Using Nanospray Liquid Chromatography and Mass Spectrometry to

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Food Safety and Toxicology

Using Nanospray Liquid Chromatography and Mass Spectrometry to Quantitate Shiga Toxin Production in Environmental Escherichia coli Recovered from a Major Produce Production Region in California Christopher J. Silva, Bertram G. Lee, Jaszemyn C. Yambao, Melissa L. Erickson-Beltran, and Beatriz Quinones J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05324 • Publication Date (Web): 28 Nov 2018 Downloaded from http://pubs.acs.org on November 30, 2018

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Journal of Agricultural and Food Chemistry

Using Nanospray Liquid Chromatography and Mass Spectrometry to Quantitate Shiga Toxin Production in Environmental Escherichia coli Recovered from a Major Produce Production Region in California

Christopher J. Silva*, Bertram G. Lee, Jaszemyn C. Yambao, Melissa L. Erickson-Beltran, and Beatriz Quiñones*

U.S. Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Produce Safety & Microbiology Research Unit, Albany, California 94710, United States

AUTHOR INFORMATION Corresponding Authors *Telephone:

+1-510-559-6135; E-mail: [email protected] (C. J. S.).

*Telephone:

+1-510-559-6097; E-mail: [email protected] (B. Q.).

ORCID Christopher J. Silva: 0000-0003-4521-6377 Beatriz Quiñones:

0000-0001-5010-9889

Funding This work was supported by the U.S. Department of Agriculture, Agricultural Research Service, CRIS Project Numbers 2030-42000-050-00D and 2030-42000-051-00D.

1 ACS Paragon Plus Environment


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ABSTRACT A set of 45 environmental strains of Shiga toxin producing E. coli (STEC) from three

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California counties were analyzed for Shiga toxin production by nanospray liquid

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chromatography-mass spectrometry and Vero cell bioassay. The STEC in this set comprised six

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serotypes ((O113:H21, O121:H19, O157:H7, O6:H34, O177:H25, and O185:H7) each

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containing either the stx2a or stx2c operon. Six of the seven O113:H21 were found to contain two

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distinct stx2a operons. Eight strains of O157:H7 possessed a stx2c operon whose A subunit gene

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was interrupted by an insertion sequence (IS1203v). Shiga toxin production was induced by

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nutrient depletion and quantitated by mass spectrometry. The 37 strains produced Shiga toxins

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in a fifty-fold range (1.4 ng/ml to 49 ng/ml). The IS-interrupted strains expressed low but

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measurable amounts of the B subunits (0.5 -1.9 ng/ml). Another strain possessed an identical

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stx operon without an IS interruption and produced intact Stx2c (5.7 ng/ml).

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KEYWORDS: Escherichia coli, food safety, liquid chromatography, mass spectrometry,

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multiple reaction monitoring, prophage, Shiga toxin


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INTRODUCTION Shiga toxin producing Escherichia coli (STEC) is a waterborne and foodborne pathogen

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responsible for human gastroenteritis with diverse clinical symptoms.1 STEC are responsible for

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approximately 260,000 infections, at least 3,600 hospitalizations, and direct costs exceeding 1

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billion dollars per year in the United States alone.2-4 Indirect costs can be substantial; bagged

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spinach producers lost approximately $200 million (2006 USD) in sales as a result of an STEC

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outbreak in 2006.5 Starting in the early 1980s STEC have been responsible for many significant

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foodborne disease outbreaks.6, 7 The outbreak in 1992-1993 was associated with a quick serve

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restaurant chain.8 The largest occurred in Sakai, Japan, sickening 8,300 school children.9 In

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2011, an STEC outbreak in Germany, sickened 3,800 and killed 53.10

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The early outbreaks were mostly associated with undercooked meat.8 The awareness of

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this hazard has made consumers and food processors more aware of the need to properly cook

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meat. As a result, the foods associated with outbreaks have changed from meat products to fresh

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produce. The outbreak in Sakai, Japan was caused by contaminated radish sprouts.9 The

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outbreak in Germany was caused by contaminated Fenugreek seeds.11 In 2006 an STEC

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outbreak associated with green leafy vegetables occurred in the Salinas Valley of California.12

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This is probably a reflection of changes in dietary habits, an emphasis on cooking meats at

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proper temperatures, and an increase in the consumption of fresh leafy greens.13

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Given the significance of STEC outbreaks, it is imperative to develop quantitative

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methods to characterize the relevant virulence factors contributing to foodborne illness. A key

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virulence factor contributing to STEC pathogenicity in humans is the production of Shiga toxins

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(Stx) by these bacteria. In spite of their amino acid sequence differences, all Stx are

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heterohexameric (non-covalent) protein (AB5) toxins composed of a single A subunit (toxic



40

portion) and five identical B subunits (target cell binding).14 Stx share a common operon

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structure, secondary and tertiary structures, mechanism of action, and biological activity. They

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are classified as type 1 (Stx1), type 2 (Stx2) or a variant of those types based on their amino acid

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sequences.15 The amino acid sequence of Stx1 is very similar to the toxin isolated from Shigella

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dysenteriae type 1. The amino acid sequences of Stx2 are substantially different from those of

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the Stx1 variants.15 There are at least four subtypes of Stx1 and eight Stx2 (Stx2a-Stx2h)

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subtypes.15, 16 Some, such as Stx2b (formerly referred to as Stx2d15) and Stx2e are associated

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with mild disease in humans.17, 18 In contrast, Stx2a and Stx2c are more commonly associated

48

with more severe sequelae than are the other Stx1 and Stx2 subtypes.17, 19, 20 It is therefore

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important to be able to detect and distinguish among the Stx.

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Stx have been detected by cell-based assay, PCR, immunological methods and mass

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spectrometry.21, 22 PCR has been used to detect the presence of stx genes15, and the subsequent

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use of a fluorescent Vero cell-based assay to measure the loss of protein synthesis can be used to

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detect the active toxin subtype in a sample.23, 24 Antibody-based methods have been used to

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detect and distinguish among Stx, but quantitation is complicated by the polymorphisms present

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in the A subunits of the Stx variants.25 Although Stx have been previously detected and

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distinguished using top down-based mass spectrometry-based methods,26 the use of a multiple

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reaction monitoring (MRM)-based mass spectrometry method enables the quantification of the

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amount of Stx subtypes present in a sample without purification.27 The MRM-based method

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provides a convenient means of simultaneously distinguishing the Stx subtypes present in a

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sample.

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In STEC, Stx production is controlled not by the E. coli host, but by a temperate

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lambdoid phage (stx phage) or phages that infect the E. coli host.28-31 Each stx phage produces a


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single Stx subtype from a single stx operon during the lytic growth. Bacterial genomes also

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possess insertion sequences (IS), which have been shown to also be inserted into the stx operon

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and consequently disrupt the production of Stx.32-37 An enzyme, acting as an IS excision

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enhancer, has been identified in STEC strains with the O157:H7 serotype, and the removal of the

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IS from the stx operon would then restore the expression of a functional Stx.38

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Here we report our results using a previously described mass spectrometry-based method

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of detecting Stx in complex media and apply it to detect Stx produced by 45 STEC strains.

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These strains were selected from a collection of strains, previously recovered from a large survey

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to identify relevant animal and environmental sources of STEC in a major agricultural region in

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California.39 Each strain was induced to express Stx and the amount produced was quantitated by

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mass spectrometry. Eight of these strains were found to possess a stx prophage with an IS

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inserted into the stx2c operon, resulting in a non-functional Stx. One STEC strain possessed a

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stx2c operon with an identical sequence as those other eight strains with a non-functional Stx, but

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without the IS inserted. Interestingly, the use of the MRM-based mass spectrometry method

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revealed that the IS-positive strains produced detectable and quantifiable levels of the B subunit.

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The findings from this study demonstrate the usefulness of MRM-based quantitative mass

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spectrometry for the direct detection and quantitation of the Stx produced by STEC strains, since

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the detection of the stx gene does not mean that a functional Stx will be expressed.



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MATERIALS AND METHODS Bacterial Strain Isolation and Characterization. The STEC strains examined in the

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present study are shown in Table 1. For the recovery of STEC strains from environmental

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sources in a major agricultural region in Monterey, San Benito, and San Luis Obispo Counties in

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the Central Coast of California, watershed sediment or animal feces were subjected to an

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enrichment step, as previously documented.39 The enrichments were further screened for stx1

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and stx2 by real-time PCR, and STEC strains were recovered after plating samples from positive

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enrichments on different selective chromogenic media CHROMagar O157 media (DRG

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International, Mountainside, NJ), Rainbow O157 agar (Biolog, Inc., Hayward, CA) or modified

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sheep’s blood agar, as reported previously.39 The recovered STEC strains were further serotyped

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by a PCR assay using sequence specific primers to target either wxz or wxy genes in the O-

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antigen gene clusters of twelve serogroups (O26, O45, O55, O91, O103, O104, O111, O113,

93

O121, O128, O145 and O157) and seven flagellar antigens H2, H7,H8, H11, H19, H21, and H28

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(fliC genes).40, 41 As PCR template, crude lysates were prepared from bacterial cultures

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propagated on Luria-Bertani (LB) agar (Difco, Detroit, MI). A 1L loop sample was taken from

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a colony and suspended in 55 l of HyPure™ molecular biology-grade water (HyClone

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Laboratories, Inc., Logan, UT). The suspension was thoroughly mixed by vortexing and then

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incubated at 95°C for 20 minutes, as in previous studies.41 PCR amplifications consisted of a 25

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l reaction mixture, each containing 3 L of the bacterial crude lysate, 0.5 M of each primer

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(Eurofins MWG Operon, Huntsville, AL), and 12.5 l of 2× GoTaq® Green Master Mix

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(Promega Corporation, Madison, WI). The reaction mixtures were placed in a Dyad Peltier

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Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). The primer sequences, PCR cycling

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conditions and strains used as controls were described in a previous study.40, 41 Amplified 6 ACS Paragon Plus Environment

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products were analyzed in 1% agarose gels containing 0.04 l/ml GelRed Nucleic Acid Stain

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(Phenix Research, Candler, NC). Strains untypeable by PCR to identify the O- and H-antigens

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were sent for serotyping by the Escherichia coli Reference Center (The Pennsylvania State

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University, University Park, PA).

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Screening and Gene Sequencing of stx2 Subtypes in the Examined STEC Strains. To

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determine the stx2 subtype of the tested environmental STEC strains, crude lysates were prepared

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from STEC strains grown on Luria-Bertani (LB) agar plates, as described above (Difco, Detroit,

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MI) for use as PCR template. The primer sequences, PCR reaction and cycling conditions and

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strains used as controls for each specific stx2 subtypes were described in a previous study.42 To

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determine the full-length gene sequence encoding the Stx A- and B-subunits, the stx2 operon was

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amplified using primers Stx2F-21328 (5’-TTCTGAGCAATCGGTCACTG-3’) and Stx2R-

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22087 (5’- CGGCGTCATCGTATACACAG -3’) and primers Stx2F-22017 (5’-

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GTCACAGCAGAAGCCTTACG -3’) and Stx2R-22711 (5’- ACCCACATACCACGAATCAG

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-3’).43 To determine the insertion element in the stx2 A-subunit gene in the STEC strains

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RM10641, RM10645, RM10646, RM10649, RM10716, RM10718, RM10719, RM10720 (Table

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1), PCR amplifications were performed using primers iZ1 (5’-

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TACCTCCGCTTTGTAAAGACCATTG-3’) and iZ2 (5’-CGTGAAAATCGTGAACTGCG-3’).

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PCR amplifications were performed using genomic DNA extracted with DNeasy Blood & Tissue

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Kits (Qiagen, Valencia, CA). Cycling reactions were performed using 500 ng of genomic DNA,

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0.5 M (each) of the forward and reverse primer, and 25 l of Phusion® High-Fidelity PCR

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Master Mix (New England Biolabs, Ipswich, MA) in a final volume of 50 l, and amplifications

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were performed with an initial denaturation at 94 °C for 5 minutes, followed by 30 cycles of

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denaturation at 94 °C for 45 seconds, annealing at 60 °C for 1 minute, and primer extension at 72



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°C for 1 minute with a final extension at 72 °C for 7 minutes. The nucleotide sequences of the

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amplicons were further determined by conventional Sanger DNA sequencing (Elim

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Biopharmaceuticals, Inc., Hayward, CA, USA) to confirm the stx2 subtype. The nucleotide

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accession number for the stx2 operon in each tested STEC strain are listed in Table 1. Given that

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STEC strains RM7788, RM7806, RM7807 had double traces by Sanger sequencing, high

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resolution genome sequencing was performed using an Illumina MiSeq sequencer (Illumina,

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Inc., San Diego, CA), as previously described.44 Genomic DNA was extracted from overnight

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cultures using the Wizard genomic DNA purification kit (Promega Corp., Madison, WI). The

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quantity of DNA was assessed by fluorometric measurement using the Quant-iT PicoGreen DNA

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assay kit (Invitrogen, Carlsbad, CA). DNA sequencing libraries with 575 to 675 bp inserts were

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prepared using the KAPA LTP library preparation kit (KAPA Biosystems, Wilmington, MA).

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The pooled amplicon libraries were loaded into a MiSeq System and sequenced using a MiSeq

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reagent kit v2 with 2 x 250 cycles (Illumina, Inc.). Draft genomes were assembled using

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Newbler assembler (version 2.6, Roche) for generating a contig graph file, as in previous

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reports.44

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Fluorescent Cell-Based Assay to Detect Stx Activity. The Stx activity of the examined

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STEC strains was measured using a Vero cell line, Vero-d2EGFP, that harbored a destabilized

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variant (t1/2 = 2 hour) of the enhanced green fluorescent protein (EGFP).23, 24, 45 To monitor the

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Stx-induced inhibition of protein synthesis, cell-free culture supernatants from the STEC strains

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(Table 1) were determined. The Stx2a-expressing O157:H7 strain RM2084 was used as a

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positive control. The Stx-negative O157:H7 strains RM1273, RM4876 and the K12 strain

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RM5034 (Stx-negative) were used as negative controls. All E. coli strains were inoculated in 1

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ml of sterile LB broth (Difco, Detroit, MI) and were grown aerobically for 24 hours at 37 °C


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(nutrient depletion) with shaking at 200 rpm, then centrifuged at 2,000 × g for 15 minutes.

The

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culture supernatants were filter-sterilized using 0.45 m polyvinylidene fluoride syringe filters

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(Durapore® membranes, Millipore Corporation, Billerica, MA) and were frozen at -20 °C until

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further use.24, 42 A day before intoxication, Vero-d2EGFP cells were seeded at 10,000 cells per

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well in Greiner black 96-well microplates with clear bottoms (VWR International, Aurora, CO,

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USA) and incubated at 5% CO2 and 37 °C under humidified conditions in Ham's F-12 medium

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(Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (American

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Type Culture Collection, Manassas, VA, USA) and 1% penicillin-streptomycin-amphotericin B

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(Life Technologies).23, 24, 45 The Vero-d2EGFP cells were then intoxicated with Ham's F-12

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complete medium (Life Technologies) containing 10-fold dilutions of the cell-free culture

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supernatants from each strain and were incubated at 37 °C for 16-18 hours in a 5% CO2

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humidified incubator. After, the cells were washed with 1× Dulbecco’s phosphate buffered

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saline (Life Technologies). EGFP fluorescence from the Vero-d2EGFP cells was measured

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using a Synergy HT Multi-Detection microplate reader (BioTek, Winooski, VT, USA) with a

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485/20-nm excitation filter and a 528/20-nm emission filter, as in previous studies.23, 24 All

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measurements were performed with replicates, and the results were expressed as percentages of

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the fluorescence values for culture supernatant-treated Vero-d2EGFP cells compared to the

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fluorescence values for control Vero-d2EGFP cells incubated without bacterial supernatants.

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Peptide Optimization. The FVTVTAEALR peptide was obtained from Elim

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Biopharmaceuticals. It was at least 95% pure and its structure was verified by mass

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spectrometry. The other peptides used in this study were previously optimized.27 The

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FVTVTAEALR peptide fragmented to yield the characteristic a2, y6, or y8 ions. The signal

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intensity of each ion was optimized by adjusting the Q2 oﬀset voltage (collision energy) for each



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ion. The mass settings for the FVTVTAEALR peptide were empirically determined [precursor

174

ion at m/z 553.8, product ion at m/z 219.5 (a2 ion, CE 33), 660.4 (y6 ion, CE 29), or 860.5 (y8

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ion, CE 26.2)]. For each ion the declustering potential (DP, 142), entrance potential (EP, 10.9)

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and collision cell exit potential (CXP, 15) were identical. Quantitation was done with the

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Intelliquan quantitation algorithm using Analyst version 1.4.

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Reduction, Alkylation, and Tryptic cleavage of Stxs. The preparation of samples for

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mass spectrometry using reduction/alkylation/trypsin cleavage has been described previously.27,

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46

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concentrated approximately 10-fold by SpeedVac (Thermo Scientific, Waltham, MA USA). The

182

exact amount of each concentrate was measured with a pipette, recorded, and used in

183

calculations of toxin amounts. 8 l of concentrated bacterial supernatant was brought up to 40 l

184

with denaturing buffer (7.5M guanidine hydrochloride (GuCl) in 25 mM ammonium

185

bicarbonate, pH8) and thoroughly mixed. 5 l of reducing buffer (45 mM dithiothreitol in

186

25mM Ammonium Bicarbonate pH 8.0, 0.01% -octylglucopyranoside (BOG), 0.9 pmol/l L-

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methionine (Met), and 8% Acetonitrile (ACN)) was added and incubated at 37 °C with shaking

188

(300 rpm). After 0.5 hour the solution was cooled to room temperature and 5 l of alkylating

189

buffer (125 mM iodoacetamide in 25mM ammonium bicarbonate pH 8.0, 0.01% -

190

octylglucopyranoside (BOG), 0.9 pmol/l L-methionine (Met), and 8% Acetonitrile (ACN)) was

191

added and incubated at room temperature for 0.5 hour in the dark. The excess alkylating agent

192

was quenched by the addition of 6.5 l of reducing buffer followed by brief (30 sec.) vortexing.

193

540 l of chilled methanol (-20 °C) was added to the solution and chilled at -20 °C for 1 hour.

194

The chilled solution was centrifuged for 10 minutes (20,000 x g; -11 °C). The supernatant was

195

discarded, the pellet washed with 200 l of chilled methanol and centrifuged for an additional 10

Freshly prepared solutions were used. 500 l of each sterile filtered bacterial supernatant was


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minutes (20,000 x g; -11 °C). The resulting supernatant was discarded and the pellet was dried

197

for 10 minutes at room temperature to remove residual methanol. 90 l of digestion buffer

198

(25mM ammonium bicarbonate pH 8.0, 0.01% -octylglucopyranoside (BOG), 0.9 pmol/l L-

199

methionine (Met), and 8% acetonitrile (ACN)) was added to solubilize the dried pellet. 10 l of

200

trypsin solution (100 g/ml trypsin in water) was added to the solubilized pellet and incubated

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for 2 hours with periodic sonication. After the digestion, each sample was filtered through a

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10,000 MWCO filter (12 min; 14,000 x g) and then analyzed or stored (-80 °C) for eventual

203

analysis.

204 205

Mass Spectrometry. The instrument response was optimized by a previously described

206

method.27 The qualitative mass spectrometry required to verify that the synthetic peptides were

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correctly synthesized was performed using a Thermo Scientific model Orbitrap Elite instrument

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equipped with a nanoelectrospray source (Supporting Information). An Applied Biosystems

209

(ABI/MDS Sciex, Toronto, ON) model 4000 Q-Trap instrument equipped with a

210

nanoelectrospray source was used for quantification. This mass spectrometer was operated in

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multiple reaction monitoring (MRM) mode, alternating between detection of analyte peptides

212

and the corresponding 15N-labeled internal standards. The mass settings for the peptides and the

213

retention times of the peptides have been previously reported. Quantification was done with the

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IntelliQuan quantification algorithm using Analyst 1.4 software (Applied Biosystems).

215 216

RESULTS AND DISCUSSION

217



218

Description of environmental samples. Environmental samples were subjected to a

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robust isolation procedure designed to recover representative STEC from diverse environmental

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sources in a major produce production region in California.39 As summarized in Table 1, a

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subset of strains was selected based on the presence of the stx2a or the stx2c genes, encoding for

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clinically-relevant Stx2 subtypes. Each of these strains was serotyped, and these efforts yielded

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45 STEC strains with 6 different serotypes, recovered from soil (3), water (3), domestic cattle

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(27), birds (2), wild deer (2), domestic swine (5), wild swine (2), and produce (1).39 Three of the

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identified serotypes, O113:H21, O121:H19, and O157:H7, have been associated with severe

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disease symptoms in humans and STEC outbreaks.47-49

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Sequencing of the stx operons. High-resolution sequencing of each stx operon in the

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tested 45 STEC revealed that six of O113:H21 STEC strains containing the stx2a operon

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(RM7788, RM7806, RM7807, RM9244, RM9245, and RM9246) possessed two distinct stx2a

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operons, suggesting they were infected with two individual stx phages (Tables 1 and Supporting

231

Information, Figure S-1). Those stx2a operon sequences in the STEC strains with the O121:H19

232

(4) and O157:H7 (10) serotypes showed no nucleotide polymorphisms within the respective

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serotypes (Supporting Information, Figure S-1). Sequence analysis of three STEC serotypes

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possessing the stx2c operon, O6:H34 (3), O177:H25 (2), or O185:H7 (9), showed no nucleotide

235

polymorphisms within strains with those three serotypes (Supporting Information, Figure S-1),

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which indicates that each serotype expresses a distinct stx2c gene. The stx2c operon in the two

237

O157:H7 serotypes (RM10024 and RM10058) showed 10 nucleotide polymorphisms

238

(Supporting Information, Figure S-1), indicating the stx2c gene diversity within O157:H7

239

serotype.


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Amplification of stx operons by PCR revealed a 1.3-kb IS present in the coding region of

241

the A subunit of the stx2c operon (Figure 1). The ISs were identified in eight strains of the

242

O157:H7 isolated from four distinct samples (F-1858, F-1884, F-1900, and F-1887) of feces

243

from domestic cattle (strains RM10641, RM10645, RM10646, RM10649, RM10716, RM10718,

244

RM10719, and RM10720). A BLAST search analysis revealed the IS in the tested STEC

245

O157:H7 strains resembled the sequence of the insertion sequence IS1203 variant (IS1203v).

246

Previous reports documented the IS1203v inserted in the stx2c operon in O157:H7 strains.32-37 In

247

the present study, the IS1203v was found inserted in the stx2c operon in the O157:H7 cattle

248

strains, and the IS disrupted the expression of the A subunit but not the B subunit (Figure 1).

249

Sequence analysis, after subtraction of the IS sequence, showed that the eight stx2c operons in the

250

O157:H7 strains were identical.

251

Sequence comparison of the intact stx2c operon from another O157:H7 strain (RM10024)

252

with the non-IS portions of the stx2c operon from the eight O157:H7 strains were found to be

253

identical. The stx2c sequence of the other O157:H7 stx2c-positive strain (RM10058) was found to

254

have ten nucleotide polymorphisms when compared to strain RM10024 (Supporting Information,

255

Figure S-1). This indicates that the eight IS interrupted stx2c operons and the one non-IS

256

interrupted stx2c operon (RM10024) may reside on a similar phage. This suggests that RM10024

257

could have been infected with the progenitor phage whose stx2c operon was inactivated by the

258

ISs, which would consequently convert an STEC strain into a non-functional Stx but still show

259

as positive for the stx2c gene by conventional PCR-based typing assays, as observed for the eight

260

O157:H7 strains.

261 262

Vero cytotoxicity assay on induced Stx. Stx production was induced by stress and nutrient depletion, and the toxin activity was further monitored by a fluorescent Vero cell-based



263

assay. The nutrient depletion method results in induction of the phage lytic cycle with

264

concomitant Stx expression as was previously shown with this fluorescent Vero cytotoxicity

265

assay.23, 24

266

(N=6) by using this Vero cell line that expresses an unstable version of the enhanced green

267

fluorescent protein, which is used as a marker to measure the inhibition of protein synthesis by

268

Stx.23 As shown in Table 1, the inhibition of protein synthesis with this fluorescent Vero cell-

269

based assay was reported as a value of 100, indicating no reduction of protein

270

synthesis/cytotoxicity, while a value of 0 indicates high cytotoxicity. This is a very specific and

271

sensitive way of measuring the cytotoxicity of Stx produced by a strain. The limit of detection

272

for a Vero cell-based assay is at most 0.005 ng/ml.27 For 37 of the 45 samples, the results

273

indicated a significant amount of toxin was present in the cell-free culture supernatant at the

274

tested 10-fold and 100-fold dilutions (values much less than 100) (Table 1). Among the eight

275

supernatants collected from the strains with the IS-interrupted stx2c operon, only five culture

276

supernatants from strains RM10645, RM10646, RM10649, RM10716, and RM10718 were

277

found to still produce very low levels of active Stx at the 10-fold dilution. Other researchers

278

have shown in experimental systems that ISs can relocate from a stx operon and thereby convert

279

a non-Stx producing into a Stx producing strain.38, 50

280

The supernatants, after removal of the bacteria and sterile filtration, were assayed

Mass spectrometry-based quantitation of Stx production. An MRM mass

281

spectrometry method was used to quantitate the amount of Stx produced by 37 of the 45

282

STEC.27, 46 This method is based on the detection of characteristic peptides derived from the

283

tryptic digestion of the five identical B subunits present in the AB5 holotoxin and has a limit of

284

detection below 100 attomoles (10-18 moles). A schematic representation of the MRM method is

285

shown (Supporting Information, Figure S-2). The mass settings for the Q1 and Q3 mass filters


Page 14 of 34

Page 15 of 34


286

are listed (Supporting Information, Figure S-3). Of the five possible analyte peptides, only

287

YNEDDTFTVK and YNENDTFTVK were detected (Figure 2). Of the two peptides used to

288

discriminate among the Stx2 subtypes, EYWTSR and EYWTNR, only EYWTSR was present.

289

This indicated that only Stx2a, or Stx2c or Stx2d could be present. The lack of stx2d sequence

290

meant that only Stx2a or Stx2c could be present in the samples. For a sample to be a true Stx2a

291

or Stx2c positive, each sample must show a signal for both the b2 and y8 ions of the analyte

292

peptide for three replicate injections (Figures 3 and 4) and show a signal consistent with the

293

EYWTSR peptide (Supporting Information, Figures S-4 and S-5). The three negative controls

294

showed no signals above background for any of the five analyte peptides, EYWTNR, or

295

EYWTSR (Supporting Information Figure S-6). Only Stx2a or Stx2c toxins were detected and

296

only one was detected for each strain (Table 1). The results using the mass spectrometry method

297

were consistent with the stx operon sequencing and the Vero cell assay results. This consistency

298

is not always observed for environmental strains, since the detection of the stx gene subtype does

299

not imply that Stx will be expressed.51

300

15N-labeled

analogs of the two analyte peptides were used to quantitate the amount of

301

toxin present in each sample. Calibration curves were prepared to empirically relate a fixed

302

amount of an15N-labeled analyte peptide (YNEDDTFTVK or YNENDTFTVK) to a variable

303

amount of the natural abundance (14N) peptide (Supporting Information, Figures S-7, S-8, and S-

304

9). These calibration curves were linear with excellent correlation coefficients (> 0.999) and

305

were used to quantitate the amount of toxin present in each sample. The amounts of toxin

306

detected (N=3 for each sample) varied from between 1.4 ± 0.3 and 49 ± 8 ng/ml (Table 1,

307

Figures 3 and Figures 4). Using the MRM mass spectrometry method, the amount produced by

308

the depletion of nutrients method is less than that observed when Stx expression is induced by



309

the antibiotic mitomycin C.27, 46 The Stx2a amounts detected in the tested STEC strains varied

310

from 1.4 ± 0.3 to 49 ± 8 ng/ml, while the detection of Stx2c varied between 5.6 ± 0.5 and 39 ± 2

311

ng/ml (Table 1). The six strains of O113:H21 infected with two Stx2a producing phages

312

generated between 12 ± 3 and 49 ± 8 ng/ml, resulting in higher levels of Stx detected (Table 1).

313

These results demonstrate the sensitivity of this MRM method to discriminate and quantitate the

314

Stx produced by environmental STEC strains which have been induced to produce Stx by

315

nutrient depletion.

316

Analysis of strains with insertion sequences in the stx operon. Of the eight strains

317

possessing the IS-inserted stx2c operon, six produced detectable amounts of B subunits, as

318

determined by MRM-based quantitation. The amounts were considerably lower (0.5 ± 0.3 to 1.9

319

± 0.1 ng/ml) than what was observed for the Stx2c-producing strain RM10024 at 5.7 ± 0.8 ng/ml.

320

When these strains were probed with ELISA or immunoblot, using antibodies against the A

321

subunits, the results were negative (data not shown). Similar results have been seen in other

322

studies with IS-inserted into stx operons.34 Given that only truncated forms of the A subunit are

323

produced, they are not detected by the A subunit specific antibodies. In the present study, the IS

324

did not prevent expression of the B subunits in the O157:H7 strains (Table 1), and these

325

observations were in agreement with a recent report that employed a top-down/middle-down

326

mass spectrometry method for detecting Stx expression in STEC strains.52 Reports have

327

documented that B-subunits have their own promoter,53 and in the examined STEC strains, the B

328

subunit promoter seemed to be weaker than the promoter for the A subunit since the expression

329

levels are much lower than that observed for an intact stx operon.

330 331

Detecting a conserved decapeptide containing the active site of the Shiga toxin A subunit. A decapeptide, FVTVTAEALR, was predicted to be produced by trypsin cleavage of


Page 16 of 34

Page 17 of 34


332

the A subunit of Stx. The peptide contains the highly conserved active site of the toxic enzyme,

333

and is common to both Stx2a and Stx2c.15 An analysis of the sequences of the stx genes from the

334

37 strains used in this study (those without IS) showed they all possess the FVTVTAEALR

335

peptide (Supporting Information Figures S-10, S-11, and S-12). Instrument parameters were

336

optimized for detection of the peptide (m/z = 553.1; z = 2). The signal intensity of the a2 ion

337

(m/z = 219.2; z = 1; FV) was comparable to that of the y8 ion (m/z = 860.5; z = 1; TVTAEALR),

338

and both were greater than that of the y6 ion (m/z = 660.4; z = 1; TAEALR). The ratio of the

339

signal intensities of the a2, y6, and y8 ions is 1:0.5:1 (Supporting Information Figure S-13).

340

The area ratio of the y8 ion of FVTVTAEALR to the y8 ion of YNEDDTFTVK from a digest of

341

the supernatant of a Stx2a producing STEC was determined to be 0.51 ± 0. 05 (N=12)

342

(Supporting Information Figure S-14). This indicates that the FVTVTAEALR peptide can be

343

used to detect the presence of A subunits in a Stx-containing sample provided there is a sufficient

344

amount of toxin present in the sample.

345

By including the FVTVTAEALR peptide in the MRM method, its detection can be used

346

to demonstrate the presence of an intact Stx. Once added, the method can be used to detect both

347

the presence of B subunits and the active site in the A subunit. Samples containing either Stx2a

348

(RM7807), Stx2c (RM8091), a negative control (RM1273), or an IS interrupted Stx2c

349

(RM10716) were analyzed using a MRM method including the FVTVTAEALR peptide. As

350

expected, the FVTVTAEALR peptide was detectable in the Stx2a and Stx2c samples, but not the

351

negative control or IS interrupted sample (Supporting Information Figure S-15). This will allow

352

researchers to distinguish between the expressed proteins of IS interrupted stx operon, either the

353

A subunit or the B subunit, and that of the intact stx operon, both A and B subunits. In principle,

354

it can detect the interruption of the B subunit gene by an IS since the A subunit would still be



355

produced. In this way, an intact holotoxin can be detected by observing the presence of the

356

FVTVTAEALR peptide and the appropriate analyte peptide from the B subunit. Interruption of

357

the B subunit would be observable by the presence of the FVTVTAEALR peptide and the

358

absence of the analyte peptide from the B subunit. Detecting the B subunits, but not an A

359

subunit, implies an IS disruption of the A subunit, provided the samples are concentrated enough

360

to permit the detection of the FVTVTAEALR signal.

361

In summary, the present study optimized the use of an MRM mass spectrometry method

362

to measure the Stx2a and Stx2c amounts produced by environmental STEC strains recovered

363

from relevant sources and locations in a major agricultural region in California. The findings

364

using the MRM mass spectrometry method correlated with the fluorescent Vero cytotoxicity

365

assay when examining STEC O113:H21 strains harboring two stx2a-encoding prophages,

366

resulting in the highest levels of Vero cytotoxicity and Stx2a production. Interestingly, the

367

MRM mass spectrometry method enabled the detection of the Stx2c B-subunit in STEC strains

368

that were found to be PCR-positive for the stx2c gene but still negative with the Vero cytotoxicity

369

assay due to presence of the IS1203v insertion sequence in the Stx2c A-subunit coding sequence.

370

Future studies are aimed at the combinatorial use of proteomics and genomics to assess the

371

induction of multiple stx-encoding prophages in a single host strain as well as the development

372

of quantitative detection methods to determine the prevalence of STEC phages isolated from

373

agricultural environments.

374 375 376 377

SUPPORTING INFORMATION Extra experimental details, tables of gene polymorphisms, cartoon of the MRM method, table of instrument parameters, signal intensity graphs, calibration curves, protein sequences,


Page 18 of 34

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tables protein polymorphisms, and signal intensity graphs of active site peptides This material is

379

available free of charge via the Internet at http://pubs.acs.org.



380

REFERENCES

381

(1) Law, D. Virulence factors of Escherichia coli O157 and other Shiga toxin-producing E. coli.

382

J. Appl. Microbiol. 2000, 88, 729-745.

383

(2) Frenzen, P. D.; Drake, A.; Angulo, F. J. Economic cost of illness due to Escherichia coli O157

384

infections in the United States. J. Food Prot. 2005, 68, 2623-2630.

385

(3) Hoffmann, S.; Maculloch, B.; Batz, M. Economic burden of major foodborne illnesses

386

acquired in the United States, EIB-140. In U.S. Department of Agriculture, Economic Research

387

Service, 2015.

388

(4) Scallan, E.; Hoekstra, R. M.; Angulo, F. J.; Tauxe, R. V.; Widdowson, M. A.; Roy, S. L.;

389

Jones, J. L.; Griffin, P. M. Foodborne illness acquired in the United States--major pathogens.

390

Emerg. Infect. Dis. 2011, 17, 7-15.

391

(5) Arnade, C.; Calvin, L.; Kuchler, F. Consumer response to a food safety shock: The 2006 food-

392

borne illness outbreak of E. coli O157:H7 linked to spinach. Rev. Agric. Econ. 2009, 31, 734-750.

393

(6) Heiman, K. E.; Mody, R. K.; Johnson, S. D.; Griffin, P. M.; Gould, L. H. Escherichia coli

394

O157 outbreaks in the United States, 2003-2012. Emerg. Infect. Dis. 2015, 21, 1293-1301.

395

(7) Rangel, J. M.; Sparling, P. H.; Crowe, C.; Griffin, P. M.; Swerdlow, D. L. Epidemiology of

396

Escherichia coli O157:H7 outbreaks, United States, 1982-2002. Emerg. Infect. Dis. 2005, 11, 603-

397

609.

398

(8) Bell, B. P.; Goldoft, M.; Griffin, P. M.; Davis, M. A.; Gordon, D. C.; Tarr, P. I.; Bartleson, C.

399

A.; Lewis, J. H.; Barrett, T. J.; Wells, J. G.; et al. A multistate outbreak of Escherichia coli

400

O157:H7-associated bloody diarrhea and hemolytic uremic syndrome from hamburgers. The

401

Washington experience. JAMA 1994, 272, 1349-1353.


Page 20 of 34

Page 21 of 34


402

(9) Michino, H.; Araki, K.; Minami, S.; Takaya, S.; Sakai, N.; Miyazaki, M.; Ono, A.; Yanagawa,

403

H. Massive outbreak of Escherichia coli O157:H7 infection in schoolchildren in Sakai City, Japan,

404

associated with consumption of white radish sprouts. Am. J. Epidemiol. 1999, 150, 787-796.

405

(10) Frank, C.; Faber, M. S.; Askar, M.; Bernard, H.; Fruth, A.; Gilsdorf, A.; Hohle, M.; Karch,

406

H.; Krause, G.; Prager, R.; Spode, A.; Stark, K.; Werber, D. Large and ongoing outbreak of

407

haemolytic uraemic syndrome, Germany, May 2011. Euro. Surveill. 2011, 16.

408

(11) Buchholz, U.; Bernard, H.; Werber, D.; Bohmer, M. M.; Remschmidt, C.; Wilking, H.;

409

Delere, Y.; an der Heiden, M.; Adlhoch, C.; Dreesman, J.; Ehlers, J.; Ethelberg, S.; Faber, M.;

410

Frank, C.; Fricke, G.; Greiner, M.; Hohle, M.; Ivarsson, S.; Jark, U.; Kirchner, M.; Koch, J.;

411

Krause, G.; Luber, P.; Rosner, B.; Stark, K.; Kuhne, M. German outbreak of Escherichia coli

412

O104:H4 associated with sprouts. N. Engl. J. Med. 2011, 365, 1763-1770.

413

(12) Centers for Disease Control and Prevention (CDC) Ongoing multistate outbreak of

414

Escherichia coli serotype O157:H7 infections associated with consumption of fresh spinach--

415

United States, September 2006. MMWR Morb. Mortal. Wkly. Rep. 2006, 55, 1045-1046.

416

(13)

417

(http://wwwn.cdc.gov/foodborneoutbreaks/).

418

(14) Bergan, J.; Dyve Lingelem, A. B.; Simm, R.; Skotland, T.; Sandvig, K. Shiga toxins. Toxicon

419

2012, 60, 1085-1107.

420

(15) Scheutz, F.; Teel, L. D.; Beutin, L.; Pierard, D.; Buvens, G.; Karch, H.; Mellmann, A.;

421

Caprioli, A.; Tozzoli, R.; Morabito, S.; Strockbine, N. A.; Melton-Celsa, A. R.; Sanchez, M.;

422

Persson, S.; O'Brien, A. D. Multicenter evaluation of a sequence-based protocol for subtyping

423

Shiga toxins and standardizing Stx nomenclature. J. Clin. Microbiol. 2012, 50, 2951-2963.

FOODTool

The

foodborne

outbreak

online


database

(FOOD

Tool)


424

(16) Bai, X.; Fu, S.; Zhang, J.; Fan, R.; Xu, Y.; Sun, H.; He, X.; Xu, J.; Xiong, Y. Identification

425

and pathogenomic analysis of an Escherichia coli strain producing a novel Shiga toxin 2 subtype.

426

Sci. Rep. 2018, 8, 6756.

427

(17) Friedrich, A. W.; Bielaszewska, M.; Zhang, W. L.; Pulz, M.; Kuczius, T.; Ammon, A.; Karch,

428

H. Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical

429

symptoms. J. Infect. Dis. 2002, 185, 74-84.

430

(18) Sonntag, A. K.; Bielaszewska, M.; Mellmann, A.; Dierksen, N.; Schierack, P.; Wieler, L. H.;

431

Schmidt, M. A.; Karch, H. Shiga toxin 2e-producing Escherichia coli isolates from humans and

432

pigs differ in their virulence profiles and interactions with intestinal epithelial cells. Appl. Environ.

433

Microbiol. 2005, 71, 8855-8863.

434

(19) Bielaszewska, M.; Friedrich, A. W.; Aldick, T.; Schurk-Bulgrin, R.; Karch, H. Shiga toxin

435

activatable by intestinal mucus in Escherichia coli isolated from humans: predictor for a severe

436

clinical outcome. Clin. Infect. Dis. 2006, 43, 1160-1167.

437

(20) Persson, S.; Olsen, K. E.; Ethelberg, S.; Scheutz, F. Subtyping method for Escherichia coli

438

Shiga toxin (verocytotoxin) 2 variants and correlations to clinical manifestations. J. Clin.

439

Microbiol. 2007, 45, 2020-2024.

440

(21) Silva, C. J.; Brandon, D. L.; Skinner, C. B.; He, X., Shiga toxins: A Review of Structure,

441

Mechanism, and Detection. Springer International Publishing: Cham Switzerland, 2017; p 118.

442

(22) Silva, C. J. Food forensics: Using mass spectrometry to detect foodborne protein

443

contaminants, as exemplified by Shiga toxin variants and prion strains. J. Agric. Food Chem. 2018,

444

66, 8435-8450.


Page 22 of 34

Page 23 of 34


445

(23) Quiñones, B.; Massey, S.; Friedman, M.; Swimley, M. S.; Teter, K. Novel cell-based method

446

to detect Shiga toxin 2 from Escherichia coli O157:H7 and inhibitors of toxin activity. Appl.

447

Environ. Microbiol. 2009, 75, 1410-1416.

448

(24) Quiñones, B.; Swimley, M. S. Use of a Vero cell-based fluorescent assay to assess relative

449

toxicities of Shiga toxin 2 subtypes from Escherichia coli. Methods Mol. Biol. 2011, 739, 61-71.

450

(25) He, X.; Kong, Q.; Patfield, S.; Skinner, C.; Rasooly, R. A new immunoassay for detecting

451

all subtypes of Shiga toxins produced by Shiga toxin-producing E. coli in ground beef. PLoS ONE

452

2016, 11, e0148092.

453

(26) Fagerquist, C. K.; Zaragoza, W. J.; Sultan, O.; Woo, N.; Quiñones, B.; Cooley, M. B.;

454

Mandrell, R. E. Top-down proteomic identification of Shiga toxin 2 subtypes from Shiga toxin-

455

producing Escherichia coli by matrix-assisted laser desorption ionization-tandem time of flight

456

mass spectrometry. Appl. Environ. Microbiol. 2014, 80, 2928-2940.

457

(27) Silva, C. J.; Erickson-Beltran, M. L.; Skinner, C. B.; Dynin, I.; Hui, C.; Patfield, S. A.; Carter,

458

J. M.; He, X. Safe and effective means of detecting and quantitating Shiga-like toxins in attomole

459

amounts. Anal. Chem. 2014, 86, 4698-4706.

460

(28) O'Brien, A. D.; Newland, J. W.; Miller, S. F.; Holmes, R. K.; Smith, H. W.; Formal, S. B.

461

Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or

462

infantile diarrhea. Science 1984, 226, 694-696.

463

(29) Scotland, S. M.; Smith, H. R.; Willshaw, G. A.; Rowe, B. Vero cytotoxin production in strain

464

of Escherichia coli is determined by genes carried on bacteriophage. Lancet 1983, 2, 216.

465

(30) Smith, H. W.; Green, P.; Parsell, Z. Vero cell toxins in Escherichia coli and related bacteria:

466

transfer by phage and conjugation and toxic action in laboratory animals, chickens and pigs. J.

467

Gen. Microbiol. 1983, 129, 3121-3137.



468

(31) Tyler, J. S.; Livny, J.; Friedman, D. I., Lambdoid Phages and Shiga Toxin. In Phages: Their

469

role in Pathogenesis and Biotechnology., Waldor, M. K.; Friedman, D. I.; Adhya, S. L., Eds. ASM

470

Press: Washington, D. C., 2005; pp 131-164.

471

(32) Ding, H.; Huang, L.; Mao, X.; Zou, Q. Characterization of stx2 and its variants in Escherichia

472

coli O157:H7 isolated from patients and animals. Afr. J. Biotechnol. 2011, 10, 2991-2998.

473

(33) Harada, T.; Hirai, Y.; Itou, T.; Hayashida, M.; Seto, K.; Taguchi, M.; Kumeda, Y. Laboratory

474

investigation of an Escherichia coli O157: H7 strain possessing a vtx2c gene with an IS1203

475

variant insertion sequence isolated from an asymptomatic food handler in Japan. Diagn. Microbiol.

476

Infect. Dis. 2013, 77, 176-178.

477

(34) Jinneman, K. C.; Weagant, S. D.; Johnson, J. M.; Abbott, S. L.; Hill, W. E.; Tenge, B. J.;

478

Dang, N.-L.; Ramsden, R.; Omiecinski, C. J. A large insertion in the Shiga-like toxin 2 gene (stx)

479

of an 2 Escherichia coli O157:H7 clinical isolate. Int. J. Food. Microbiol. 2000, 57 115–124.

480

(35) Kusumoto, M.; Nishiya, Y.; Kawamura, Y.; Shinagawa, K. Identification of an insertion

481

sequence, IS1203 variant, in a Shiga toxin 2 gene of Escherichia coli O157:H7. J. Biosci. Bioeng.

482

1999, 87, 93-96.

483

(36) Park, D.; Stanton, E.; Ciezki, K.; Parrell, D.; Bozile, M.; Pike, D.; Forst, S. A.; Jeong, K. C.;

484

Ivanek, R.; Döpfer, D.; Kaspar, C. W. Evolution of the stx2-encoding prophage in persistent bovine

485

Escherichia coli O157: H7 strains. Appl. Environ. Microbiol. 2013, 79, 1563-1572.

486

(37) Vasant, B. R.; Stafford, R. J.; Jennison, A. V.; Bennett, S. M.; Bell, R. J.; Doyle, C. J.; Young,

487

J. R.; Vlack, S. A.; Titmus, P.; Saadi, D. E.; Jarvinen, K. A. J.; Coward, P.; Barrett, J.; Staples, M.;

488

Graham, R. M. A.; Smith, H. V.; Lambert, S. B. Mild illness during outbreak of Shiga toxin–

489

producing Escherichia coli O157 infections associated with agricultural show, Australia. Emerg.

490

Infect. Dis. 2017, 23, 1686-1689.


Page 24 of 34

Page 25 of 34


491

(38) Kusumoto, M.; Ooka, T.; Nishiya, Y.; Ogura, Y.; Saito, T.; Sekine, Y.; Iwata, T.; Akiba, M.;

492

Hayashi, T. Insertion sequence-excision enhancer removes transposable elements from bacterial

493

genomes and induces various genomic deletions. Nat. Commun. 2011, 2, 152.

494

(39) Cooley, M. B.; Jay-Russell, M.; Atwill, E. R.; Carychao, D.; Nguyen, K.; Quiñones, B.; Patel,

495

R.; Walker, S.; Swimley, M.; Pierre-Jerome, E.; Gordus, A. G.; Mandrell, R. E. Development of a

496

robust method for isolation of Shiga toxin-positive Escherichia coli (STEC) from fecal, plant, soil

497

and water samples from a leafy greens production region in California. PLoS ONE 2013, 8.

498

(40) Bugarel, M.; Beutin, L.; Martin, A.; Gill, A.; Fach, P. Micro-array for the identification of

499

Shiga toxin-producing Escherichia coli (STEC) seropathotypes associated with hemorrhagic

500

colitis and hemolytic uremic syndrome in humans. Int. J. Food. Microbiol. 2010, 142, 318-329.

501

(41) Quiñones, B.; Swimley, M. S.; Narm, K. E.; Patel, R. N.; Cooley, M. B.; Mandrell, R. E. O-

502

antigen and virulence profiling of Shiga toxin-producing Escherichia coli by a rapid and cost-

503

effective DNA microarray colorimetric method. Front. Cell. Infect. Microbiol. 2012, 2, 61.

504

(42) Amézquita-López, B. A.; Quiñones, B.; Lee, B. G.; Chaidez, C. Virulence profiling of Shiga

505

toxin-producing Escherichia coli recovered from domestic farm animals in Northwestern Mexico.

506

Front. Cell. Infect. Microbiol. 2014, 4.

507

(43) Zheng, J.; Cui, S.; Teel, L. D.; Zhao, S.; Singh, R.; O'Brien, A. D.; Meng, J. Identification

508

and characterization of Shiga toxin type 2 variants in Escherichia coli isolates from animals, food,

509

and humans. Appl. Environ. Microbiol. 2008, 74, 5645-5652.

510

(44) Quiñones, B.; Yambao, J. C.; Lee, B. G. Draft genome sequences of Escherichia coli O113:

511

H21 strains recovered from a major produce production region in California. Genome Announc.

512

2017, 5.



513

(45) Cherubin, P.; Quiñones, B.; Elkahoui, S.; Yokoyama, W.; Teter, K. A cell-based fluorescent

514

assay to detect the activity of AB toxins that inhibit protein synthesis. In Methods Mol. Biol., 2017;

515

Vol. 1600, pp 25-36.

516

(46) Silva, C. J.; Erickson-Beltran, M. L.; Skinner, C. B.; Patfield, S. A.; He, X. Mass

517

spectrometry-based method of detecting and distinguishing type 1 and type 2 Shiga-like toxins in

518

human serum. Toxins (Basel) 2015, 7, 5236-5253.

519

(47) Bettelheim, K. A. The non-O157 Shiga-toxigenic (Verocytotoxigenic) Escherichia coli;

520

under-rated pathogens. Crit. Rev. Microbiol. 2007, 33, 67-87.

521

(48) Beutin, L.; Krause, G.; Zimmermann, S.; Kaulfuss, S.; Gleier, K. Characterization of Shiga

522

toxin-producing Escherichia coli strains isolated from human patients in Germany over a 3-year

523

period. J. Clin. Microbiol. 2004, 42, 1099-1108.

524

(49) Brooks, J. T.; Sowers, E. G.; Wells, J. G.; Greene, K. D.; Griffin, P. M.; Hoekstra, R. M.;

525

Strockbine, N. A. Non-O157 Shiga toxin-producing Escherichia coli infections in the United

526

States, 1983-2002. J. Infect. Dis. 2005, 192, 1422-1429.

527

(50) Kusumoto, M.; Okitsu, T.; Nishiya, Y.; Suzuki, R.; Yamai, S.; Kawamura, Y. Spontaneous

528

reactivation of Shiga toxins in Escherichia coli O157:H7 cells caused by transposon excision. J.

529

Biosci. Bioeng. 2001, 92, 114-120.

530

(51) Martínez-Castillo, A.; Allué-Guardia, A.; Dahbi, G.; Blanco, J.; Creuzburg, K.; Schmidt, H.;

531

Muniesa, M. Type III effector genes and other virulence factors of Shiga toxin-encoding

532

Escherichia coli isolated from wastewater. Environ. Microbiol. Rep. 2012, 4, 147-155.

533

(52) Fagerquist, C. K.; Zaragoza, W. J.; Lee, B. G.; Yambao, J. C.; Quiñones, B. Clinically-

534

relevant Shiga toxin 2 subtypes from environmental Shiga toxin-producing Escherichia coli


Page 26 of 34

Page 27 of 34


535

identified by top-down/middle-down proteomics and DNA sequencing. Clin. Mass Spec. 2018, In

536

review.

537

(53) Habib, N. F.; Jackson, M. P. Identification of a B subunit gene promoter in the Shiga toxin

538

operon of Shigella dysenteriae 1. J. Bacteriol. 1992, 174, 6498-6507.

539



540

FIGURE CAPTIONS

541

Figure 1. Schematic of the stx2c operon (▄▄) containing the insertion sequence (▄▄▄▄) from

542

an E. coli O157:H7 strain.

543 544

Figure 2. Alignment of the expressed protein sequences from the B subunits of Shiga toxin

545

from Shigella dysentariae type 1 (Stx*) and Stx1a,c,d, Stx2a,f, Stx2b,c,d, Stx2e, and Stx2g.15

546

The five distinct analyte peptides (YNDDDTFTVK, YNEDDTFTVK, YNENDTFTVK,

547

YNEDNTFTVK, and YNGDNTFTVK) are indicated in bold and underlined. The peptides

548

used to distinguish among the Stx2 subtypes (EYWTNR and EYWTSR) are indicated in bold

549

only.

550 551

Figure 3. Chromatograms showing the signal intensity of the analyte peptide

552

(YNEDDTFTVK) from the Stx2a present in the sample. The Q1 (m/z) parameter is set at 616.3

553

and the Q3 parameter (m/z) cycles between a m/z value of 278.1 (b2 ion; YN) and 954.4 (y8 ion;

554

EDDTFTVK). The shaded region indicates the chromatographic retention time of the internal

555

standard (15N-labeled YNEDDTFTVK).

556 557

Figure 4. Chromatograms showing the signal intensity of the analyte peptide

558

(YNENDTFTVK) from the Stx2c present in the sample. The Q1 (m/z) parameter is set at 615.8

559

and the Q3 parameter (m/z) cycles between a m/z value of 278.2 (b2 ion; YN) and 953.4 (y8 ion;

560

ENDTFTVK). The shaded region indicates the chromatographic retention time of the internal

561

standard (15N-labeled YNENDTFTVK).

562


Page 28 of 34

Page 29 of 34


TABLES Table 1. Characterization of Stx Expression by Mass Spectrometry and Vero Cell Assay in the Tested STEC Strains, Recovered from Agricultural Regions in California.39 Strain

Fecal sample Source

Serotype

Mass spectrometry assay (ng/ml)

stx genotype

GenBank accession number for the stx nucleotide sequence

Vero assay (1:10)

Vero assay (1:100)

RM7405

CR-10B

Deer

O6:H34

stx 2c

Stx1a,c,d ---

RM7406

CR-10B

Deer

O6:H34

stx 2c

---

RM7787

SP-0104

Pig

O185:H7

stx 2c

---

---

---

27 ± 1

---

---

---

MH822843

30.3 ± 10.9

56.7 ± 9.1

RM7788

W-0100

Water

O113:H21

stx 2a

---

38 ± 2

---

---

---

---

---

MH822844,MH822845

6.8 ± 2.3

12.6 ± 1.4

RM7804

P-0794

Lettuce

O185:H7

stx 2c

---

---

---

6±2

---

---

---

MH780102

17.7 ± 6.8

41 ± 7.9

RM7805

F-0281

Pig

O185:H7

stx 2c

---

---

---

25 ± 1

---

---

---

MH780103

10.5 ± 6.3

38.9 ± 6.9

RM7806

F-0281

Pig

O113:H21

stx 2a

---

41 ± 6

---

---

---

---

---

MH822846,MH822847

7.2 ± 2.1

11.9 ± 2.6

RM7807

F-0280

Pig

O113:H21

stx 2a

---

49 ± 8

---

---

---

---

---

MH822848,MH822849

5.8 ± 1.4

9.1 ± 1.6

RM7811

F-0282

Pig

O185:H7

stx 2c

---

---

---

14 ± 2

---

---

---

MH780104

46 ± 4.6

77.2 ± 9.5

RM7812

S-0881

Soil

O6:H34

stx 2c

---

---

---

11 ± 2

---

---

---

MH780105

51 ± 11.6

59.5 ± 7.6

RM8091

F-0525

Cattle

O185:H7

stx 2c

---

---

---

39 ± 2

---

---

---

10 ± 0

42.3 ± 7.0

RM9244

F-1200

Cattle

O113:H21

stx 2a

---

5.3 ± 0.8

---

---

---

---

---

MH822850 MH780124, MH780125

7.8 ± 1.3

11.5 ± 1.7

RM9245

F-1222

Cattle

O113:H21

stx 2a

---

13 ± 2

---

---

---

---

---

MH780126, MH780127

5.3 ± 2.9

9 ± 4.8

RM9246

F-1240

Cattle

O113:H21

stx 2a

---

12 ± 3

---

---

---

---

---

MH780128, MH780129

14.6 ± 3.8

18.4 ± 5.3

RM9482

F-1510

Cattle

O185:H7

stx 2c

---

---

---

15 ± 0.3

---

---

---

MH822851

23.5 ± 10.4

39.2 ± 8.1

RM9483

F-1510

Cattle

O185:H7

stx 2c

---

---

---

12 ± 1

---

---

---

MH822852

14.7 ± 7

43.2 ± 7.3

RM9880

F-1391

Cattle

O157:H7

stx 2a

---

5.7 ± 0.4

---

---

---

---

---

MH780106

15.2 ± 3

46.5 ± 2.6

RM9885

F-1398

Cattle

O157:H7

stx 2a

---

6.6 ± 0.8

---

---

---

---

---

MH780107

29.2 ± 3

45.9 ± 5.1

RM9888

F-1404

Cattle

O157:H7

stx 2a

---

12 ± 0.6

---

---

---

---

---

MH780108

30.1 ± 4.7

37.5 ± 5.4

RM9898

F-1493

Cattle

O177:H25

stx 2c

---

---

---

31 ± 4

---

---

---

MH780109

38.1 ± 5.8

49.3 ± 5.9

RM9908

S-1718

Soil

O157:H7

stx 2a

---

5 ± 0.3

---

---

---

---

---

MH780110

13.9 ± 4.1

41.2 ± 2.6

RM9982

W-0579

Water

O121:H19

stx 2a

---

7.2 ± 0.8

---

---

---

---

---

MH780111

24.6 ± 4.6

45.3 ± 3.5

RM10024

F-1462

Cattle

O157:H7

stx 2c

---

---

---

5.7 ± 0.8

---

---

---

MH780112

49.5 ± 4.9

73.5 ± 3.3

RM10046

F-1588

Cattle

O121:H19

stx 2a

---

8.1 ± 0.5

---

---

---

---

---

MH780113

21.2 ± 3.8

42.4 ± 4.5

RM10056

S-1760

Soil

O157:H7

stx 2a

---

5.5 ± 0.7

---

---

---

---

---

MH780114

22.9 ± 3.7

38.2 ± 9.3

RM10058

SBB-0124

Cowbird

O157:H7

stx 2c

---

---

---

14 ± 0.7

---

---

---

29.6 ± 11.1

40.8 ± 10.3

RM10068

W-0579

Water

O121:H19

stx 2a

---

5.4 ± 0.3

---

---

---

---

---

MH822853 MH780115

21.4 ± 7.3

30.4 ± 9.0

RM10361

F-1691

Cattle

O121:H19

stx 2a

---

11 ± 0.3

---

---

---

---

---

MH780116

22.8 ± 4.5

33.7 ± 6.6

RM10638

F-1848

Cattle

O157:H7

stx 2a

---

3.5± 0.5

---

---

---

---

---

26.3 ± 4.4

57.2 ± 2.9

RM10641

F-1858

Cattle

O157:H7

stx 2c

---

---

---

0.5 ± 0.3

---

---

---

MH822854 MH780130

100 ± 0

100 ± 0

RM10642

F-1859

Cattle

O157:H7

stx 2a

---

8.8 ± 0.4

---

---

---

---

---

MH780117

3.5 ± 10.1

18.1 ± 5.9

RM10645

F-1884

Cattle

O157:H7

stx 2c

---

---

---

0.5 ± 0.3

---

---

---

MH780131

92.9 ± 8.5

100 ± 0

RM10646

F-1884

Cattle

O157:H7

stx 2c

---

---

---

---

---

---

---

MH780132

96.9 ± 7.4

100 ± 0

RM10649

F-1900

Cattle

O157:H7

stx 2c

---

---

---

0.7 ± 0.2

---

---

---

MH780133

95 ± 5.3

97.8 ± 4

RM10668

SP-0197

Feral Pig

O157:H7

stx 2a

---

6.6 ± 0.3

---

---

---

---

---

MH780118

15 ± 3.4

27.9 ± 5.5

RM10716

F-1858

Cattle

O157:H7

stx 2c

---

---

---

1.9 ± 0.1

---

---

---

MH780134

76.6 ± 3.6

100 ± 0.

RM10717

F-1859

Cattle

O157:H7

stx 2a

---

7.3 ± 0.3

---

---

---

---

---

MH780119

9 ± 3.4

18.1 ± 14.7

RM10718

F-1884

Cattle

O157:H7

stx 2c

---

---

---

---

---

---

---

MH780135

80 ± 9.1

100 ± 0

RM10719

F-1884

Cattle

O157:H7

stx 2c

---

---

---

1.6 ± 0.6

---

---

---

MH780136

100 ± 0

100 ± 0

RM10720

F-1887

Cattle

O157:H7

stx 2c

---

---

---

0.5 ± 0.1

---

---

---

MH780137

100 ± 0

100 ± 0

RM10736

SCR-0089

Crow

O185:H7

stx 2c

---

---

---

5.6 ± 0.5

---

---

---

MH780120

19.4 ± 10.6

26.4 ± 11.5

RM10786

F-2065

Cattle

O177:H25

stx 2c

---

---

---

18 ± 1

---

---

---

MH780121

16.7 ± 12.2

28.9 ± 11

RM10806

MA0409

Feral Pig

O157:H7

stx 2a

---

3.6 ± 0.3

---

---

---

---

---

MH780122

2.2 ± 0

8.6 ± 1.2

RM10936

F-2004

Cattle

O185:H7

stx 2c

---

---

---

8.8 ± 1

---

---

---

29.6 ± 6.3

40.9 ± 4.5

RM10940

F-2024

Cattle

O113:H21

stx 2a

---

1.4 ± 0.3

---

---

---

---

---

MH822857 MH780123

9.7 ± 3.1

29.1 ± 5.8

Meat

O157:H7

stx 2a

---

28 ± 4

---

---

---

---

---

AF461165

Human Human Human

O157:H7 O157:H7 NA

stxstxstx-

-------

-------

-------

-------

-------

-------

-------

NA NA NA

11.4 ± 1.2 90.9 ± 2.1 81.4 ± 2.3 92.3 ± 2.7

24.4 ± 2.0 99 ± 3.6 99.1 ± 2.3 100 ± 1.6

a

b

Stx2a ---

Stx2b ---

Stx2c 11 ± 0.9

Stx2e ---

Stx2f ---

Stx2g ---

MH780100

23.9 ± 10.2

47 ± 8.4

---

---

13 ± 4

---

---

---

MH780101

29.6 ± 7.7

51.2 ± 9.5

RM2084 RM1273 RM4876 RM5034

NA NA NA NA

aOther

strain name designations: strain RM2084 (EDL-933, DEC 4f); strain RM1273 (ATCC

43888); strain RM5034 (K12, ATCC 29425). bNA,

Not applicable.



Page 30 of 34

FIGURE 1

Transposase ORF Insertion sequence IS1203v

A subunit

A subunit


B subunit

Page 31 of 34


FIGURE 2 Stx*

TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR—

Stx1a

TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR--

Stx1c

APDCVTGNVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGRGFSEVIFR--

Stx1d

APDCVTGKVEYTKYNDDDTFTVKVADKELFTNRWNLQSLLLSAQITGMTVTIKTTACHNGGGFSEVIFR--

Stx2a

-ADCAKGKIEFSKYNEDDTFTVKVDGKEYWTSRWNLQPLLQSAQLTGMTVTIKSSTCESGSGFAEVQFNND

Stx2b

-ADCAKGKIEFSKYNENDTFTVKVAGKEYWTNRWNLQPLLQSAQLTGMTVTIKSNTCASGSGFAEVQFN--

Stx2c

-ADCAKGKIEFSKYNENDTFTVKVAGKEYWTSRWNLQPLLQSAQLTGMTVTIKSSTCESGSGFAEVQFNND

Stx2d

-ADCAKGKIEFSKYNENDTFTVKVAGKEYWTSRWNLQPLLQSAQLTGMTVTIKSSTCASGSGFAEVQFNND

Stx2e

-ADCAKGKIEFSKYNEDNTFTVKVSGREYWTNRWNLQPLLQSAQLTGMTVTIISNTCSSGSGFAQVKFN--

Stx2f

-ADCAVGKIEFSKYNEDDTFTVKVSGREYWTNRWNLQPLLQSAQLTGMTVTIISNTCSSGSGFAQVKFN--

Stx2g

-ADCAKGKIEFSKYNGDNTFTVKVDGKEYWTNRWNLQPLLQSAQLTGMTVTIKSNTCESGSGFAEVQFNND



Page 32 of 34

MRM intensity

FIGURE 3

50000

11000

5400

2600

25000

5500 y8

2700 y8

1300 y8

y8

b2

b2

b2

b2

0 10

15

RM7807

0 10

15

RM9245

0 9

0

14

RM10668

Retention time (minutes)


1200

9

14

RM10806

600

y8

b2

0 9

14

RM10940

Page 33 of 34


MRM intensity

FIGURE 4

7000

4500

2100

500

3500

2250

y8 1050

250

600

y8 y8

b2

0 10

15

RM8091

300

y8

y8

b2

0 10

15

RM9482

b2

0 9

14

b2

0 9

RM10024

Retention time (minutes)


14

RM10641

b2

0 9

14

RM10716

Shiga Toxin Analysis Results Agricultural Journal of Agricultural and Food Chemistry Page 34 of 34 Environments Gene sequencing

California

….GGTACTGTGCCTGTTACT…..

STEC Strain Isolation

Strain

Fecal sample Source

Serotype

stx genotype

RM7405

CR-10B

O6:H34

stx 2c

CR-10B SP-0104

RM7788

W-0100

RM7804

P-0794

RM7805

YN+ YNENDTFTVK+2 ENDTFTVK+

F-0281

Deer Deer Pig Water Lettuce Pig

O6:H34

stx 2c

O185:H7

stx 2c

---

38 ± 2

stx 2c

---

Stx2b ---------

Stx2c

Stx2e

11 ± 0.9 --13 ± 4 27. ± 1 ---

-------

Genbank accession No. Stx2f

---------

Vero assay (1:10)

Vero assay (1:100)

Stx2g ---------

MH780100

23.9 ± 10.2 47 ± 8.4

MH780101

29.6 ± 7.7

MH822843

30.3 ± 10.9 56.7 ± 9.1

MH822844,MH822845

6.8 ± 2.3

---

---

6±2

---

---

---

MH780102

17.7 ± 6.8

---

---

25 ± 1

---

---

---

MH780103

10.5 ± 6.3

51.2 ± 9.5 12.6 ± 1.4 41 ± 7.9 38.9 ± 6.9

stx 2a

---

41 ± 6

---

---

---

---

---

MH822846,MH822847

7.2 ± 2.1

11.9 ± 2.6

stx 2a

---

49 ± 8

---

---

---

---

---

MH822848,MH822849

5.8 ± 1.4

9.1 ± 1.6

RM7811

F-0282

Pig

O185:H7

stx 2c

---

---

---

14 ± 2

---

---

---

MH780104

RM7812

S-0881

Soil

O6:H34

stx 2c

---

---

---

11 ± 2

---

---

---

MH780105

51 ± 11.6

59.5 ± 7.6

MH822850 MH780124, MH780125

10 ± 0 7.8 ± 1.3

42.3 ± 7.0 11.5 ± 1.7

MH780126, MH780127

5.3 ± 2.9

9 ± 4.8

F-0525

Cattle

O185:H7

stx 2c

---

---

RM9244

F-1200

Cattle

O113:H21

stx 2a

---

12 ± 3

RM9245

F-1222

Cattle

O113:H21

stx 2a

---

13 ± 2

RM9246

F-1240

Cattle

RM9482

F-1510

Cattle

RM9483

F-1510

Cattle

RM9880

F-1391

O113:H21

stx 2a

---

12 ± 3

---------

39 ± 2 -------

---------

---------

---------

MH780128, MH780129

46 ± 4.6

14.6 ± 3.8

77.2 ± 9.5

18.4 ± 5.3

O185:H7

stx 2c

---

---

---

15 ± 0.3 ---

---

---

MH822851

23.5 ± 10.4 39.2 ± 8.1

O185:H7

stx 2c

---

---

---

12 ± 1

---

---

MH822852

14.7 ± 7

stx 2a

---

43.2 ± 7.3

Cattle

O157:H7

5.7 ± 0.4 ---

---

---

---

---

MH780106

15.2 ± 3

46.5 ± 2.6

RM9885

F-1398

Cattle

O157:H7

stx 2a

---

6.6 ± 0.8 ---

---

---

---

---

MH780107

29.2 ± 3

45.9 ± 5.1

RM9888

F-1404

Cattle

O157:H7

stx 2a

---

12 ± 0.6 ---

---

---

---

---

MH780108

31 ± 4

---

---

---

MH780109

38.1 ± 5.8

---

---

---

---

MH780110

13.9 ± 4.1

---

RM9898

F-1493

Cattle

O177:H25

stx 2c

---

---

RM9908

S-1718

Soil

O157:H7

stx 2a

---

5 ± 0.3

RM9982

W-0579

Water

O121:H19

stx 2a

---

-----

stx 2c

---

---

5.7 ± 0.8 ---

---

---

MH780112

49.5 ± 4.9

stx 2a

---

8.1 ± 0.5 ---

---

---

---

---

MH780113

21.2 ± 3.8

5.5 ± 0.7 ---

---

---

---

---

MH780114

22.9 ± 3.7

---

14 ± 0.7 ---

MH822853

29.6 ± 11.1 40.8 ± 10.3

S-1760

Soil

O157:H7

stx 2a

---

SBB-0124

Cowbird O157:H7

stx 2c

---

RM10068

W-0579

Water

O121:H19

stx 2a

---

---

---

MH780111

37.5 ± 5.4

O157:H7 O121:H19

RM10056

---

49.3 ± 5.9

Cattle Cattle

RM10058

---

41.2 ± 2.6

---

F-1462 F-1588

---

---

30.1 ± 4.7

7.2 ± 0.8 ---

RM10024 RM10046

24.6 ± 4.6

45.3 ± 3.5 73.5 ± 3.3 42.4 ± 4.5 38.2 ± 9.3

---

5.4 ± 0.3 ---

---

---

---

---

MH780115

RM10361

F-1691

Cattle

O121:H19

stx 2a

---

11 ± 0.3 ---

---

---

---

---

MH780116

22.8 ± 4.5

33.7 ± 6.6

RM10638

F-1848

Cattle

O157:H7

stx 2a

---

8±2

---

---

---

---

MH822854

26.3 ± 4.4

57.2 ± 2.9

F-1858

Cattle

O157:H7

stx 2c

0.5 ± 0.3 ---

---

---

MH780130

100 ± 0

100 ± 0.0

F-1859

Cattle

O157:H7

stx 2a

---

8.8 ± 0.4 ---

---

---

---

---

MH780117

3.5 ± 10.1

18.1 ± 5.9

Cattle

O157:H7

stx 2c

---

---

0.5 ± 0.3 ---

---

---

MH780131

92.9 ± 8.5

100 ± 0

---

---

F-1884 F-1900

Cattle

SP-0197

Feral Pig O157:H7

RM10716

F-1858

RM10717

F-1859 F-1884

RM10719

F-1884

RM10720

Cattle

Cattle

Cattle Cattle

---

O157:H7

stx 2c

---

---

---

---

O157:H7

stx 2c

---

---

---

0.7 ± 0.2 -----

stx 2c

---

---

1.9 ± 0.1 ---

O157:H7

stx 2a

---

7.3 ± 0.3 ---

O157:H7

stx 2a

stx 2c

---

---

6.6 ± 0.3 ---

O157:H7

---

---

-----

---

MH780132

96.9 ± 7.4

100 ± 0

---

---

MH780133

95 ± 5.3

97.8 ± 4

---

---

MH780118

15 ± 3.4

27.9 ± 5.5

---

---

MH780134

76.6 ± 3.6

100 ± 0.

MH780119

9 ± 3.4

---

---

---

---

---

---

---

---

MH780135

80 ± 9.1

MH780136

100 ± 0

18.1 ± 14.7 100 ± 0

O157:H7

stx 2c

---

---

---

1.6 ± 0.6 ---

F-1887

Cattle

O157:H7

stx 2c

---

---

---

0.5 ± 0.1 ---

RM10736

SCR-0089

Crow

O185:H7

stx 2c

---

---

---

5.6 ± 0.5 ---

---

---

MH780120

19.4 ± 10.6 26.4 ± 11.5

RM10786

F-2065

Cattle

O177:H25

stx 2c

---

---

---

18 ± 1

---

---

MH780121

16.7 ± 12.2 28.9 ± 11

MA0409

Feral Pig O157:H7

RM10806

Cattle

---

30.4 ± 9.0

F-1884

RM10646

---

21.4 ± 7.3

RM10642

RM10649

---

---

RM10645

RM10718

Fluorescence

---

O113:H21 O113:H21

RM10668

Phase Contrast

stx 2c

Pig Pig

Vero cell assay ACS Paragon Plus Environment + Shiga toxin

Stx2a -------

stx 2a

O185:H7

F-0281 F-0280

RM10641

- Shiga toxin

-------

O113:H21 O185:H7

RM7806 RM7807

RM8091

Mass spectrometry

Mass spectrometry assay (ng/ml) Stx1a

RM7406 RM7787

stx 2a

---

---

---

-----

---

MH780137

100 ± 0

100 ± 0 100 ± 0

3.6 ± 0.3 ---

---

MH780122

2.2 ± 0

8.6 ± 1.2

RM10936

F-2004

Cattle

O185:H7

stx 2c

---

---

8.8 ± 1

---

---

---

MH822857

29.6 ± 6.3

40.9 ± 4.5

RM10940

F-2024

Cattle

O113:H21

stx 2a

---

1.4 ± 0.3 ---

---

---

---

---

MH780123

9.7 ± 3.1

29.1 ± 5.8

---

---

-----

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