA TOXb

BacTrace Non-O157 STEC Antibodies Captivate IMS beads Dynabeads EHEC IMS (Seiken Particles) RapidChek CONFIRM non-O157 STEC IMS Duopath Verotoxins Gold Labeled Immunosorbent Assay (GLISA)b,c ImmunoCard STAT! EHECb Dryspot E. coli Seroscreen All STEC O26, O91, O103, O111, O128, and O145 All STEC All STEC All STEC

4 VTEC-RPLA VTEC-RPLA SEIKENb Assurance GDS Top 7 STECd BAX System Real-Time PCR STEC Suited GeneDisc STECc GeneDisc EHEC 5 IDc iQ-Check STEC VirX and SerOd VTEC MP-0510 Loopamp VTEC Detection Kit NeoSEEK STEC Conrmationd Assurance GDS MPX Top 7 STECd RapidFinder STECd Top 6 Top 6 Top 6

Microplate enzyme immunoassay (EIA) Optical immunoassay

Molecular

Reverse passive latex agglutination (RPLA) Real-time polymerase chain reaction (PCR)

All STEC All STEC All STEC harboring the eae gene Top 6 All STEC O103, O111, O26, O145 All STEC particularly top 6 O26, O103, O111, O145 All STEC

Meridian BioScience, Inc., Cincinnati, OH Remel, Lenexa, KS Inverness Medical Professional Diagnostics, Inc., Boston, MA Oxoid Ltd., Hampshire, UK Denka Seiken Co., Ltd., Tokyo, Japan BioControl Systems, Inc., Bellevue, WA DuPont Qualicon, Wilmington, DE Pall Corporation, Port Washington, NY Bio-Rad Laboratories, Hercules, CA Multiplicom N.V., Niel, Belgium Eiken Chemical Co., Tokyo, Japan Neogen Corp., Lansing, MI BioControl Systems, Inc., Bellevue, WA Life Technologies, Foster City, CA

Immunological and molecular

Loop-mediated isothermal amplication PCR-mass spectrometry IMS and PCR

IMS and real-time PCR

Top 6 non-O157 STEC serogroups consist of O26, O45, O103, O111, O121, O145. The assays have been approved by the U.S. Food and Drug Administrations Center for Devices and Radiological Health for in vitro diagnosis of STEC infections. c The assays have been validated by the AOACs Performance Tested Methods Program. d No-objection letters were issued by U.S. Department of Agriculture Food Safety and Inspection Service for these non-O157 STEC assays as of January 8, 2013 (USDA, 2012d).

DETECTION METHODS FOR NON-O157 STEC high-level tellurite resistance observed among these serogroups (Hiramatsu et al., 2002; Catarame et al., 2003; Orth et al., 2007). There are also several commercially available chromogenic agars for non-O157 STEC isolation, e.g., modied rainbow agar (mRBA; Biolog, Inc., Hayward, CA) (FDA, 2012a; USDA, 2012a) and CHROMagar STEC (CHROMagar Microbiology, Paris, France) (Hirvonen et al., 2012; Tzschoppe et al., 2012). medium based on a mixture of carbohyRecently, Posse drate sources (sucrose and sorbose), b-D-galactosidase activity, and selective compounds (bile salts, novobiocin, and potassium tellurite) has been described for color-based differentiation of STEC O26, O103, O111, and O145. Conrmation of suspected colonies would require plating on additional agars containing dulcitol, rhamnose, rafnose, or arabinose et al., 2008). The isolation efciency of Posse medium (Posse for low levels of non-O157 STEC in various dairy and meat et al., 2008). However, products ranged from 64.3-100% (Posse medium in food applione independent evaluation of Posse cations reported ambiguity in the color differentiation between non-O157 STEC serogroups (Mathusa et al., 2010). Another recent development is the STEC heart infusion washed blood agar with mitomycin C (SHIBAM agar) (Lin et al., 2012) adopted by the FDAs BAM (Fig. 1). SHIBAM agar modies washed sheep blood agar by adding mitomycin C and optimizes both the washed blood and base agar to facilitate STEC isolation (Beutin et al., 1996; Kimura et al., 1999; Sugiyama et al., 2001; Lin et al., 2012). Evaluation of SHIBAM agar showed that 89% (365 of 410) of STEC strains were hemolytic, while 86.3% (63 of 73) of non-STEC strains were not; SHIBAM also greatly improved the recovery of non-O157 STEC from articially inoculated produce at < 10 colonyforming units (CFU)/25 g when compared with Levines eosin-methylene blue (L-EMB) agar (Lin et al., 2012). For non-O157 enrichment, base media including trypticase soy broth (TSB), buffered peptone water (BPW), and E. coli broth (EC) are commonly used and frequently supplemented with novobiocin, cexime, potassium tellurite, vancomycin, acriavin, and cefsulodin to suppress the growth of background ora (Vimont et al., 2006; Hussein and Bollinger, 2008). The effects of enrichment broth, selective agent, and incubation protocol on the outcome of non-O157 STEC enrichment in various foods have been evaluated (Catarame et al., 2003; Vimont et al., 2006; Baylis, 2008; Hussein and Bollinger, 2008; Verstraete et al., 2010; Kanki et al., 2011; Gill et al., 2012). Catarame et al. (2003) reported that the optimum condition to recover E. coli O26 and O111 from minced beef was enrichment in TSB supplemented with cexime (50 lg/L), vancomycin (40 lg/mL), and potassium tellurite (2.5 lg/mL; this compound was omitted for E. coli O111). In contrast, BPW was found to give increased recovery rates of both STEC O26 and O111 compared with TSB and EC broth (Drysdale et al., 2004). Baylis (2008) compared 10 enrichment broths and found non-O157 STEC strains only grew poorly in EC or modied EC (mEC) supplemented with novobiocin (20 lg/mL), likely due to the inhibitory effect of novobiocin at 20 lg/mL as mentioned above. More recently, Gill et al. (2012) examined six selective agents (cexime, cefsulodin, mitomycin C, novobiocin, tellurite, and vancomycin) either singly or in combination for the optimal enrichment of a panel of 18 STEC serogroups in modied trypticase soy broth (mTSB), and found a combination of vancomycin (10 lg/mL) and cefsulodin (3 lg/mL) en-

5 abled the growth of all strains. Current FDAs BAM procedure for STEC enrichment in food uses modied BPW (containing casamino acids) with pyruvate for 5 h then supplemented with acriavin (10 lg/mL), cefsulodin (10 lg/mL), and vancomycin (8 lg/mL) (FDA, 2012a), while USDA uses mTSB supplemented with novobiocin (8 lg/mL) and casamino acids (USDA, 2012a). Several studies reported that incubating samples at 42C was more effective than 37C for isolating STEC O26, O111, and other STEC from various food samples ( Hara-Kudo et al., 2000; Catarame et al., 2003; Drysdale et al., 2004; Gill et al., 2012). We (Wang and others, unpublished data) also observed that 42C enrichment was clearly more effective than 37C for recovering low levels of the top 6 STEC serogroups from various produce items (lettuce, spinach, alfalfa sprouts). The current FDAs BAM protocol uses pre-enrichment at 37C for 5 h to resuscitate injured/stressed cells followed by selective enrichment at 42C overnight (FDA, 2012a), whereas the USDA method uses incubation at 42C for 1522 h (USDA, 2012a). Recently, exposure to strong acidic conditions (TSB adjusted to pH 2 or 3) for a short period of time (30 min2 h) was used to effectively reduce background ora before enrichment (Grant, 2005; Hu et al., 2009). The USDA method also applies a similar acid treatment step postenrichment to facilitate the isolation of non-O157 STEC strains from enrichment cultures (USDA, 2012a). Immunological-Based Methods Antibodies that recognize Shiga toxins or specic O antigens have been used in the direct detection of STEC serogroups in food samples or aid in their isolation through immunomagnetic separation (IMS) or colony immunoblot. Traditional cell cytotoxicity assays for the Vero and HeLa cell lines are very sensitive in detecting Shiga toxins but they are labor intensive, time consuming, and costly (Paton and Paton, 1998b; Johnson et al., 2006). Interestingly, simple and costeffective Vero cellbased assays have been described recently (Zhao and Haslam, 2005; Quinones et al., 2009). Since rst introduced to the United States in 1995, Shiga toxin enzyme immunoassays (EIAs) have gradually gained acceptance by clinical laboratories (Hoefer et al., 2011). To date, six Shiga toxin EIAs (Table 1) have been approved by the FDA for in vitro diagnosis of STEC infections (FDA, 2012b). Other commercially available EIAs include the Ridascreen Verotoxin EIA (R-Biopharm AG, Darmstadt, Germany) and VTEC-RPLA (Oxoid Ltd., Hampshire, United Kingdom). The time required to run these assays ranges from 20 min to 4 h, although overnight enrichment prior to the analysis is strongly recommended (Gould et al., 2009). Sensitivity of the EIAs can be enhanced by adding mitomycin C or polymyxin B in enrichment broth to stimulate the production or liberation of Shiga toxins ( Johnson et al., 2006). The application of Shiga toxin EIAs in food has been evaluated. Ridascreen and Premier EHEC (Meridian BioScience, Inc., Cincinnati, OH) were found to be 10-fold more sensitive than ProSpecT (Remel, Lenexa, KS); however, all three assays failed to detect Stx2d and Stx2e variants (Willford et al., 2009). Another study reported Premier EHEC to be useful for STEC screening in various food samples including raw milk, fresh and processed meat, and dairy food (Pontello et al., 2003). Very recently, Duopath Verotoxins kit (Merck, Darmstadt,

6 Germany) was incorporated in a proposed streamlined method for STEC detection in food (Gill et al., 2012). Currently, Duopath Verotoxins kit is the only Shiga toxin EIA validated by the AOACs Performance Tested Methods Program and the use for STEC detection is limited to pure culture, in contrast to numerous E. coli O157:H7 test kits validated by the same AOAC program or adopted as AOAC Ofcial Methods to be used in a variety of foods (AOAC International, 2012). This scenario is anticipated to change in the near future, given the new regulation and increasing demand for nonO157 STEC testing. Besides EIAs, a Shiga toxin colony immunoblot is commercially available (Roche Diagnostics GmbH, Mannheim, Germany). This assay has been used for the detection and isolation of STEC colonies from a primary isolation plate (Bettelheim, 2007). The addition of mitomycin C or trimethoprimsulfamethoxazole was found to stimulate toxin production, thereby facilitating immunoblot detection (Karch et al., 1986; Hull et al., 1993). Similar to Shiga toxins, E. coli O antigens are common markers for the detection and identication of non-O157 STEC serogroups. Conventional serotyping for O serogroup identication is based on agglutination reactions caused by the antigenantibody cross-linking (DebRoy et al., 2011a). Commercial latex agglutination kits for certain non-O157 STEC serogroups are now available (Table 1), while complete serotyping is generally conducted at E. coli reference laboratories such as those at the Centers for Disease Control and Prevention or Pennsylvania State University (Atkinson et al., 2006). Latex agglutination kits are rapid and easy to use, in direct contrast to conventional serotyping, which is time consuming and labor intensive. Very recently, USDA scientists described a method to prepare latex agglutination reagents for the top 6 non-O157 STEC serogroups by covalent immobilization of polyclonal immunoglobulin G antibodies onto the polystyrene particles (lateximmunoglobulin G) (Medina et al., 2012). Inclusivity and exclusivity testing showed that all target organisms produced positive results, but three antisera (anti-O26, anti-O103, and anti-O145) crossreacted with other STEC serogroups. The immuno-afnity displayed by E. coli O antigens and corresponding antibodies has also been used widely via the IMS process to facilitate the detection and isolation of nonO157 STEC from food. During IMS, microscopic, paramagnetic beads coated with specic antibodies are used to capture target pathogens from food matrices, thereby concentrating the target cells and simultaneously removing inhibitors from complex food samples (Ge and Meng, 2009; Dwivedi and Jaykus, 2011). The recovery efciency of IMS varies depending on the target pathogen and its antigenic expression, the afnity between pathogen surface antigen and antibody, and the physicochemical properties of the food matrix (Dwivedi and Jaykus, 2011). Commercial IMS products are now available for some non-O157 STEC serogroups (Table 1). Seiken particles (Denka Seiken Co., Ltd., Tokyo, Japan) in combination with selective enrichment and plating were found to be effective for the isolation of O26 and O111 from beef and produce (Hara-Kudo et al., 2000; Catarame et al., 2003; Kanki et al., 2011). The current USDA protocol uses SDIX beads (Newark, DE) coated with antibodies for the top 6 STEC followed by acid treatment and plating on mRBA agar (USDA, 2012a). It is important to note that the number of non-O157

WANG ET AL. STEC serogroups to which IMS can be applied is limited by the availability of specic and high-afnity antibodies for those serogroups. Very recently, ow cytometry and enzyme-linked immunosorbent assays (ELISA) have been developed for the rapid detection of the top 6 non-O157 STEC in ground beef using SDIX polyclonal antibodies (Hegde et al., 2012a; Hegde et al., 2012b). The ow cytometry assay was rapid, specic, and quantitative with a sensitivity of 2 103 target cells in pure culture and 110 cells in ground beef following 8 h of enrichment (Hegde et al., 2012b). The ELISA assays exhibited 100% specicity for serogroups O103, O111, and O121, 98.2% specicity for serogroups O26 and O45, and 99.1% specicity for serogroup O145. In articially inoculated ground-beef samples, the detection limits were in the range of 110 CFU/25 g following 24-h enrichment (Hegde et al., 2012a). Another study used enzyme-labeled phages (Phazymes) in combination with IMS enrichment on an ELISA platform to detect STEC O26, O103, O111, O145, and O157 (Willford et al., 2012). Depending on the serogroups, the detection accuracy ranged from 75.9% to 93% with detection limits of 105 to 106 CFU/mL in pure culture and 1 CFU/g in spinach samples following 8-h enrichment (Willford et al., 2012). Molecular-Based Methods Nucleic acid amplication tests (NAATs) such as PCR, realtime quantitative PCR (qPCR), and isothermal amplication techniques have been adopted to detect non-O157 STEC. The assays target either common virulence genes or O serogroup specic genes. The most frequently targeted virulence gens are those coding for Shiga toxins (Stx) and intimin. Over the years, many variants of Stx1, Stx2, and intimin have been identied, which currently encompass three Stx1 subtypes (Stx1a, Stx1c, and Stx1d) and seven Stx2 subtypes (Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g) (Feng et al., 2011), as well as 21 intimin subtypes (a1, a2, b1, nR/b2B, d/b2O, j, c1, c2, h, e1, mR/e2, f, g1, g2, i1, lR/i2, k, lB, mB, nB, and p) (Blanco et al., 2006). Other targets used include hemolysin gene (hlyA) and genes on pathogenicity islands (termed O islands), such as a number of type III non-LEE-encoded effector (nle) genes (Coombes et al., 2008; Madic et al., 2011). Since the early 1990s, numerous PCR assays have been developed to detect stx1 and stx2 (Table 2). As the numbers of subtypes increase, the variety and number of PCR assays designed to detect these subtypes also increase (Bettelheim and Beutin, 2003). Although most PCR assays were fast (24 h) and sensitive (10103 CFU/reaction), performances varied when evaluated independently (Bastian et al., 1998; Ziebell et al., 2002). With the advent of qPCR technology in the 2000s, great improvement in the speed and sensitivity of STEC detection has been achieved (Bettelheim and Beutin, 2003). In addition, many of the stx PCR and qPCR assays are amenable to multiplexing with those for eae or other virulence factors, a feature not easily achievable by immunological-based methods. Both FDA and USDA methods use qPCR assays to screen for STEC (Monday et al., 2007; FDA, 2012a; USDA, 2012a). It is of note that when comparing FDAs PCR method and commercially available Shiga toxin EIAs, both types of assays failed to detect some Stx subtypes but not others (Feng et al., 2011). Other studies also reported the failure of some PCR assays to detect genetically distant Stx subtypes (Reischl et al., 2002; Beutin et al., 2009).

Table 2. A Select List of Polymerase Chain Reaction (PCR) and Real-Time Quantitative PCR (qPCR) Assays for Detecting Shiga ToxinProducing Escherichia coli (STEC) Serogroups O26, O45, O103, O111, O121, and O145 Target genes Clinical DNA Beef and bovine feces Clinical samples Swine fecal samples Seeded apple juice Minced beef DNA DNA Clinical isolates Fecal samples Clinical isolates Raw food Food samples Pure culture Cheese Raw-milk cheeses 2 CFU/reaction 1.5 103 CFU/mL 106 CFU/0.2 g N/A 12 MPN/kg, after overnight enrichment 2 CFU/25 g or mL, after 20-h enrichment N/A 5 CFU/25 g, after overnight enrichment N/A 110 CFU/mL, after overnight enrichment 103 colony-forming units (CFU)/reaction N/A 110 CFU/g, after 16-h enrichment N/A N/A 10 CFU/mL, after 18-h enrichment 1020 CFU/g, after 6-h enrichment 525 copies/reaction Matrices Sensitivity Reference (Paton and Paton, 1998a) (Wang et al., 1998) (Sharma, 2002) (DSouza et al., 2002) (Fratamico et al., 2003) (DebRoy et al., 2004) (OHanlon et al., 2004) (Perelle et al., 2004) (Perelle et al., 2005) (Fratamico et al., 2005) (DebRoy et al., 2005) (Feng et al., 2005) (Perelle et al., 2007) (Fratamico et al., 2009) (Beutin et al., 2009) (Madic et al., 2010) (Madic et al., 2011) (Valadez et al., 2011) (DebRoy et al., 2011b) (Fratamico et al., 2011) (Lin et al., 2011b) Food and clinical samples Cattle feces Ground beef (Paddock et al., 2012) (Fratamico and Bagi, 2012)

Year

Platform

Serogroup

1998 1998 2002 2002 2003 2004 2004 2004

PCR PCR qPCR PCR PCR PCR qPCR PCR

O111 O111 O26, O111 O26 O121 O26 O26, O111 O26, O103, O111, O145

2005 2005 2005 2005 2007

qPCR qPCR PCR, qPCR PCR qPCR

O103 O103 O45 O145 O26, O103, O111, O145

7 wzx stx1, stx2, eae, wzx Beef carcass swab, beef trim, and ground beef Culture, apple juice Ground beef wzxO26, wzyO45, wzxO103, wzxO111, wzyO121, wzyO145 wzx, wbqO121 O-serogroup specic genes

2009 2009

PCR, qPCR qPCR

O145 O26, O103, O111, O145

2010 2011

qPCR qPCR

O26, O103, O111, O145 O26, O103, O111, O145

2011

PCR

O26, O103, O111, O145

stx1, stx2, eae, rfbO111 wzx,wzy, wbdH, wbdM stx1, stx2, eaeO26, eaeO111 wzx, wzy, wbuA wzx, wzy wzx, wzy iCO26, iAO26, wzyO111 wzxO26, eaeO103, wbdIO111, ihp1O145 wzx, galE wzx, wzy wzx, wzy wzx, wzy wzxO26, eaeO103, wbdIO111, ihp1O145 stx1, stx2, wzx, wzy stx, eae, wzxO26, wzxO103, wbdIO111, ihp1O145 iC, eae stx, eae, wzxO26, wzxO103, wbdIO111, ihp1O145 wzxO26, wzxO103, rfbO111, wzxO145

2011

PCR

O26, O45, O103, O111, O121, O145

2011

qPCR

2011

qPCR

2012

PCR

2012

qPCR

O26, O45, O103, O111, O121, O145 O26, O45, O103, O111, O121, O145 O26, O45, O103, O111, O121, O145 O26, O45, O91, O103, O111, O113, O121, O145, O157

10 copies/reaction for TSB, 100 copies/ reaction for apple juice, after 16-h enrichment 50 CFU/reaction, 12 CFU/25 g after 24-h enrichment 30 CFU/25 g or mL for food, 1000 CFU/ 0.5 g for stools, after 24-h enrichment 4.1 105 CFU/g without enrichment, and 2.3 102 CFU/g after 6 -h enrichment 220 CFU/25 g after 24-h enrichment

N/A, not available; MPN, most probable number; TSB, trypticase soy broth.

8 As the complete sequences of many (93 to date) E. coli O-antigen gene clusters become available, corresponding NAATs for specic O-serogroups are being developed at a fast pace (DebRoy et al., 2011a). Two genes, wzx and wzy, encoding the O-antigen ippase and the O-antigen polymerase, respectively, were unique across most of the E. coli O-antigen gene clusters, promising good targets for serogroupspecic detection. PCR primers for 58 E. coli O-serogroups targeting these two genes were compiled recently (DebRoy et al., 2011a). Additional genes on the O-antigen gene cluster, such as wbd and wzm, have also been used as targets for assay design (Wang et al., 1998; DebRoy et al., 2011a). Table 2 is a select list of PCR and qPCR assays developed for the top 6 nonO157 STEC serogroups; many are multiplex assays for stx, eae, and other virulence genes. Recently, a novel NAAT termed loop-mediated isothermal amplication (LAMP) has attracted great attention as a rapid, specic, sensitive, and cost-effective pathogen detection method in food testing and clinical diagnostics (Notomi et al., 2000; Mori and Notomi, 2009). LAMP employs four to six specially designed primers and a strand-displacing Bst DNA polymerase to amplify up to 109 target DNA copies under isothermal conditions (6065C) within an hour (Notomi et al., 2000). LAMP assays targeting stx1 and stx2 have been developed and evaluated in food samples (Hara-Kudo et al., 2007; Hara-Kudo et al., 2008; Kouguchi et al., 2010; Zhao et al., 2010). A LAMP STEC detection kit (Eiken Chemical Co., Ltd., Tokyo, Japan) is commercially available (Table 1). Very recently, Wang et al. (2012a; 2012b) developed a suite of LAMP assays by targeting stx1, stx2, eae, and wzx or wzy genes of the top 6 nonO157 STEC serogroups and E. coli O157:H7 and evaluated the assays in ground beef and produce samples. The assays were rapid (40 min), specic, and sensitive (approximately 120 CFU per reaction). After 68 h of enrichment, the LAMP assays consistently achieved accurate detection of very low levels (12 CFU/25 g) of non-O157 STEC cells in ground beef, beef trim, and produce (Wang et al., 2012b). Despite the advances in developing NAATs for the rapid, specic, and sensitive detection of non-O157 STEC in food, except for those listed in Table 1, the vast majority of methods are not yet commercially available. Other caveats associated with NAATs are the presence of assay inhibitors in food and the inability to differentiate dead from viable cells (Ge and Meng, 2009). The inclusion of internal amplication controls and the addition of viability dyes such as propidium monoazide have been developed to address those concerns (Yaron and Matthews, 2002; Chen et al., 2011; Li and Chen, 2012). Finally, the detection of virulence genes is not a guarantee of gene expression (Feng et al., 2011), and in the case of multiple STEC strains contaminating a single food sample, further conrmation is needed to determine whether the gene proles detected are from a single strain or multiple different strains (Gould et al., 2009). On several occasions, NAATs have been combined with immunological methods for the increased sensitivity. For example, a PCR assay (targeting common regions of stx1 and stx2) was coupled with ELISA to develop a highly sensitive and specic method for STEC detection in ground beef (Ge et al., 2002). On the other hand, highly sensitive immuno-PCR assays have been developed to detect Stx2 toxin and its variants based on antibody capture followed by DNA amplication (Zhang et al., 2008; He et al., 2011). Assurance GDS MPX

WANG ET AL. Top 7 STEC (BioControl Systems, Inc., Bellevue, WA) uses IMS rst to capture E. coli O157 and the top 6 non-O157 STEC serogroups, which is followed by qPCR assays (Table 1). Probe hybridization is yet another format of molecularbased detection method. Specically, colony-lift hybridization procedures have been applied in an effort to increase the isolation of non-O157 STEC strains, particularly when they are present at low levels relative to background ora, as in food samples (Grant et al., 2011). The process is similar to colony immunoblot described above but differs in that probes are used rather than antibodies. Specically, the samples are screened rst by PCR for stx1 and stx2, then positive samples are plated out and hybridized onto membranes with stxlabeled probes (Grant et al., 2011). This approach has been used successfully in multiple studies to facilitate the isolation of non-O157 STEC from various food samples (Begum et al., 1993; Cobbold and Desmarchelier, 2000; Jenkins et al., 2003; Nielsen and Andersen, 2003; Auvray et al., 2007; Stephan et al., 2008; Ju et al., 2012). Advanced Technologies Several low-density DNA oligonucleotide microarrays combining the detection of multiple O-serogroups and H types, as well as virulence genes, have been designed for the rapid detection and characterization of STEC strains (Ballmer et al., 2007; Bugarel et al., 2010; Quinones et al., 2012). For example, a GeneDisc array was used to identify 12 O-serogroups (top 6, O55, O91, O104, O113, O118, and O157) and 7 H types (H2, H7, H8, H11, H19, H21, and H28), which are the most clinically relevant STEC serotypes worldwide. Multiple virulence genes were also simultaneously targeted, including stx1, stx2, eae, ehxA (encoding enterohemolysin), and nleB and nleE (located on O island-122), among others (Bugarel et al., 2010). High specicity and concordance with conventional serotyping were reported, suggesting a valuable approach for the identication of STEC strains with a high potential for human disease. It is noteworthy that such assays have not been directly applied in food enrichment broth; rather, they are conrmation tools after pure cultures have been isolated and DNA purication is necessary to obtain reliable results. Recently, microbead-based suspension arrays using the Luminex technology have been used in an immunoassay to detect Shiga toxins (Clotilde et al., 2011) and in a probe-based assay to detect 10 clinically relevant STEC serogroups (top 6, O91, O113, O128, and O157) (Lin et al., 2011a). The immunoassay recognized Stx1 and several Stx2 subtypes and correctly identied them in 48 samples of ground beef, lettuce, and milk spiked with low levels ( < 2 CFU/g) of STEC strains (Clotilde et al., 2011). In the probe-based assay (Lin et al., 2011a), PCR primers were rst designed to target serogroup-specic wzx or wzy genes. The PCR products were hybridized to probelabeled bead sets, and uorescence signals of the beads were detected on a Luminex platform (Bio-Rad Laboratories, Hercules, CA). The assay was rapid ( < 4 h), specic, robust, and high throughput as demonstrated in a multilaboratory validation study (Lin et al., 2013). As the Luminex technology is designed to detect up to 100 analytes, the array is amenable to expansion with additional O-serogroups of interest (Lin et al., 2011a). Very recently, polymorphisms of the clustered regularly interspaced short palindromic repeat (CRISPR) regions of

DETECTION METHODS FOR NON-O157 STEC STEC strains belonging to serotypes O26:H11, O45:H2, O103:H2, O104:H4, O111:H8, O121:H19, O145:H28, and O157:H7 have been explored as unique markers for assay development (Delannoy et al., 2012a; Delannoy et al., 2012b). DNA sequences of the CRISPR loci of these strains were analyzed and unique regions were selected as targets for designing single qPCR assays for each individual serotype (O:H combination). The assays were shown to be highly specic and sensitive, although cross-activity occurred, particularly to strains of the same H type (Delannoy et al., 2012a; Delannoy et al., 2012b). Further studies are warranted to evaluate the accuracy of these CRISPR-based assays with a large strain set and also their applicability in food testing. Future Directions Given the recently implemented USDA regulation on six non-O157 STEC serogroups in raw beef products, testing demand for non-O157 STEC in food is on the upward trend. In response to this growing market, many test-kit-manufacturing companies may rapidly expand their efforts to develop and validate such kits. An ideal detection system for non-O157 STEC should be rapid, specic, sensitive, high throughput, and capable of simultaneously detecting multiple serogroups. Due to the low numbers of non-O157 STEC organisms in food, enrichment may be an indispensable step. One strategy to decrease the sample-to-result time is to design improved media formulations that would facilitate ash enrichment. This improved enrichment procedure could then be coupled with rapid screening tools (e.g., Shiga toxin EIA, PCR, LAMP) to obtain denitive negative or presumptive positive results. For presumptive positive samples, further conrmations by traditional culture-based methods are needed. It is therefore important that methods be developed to facilitate the efcient isolation of non-O157 STEC in the presence of high-level background cells by culture. Such methods would include improved IMS, immunoblot, colony hybridization protocols, as well as better-formulated selective and differential media. Any media formulation (or modication) will need to be carefully designed and validated to ensure its applicability across pertinent non-O157 serogroups while still effectively reducing interfering background ora. Also, for the purpose of public health surveillance and disease prevention and control, bacterial cultures remain critical. It is also desirable that new procedures for detecting nonO157 STEC in food be integrated as much as possible into those already in place for E. coli O157:H7. Although this review focuses on the top 6 non-O157 STEC serogroups in the United States, we acknowledge that non-O157 STEC is a diverse group that is emerging and rapidly evolving. Therefore, detection methods should be easily adaptable to accommodate newly recognized serogroups of signicant public health interest, as in the case of E. coli O104:H4, an enteroaggregative E. coli that acquired the stx gene and has been implicated in the recent German (also involving other countries) outbreak (Buchholz et al., 2011). Technologies routinely used in chemical, physical, and engineering laboratories have been adopted for STEC detection, such as biosensors (Subramanian et al., 2012), mass spectrometry (Pierce et al., 2012), and nanotechnology ( Jyoti et al., 2010). Recently, an inexpensive and user-friendly pointof-care testing device (termed Gene-Z) was demonstrated to Disclosure Statement No competing nancial interests exist. References

9 be able to carry out multiplexed genetic testing using a wireless connection (Stedtfeld et al., 2012). The rapidly evolving next-generation sequencing technology is yet another powerful tool that may potentially transform the methods used for identifying and characterizing foodborne pathogens. Although pure culture is generally required to carry out next-generation sequencing, culture-independent sequencing technologies do exist and have been used to examine the ecology of bacterial population in the environment, food, and the intestinal tracts of animals and humans (Pallen et al., 2010). With collaborative efforts among scientists in multiple disciplines, such promising technologies may be adopted in the near future for the detection of pathogens such as non-O157 STEC in food.

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Address correspondence to: Beilei Ge, PhD Division of Animal and Food Microbiology Ofce of Research Center for Veterinary Medicine U.S. Food and Drug Administration 8401 Muirkirk Road Laurel, MD 20708 E-mail: beilei.ge@fda.hhs.gov