A new pyrosequencing assay for rapid detection and genotyping of Shiga toxin, intimin and O157-specific rfbE genes of Escherichia coli.

MIMET-04531; No of Pages 13 Journal of Microbiological Methods xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth

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Noriko Goji a, Amit Mathews b, George Huszczynski b, Chad R. Laing c, Victor P.J. Gannon c, Morag R. Graham d, Kingsley K. Amoako a,⁎

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a r t i c l e

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Article history: Received 1 October 2014 Received in revised form 3 December 2014 Accepted 4 December 2014 Available online xxxx

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Keywords: STEC Pyrosequencing Genotyping Intimin SNP O157:H7/NM

Canadian Food Inspection Agency, National Centres for Animal Disease, Lethbridge Laboratory, P. O. Box 640, Township Road 9-1, Lethbridge, Alberta T1J 3Z4, Canada Canadian Food Inspection Agency, Greater Toronto Area Laboratory, 2301 Midland Ave., Scarborough, Ontario M1P 4R7, Canada Public Health Agency of Canada, Lethbridge, Laboratory for Foodborne Zoonoses, P. O. Box 640, Township Road 9-1, Lethbridge, Alberta T1J 3Z4, Canada d Public Health Agency of Canada, National Microbiology Laboratory, 1015 Arlington St., Winnipeg, Manitoba R3E 3R2, Canada b

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A new pyrosequencing assay for rapid detection and genotyping of Shiga toxin, intimin and O157-specific rfbE genes of Escherichia coli

Shiga toxin (stx)-producing Escherichia coli (STEC) contamination in food and water is one of the most recognized concerns and a major financial burden in human hygiene control worldwide. Rapid and highly reliable methods of detecting and identifying STEC causing gastroenteric illnesses are crucial to prevent foodborne outbreaks. A number of tests have been developed and commercialized to detect STEC using molecular microbiology techniques. Most of these are designed to identify virulence factors such as Shiga toxin and intimin as well as E. coli O and H antigen serotype specific genes. In order to screen pathogenic STEC without relying on O: H serotyping, we developed a rapid detection and genotyping assay for STEC virulence genes using a PCR-pyrosequencing application. We adapted the PyroMark Q24 Pyrosequencing platform for subtyping 4 major virulence genes, Shiga toxin 1 and 2 (stx1 and stx2), intimin (eae) and O157-antigen gene cluster target rfbE, using Single Nucleotide Polymorphism (SNP) analysis. A total of 224 E. coli strains including isolates from Canadian environment, food and clinical cases were examined. Based on the multiple alignment analysis of 30–80 base nucleotide pyrogram reads, three alleles of the Shiga toxin 1a gene (stx1a) (stx1a-I, stx1a-II, stx1a-III) were identified. Results of the stx1, stx2, eae and rfbE genotyping revealed that each group of O:H serotype shares distinctive characteristics that could be associated with the virulence of each genotype. O157:H7/NM carries stx1a-II (94%), stx2a (82%), λ/γ1-eae (100%) and rfbE type-H7/NM (100%). Whereas isolates of the “Top-6” serotypes (O26, O45, O103, O111, O121, O145) had a high incidence of stx1a-I (90%) and stx2a (100%), stx1a-III (60%) was only observed in non Top-7 (Top-6 plus O157) STEC and Shigella spp. The entire assay, from extracting DNA from colonies on a plate to the generation of sequence information, can be completed in 5 h. The method of profiling these 4 STEC pathogenic genotypes as demonstrated in this paper is rapid, easily performed, informative and cost-effective, and thus has a potential to be deployed in the food industry for the routine screening of potentially pathogenic STEC isolates. © 2014 Published by Elsevier B.V.

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food testing laboratories, STEC colonies are identified following enrichment and culture on selective media such as MacConkey agar and modified rainbow agar. PCR and real-time PCR have been commonly applied to pathogen detection and identification in food laboratories, replacing the traditional and time consuming method of biochemical and immunological characterization after culture. Using this nucleotide amplification and detection technology, various virulence factors associated with enterohemorrhagic E. coli (EHEC) have been investigated as pathogen-specific gene targets. Shiga toxin (stx1, stx2), intimin (eae), intimin receptor (tir), hemolysin (hlyA or ehxA), type III secreted protein (espA and espB), putative adherence protein (saa), flagellin, also known as H-antigen (fliC), and O antigen specific genes are some of the most commonly-tested genes using PCR and real-time PCR (Bai et al., 2012; Conrad et al., 2014; Marejkova et al., 2013). Due to the complexity of these virulence genes and their diverse contribution to

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1. Introduction

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Shiga toxin producing Escherichia coli (STEC) is one of the most frequent causes of food- and water-borne gastroenteritis in developing countries as well as developed countries that involves human disease outbreaks and food recalls. In a recent report 2,801,000 acute illnesses per year are estimated to be caused by STEC worldwide (Majowicz et al., 2014). Since STEC frequently originate from a ruminant reservoir and can contaminate the farm environment (Karmali et al., 2010), monitoring and identifying STEC through the supply chain from farming environments to fresh foods and food products is one of the most important control points for preventing human disease outbreaks. In

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⁎ Corresponding author. Tel.: +1 403 382 5597; fax: +1 403 381 1202. E-mail address: [email protected] (K.K. Amoako).

http://dx.doi.org/10.1016/j.mimet.2014.12.003 0167-7012/© 2014 Published by Elsevier B.V.

Please cite this article as: Goji, N., et al., A new pyrosequencing assay for rapid detection and genotyping of Shiga toxin, intimin and O157-specific rfbE genes of Escherichia coli..., J. Microbiol. Methods (2014), http://dx.doi.org/10.1016/j.mimet.2014.12.003

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2.1. Bacterial strains and DNA template preparation

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2. Materials and methods

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All bacterial strains tested in this study are listed on Table 1. E. coli strains isolated from the environment, and animal faces or human clinical samples were obtained from the Public Health Agency of Canada (PHAC), Lethbridge and Guelph Laboratories, and Canadian Food Inspection Agency (CFIA), Greater Toronto Area Laboratory (GTA) and Ottawa Laboratory Carling (OLC). The serotypes of each isolate were previously tested by each provider. Reference strains of Shiga toxin subtypes for stx1a, stx1c, stx1d and stx2a, stx2b, stx2c, stx2d, stx2e, stx2f and stx2g were generously provided by Dr. Roger Johnson, PHAC Laboratory for Foodborne Zoonoses Guelph Ontario, with permission from Dr. Scheutz, WHO Reference Laboratory, Statens Serum Institut, Denmark. The “Top-7” (O26, O45, O103, O111, O121, O145 and O157) strains were provided by the Genomic Research and Development Initiative Food and Water Safety (GRDI FWS) Pilot funding and project strain collation. The E. coli strains and 12 other food-borne bacteria, listed in Table 1, were cultured overnight at 37 °C on tryptic soy agar plates with 5% sheep blood. An individual colony was picked with a disposable plastic loop and suspended into 0.2 mL PCR tube containing 50 μL of sterile distilled water. At least three colonies were tested independently for each strain. The sample was heated at 100 °C for 5 min on a heat block. After brief vortexing and centrifugation for 2 min at 10,000 ×g at room temperature, 30 μL supernatant was mixed with an equal amount of 2× Tris EDTA buffer (pH 8.0) and stored at −20 °C until use. Some purified DNA samples obtained from E. coli O157:H7 (listed in Table 1) were also tested. The DNA concentrations were adjusted to 10 ng/μL in TE buffer based on the measurements on NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA) and stored at −20 °C until use.

156 157

2.2. PCR assay design and 2-plex PCR conditions

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Complete or partial nucleotide sequences of Shiga toxin subtypes (stx1a, stx1c, stx1d and stx2a, stx2b, stx2c, stx2d, stx2e, stx2f and stx2g), intimin gene subtypes (α1-eae, α2-eae, β-eae, γ1-eae, γ2-eae, ε-eae, ζ-eae, η-eae, θ-eae, ι-eae, κ-eae, λ-eae, μ-eae, ν-eae, ο-eae, π-eae, ρ-eae, σ-eae, τ-eae, and υ-eae), with at least two representatives from each subtype, and rfbE genes were selected from a literature search (Blanco et al., 2004; Cookson et al., 2007; Scheutz et al., 2012; Wasilenko et al., 2012) and GenBank (http://www.ncbi.nlm.nih.gov/ nucleotide/). The NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and Geneious sequence search tool on the Geneious 7R software (Biomatters, Auckland, New Zealand) were used to align multiple genes. The target regions of each stx1, stx2, eae and rfbE genes were meticulously selected as shown in Figs. 1–3. The consensus sequences selected were then imported into the PyroMark Assay Design software version 2.0.1 (Qiagen Inc., Hilden, Germany) and the PCR primers and

186 187

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93 94

140 141

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91 92

O157:H7, O26:H11, O103:H2, O111:H8 and O145:H28 and would be useful for screening STEC. In this study, we applied the combination of multiplex PCR and pyrosequencing technology for detection and genotyping of E. coli isolates from the group of “Top-7” serotypes (the seven priority serotypes that most frequently causes severe clinical cases worldwide including O26, O45, O103, O111, O121, O145 and O157) in addition to those that were randomly selected from human or animal feces and the Canadian environment, representing a total of 102 different O:H serotypes. We focused on four virulence genes – Shiga toxin 1 and 2 (stx1 and stx2), intimin (eae), and O157 specific biosynthesis gene (rfbE) and investigated the assay as a potential diagnostic tool for the screening of high-risk STEC colonies from clinical, food or environmental samples without depending on serological typing.

T

89 90

C

87 88

E

85 86

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83 84

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80

C

78 79

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76 77

human illness, determination of the existence of these genes may not provide sufficient information on the potential hazard of E. coli isolates (Feng and Reddy, 2013). Thus PCR and real-time PCR positive results have been treated as “presumptive positive” in STEC detection and used only for screening and require additional confirmation procedures. In order to provide more reliable results and a higher capacity to characterize isolates for trace-back to outbreak sources, a variety of genotyping methods have been developed. Single nucleotide polymorphism (SNP) analysis has been one of the most discriminatory genomic comparative approaches and has been widely examined using various techniques such as 454 resequencing (Kotewicz et al., 2008), MultiLocus Variable number of tandem repeat Analysis (MLVA) (Byrne et al., 2014), microarray (Laing et al., 2009), electrospray ionization mass spectrometry (Shen et al., 2013) and whole genome sequencing (Steyert et al., 2012; Underwood et al., 2013). Whole genome sequencing has become more affordable with shorter run-time in the last few years, and has been applied to routine STEC testing (Joensen et al., 2014). However, this highly robust tool requires a significant amount of training for operators, and time to prepare DNA library samples from isolated colonies. Furthermore, in order to analyze the very large volume of nucleotide sequence data generated, a high-speed computer and sequence analysis software are required. Pyrosequencing is a novel sequence-based detection technology that enables rapid and accurate quantification of sequence variations (Kreutz et al., 2013). We have recently demonstrated the application of this technique for the detection of Yersinia pestis strains and their antimicrobial resistant gene profiling, and also the detection of Y. pestis and B. anthracis from food (Amoako et al., 2013, 2012a,b). This highly sensitive and specific technology provides not only an added layer of confidence compared to a presumptive positive PCR or real-time PCR result, but also potential genotype information that could be critical for diagnosis, when PCR primers and sequencing primers are designed to detect gene mutation and/or single nucleotide polymorphisms (SNPs) (Alderborn et al., 2000). In addition, multi-base reading capacity around 50–100 bp enables the detection of multiple closely located SNPs in a single run for the identification of genetically variable genes such as stx1, stx2 and eae. Shiga toxin is the enterotoxin secreted by STEC and Shigella spp. that causes gastroenteritis in humans and other animals, and it is known to have phenotypic and genotypic variations. According to current nomenclature, there are 3 and 7 subtypes for stx1 and stx2, respectively — stx1a, stx1c, stx1d, stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, stx2g (Scheutz et al., 2012). Some STEC isolates carry both stx1 and stx2, or more than one stx2 subtype. Although there is not a clear borderline in virulence among Shiga toxin subtypes, previous reports indicate that STEC isolates associated with severe gastrointestinal disease cases like hemolytic uremic syndrome (HUS) most often carry subtype stx2a (Haugum et al., 2014) and/or stx2c (Persson et al., 2007). The stx2e and stx2g subtypes are not frequently associated with human disease cases (Beutin et al., 2008; Prager et al., 2011) and recently, Friesema et al. reported mild diarrhea cases caused by stx2f (Friesema et al., 2014). Thus, Shiga toxin subtyping could potentially provide valuable information associated with risk assessment for human illness and environmental and food surveillance. The intimin gene (eae) is part of a pathogenicity island termed the locus of enterocyte effacement (LEE) that is responsible for adhesion of the host organism to the intestinal mucosa and forming attaching and effacing (A/E) lesions. LEE is located on the chromosome and known to have considerable nucleotide variation at the 3′-end. At least 28 subtypes have been characterized (Lacher et al., 2006) and eae gene subtyping methods using restriction fragment length polymorphism (RFLP) of PCR products and/or sequencing the 3′-end variable regions have been reported (Cookson et al., 2007; Lacher et al., 2006). Recently, to replace these time-consuming detection methods, Madic et al. have developed a multiplex real-time PCR assay for β1-, γ1-, ε- and θ-eae detection (Madic et al., 2010). These 4 eae subtypes are found in the major highly-virulent STEC serogroups including

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N. Goji et al. / Journal of Microbiological Methods xxx (2014) xxx–xxx

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158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184

188 189 190 191 192 193 194 195 196 197 198 199 200


stx1 Pyro

stx1 ref

stx2 Pyro

stx2 ref

O26

Bovine feces

1a–I

1a

–

–

β

β

–

–

ECI–2703

O26

Bovine feces

1a–I

1a

–

–

β

β

–

–

ECI–2706

O26

Bovine feces

1a–I

1a

–

–

β

β

–

–

ECI–2617

O26:H11

Bovine feces

1a–I

1a

–

–

β

β

–

–

ECI–2752

O26:H11

Bovine feces

1a–I

1a

–

–

β

β

–

–

ECI–3065

O26:H11

Environment

1a–I

1a

–

–

β

β

–

–

ECI–2618

O26:H11

Human

1a–I

1a

–

–

β

β

–

–

ECI–2853

O26:H11

Human

1a–I

1a

–

–

β

β

–

–

ECI–2858

O26:H11

Human

1a–I

1a

–

–

β

β

–

–

ECI–2862

O26:H11

Human

1a–I

1a

–

–

β

β

–

–

ECI–2904

O26:H11

Water

1a–I

1a

–

–

β

β

–

–

ECI–3066

O26:H11

Water

1a–I

1a

–

–

β

β

–

–

ECI–3067

O26:H11

Water

1a–I

1a

–

–

β

β

–

–

O26 GTA

O26

Unknown

1a–I

1a

–

–

β

β

–

–

FWS_EC_0001

O26:H11

Human

1a–I

1a

2a

2a

β

β

–

–

ECI–2645

O26:NM

Bovine feces

1a–I

1a

–

–

β

β

–

–

ECI–2663

O26:NM

Bovine feces

1a–I

1a

–

–

β

β

–

–

ECI–3014

O26:NM

Bovine feces

1a–I

1a

–

–

β

β

–

–

ECI–2920

O26:NM

Environment

1a–I

1a

–

–

β

β

–

–

ECI–2852

O26:NM

Human

1a–I

1a

–

–

β

β

–

–

ECI–2664

O26:NM

Bovine feces

1a–I

1a

–

–

β

β

–

–

ECI–2699

O26+

Bovine feces

1a–I

1a

–

–

β

β

–

–

FWS_EC_0003

O45:H2

Human

1a–I

1a

–

–

ε

ε

–

– –

ε

–

–

–

–

–

–

–

ε

ε

–

–

1a

–

–

ε

ε

–

–

1a

–

–

ε

ε

–

–

1a 1a

– –

– –

ε θ/ γ2

ε θ/ γ2

– –

– –

–

–

θ/ γ2

θ/ γ2

–

–

1a

–

–

β

β

–

–

ECI–2934

O103

Unknown

1a–I

1a

FWS_EC_0007 ECI–2860

O103:H2 O103:H25

Human Human

1a–I 1a–I

ECI–2891

O103:H25

Water

1a–I

ECI–2700

O111

Bovine feces Unknown Bovine feces Human

ECI–2855

O111:HNM

Human

FWS_EC_0005

O111:HNM

Human

ECI–2579

O121:H19

Human

O121:H19

Human

O121:H19

Water

O121:H19

Water

O121:H19

Water

N C O

ECI–2797 ECI–2900

R

ECI–2850 ECI–2796

1a–I

1a

–

–

β

β

–

–

1a–I 1a–II

1a 1a

– –

– –

β θ/ γ2

β θ/ γ2

– –

– –

1a–II

1a

–

–

θ/ γ2

θ/ γ2

–

–

1a–II

1a

2a

2a

θ/ γ2

θ/ γ2

–

–

–

–

2a

2a

ε

ε

–

–

E

O111 O111:H11 O111:HNM

1a

1a–I

R

O111 GTA ECI–2701 ECI–2572

T

1a–I

C

1a–I

E

–

Human feces

F

ε

1a

–

Human

rfbE ref

–

1a–I

O103:H2

rfbE Pyro

–

Unknown Unknown

O103:H2

eae ref

–

O45 O45

ECI–2634

eae Pyro

–

EHEC–108 EHEC–114

ECI–2851

P

O:H–serotype

D

Strain names

R O

Origin

ECI–2702

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Table 1 Bacterial strains tested in this study and the results of PCR and pyrosequencing. Highlighted cells are showing discrepancy between pyrosequencing and conventional reference PCR results.

–

–

2a

2a

ε

ε

–

–

–

–

2a

2a

ε

ε

–

–

–

–

2a

2a

ε

ε

–

–

–

–

2a

2a

ε

ε

–

–

GTA–EHEC–105

O121

Unknown

–

–

2a

2a

ε

ε

–

–

GTA–EHEC–107

O121

Unknown

–

–

2a

2a

ε

ε

–

–

GTA–EHEC–110

O121

Unknown

–

–

2a

2a

ε

ε

–

–

FWS_EC_0006

O121:H19

Human

–

–

2a

2a

ε

ε

–

–

ECI–2578

O121:H7

Bovine feces

1d

1d

–

–

–

–

–

–

ECI–2849

O121:untypeable

Human

–

–

2a

2a

ε

–

–

1a–I

1a

–

–

ε λ/γ1

γ1

–

–

–

–

–

–

λ/γ1

γ1

–

–

O145 GTA ECI–2591 ECI–2585 FWS_EC_0002

O145

Unknown

O145:NM

Bovine feces

O145:NM

Human

1a–I

1a

–

–

λ/γ1

γ1

–

–

O145:NM

Human

1a–I

1a

–

–

λ/γ1

γ1

–

–

U

t1:1 t1:2 t1:3

3

GTA–EHEC–08

O157:H7

Beef trim

1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

GTA–EHEC–15

O157:H7

Beef trim

1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

GTA–EHEC–52

O157:H7

Frozen raw beef patties

1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

GTA–EHEC–55

O157:H7


1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

GTA–EHEC–57

O157:H7


1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

GTA–EHEC–59

O157:H7


1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

GTA–EHEC–44

O157:H7

Bovine liver

–

–

2a

2a

λ/γ1

γ1

H7/NM

+

GTA–EHEC–71

O157:H7

Frozen spiced beef burger

1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

(continued on next page)


4

Table 1 (continued)

stx1 ref

stx2 Pyro

stx2 ref

eae Pyro

eae ref

rfbE Pyro

1a–II

1a

2a*

2a, 2c

λ/γ1

γ1

H7/NM

+

–

–

2a

2a, 2c

λ/γ1

γ1

H7/NM

+

1a–II

1a

2a

2a, 2c

λ/γ1

γ1

H7/NM

+

Beef burger

–

–

2a, 2c

2a, 2c

λ/γ1

γ1

H7/NM

+

O157:H7

Beef burger

–

–

2a, 2c

2a, 2c

λ/γ1

γ1

H7/NM

+

GTA–EHEC–198

O157:H7

Veal cheek meat

–

–

2a

2a

λ/γ1

γ1

H7/NM

+

ECI–1375

O157:H7

Bovine feces

1a–II

1a

2a

2a

λ/γ1

+

H7/NM

+

ECI–1379

O157:H7

Bovine feces

1a–I

1a

2c–d

2c

λ/γ1

+

H7/NM

+

ECI–563

O157:H7

Bovine feces

1a–II

1a

2a

2a

λ/γ1

+

H7/NM

+

ECI–585

O157:H7

Bovine feces

1a–II

1a

2a

2a

λ/γ1

+

H7/NM

+

ECI–590

O157:H7

Bovine feces

1a–II

1a

2a

2a

λ/γ1

+

H7/NM

+

ECI–634

O157:H7

Bovine feces

1a–II

1a

2a

2a, 2c

λ/γ1

+

H7/NM

+

ECI–654

O157:H7

Bovine feces

1a–II

1a

2a

2a

λ/γ1

+

H7/NM

+

Origin Frozen spiced beef burger

GTA–EHEC–101

O157:H7


GTA–EHEC–75

O157:H7

Frozen spiced beef burger

GTA–EHEC–179

O157:H7

GTA–EHEC–183

rfbE ref

O157:H7

Bovine feces

1a–II

1a

2a

2a, 2c

λ/γ1

+

H7/NM

+

ECI–1757

O157:H7

Human

1a–II

1a

2a

2a

λ/γ1

+

H7/NM

+

2a

λ/γ1

+

H7/NM

+

2a

λ/γ1

+

H7/NM

+

2a

λ/γ1

+

H7/NM

+

2a

λ/γ1

+

H7/NM

+

2a

λ/γ1

+

H7/NM

+

2a

λ/γ1

+

H7/NM

+

2a

λ/γ1

+

H7/NM

+

2c

λ/γ1

+

H7/NM

+ +

ECI–1907

O157:H7

Human

–

–

2a

ECI–233

O157:H7

Human

–

–

2a

ECI–280

O157:H7

Human

1a–II

1a

2a

ECI–320

O157:H7

Human

1a–II

1a

2a

ECI–539

O157:H7

Human

1a–II

1a

2a

ECI–853

O157:H7

Human

1a–II

1a

2a

ECI–838

O157:H7

Human

–

–

2a

ECI–1745

O157:H7

Unknown

1a–I

1a

2c–d

R O

ECI–683

P

O157:H7

O

O:H–serotype

GTA–EHEC–73

F

stx1 Pyro

Strain names

O157:H7

Unknown

1a–II

1a

2a

2a

λ/γ1

+

H7/NM

O157:H7

Unknown

1a–II

1a

2a

2a

λ/γ1

+

H7/NM

+

FWS_EC_0004

O157:H7

Human

1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

OLC–797

O157:H7

Unknown

1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

ECI–2152

O157:NM

Bovine feces

1a–II

1a

2c–d

2c

λ/γ1

γ1

H7/NM

+

ECI–1655

O157:NM

Water

1a–II

1a

2c–d

2c

λ/γ1

γ1

H7/NM

+

ECI–1784

O157:NM

Water

1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

ECI–1788

O157:NM

Water

1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

ECI–2337

O157:NM

Water

1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

ECI–2341

O157:NM

Water

1a–II

1a

2c–d

2c

λ/γ1

γ1

H7/NM

+

ECI–2350

O157:NM

Water

1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

ECI–2351

O157:NM

Water

1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

+

ECI–2822

O157:NM

Water

–

–

2c–d

2a, 2c

λ/γ1

γ1

H7/NM

+

EC19970633

O157:H12

Beef salami

–

–

–

–

–

–

non–H7

+

EC20050147

O157:H12

Bovine feces

–

–

–

–

–

–

non–H7

+

E

T

C

E

Bovine feces

O157:H19

Unknown

ECI–2181

O157:H19

Unknown

EC20050096

O157:H2

Porcine feces

EC19970636

O157:H29

EC20060754

O157:H29

EC20070110

O157:H42

EC19970524

O157:H42

EC19970706

O157:H45

EC19970532

R

O157:H19

ECI–2180

–

–

–

–

–

–

non–H7

+

–

–

–

–

–

–

non–H7

+

–

–

–

–

–

–

non–H7

+

–

2e

2e

–

–

non–H7

+

Beef salami

–

–

–

–

–

–

non–H7

+

Bovine feces

O

–

–

–

–

–

–

non–H7

+

Bovine feces

–

–

–

–

–

–

non–H7

+

Beef

–

–

–

–

–

–

non–H7

+

Ground beef

–

–

–

–

–

–

non–H7

+

O157:H45

Beef

–

–

–

–

–

–

non–H7

+

EC19970465–1

O157:H45

Beef

–

–

–

–

–

–

non–H7

+

ECI–2327

O157:H45

Water

–

–

–

–

–

–

non–H7

+

ECI–2673

O1:H7

Bovine feces

–

–

–

–

–

–

–

–

ECI–2838

O2:H27

Water

–

–

2a

2a

–

–

–

–

ECI–2929

O2:H6

Bovine feces

–

–

2g?

untypeable

–

–

–

–

ECI–2737

O5:K4:H4

Unknown

–

–

–

–

–

–

–

–

ECI–2795

O5:NM

Environment

1a–I

1a

–

–

β

β

–

–

ECI–2667

O6:H34

Bovine feces

–

–

2c–d

2c

–

–

–

–

ECI–2668

O6:H34

Bovine feces

–

–

2c–d

2c

–

–

–

–

ECI–2774

O7:H4

Bovine feces

1a–III

1a

2a

untypeable

–

–

–

–

ECI–2643

O8:H16

Bovine feces

–

–

–

–

–

–

–

–

ECI–0716

O8:H19

Environment

–

–

2a

2a

–

–

–

–

EC20000890

O8:NM

Human

–

–

2e

2f

ε

untypeable

–

–

ECI–2917

O9:NM

Bovine feces

–

–

–

–

–

–

–

–

ECI–2740

O11:K10:H10

Unknown

–

–

–

–

–

–

–

–

N

–

C

R

EC20070204

D

ECI–1863 ECI–738

U

t1:5




Table 1 (continued)

stx1 Pyro

stx1 ref

stx2 Pyro

stx2 ref

ECI–2741

O13:K11:H11

Unknown

–

–

–

–

–

–

–

–

ECI–2916

O15:H45

Bovine feces

–

–

–

–

–

–

–

–

ECI–2244

O17:H19

Environment

–

–

–

–

–

–

–

–

ECI–2736

O18a,18c:K77:H7

Unknown

–

–

–

–

–

–

–

–

ECI–2753

O22:H8

Bovine feces

1a–III

1a

2a, 2c–d

2a, 2c

–

–

–

–

ECI–2726

O22:H8

Environment

1a–III

1a

2a

2a

–

–

–

–

ECI–2837

O22:H8

Water

–

–

2a

2a

–

–

–

–

ECI–2964

O28ac:H25

Bovine feces

–

–

2a, 2c–d

2a, 2c, 2d

–

–

–

–

ECI–2971

O28ac:H25

Environment

–

–

2a

2a, 2c

–

–

–

–

Strain names

O:H–serotype

Origin

eae Pyro

eae ref

rfbE Pyro

rfbE ref

O36:H19

Porcine feces

–

–

2e

2e

–

–

–

–

ECI–2748

O36:K:H9

Unknown

–

–

–

–

–

–

–

–

ECI–2895

O46:H38

Environment

1a–III

1a

2c–d

2d

–

–

–

–

ECI–2995

O46:H38

Environment

1a–III

1a

2c–d

2d

–

–

–

–

EC20000924

O65:H9

Porcine feces

–

–

2e

2e

–

–

–

ECI–2992

O68:H12

Environment

ECI–2901

O69:H11

Water

ECI–2743

O70:K:H42

ECI–2749

O71:K:H12

ECI–2859

–

2f

2f

–

–

–

–

1a

–

–

β

β

–

–

Unknown

–

–

–

–

–

–

–

–

Unknown

–

–

–

–

–

–

–

–

O73:H2

Human

–

–

–

–

–

–

–

–

ECI–2682

O79:H2

Bovine feces

1a–III

1a

2c–d

ECI–2887

O79:H2

Water

–

–

2a

R O

– 1a–I

O

F

EC20060727

–

–

–

–

–

2a, 2c

–

–

–

–

P

2c, 2d

O84:H2

Environment

1a–I

1a

–

–

–

ζ

–

–

O88:H25

Bovine feces

1a–III

1a

2a

2a

–

–

–

–

ECI–2685

O88:H25

Bovine feces

1a–III

1a

2a

2a

–

–

–

–

ECI–2801

O88:H25

Bovine feces

1a–III

1a

2a

–

–

–

–

ECI–2924

O91:H21

Water

–

–

FWS_EC_0008

O91:H21

Human

–

–

ECI–2648

O93:H28

Bovine feces

–

EC20000908

O100:NM

Human

–

ECI–2607

O104:H21

Bovine feces

–

FWS_EC_0009

O104:H4

Human

–

ECI–2603

O104:H7

Bovine feces

ECI–2991

O107:H28

Water

ECI–2836

O107:H7

Environment

ECI–2681

O109:H5

Bovine feces

ECI–2886

O109:H5

Water

R

2a

2a, 2c, 2d

–

–

–

–

2a, 2c, 2d

–

–

–

–

–

2a, 2c–d

2a, 2c

–

–

–

–

–

2e

untypeable

–

–

–

–

T

E

2a

2a, 2c–d

–

–

–

–

–

–

–

2a

2a

–

–

–

–

C

– –

2a

2a

–

–

–

–

1a–III

1a

2a

2a

–

–

–

–

–

–

2c–d

2b

–

–

–

–

1a–III

1a

–

–

–

–

–

–

1a–III

1a

2a

2a

–

–

–

–

E

–

D

ECI–2889 ECI–2677

1a–I

1a

–

–

β

β

–

–

1a–I

1a

–

–

β

β

–

–

O112

Bovine feces

O113

Bovine feces

ECI–2756

O113:H21

Bovine feces

–

–

2c–d

2d

–

–

–

–

ECI–2595

O113:H21

Human

–

–

2c–d

2d

–

–

–

–

FWS_EC_0010

O113:H21

Human

–

–

2a, 2c–d

2a, 2c, 2d

–

–

–

–

FWS_EC_0011

O113:H21

Bovine

–

–

–

–

–

–

–

–

ECI–3070

O113:H4

Environment

1a–III

1a

2c–d

2d

–

–

–

–

N C O

R

ECI–2704 ECI–2705

ECI–2707

O114

Bovine feces

1a–I

1a

–

–

β

β

–

–

ECI–2708

O115

Bovine feces

1a–I

1a

–

–

β

β

–

–

ECI–2710

O116:H28

Bovine feces

1a–I

1a

–

–

β

β

–

–

ECI–2940

O116:H28

Water

1a–I

1a

–

–

–

–

–

–

ECI–2750

O117:H16

Bovine Feces

1a–III

1a

–

–

–

–

–

–

ECI–2854

O118:H16

Human

1a–I

1a

–

–

β

β

–

–

O126:H8

Water

1a–III

1a

–

–

–

–

–

–

O126:H8

Water

1a–III

1a

–

–

–

–

–

–

O128a,128b:B12

unknown

–

–

–

–

β

β

–

–

ECI–0719 ECI–0720 ECI–2745

U

t1:7

5

ECI–2922

O130:H11

Environment

–

–

2a

2a

–

–

–

–

ECI–3090

O130:H38

Bovine Feces

1a–III

1a

2a, 2c–d

2a, 2d

–

–

–

–

ECI–2865

O132:NM

Bovine Feces

–

–

2a

2a

–

–

–

–

ECI–3036

O136:H12

Environment

1a–III

1a

–

–

–

–

–

–

ECI–2885

O136:H16

Environment

1a–III

1a

–

–

–

–

–

–

ECI–2907

O136:H16

Environment

1a–III

1a

–

–

–

–

–

–

ECI–2644

O139:H19

Bovine Feces

1a–III

1a

2a

2a

–

–

–

–

ECI–2693

O139:H19

Bovine Feces

1a–III

1a

2a

2a

–

–

–

–

ECI–2960

O139:H19

Bovine Feces

1a–III

1a

2a

2a

–

–

–

–

ECI–3080

O139:H19

Bovine Feces

1a–III

1a

2a

2a

–

–

–

–

(continued on next page)


6

Table 1 (continued)

O:H–serotype

Origin

stx1 Pyro

stx1 ref

stx2 Pyro

stx2 ref

ECI–2690

O141ac:H2

Bovine Feces

1a–III

1a

2c–d

2c, 2d

–

–

–

–

ECI–2722

O141ac:H49

Bovine Feces

1a–III

1a

–

–

–

–

–

–

ECI–2863

O146:H21

Human

1c

1c

–

–

–

–

–

–

ECI–0717

O150:H8

Water

–

–

–

–

–

–

–

–

ECI–3007

O153:NM

Environment

–

–

2f

2f

β

β

–

–

ECI–2926

O163:H19

Water

1a–III

1a

2c–d

2d

–

–

–

–

ECI–2713

O165:H25

Bovine Feces

1a–I

1a

2a

2a

ε

ε

–

–

Strain names

eae ref

rfbE Pyro

rfbE ref

ECI–2715

O165:H25

Bovine Feces

1a–I

1a

2a

2a

ε

ε

–

–

O165:H25

Bovine Feces

1a–I

1a

2a

2a

ε

ε

–

– –

O165:NM

Bovine Feces

1a–I

1a

2a

2a

ε

ε

–

O168:H8

Environment

–

–

2a

2a

–

–

–

–

ECI–3062

O171:H2

Water

–

–

2c–d

2c

–

–

–

–

ECI–1648

O174:H4

Environment

–

–

–

–

ECI–1106

O174:H43

Water

–

–

–

–

ECI–2939

O175:NM

Environment

1a–III

1a

2a

2a

ECI–2683

O178:H19

Bovine Feces

1a–III

1a

2c–d

2d

ECI–2840

O178:H19

Bovine Feces

1a–III

1a

2a, 2c–d

2a, 2c, 2d

ECI–2126

O179:H7

Bovine Feces

1a–II

1a

2a

2a

ECI–3012

O179:H8

Environment

–

–

2a

ECI–2767

O182:H25

Bovine Feces

–

–

2a

ECI–2805

O182:H25

Bovine Feces

1a–III

1a

–

ECI–2913

(O109), O182:H5

Water

1a–III

1a

–

ECI–2614

unknown

Bovine Feces

1a–III

1a

ECI–2627 ECI–2570

unknown not tested

Bovine Feces Bovine Feces

1a–I 1a–II

1a 1a

ECI–2593

not tested

Bovine Feces

1a–III

1a

ECI–2611

not tested

unknown

1a–III

1a

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

– λ/γ1

–

–

–

γ1

H7/NM

–

–

–

–

–

–

ζ

–

–

–

–

ζ

–

–

–

–

–

–

–

2a, 2c–d

2a, 2c, 2d

–

–

–

–

– –

– –

ε θ/γ2

ε θ/γ2

– –

– –

E

D

2a

2a

P

R O

O

–

F

ECI–2714 ECI–2692

2c–d

2d

–

–

–

–

2b

2b

–

–

–

–

not tested

Bovine Feces

1a–I

1a

–

–

ε

ε

–

–

not tested

Human

1a–I

1a

–

–

ε

ε

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

Z22

–

01–948

–

Citrobacter braakii

ATCC 12012

–

Enterococcus faecalis

ATCC 29212

Klebsiella pneumoniae

ATCC 13883

Proteus vulgaris

ATCC 8427

Pseudomonas aeruginosa

ATCC 27853

Salmonella enterica subsp. Enteritidis

12–6172 (NML) unknown

Shigella dysenteriae

ATCC 11835

Vibrio vulnificus

Z86

E

O

D2653

R

Shigella sonnei

C

Aeromonas hydrophila Campylobacter jejuni

CP008957a

O157:H7

T

ECI–2639 ECI–2628

Salmonella enterica subsp. Typhimurium 71–471

–

–

–

–

–

–

–

–

1a–III

1a

–

–

–

–

–

–

1a–III

1a

–

–

–

–

–

–

–

–

–

–

–

–

–

1a–II

1a

2a

2a

λ/γ1

γ1

H7/NM

– +

O174:H8

Z36901a (for stx1c)

1c

1c

2b

2b

–

–

–

–

O8:K85ab:Hrough

AY170851a

1d

1d

–

–

–

–

–

–

C

D3602 D3522

Z37725a (for stx2a)

1a–III

1a

2a

2a

–

–

–

–

AF043627a

–

–

2b

2b

–

–

–

–

L11079a

–

–

2c–d

2c

–

–

–

–

O73:H18

DQ059012a

–

–

2d

2d

–

–

–

–

D3648

O139:K12:H1

M21534a

–

–

2e

2e

–

–

–

–

D3546

O128ac:[H2]

AJ010730a (for stx2f)

–

–

2f

2f

β

β

–

–

D3509

O2:H25

AY286000a

–

–

2g

2g

–

–

–

–

O48:H21 O118:H12

D2587

O174:H21

D3435

N

D2435 D3428

U

a

eae Pyro

ECI–2806

R

t1:9


GenBank accession number.

t1:11

201 202 203 204 205 206 207 208 209

sequencing primers were designed based on the sequence analysis method (SQA) for each target. All primers designed in this study (gene name_NCAD_fwd, rvs and seq) are listed in Table 2. The specificity of the primers was verified in silico using the NCBI BLAST. Two-multiplex PCR reactions were set up: the first set to amplify stx1 and stx2, and the second set, eae and rfbE. The PCR reaction for subsequent pyrosequencing was performed following the manufacturer's instructions. Briefly, 1 μL of DNA template, prepared by the colony-boiling method described above, was added to

24 μL of PCR reaction mix containing 2× PyroMark Master Mix (Qiagen Inc.) and primers with the final concentration listed in Table 2. PCR amplification was performed using a peqSTAR 96 Universal Gradient Thermocycler (PeqLab, Erlangen, Germany) with the hotstart program starting at 95 °C for 15 min, 45 cycles of 94, 56, 72 °C for 30 s each and final extension of 72 °C for 5 min. PCR amplicons were visualized using a QIAxcel DNA Kit on the QIAxcel capillary gel electrophoresis system (Qiagen Inc.). Only the samples with PCR amplicons of expected size were used for subsequent pyrosequencing.


210 211 212 213 214 215 216 217 218


241

2.4. Sensitivity and specificity tests

242

253 254

In order to evaluate the sensitivity of our PCR assays for detection of each target, conventional PCR was performed on the same DNA samples using primer sets previously reported (Blais et al., 2012; Jenkins et al., 2012; Wasilenko et al., 2012), as shown on Table 2. The PCR conditions were the same as those used for generating amplicons for pyrosequencing described above except for the annealing temperature and primer concentration (Table 2). To further confirm the genotype information generated by pyrosequencing, another conventional PCR for subtyping stx1, stx2, and eae genes was also performed. on the samples that were positive for the PCR above. PCR conditions and subtype specific primers for stx1, stx2 and eae genes were used as reported (Blanco et al., 2004; Scheutz et al., 2012). Targeted gene amplification was confirmed by observing the expected size band on QIAxcel.

255

2.5. Data analysis

256 257

260

The raw pyrogram data from the instrument was transferred to the PyroMark Q24 software (version 2.0; Qiagen Inc.) for analysis. The FASTA sequences obtained were then aligned to the reference sequences using the Geneious software and sequence search using megablast on BLAST to determine the subtypes of each gene.

261

3. Results

262

3.1. stx1 and stx2, eae and rfbE PCR amplification

263 264

Two sets of duplex PCR reactions were performed to amplify 4 different virulence related genes of STEC and subsequent pyrosequencing. A total of 224 E. coli strains including 10 Shiga toxin subtype reference strains as well as 12 members of other foodborne pathogenic bacterial species were tested; 134 E. coli and 2 Shigella spp. isolates were positive for stx1 and 126 E. coli for stx2 (Tables 1 and 3). The accuracy of the new multiplex PCR amplification was confirmed by other PCR assays using primers previously reported (Table 2) and both results were 100% identical for the detection of stx1, stx2 and rfbE and 98% (117/119 positive) for eae detection. These new multiplex PCR assays were successfully applied to the analysis of targets in complex DNA samples extracted from enriched culture media of environmental swabs or manure samples provided by Dr. Keith Warriner of University of Guelph (data not

243 244 245 246 247 248 249 250 251 252

258 259

265 266 267 268 269 270 271 272 273 274 275

3.2. Pyrosequencing analysis of stx1 gene

295

After confirming the amplification of the genes by observing the expected amplicons size (Table 2) with QIAxcel, subsequent pyrosequencing assays were performed on the PCR positive amplicons for stx1 and stx2bcd, and/or stx2aefg. The pyrogram sequence reads obtained were then imported into the Geneious software and aligned to the reference gene sequences of each Shiga toxin subtype for genotype callings (Fig. 1a and b). The results show that there are 9 SNPs identified from sequence 661 to 702 in the stx1 gene. Location 666 (A-G) to distinguish stx1c, and 672 (C-T), 681,682 (GT-AC), 691 (G-A), 693 (C-T) and 699 (T-C) are for stx1d. stx1a is further subtyped into stx1a type I (690, C-T), stx1a type II (702, T-C), and stx1a type III (no SNPs). The distribution of stx1a subtypes to O-serotypes based on the Top-7 priority O serotypes (O26, O45, O103, O111, O121, O145 and O157) revealed that 90.0% (36 isolates) of the strains from the Top-6 (excluding O157) serotypes carry stx1a-I, while 94.3% (33 isolates) of O157:H7/NM serogroup possess stx1a-II (Tables 1 and 3). It is noteworthy that besides the O157: H7/NM serogroup, only O111:NM isolates tested in this study (Table 1), some Shigella dysenteriae and Aeromonas caviae from the GenBank database (data not shown) carried stx1a -II. The stx1a -III was not observed in any STEC from the Top-7 serogroups tested, nor by BLAST search (data not shown). The subtype most commonly observed in the non Top-7 STEC group and Shigella spp. was stx1a-III (60.3%, 34 samples) (Table 3).

296 297

3.3. Pyrosequencing analysis of stx2 gene

319

In order to amplify more than one subtype of Shiga toxin 2 genes, 3 reverse primers and two sequencing primers (stx2_seq-aefg for stx2a, stx2e, stx2f and stx2g and stx2_seq-bcd for stx2b, stx2c, and stx2d) were designed for this study (Table 2). As a result of this multiplex strategy, all the stx2 subtypes were amplified in one reaction tube in multiplex with stx1 including stx2f. The subtype stx2f is very often left out of PCR based assays due to the low identity to other stx2 subtypes (pairwise to stx2a 70%, stx2c 72%). We chose this combination of sequencing primers because, to our knowledge, carriage of multiple stx2 genes in a single strain occurs only among stx2a, stx2b, stx 2c and stx 2d, not stx2e, stx2f and stx2g (Feng and Reddy, 2013). Subtype stx2a is identified as the SNP (C-T) at base position 1079 of stx2a gene and the other SNP locations for identifying each subtype are shown in Fig. 1b. The results from PCR subtyping showed that out of 107 isolates carrying only one Shiga toxin 2 subtype, only 5 isolates (1.1%) were in conflict with the pyrosequencing subtyping results. Three of them were positive with universal stx2 PCR but couldn't be amplified using any subtyping primers (EC20000908, ECI-2929 and

320

D

237 238

E

235 236

T

233 234

C

231 232

E

229 230

R

227 228

R

225 226

N C O

224

U

222 223

F

239 240

Sequencing primers were designed for stx1, stx2, rfbE and eae genotyping as described in Table 2. In order to read two sequences from an isolate carrying stx2a plus stx2b, stx2c or stx2d simultaneously, stx2-NCAD-seq-aefg primer was selected for sequencing of stx2a, stx2e, stx2f and stx2g, and stx2-NCAD-seq-bcd for stx2b, stx2c and stx2d. Sequencing primers for stx2 were selected based on the amplicon size. The amplicons of 269 bp are tested for stx2bcd and 273–4 bp for stx2aefg and both sequencing primers were tested on those samples with 2 PCR bands or ambiguous size amplicons. Pyrosequencing using the PyroMark Q24 system (Qiagen Inc. [http://www.qiagen.com/products/pyromarkq24.aspx]) was performed following the manufacturer's instructions. Briefly, 7 μL of PCR amplicon was placed in 24-well plates and mixed with streptavidin coated sepharose beads (GE Healthcare, Pitcataway, NJ, USA). After denaturing the PCR amplicon/sepharose beads conjugate, non-biotin-labeled DNA was washed away using the PyroMark Q24 vacuum workstation (Qiagen Inc.). Sepharose beads were then resuspended and heated at 80 °C for 1 min in annealing buffer containing 0.3 μM sequencing primer. After cooling back to ambient temperature, pyrosequencing reaction was performed using the PyroGold Q24 reagents with a dispensation specific to each gene using the PyroMark Q24 AQ assay system.

276 277

O

220 221

shown). The gene detection results were 100% in agreement with the ones obtained from the reference primers (data not shown). In order to distinguish between multiple subtypes by subsequent pyrosequencing, the PCR assay was designed from variable regions of the genes. However, in the design of eae assays, in order to focus on identifying the four major intimin subtypes, β-, γ1, ε-, and θ-eae, some minor subtypes such as τ-, ο-, ν-, and η-eae were disregarded due to the diversity of the eae gene. Thus, the eae primers designed for pyrosequencing in this paper couldn't amplify eae genes in two isolates (ECI2767 and ECI-2889) whereas the confirmatory PCR that amplifies the conserved region of eae were positive (Table 1). Individual eae subtyping PCR confirmed that these isolates had zeta intimin (ζ-eae). Isolate ECI-2805 was eae positiveby the multiplex PCR with correct amplicon size but the subsequent pyrosequencing run failed to generate a sequence read, and this also carried ζ-eae (Table 1). The amplification of a 106 bp fragment from the rfbE gene was observed in all the 58 O157 isolates tested and neither from the other O subtypes nor the other food-borne pathogens, except one isolate of O179:H7 (ECI-2126) (Table 1).

R O

2.3. Pyrosequencing

P

219

7


278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294

298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318

321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337


338 339 340 341 342 343 344 345 346 347

Fig. 1. Multiple alignment of the stx1 (1a) and stx2 (1b) genes and location of primers. Sequences were selected from GenBank and aligned using the Geneious Software. Consensus sequences showing unique SNPs for subtyping were imported into the PyroMark Assay Design software for the design of amplification and pyrosequencing primers. The numbers of consensus sequence on the top of each figure are of the ORF sequences of O157:H7, EDL933 reference strain.

U

Q3

N

C

O

R

R

E

C

T

E

D

P

R O

O

F

8

ECI-2774), while the pyrosequencing results clearly presented their subtypes as 2e, 2 g and 2a, respectively (Table 1). One isolate (EC20000890), previously identified as carrying stx2e and confirmed by pyrosequencing analysis, was only amplified with the stx2f subtyping primers. The isolate ECI-2929 (O2:H6) possesses a very unique sequence (Fig. 1b), with a consensus read of a total of 67 bases obtained from both sequencing primers, stx2_seq-aefg and stx2_seq-bcd, indicating that this isolate is 90% and 88% identical to stx2g and stx2e, respectively. There was not any stx2g isolate other than the reference strain D3509 in this study, although the sequence analysis showed 100%

identity to the Sanger sequence data of the strain up to 56 bp (Fig. 1b), and that read length is long enough to distinguish stx2g from other subtypes. The only one isolate, ECI-2836 (O107:H7), out of 110 tested show incongruent results between pyrosequencing and conventional PCR subtyping for stx2a, stx2c and stx2d (Tables 1 and 3). The pyrosequencing result of ECI-2836 was stx2c–d while the PCR results subtyped as stx2b. Isolate ECI-2774 (O7:H4) was untypeable with the PCR subtyping while pyrosequencing indicated it was stx2a. Nine isolates, including five O157:H7 (ECI-634, -683, GTA-EHEC-73, -75, and -101) and ECI-


348 349 350 351 352 353 354 355 356 357

9

O

F


363

Among all the isolates tested, 119 strains were eae positive using the primer sets designed in this study and only one of the isolates (ECI2745, O128ab:B12) was non STEC (Table 1). Multiple alignment analysis of 50–78 bases between 1822th to 1899th nucleotide positions could differentiate between at least four different gene subtypes, λ/γ1-eae, β-eae, ε-eae, and θ/γ2-eae (Fig. 2). All the O26 isolates were β-eae positive, O45, O103:H2 and O121:H19 were ε-eae, and O103:H25 and O111:NM were θ/γ2-eae. The λ/γ1-eae target was only detected among the O145 and O157:H7/NM isolates, except one O179:H7 isolate, ECI-2126, while those in the O157 non-H7 group didn't possess any eae subtypes. All these genotyping results generated by pyrosequencing were 100% identical to the individual eae PCR subtyping (Table 1). All positive isolates of θ/γ2-eae were γ2-eae by PCR subtyping. The ζ-eae was the only subtype not detected by the PCR-pyrosequencing assay among the STEC isolates tested in this study.

374 375 376 377 378

C

E

372 373

R

370 371

R

368 369

N C O

366 367

Multiple alignment analysis data of rfbE genes retrieved from GenBank revealed that the T-C SNP at base position 1021 of the rfbE gene was found in all O157:H7 but not non-H7 O157 (Fig. 3). A total of 58 O157 isolates including O157:H7, NM (no motility was confirmed with motility test medium, data not shown), H2, H12, H19, H29, H42 and H45 were all PCR positive using rfbE primers and the amplicons were pyrosequenced. The analysis showed that the 1021-T SNP was observed among all the O157:H7 including the non-motile isolates, while all the other H serotypes tested carried the 1021-C SNP (Table 1 and Fig. 3). Outside of the O157 STEC group, only one STEC (ECI-2126, O179:H7) isolated from bovine feces had the rfbE gene with 1021-T SNP (Table 1).

380

4. Discussion

392

When cases of foodborne illness are reported, a quick response in the recall of the causative food item is key to minimizing the outbreak. Thus, fast and robust detection methods for the identification of the implicated pathogen are required in food testing laboratories. The combination of PCR and PyroMark pyrosequencing technology developed in this study is a rapid, confirmatory, cost-effective and

393

U

364 365

379

P

3.4. Pyrosequencing analysis of eae subtypes

3.5. rfbE gene SNP analysis

D

362

T

361

2822 (O157:NM) recognized as carrying multiple stx2 genes, particularly stx2a gene with stx2c and/or stx2d by PCR subtyping, showed only one amplicon by PCR for pyrosequencing and were identified as isolates carrying only stx2a (Tables 1 and 3).

359 360

E

358

R O

Fig. 2. Multiple alignment of the eae gene and location of primers. Sequences were selected from GenBank and aligned using the Geneious Software. Consensus sequences showing unique SNPs for subtyping were imported into the PyroMark Assay Design software for the design of amplification and pyrosequencing primers.

Fig. 3. Multiple alignment of the rfbE gene and location of primers. Sequences were selected from GenBank and aligned using the Geneious Software. Consensus sequences showing unique SNPs for subtyping were imported into the PyroMark Assay Design software for the design of amplification and pyrosequencing primers.


381 382 383 384 385 386 387 388 389 390 391

394 395 396 397 398

10 t2:1 t2:2


Table 2 PCR primers used in this study for detection of target genes and pyrosequencing.

t2:3

primer names

Sequence

Amplicon size (bp)

Concentration in reaction (μM)

Annealing temperature (°C)

Location on EDL933a

References

t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26

stx1_NCAD_fwd stx1_NCAD_rev stx1_R_fwd stx1_R_rvs stx2_NCAD_fwd stx2_NCAD_rvs-bcd stx2_NCAD_rvs-ag stx2_NCAD_rvs-ef stx2_R_fwd stx2_R_rvs eae_NCAD_fwd eae_NCAD_rev eae_R_fwd eae_R_rvs rfbE_NCAD_fwd rfbE_NCAD_rvs rfbE_R_fwd rfbE_R_rvs stx1_NCAD_seq stx2_NCAD_seq-bcd stx2_NCAD_seq-aefg eae_NCAD_seq rfbE_NCAD_seq

ATC TCA GTG GGC GTT CTT ATG /5Biotin/ CAT CTG CCG GAC ACA TAG AAG GTG GCA AGA GCG ATG TTA CGG TTTG ATG ATA GTC AGG CAG GAC GCT ACT C /5Biotin/ ATG TCA GAT WRY TGG MGA CAG G AYT CTT TYC CGG CCA CTT TTA CT CCA GTA TTC TTT CCC GTC AAC CT CCA GTA TTC TCT TCC TGA CAC CT ACG AGG GCT TGA TGT CTA TCA GGC G GCG ACA CGT TGC AGA GTG GTA TAA C /5Biotin/ GCT CAG GCT AAT GYC CCT GT TCT CAG TAA TRC TGG CCT KR CAT TGA TCA GGA TTT TTC TGG TGA TA CTC ATG CGG AAA TAG CCG TTM TCA TTC GAT AGG CTG GGG AAA /5Biotin/ TCC ACA CGA TGC CAA TGT ACT C CGA TGA GTT TAT CTG CAA GGT GAT TTT CAC ACT TAT TGG ATG GTC TCA A CTG CTG AAG ATG TTG ATC CCA CTT TTA CTG TGA ATG YTY CCK KMM ACC TTY AC CGG TTT TAG CAG ACA CG ATA GGC TGG GGA AAC

243

0.2 0.2 0.2 0.2 0.5 0.2 0.2 0.2 0.2 0.2 1 0.5 1 1 0.2 0.2 0.2 0.2 N/A N/A N/A N/A N/A

56

3013419–3013399 3013177–3013197 3013510–3013486 3013328–3013352 1355787–1355808 1287–1265c 1356059–1356037 1357–1335d 1355172–1355196 1355372–1355348 4685119–4685100 4684900–4684919 4686056–4686031 4685955–4685975 2870481–2870501 2870586–2870565 2870611–2870634 2870698–2870674 3013392–3013375 1275–1258c 1356049–1356033 4684972–4684988 2870488–2870502

This study This study Blais et al. Blais et al. This study This study This study This study Blais et al. Blais et al. This study This study Wasilenko et al. Wasilenko et al. This study This study Jenkins et al Jenkins et al This study This study This study This study This study

t2:27 t2:28 t2:29 t2:30

a

d

106 88

t3:1 t3:2

Table 3 stx1 and stx2 subtype distributions in each serogroup of E. coli tested in this study.

t3:3

F

pyrograms or online BLAST analysis of sequences generated. The procedure – from isolated single colonies on culture plates, to the generation of genotype information – can all be completed in 5 h. Furthermore, multiple samples can be tested simultaneously, up to 24 samples with the PyroMark Q24 system and 96 with the Q96 platform. Thus the running cost per sample is very economical, estimated less than 100 US$ for 24 samples, for all of the procedures from PCR amplification to pyrosequencing. Pyrosequencing has been previously used for the detection and/or subtyping of other pathogens (Achazi et al., 2011; Amoako et al., 2013, 2012a,b; Deng et al., 2011; Haanpera et al., 2005; Jonasson et al., 2002). To our knowledge, this is the first application of pyrosequencing for subtyping of virulence genes of E. coli including Shiga toxin 1 and 2, intimin and rfbE.

C

E

R

R

(Primers)

t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19 t3:20 t3:21 t3:22 t3:23 t3:24

N/A N/A N/A N/A N/A

O

410

Total NCAD

1a total 1a–I 1a–II 1a–III 1c 1d Total positive stx1 2a 2e 2f 2g 2b 2c 2d 2c and 2d 2a and 2c and/or 2d Untypeable stx2 Total positive stx2 Tested total

C

408 409

N

406 407

130 55 40 35 2 2 134

U

404 405

60

T

411 412

402 403

56

GenBank accession number: CP008957. Not applicable. GenBank accession number: AF043627, SSI reference strain for stx2b, D3428. GenBank accession number: M21534, SSI reference strain for stx2e, D3648.

labor-saving subtyping method for the detection of the presence and characterization of STEC virulence genes, thus could quickly provide information on the potential pathogenicity and key genomic profile of the isolates. The sample preparation is very simple and fast, as no DNA purification or library preparation and quantification is required. This genotyping method is based on the detection of novel SNPs using PyroMark pyrosequencing technology, which is known to generate reproducible sequence results similar to Sanger sequencing (Ronaghi, 2001). Pyrosequencing is a sequencing-by-synthesis application, and the nucleotide base call can be observed in real time. Data analysis of pyrosequencing results is also very easily performed, and does not require a large investment in data analysis software or operator training. Sufficient information for genotyping can be obtained by simply reading peak heights at SNP sites on

400 401

59

P

N/A N/A N/A N/A N/A

b

56

O

102

55

R O

220

56

D

c

269 273 274 201

55

E

399

b

183

80 6 3 2 3 18 14 0 126 224

Top-6

Ref

NCAD

130 N/A

39 36 3 0 0 1 40

2 2 134 71 4 4 1 4 9 9 2 19 3 126 224

12 0 0 0 0

O157:H7/NM

(%) (90) (7.5) (0) (0) 2.5 (%) (100) (0) (0) (0) (0)

0

(0)

0 0 12 52

(0) (0)

Ref

NCAD

39 N/A

35 2 33 0 0 0 35

0 1 40 12 0 0 0 0 0 0 0 0 0 12 52

(%) (5.7) (94.3) (0) (0) (0)

36 0 0 0 0

(%) (81.8) (0) (0) (0) (0)

6

(13.6)

2 0 44 44

(4.5) (0)

Non Top-7 Ref

NCAD

35 N/A

53 17 2 34 1 0 55

0 0 35 31 0 0 0 0 5 0 0 8 0 44 44

Ref (%) (29.3) (5.2) (60.3) (3.4) (1.7)

30 4 2 1 1

(%) (50.0) (6.7) (3.3) (1.6) (1.6)

10

(16.7)

12 0 60 104

(20.0) (0)

54 N/A

1 0 55 26 2 3 0 2 3 8 2 11 3 60 104


413 414 415 416 417 418 419 420 421 422 Q2 423 424 425 426


448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492

F

O

R O

446 447

P

444 445

D

442 443

carrying eae genes reported (Feng et al., 2010). Of those that were reported, none were λ/γ1-eae, but either β-, ε- or κ/δ-eae. Strain O55: H7, which is not included in this report, is also known to carry γ1-eae (McGraw et al., 1999). Foodborne illness involving O157:H7 is considered one of the most important public health concerns compared to other E. coli serotypes, and thus a new PCR/pyrosequencing assay was designed alongside the virulence related genes to distinguish O157:H7 from other E. coli serotypes. The rfbE gene located on the O157 antigen gene cluster has been widely used as a target for real-time PCR and PCR applications (Anklam et al., 2012; Huszczynski et al., 2013). The T-C SNP at base position 1021 of the rfbE gene highlighted in this study (Fig. 3) could distinguish O157:H7/NM from other H serotypes and one Escherichia fergusonii isolate that is reported to carry the O157 gene cluster (Fegan et al., 2006). To our knowledge, this is the first report describing this SNP. This rfbE gene detection and simultaneous SNP identification by pyrosequencing would be a great asset for O157:H7/NM identification, since the culture methods using selective medium widely applied in diagnostic laboratories depends on the biochemical characteristics of the isolates, which can sometimes be ambiguous. Although the BLAST results showed that two O157:NM/H-isolates (PV276 and 493/ 89 EC1367, accession numbers AB602250 and AGTG01000200) possess 1021-C (Fig. 3), further testing needs to be carried out to characterize these isolates and clarify the relationship between this SNP genotype and pathogenicity. Profiling STEC virulence related genes has been reported, since genetic profiling of STEC has a high potential to be used as a reliable tool for environmental and food surveillance, outbreak trace back and detection of pathogenic E. coli. The selection of appropriate target genes for assay development is critical in distinguishing between potentially pathogenic and non-pathogenic STEC. Virulence gene profiles in relation to serotypes have been investigated using various genotyping techniques, such as Sanger sequencing (Mora et al., 2012), PFGE (Bibbal et al., 2014) and PCR coupled to electrospray ionization mass spectrometry (PCR-MS) (Shen et al., 2013). Lorenz et al. reported hemolysin gene ehxA subtyping of STEC and non-STEC associated with different isolates from food products, clinical, animal and water sources (Lorenz et al., 2013). Karmali et al. proposed the five seropathotypes (A to E) for the virulence level classification of E. coli isolates, by analyzing O island 122 gene profile (Karmali et al., 2003) and Kobayashi et al. expanded the analysis using 17 major virulence related genes and investigated the potential risk associated with the O serotypes (Kobayashi et al., 2013). Joensen et al. targeted 6 genes (eae, vtx1, vtx2, ehxA, saa and bfpA) for their quick typing program from whole genome sequencing data (Joensen et al., 2014). Shen et al. successfully identified serotypes and clinically important serogroups of STEC using PCR-MS focusing on 5 virulence genes including eae, stx1, stx2, hlyA, and aggA. Considering the results from these previous studies, we targeted 3 virulence genes, stx1, stx2, eae that are known to have genetic variations, and also the rfbE, which is important for detecting O157:H7/NM isolates. Considering that stx2 and eae subtypes are known to be associated with pathogenicity and certain serotypes, we came up with a hypothesis that genotyping based on these four genes of the Top-7 priority serotypes may provide reliable information to identify and differentiate non- or less-pathogenic STEC from potentially harmful isolates, without being biased by O:H serotypes. This can potentially reveal the association between the genotypes and the pathogenicity of E. coli. The results presented in this report imply that the genotypes frequently found among the Top-7 group of STEC (stx1a -I, stx 1a -II, stx 2a , stx 2c , stx 2d , β-eae, λ/γ1-eae, ε-eae, θ/γ2-eae and H7/NM type rfbE) are likely related to the virulence of the STEC isolates. Since some non Top-7 STEC isolates have been reported to cause human illness, the isolates carrying these genotypes should also be flagged as high-risk STEC in environmental and food surveillance. For example, the 4 O165:H25/ NM isolates tested in this study all carried stx1a-I, stx2a and ε-eae, and this serotype has been reported in relation to human diarrhea cases

E

440 441

T

438 439

C

436 437

E

434 435

R

433

R

431 432

N C O

429 430

In this report, we demonstrated that Shiga toxin 1a gene was further subgrouped into stx1a-I, stx1a-II and stx1a-III by two SNPs 12 bp apart. Although both SNPs are silent mutations that are unlikely to affect the phenotype, these subgroups share characteristics of the high-priority Top-6 and O157:H7/NM groups of STEC and thus the associated SNPs have a good potential to be a virulence marker for STEC. For example, isolates tested in this study that belong to non-Top-7 serogroups and possess stx1a-I (Table 1), such as O118:H16, O165:H25/NM, have been reported as causative agents of HUS (Bielaszewska et al., 2009; Mora et al., 2005). Furthermore, the BLAST search for stx1a-II subgroup targeted region showed 100% homology to Aeromonas caviae and Shigella dysenteriae that are isolates from human HUS and epidemic diarrhea (Alperi and Figueras, 2010; Yang et al., 2005). Thus, we presume that stx1a-I (690C/702C) and stx1a-II (690 T/702 T) are the subtypes highly related to the severe illness cases and needs to be flagged if this subtype was detected from food and environmental isolates. There are a few isolates reported carrying multiple stx1 subtypes (Feng and Reddy, 2013), and these could potentially cause pyrosequencing ambiguity in stx1 genotyping. However, it should be noted that isolates carrying multiple stx1 subtypes are very rarely reported and our investigation indicates that the pyrosequencing assay may be able to identify them. An experiment with mixed DNA samples from two isolates carrying either stx1a and stx1c demonstrated that the pyrogram of SNP (A-G) at base position 666 showed doublet peaks for both G and A of half peak magnitude and thereby could be recognized as a multiple stx1 gene carrier (data not shown). A total of 19 isolates tested in this report were identified by PCR subtyping as carrying stx2b, stx2c and/or stx2d along with stx2a, which is the most common combination of stx2 subtype multiplicity in human HUS cases (Persson et al., 2007). The pyrosequencing assay could detect the multiplicity in 10 strains, the remaining 9 isolates were only stx2a or stx2c–d positive. This may be due to the high sequence similarity among the 3 subtypes as described in a previous publication (Scheutz et al., 2012). The comparison of pyrosequence reads to the Illumina sequence reads from part of ongoing work (GRDI FWS Pilot), of one of the 9 isolates, ECI-2921 (O28ac:H25), supported this hypothesis. Illumina sequence reads of this strain showed that stx2c didn't carry the SNP at base 1074 (A-G) that we set as the SNP to differentiate 2b, 2c, and 2d from others (Illumina sequence data not shown). Since stx2a, stx2c and/ or stx2d have all been associated with human illness (Persson et al., 2007), detecting multiplicity of stx2 gene in a single isolate is not more informative than identifying one of three subtypes, thus further analysis of the additional targets may not be necessary. The eae subtyping, we focused on subtypes β1-, γ1-, ε- and θ-eae, since our final objective of genotyping is to distinguish highly-virulent STEC and these eae subtypes are commonly reported in EHEC serotypes. The region selected in this study has identical sequences between θ- and γ2-eae, and λ and γ1-eae, thus the eae subtyping results in this study are shown as λ/γ1-eae, β-eae, ε-eae, and θ/γ2-eae (Table 1). According to the multiple alignment analysis shown in Fig. 2, other eae subtypes, including α-, ι-, κ-, δ-, μ-, σ-, υ-, π-eae should be detectable by the pyrosequencing assay, although none of the isolates tested in this study possessed these subtypes. Subtypes κ-, δ-, μ-, and π-eae have an identical sequence in this region. The primer sets we designed in this study were unlikely to amplify subtypes τ-, ο-, ν-, ζ-, and η-eae. Testing more isolates from various locations and sources will help to determine if these minor eae subtypes are associated with pathogenicity in humans, and are detectable using this newly developed pyrosequencing assay. Previous publications indicated that some eae subtypes are closely associated with O:H serotypes (Bibbal et al., 2014; Blanco et al., 2004; Madic et al., 2010) and our results supported their findings. For example, O26 isolates possessed β-eae, O103:H2 ε-eae, and O145 and O157: H7/NM carried γ1-eae. O111:NM θ/γ2-eae were reported to carry θ-eae in some previous reports (Bibbal et al., 2014; Madic et al., 2010). Non H7/NM isolates of O157 were all eae negative in our results. There have been very few isolates of O157 non H7/NM STEC

U

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The authors thank Drs. Roger Johnson (PHAC, Guelph), Keith Warriner (University of Guelph) and Burton Blais (CFIA, OLC) for providing E. coli strains used in this study. This work was funded by the Canadian Federal Genomics Research and Development Initiative (GRDI). We also acknowledge the federal GRDI interdepartmental Food and Water Safety project consortium (comprised of researchers from Agriculture and Agri-Food Canada, the Canadian Food Inspection Agency, Environment Canada, Health Canada, the National Research Council of Canada, and the Public Health Agency of Canada) for useful discussions and contributions. The authors thank Fanliang Kong and

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Achazi, K., Nitsche, A., Patel, P., Radonic, A., Donoso Mantke, O., Niedrig, M., 2011. Detection and differentiation of tick-borne encephalitis virus subtypes by a reverse transcription quantitative real-time PCR and pyrosequencing. J. Virol. Methods 171, 34–39. Alderborn, A., Kristofferson, A., Hammerling, U., 2000. Determination of single-nucleotide polymorphisms by real-time pyrophosphate DNA sequencing. Genome Res. 10, 1249–1258. Aldick, T., Bielaszewska, M., Zhang, W., Brockmeyer, J., Schmidt, H., Friedrich, A.W., Kim, K.S., Schmidt, M.A., Karch, H., 2007. Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium. Microbes Infect. 9, 282–290. Alperi, A., Figueras, M.J., 2010. Human isolates of Aeromonas possess Shiga toxin genes (stx1 and stx2) highly similar to the most virulent gene variants of Escherichia coli. Clin. Microbiol. Infect. 16, 1563–1567. Amoako, K.K., Shields, M.J., Goji, N., Paquet, C., Thomas, M.C., Janzen, T.W., Bin Kingombe, C.I., Kell, A.J., Hahn, K.R., 2012a. Rapid detection and identification of Yersinia pestis from food using immunomagnetic separation and pyrosequencing. J. Pathog. 2012, 781652. Amoako, K.K., Thomas, M.C., Kong, F., Janzen, T.W., Hahn, K.R., Shields, M.J., Goji, N., 2012b. Rapid detection and antimicrobial resistance gene profiling of Yersinia pestis using pyrosequencing technology. J. Microbiol. Methods 90, 228–234. Amoako, K.K., Janzen, T.W., Shields, M.J., Hahn, K.R., Thomas, M.C., Goji, N., 2013. Rapid detection and identification of Bacillus anthracis in food using pyrosequencing technology. Int. J. Food Microbiol. 165, 319–325. Anklam, K.S., Kanankege, K.S., Gonzales, T.K., Kaspar, C.W., Dopfer, D., 2012. Rapid and reliable detection of Shiga toxin-producing Escherichia coli by real-time multiplex PCR. J. Food Prot. 75, 643–650. Bai, J., Paddock, Z.D., Shi, X., Li, S., An, B., Nagaraja, T.G., 2012. Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces. Foodborne Pathog. Dis. 9, 541–548. Beutin, L., Kruger, U., Krause, G., Miko, A., Martin, A., Strauch, E., 2008. Evaluation of major types of Shiga toxin 2E-producing Escherichia coli bacteria present in food, pigs, and the environment as potential pathogens for humans. Appl. Environ. Microbiol. 74, 4806–4816. Bibbal, D., Loukiadis, E., Kerouredan, M., Peytavin de Garam, C., Ferre, F., Cartier, P., Gay, E., Oswald, E., Auvray, F., Brugere, H., 2014. Intimin gene (eae) subtype-based real-time PCR strategy for specific detection of Shiga toxin-producing Escherichia coli serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in cattle feces. Appl. Environ. Microbiol. 80, 1177–1184. Bielaszewska, M., Prager, R., Vandivinit, L., Musken, A., Mellmann, A., Holt, N.J., Tarr, P.I., Karch, H., Zhang, W., 2009. Detection and characterization of the fimbrial sfp cluster in enterohemorrhagic Escherichia coli O165:H25/NM isolates from humans and cattle. Appl. Environ. Microbiol. 75, 64–71. Blais, B.W., Gauthier, M., Descheenes, M., Huszczynski, G., 2012. Polyester cloth-based hybridization array system for identification of enterohemorrhagic Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157. J. Food Prot. 75, 1691–1697. Blanco, M., Blanco, J.E., Mora, A., Dahbi, G., Alonso, M.P., Gonzalez, E.A., Bernardez, M.I., Blanco, J., 2004. Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from cattle in Spain and identification of a new intimin variant gene (eae-xi). J. Clin. Microbiol. 42, 645–651. Byrne, L., Elson, R., Dallman, T.J., Perry, N., Ashton, P., Wain, J., Adak, G.K., Grant, K.A., Jenkins, C., 2014. Evaluating the use of multilocus variable number tandem repeat analysis of Shiga toxin-producing Escherichia coli O157 as a routine public health tool in England. PLoS One 9, e85901. Conrad, C.C., Stanford, K., McAllister, T.A., Thomas, J., Reuter, T., 2014. Further development of sample preparation and detection methods for O157 and the top 6 nonO157 STEC serogroups in cattle feces. J. Microbiol. Methods 105C, 22–30. Cookson, A.L., Bennett, J., Thomson-Carter, F., Attwood, G.T., 2007. Intimin subtyping of Escherichia coli: concomitant carriage of multiple intimin subtypes from forage-fed cattle and sheep. FEMS Microbiol. Lett. 272, 163–171. Deng, Y.M., Caldwell, N., Barr, I.G., 2011. Rapid detection and subtyping of human influenza A viruses and reassortants by pyrosequencing. PLoS One 6, e23400. Fegan, N., Barlow, R.S., Gobius, K.S., 2006. Escherichia coli O157 somatic antigen is present in an isolate of E. fergusonii. Curr. Microbiol. 52, 482–486. Feng, P.C., Reddy, S., 2013. Prevalences of Shiga toxin subtypes and selected other virulence factors among Shiga-toxigenic Escherichia coli strains isolated from fresh produce. Appl. Environ. Microbiol. 79, 6917–6923. Feng, P.C., Keys, C., Lacher, D., Monday, S.R., Shelton, D., Rozand, C., Rivas, M., Whittam, T., 2010. Prevalence, characterization and clonal analysis of Escherichia coli O157: nonH7 serotypes that carry eae alleles. FEMS Microbiol. Lett. 308, 62–67. Friesema, I., van der Zwaluw, K., Schuurman, T., Kooistra-Smid, M., Franz, E., van Duynhoven, Y., van Pelt, W., 2014. Emergence of Escherichia coli encoding Shiga toxin 2f in human Shiga toxin-producing E. coli (STEC) infections in the Netherlands, January 2008 to December 2011. Euro Surveill. 19, 26–32. Haanpera, M., Huovinen, P., Jalava, J., 2005. Detection and quantification of macrolide resistance mutations at positions 2058 and 2059 of the 23S rRNA gene by pyrosequencing. Antimicrob. Agents Chemother. 49, 457–460. Haugum, K., Brandal, L.T., Lindstedt, B.A., Wester, A.L., Bergh, K., Afset, J.E., 2014. PCR based detection of Shiga toxin-producing Escherichia coli (STEC) in a routine microbiology

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The new PCR/Pyrosequencing assay developed in this study for the simultaneous detection and subtyping of Shiga toxin 1 and 2, intimin and rfbE genes in E. coli demonstrates the potential of pyrosequencing for STEC genotyping assay as a quick diagnostic tool. Although the assay platform using this PCR-pyrosequencing requires isolated colonies, since sequencing multiple subtypes in a DNA sample from complex environmental/food samples that may harbor multiple STEC isolates with different genotypes could be a challenge, this method has a great potential in E. coli colony screening without the serotype classification. Up to 96 samples could be analyzed in one pyrosequencing assay, thus it can be deployed in food or public health laboratories, in particular those without whole genome sequencing and high computing hardware and software skills capability, as a genomic application tool for the rapid genotyping of pathogenic E. coli isolates. However, we understand that further investigation of these novel SNPs is required, because of the high genomic plasticity of E. coli.

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Gracia Windiasti for their dedicated technical assistance and Matthew 621 Thomas and Timothy Janzen for the valuable technical suggestions. 622

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(Bielaszewska et al., 2009). Serotypes O69:H11, O116:H28, O118: H16 possess stx1a-I and β-eae, which is the same gene profile as O26 and O111 isolates, thus these serotypes should also be noted in the STEC surveillance. Among the isolates with unknown serotypes tested in this study, ECI-2627, 2628 and 2639 carry stx1a-I and ε-eae, the same genotype as O45 and O103:H2 isolates and the genotype of ECI2570 was the same as O111:NM, implying that they may belong to these serotypes and have high potential to cause human illnesses. Only one isolate (ECI-2126, O179:H7) that is outside of the O157 group was found to have the same genetic profile as O157:H7 (Table 1). Further analysis needs to be performed on this isolate to determine if the isolate is a true O179 or if the serotype was incorrectly determined, since the whole genome mapping data of this isolate was 98% identical to O157:H7 Sakai (data not shown). The O179 is a rather new serotype and there are only a few studies on this serotype. One report showed that O179:H8 was isolated from a human hemolytic diarrhea case (Scheutz et al., 2004), while an isolate tested in this study (ECI-3012, O179:H8) was positive for only stx2a and didn't carry any eae gene. Combinations of various virulence genes with broad phylogenetic diversities are related to the causative E. coli isolates. The frequent transmission and mutation of virulence genes on bacteriophage create a perpetual concern of new emerging pathogens, including the LEE-negative O104:H4 isolate that caused an outbreak in Europe in 2011 (Scheutz et al., 2011) and HUS cases with Shiga toxin-negative O26 strains (Aldick et al., 2007). Thus the investigation of entire genome sequence including plasmid profiling is essential for human outbreak cases in order to delineate the pathogenicity of E. coli. However, the PCR/pyrosequencing assay described in this paper for the detection and subtyping of the 4 major virulence genes (stx1, stx2, eae and rfbE) could be performed quickly and cost-effectively before the sample preparation for whole genome sequencing is even completed. This should provide testing laboratories and investigators with a tool for rapid screening of STEC before further testing.

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Characterization of Shiga Toxin Subtypes and Virulence Genes in Porcine Shiga Toxin-Producing Escherichia coli.

Detection, Characterization, and Typing of Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli.

Detection of five Shiga toxin-producing Escherichia coli genes with multiplex PCR.

Development of 11-Plex MOL-PCR Assay for the Rapid Screening of Samples for Shiga Toxin-Producing Escherichia coli.

A Rapid Immunoassay for Detection of Shiga Toxin-Producing Escherichia coli Directly from Human Fecal Samples and Its Performance in Detection of Toxin Subtypes.

Real-Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical Samples.

A Poly-N-acetylglucosamine-Shiga toxin broad-spectrum conjugate vaccine for Shiga toxin-producing Escherichia coli.

Comparative possession of Shiga toxin, intimin, enterohaemolysin and major extended spectrum beta lactamase (ESBL) genes in Escherichia coli isolated from backyard and farmed poultry.

Shiga-toxin genes and genetic diversity of Escherichia coli isolated from pasteurized cow milk in Brazil.

Evaluation and optimization of a latex agglutination assay for detection of cholera toxin and Escherichia coli heat-labile toxin.

Geographic divergence of bovine and human Shiga toxin–producing Escherichia coli O157:H7 genotypes, New Zealand.

Advances in detection methods for Shiga toxin-producing Escherichia coli (STEC).

Rapid, Multiplexed Characterization of Shiga Toxin-Producing Escherichia coli (STEC) Isolates Using Suspension Array Technology.

Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coli upon subcultivation.

In silico analysis of Shiga toxins (Stxs) to identify new potential vaccine targets for Shiga toxin-producing Escherichia coli.

Rapid Detection of Escherichia coli O157 and Shiga Toxins by Lateral Flow Immunoassays.

Transcription of the Shiga-like toxin type II and Shiga-like toxin type II variant operons of Escherichia coli.

Evaluation of a loop-mediated isothermal amplification suite for the rapid, reliable, and robust detection of Shiga toxin-producing Escherichia coli in produce.

DNA hybridization and latex agglutination for detection of heat-labile- and shiga-like toxin-producing Escherichia coli in meat.

Comparative genomic analysis of Shiga toxin-producing and non-Shiga toxin-producing Escherichia coli O157 isolated from outbreaks in Korea.

Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification.

Detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) in bovine dairy herds in Northern Italy.

Evaluation of DNA probes for detection of Shiga-like-toxin-producing Escherichia coli in food and calf fecal samples.