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      Multiplex PCR Assays for the Detection of One Hundred and Thirty Seven Serogroups of Shiga Toxin-Producing Escherichia coli Associated With Cattle

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          Abstract

          Escherichia coli carrying prophage with genes that encode for Shiga toxins are categorized as Shiga toxin-producing E. coli (STEC) pathotype. Illnesses caused by STEC in humans, which are often foodborne, range from mild to bloody diarrhea with life-threatening complications of renal failure and hemolytic uremic syndrome and even death, particularly in children. As many as 158 of the total 187 serogroups of E. coli are known to carry Shiga toxin genes, which makes STEC a major pathotype of E. coli. Seven STEC serogroups, called top-7, which include O26, O45, O103, O111, O121, O145, and O157, are responsible for the majority of the STEC-associated human illnesses. The STEC serogroups, other than the top-7, called “non-top-7” have also been associated with human illnesses, more often as sporadic infections. Ruminants, particularly cattle, are principal reservoirs of STEC and harbor the organisms in the hindgut and shed in the feces, which serves as a major source of food and water contaminations. A number of studies have reported on the fecal prevalence of top-7 STEC in cattle feces. However, there is paucity of data on the prevalence of non-top-7 STEC serogroups in cattle feces, generally because of lack of validated detection methods. The objective of our study was to develop and validate 14 sets of multiplex PCR (mPCR) assays targeting serogroup-specific genes to detect 137 non-top-7 STEC serogroups previously reported to be present in cattle feces. Each assay included 7–12 serogroups and primers were designed to amplify the target genes with distinct amplicon sizes for each serogroup that can be readily identified within each assay. The assays were validated with 460 strains of known serogroups. The multiplex PCR assays designed in our study can be readily adapted by most laboratories for rapid identification of strains belonging to the non-top-7 STEC serogroups associated with cattle.

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          Most cited references115

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          German outbreak of Escherichia coli O104:H4 associated with sprouts.

          A large outbreak of the hemolytic-uremic syndrome caused by Shiga-toxin-producing Escherichia coli O104:H4 occurred in Germany in May 2011. The source of infection was undetermined. We conducted a matched case-control study and a recipe-based restaurant cohort study, along with environmental, trace-back, and trace-forward investigations, to determine the source of infection. The case-control study included 26 case subjects with the hemolytic-uremic syndrome and 81 control subjects. The outbreak of illness was associated with sprout consumption in univariable analysis (matched odds ratio, 5.8; 95% confidence interval [CI], 1.2 to 29) and with sprout and cucumber consumption in multivariable analysis. Among case subjects, 25% reported having eaten sprouts, and 88% reported having eaten cucumbers. The recipe-based study among 10 groups of visitors to restaurant K included 152 persons, among whom bloody diarrhea or diarrhea confirmed to be associated with Shiga-toxin-producing E. coli developed in 31 (20%). Visitors who were served sprouts were significantly more likely to become ill (relative risk, 14.2; 95% CI, 2.6 to ∞). Sprout consumption explained 100% of cases. Trace-back investigation of sprouts from the distributor that supplied restaurant K led to producer A. All 41 case clusters with known trading connections could be explained by producer A. The outbreak strain could not be identified on seeds from the implicated lot. Our investigations identified sprouts as the most likely outbreak vehicle, underlining the need to take into account food items that may be overlooked during subjects' recall of consumption.
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            Non-O157 Shiga toxin-producing Escherichia coli infections in the United States, 1983-2002.

            Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a well-recognized cause of bloody diarrhea and hemolytic-uremic syndrome (HUS). Non-O157 STEC contribute to this burden of illness but have been underrecognized as a result of diagnostic limitations and inadequate surveillance. Between 1983 and 2002, 43 state public health laboratories submitted 940 human non-O157 STEC isolates from persons with sporadic illnesses to the Centers for Diseases Control and Prevention reference laboratory for confirmation and serotyping. The most common serogroups were O26 (22%), O111 (16%), O103 (12%), O121 (8%), O45 (7%), and O145 (5%). Non-O157 STEC infections were most frequent during the summer and among young persons (median age, 12 years; interquartile range, 3-37 years). Virulence gene profiles were as follows: 61% stx(1) but not stx(2); 22% stx(2) but not stx(1); 17% both stx(1) and stx(2); 84% intimin (eae); and 86% enterohemolysin (E-hly). stx(2) was strongly associated with an increased risk of HUS, and eae was strongly associated with an increased risk of bloody diarrhea. STEC O111 accounted for most cases of HUS and was also the cause of 3 of 7 non-O157 STEC outbreaks reported in the United States. Non-O157 STEC can cause severe illness that is comparable to the illness caused by STEC O157. Strains that produce Shiga toxin 2 are much more likely to cause HUS than are those that produce Shiga toxin 1 alone. Improving surveillance will more fully elucidate the incidence and pathological spectrum of these emerging agents. These efforts require increased clinical suspicion, improved clinical laboratory isolation, and continued serotyping of isolates in public health laboratories.
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              Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent.

              There are four major categories of diarrheagenic Escherichia coli: enterotoxigenic (a major cause of travelers' diarrhea and infant diarrhea in less-developed countries), enteroinvasive (a cause of dysentery), enteropathogenic (an important cause of infant diarrhea), and enterohemorrhagic (a cause of hemorrhagic colitis and hemolytic uremic syndrome). Besides manifesting distinct clinical patterns, these categories of E. coli differ in their epidemiology and pathogenesis and in their O:H serotypes. Common features (albeit distinct for each category) include plasmid-encoded virulence properties, characteristic interactions with intestinal mucosa, and elaboration of various types of enterotoxins or cytotoxins. A less-well-defined fifth category of diarrheagenic E. coli is that of enteroadherent E. coli, so far identifiable only by their pattern of adherence to Hep-2 cells in tissue culture.
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                Author and article information

                Contributors
                Journal
                Front Cell Infect Microbiol
                Front Cell Infect Microbiol
                Front. Cell. Infect. Microbiol.
                Frontiers in Cellular and Infection Microbiology
                Frontiers Media S.A.
                2235-2988
                29 July 2020
                2020
                : 10
                : 378
                Affiliations
                [1] 1Department of Diagnostic Medicine/Pathobiology, Kansas State University , Manhattan, KS, United States
                [2] 2E. coli Reference Center, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University , University Park, PA, United States
                [3] 3Department of Animal Sciences and Industry/Food Science Institute, Kansas State University , Manhattan, KS, United States
                [4] 4Veterinary Diagnostic Laboratory, Industry/Food Science Institute, Kansas State University , Manhattan, KS, United States
                Author notes

                Edited by: Roxane M. Piazza, Butantan Institute, Brazil

                Reviewed by: Nora Lía Padola, National University of Central Buenos Aires, Argentina; Beatriz Arellano Reynoso, National Autonomous University of Mexico, Mexico

                *Correspondence: Jianfa Bai jbai@ 123456vet.k-state.edu

                This article was submitted to Bacteria and Host, a section of the journal Frontiers in Cellular and Infection Microbiology

                Article
                10.3389/fcimb.2020.00378
                7403468
                32850480
                6d6a03e9-9df1-4336-8ccc-c13453a6c6db
                Copyright © 2020 Ludwig, Shi, Shridhar, Roberts, DebRoy, Phebus, Bai and Nagaraja.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 06 April 2020
                : 18 June 2020
                Page count
                Figures: 1, Tables: 4, Equations: 0, References: 132, Pages: 12, Words: 10935
                Funding
                Funded by: National Institute of Food and Agriculture 10.13039/100005825
                Categories
                Cellular and Infection Microbiology
                Methods

                Infectious disease & Microbiology
                shiga toxin-producing escherichia coli (stec),top-7 stec,non-top-7 stec,multiplex pcr assays,cattle,feces

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