Lori K. Bagi

Eastern Regional Research Center

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Pina M. Fratamico

Eastern Regional Research Center

Gian Marco Baranzoni

Eastern Regional Research Center

Robert L. Buchanan

University of Maryland, College Park

Yanhong Liu

Eastern Regional Research Center

Tiziana Pepe

University of Naples Federico II

Nereus W. Gunther

Eastern Regional Research Center

Gaylen A. Uhlich

Eastern Regional Research Center

Aisha Abdul-Wakeel

Eastern Regional Research Center

Aniello Anastasio

University of Naples Federico II

Patrick Fach

Agence Nationale de Sécurité Sanitaire de l’Alimentation, de l’Environnement et du Travail

Cooperative Institutions

Agricultural Research Service

Eastern Regional Research Center

United States Department of Agriculture

Center for Food Safety and Applied Nutrition

United States Food and Drug Administration

Roman L. Hruska U.S. Meat Animal Research Center

Pennsylvania State University

Michigan State University

Centers for Disease Control and Prevention

University of California, Davis

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Interlaboratory Evaluation of the U.S. Food and Drug Administration Escherichia coli Identification Microarray for Profiling Shiga Toxin–Producing Escherichia coli

Journal of Food Protection (2018)

Isha R. Patel Jayanthi Gangiredla David W. Lacher Mark K. Mammel Lori K. Bagi

Subtyping

STX2

Intimin

Phage typing

10.4315/0362-028x.jfp-18-052

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Citations (6)

Prevalence and Characterization of Shiga Toxin-Producing Escherichia coli in Swine Feces Recovered in the National Animal Health Monitoring System's Swine 2000 Study

Applied and Environmental Microbiology (2004)

Pina M. Fratamico Lori K. Bagi Eric J. Bush Barbara T. Solow

A study was conducted to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) in swine feces in the United States as part of the National Animal Health Monitoring System's Swine 2000 study. Fecal samples collected from swine operations from 13 of the top 17 swine-producing states were tested for the presence of STEC. After enrichment of swine fecal samples in tryptic soy broth, the samples were tested for the presence of stx1 and stx2 by use of the TaqMan E. coli STX1 and STX2 PCR assays. Enrichments of samples positive for stx1 and/or stx2 were plated, and colony hybridization was performed using digoxigenin-labeled probes complementary to the stx1 and stx2 genes. Positive colonies were picked and confirmed by PCR for the presence of the stx1, stx2, or stx2e genes, and the isolates were serotyped. Out of 687 fecal samples tested using the TaqMan assays, 70% (484 of 687) were positive for Shiga toxin genes, and 54% (370 of 687), 64% (436 of 687), and 38% (261 of 687) were positive for stx1, stx2, and both toxin genes, respectively. Out of 219 isolates that were characterized, 29 (13%) produced stx1, 14 (6%) produced stx2, and 176 (80%) produced stx2e. Twenty-three fecal samples contained at least two STEC strains that had different serotypes but that had the same toxin genes or included a strain that possessed stx1 in addition to a strain that possessed stx2 or stx2e. The STEC isolates belonged to various serogroups, including O2, O5, O7, O8, O9, OX10, O11, O15, OX18, O20, O57, O65, O68, O69, O78, O91, O96, O100, O101, O120, O121, O152, O159, O160, O163, and O untypeable. It is noteworthy that no isolates of serogroup O157 were recovered. Results of this study indicate that swine in the United States harbor STEC that can potentially cause human illness.

STX2

Shiga-like toxin

TaqMan

Tryptic soy broth

10.1128/aem.70.12.7173-7178.2004

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Citations (126)

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

Frontiers in Microbiology (2016)

Gian Marco Baranzoni Pina M. Fratamico Jayanthi Gangiredla Isha R. Patel Lori K. Bagi

Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by PCR and a high-throughput real-time PCR system to determine the Shiga toxin subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip (FDA-ECID). As expected, stx2e (81%) was the most common Shiga toxin variant, followed by stx1a (14%), stx2d (3%) and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative Escherichia coli heat stable enterotoxin-1 (astA) were commonly found in 50% and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e- or Stx2d-producing E. coli with virulence gene profiles associated with human infections.

Shiga-like toxin

Hemolysin

10.3389/fmicb.2016.00574

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Citations (68)

Cloning vectors for gene delivery, integration and expression in Campylobacter jejuni

BioTechniques (2022)

Gaylen A. Uhlich Lori K. Bagi Nereus W. Gunther

Campylobacter genetics research is negatively impacted by a shortage of molecular tools for expressing DNA elements. A previous technique coupled an antibiotic resistance gene and its promoter to a gene of interest, inserting this expression unit into a conserved chromosomal location. Here the authors describe two new plasmids for construction and gene integration utilizing aspects of the previous type of expression unit. pBlueKan+cysMPro allows for the assembly of amplified DNA targets behind a kanamycin resistance marker and a constitutively transcribed cysM promoter. Transfer of the transcription unit to plasmid pCJR01 adds flanking regions of Campylobacter rRNA homology for recombination into conserved rRNA regions. System utility was demonstrated by restoring function of a flaAB deletion (RM3194ΔflaAB::tet) with a flaA gene or flaA/flaB combination.

Homology

Kanamycin

10.2144/btn-2021-0096

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Citations (2)

A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins

Toxins (2014)

Andrew G. Gehring Xiaohua He Pina M. Fratamico Joseph Lee Lori K. Bagi

Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and pooled horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic) and/or B-PER (a cell-disrupting, protein extraction reagent). Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.

Horseradish peroxidase

STX2

Chromogenic

10.3390/toxins6061855

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Citations (11)

Diverse Virulence Gene Content of Shiga Toxin-Producing Escherichia coli from Finishing Swine

Applied and Environmental Microbiology (2014)

Marion Tseng Pina M. Fratamico Lori K. Bagi Sabine Delannoy Patrick Fach

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) infections are a critical public health concern because they can cause severe clinical outcomes, such as hemolytic uremic syndrome, in humans. Determining the presence or absence of virulence genes is essential in assessing the potential pathogenicity of STEC strains. Currently, there is limited information about the virulence genes carried by swine STEC strains; therefore, this study was conducted to examine the presence and absence of 69 virulence genes in STEC strains recovered previously from finishing swine in a longitudinal study. A subset of STEC strains was analyzed by pulsed-field gel electrophoresis (PFGE) to examine their genetic relatedness. Swine STEC strains ( n = 150) were analyzed by the use of a high-throughput real-time PCR array system, which included 69 virulence gene targets. Three major pathotypes consisted of 16 different combinations of virulence gene profiles, and serotypes were determined in the swine STEC strains. The majority of the swine STEC strains ( n = 120) belonged to serotype O59:H21 and carried the same virulence gene profile, which consisted of 9 virulence genes: stx 2e , iha , ecs1763 , lpfA O113 , estIa ( STa ), ehaA , paa , terE , and ureD . The eae , nleF , and nleH1-2 genes were detected in one swine STEC strain (O49:H21). Other genes encoding adhesins, including iha , were identified ( n = 149). The PFGE results demonstrated that swine STEC strains from pigs raised in the same finishing barn were closely related. Our results revealed diverse virulence gene contents among the members of the swine STEC population and enhance understanding of the dynamics of transmission of STEC strains among pigs housed in the same barn.

Intimin

10.1128/aem.01761-14

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Citations (40)

Detection of Shiga toxin-producing Escherichia coli in ground beef using the GeneDisc real-time PCR system

Frontiers in Cellular and Infection Microbiology (2012)

Pina M. Fratamico Lori K. Bagi

Escherichia coli O157:H7 and certain non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups have emerged as important public health threats. The development of methods for rapid and reliable detection of this heterogeneous group of pathogens has been challenging. GeneDisc real-time PCR assays were evaluated for detection of the stx1, stx2, eae, and ehx genes and a gene that identifies the O157 serogroup followed by a second GeneDisc assay targeting serogroup-specific genes of STEC O26, O45, O91, O103, O111, O113, O121, O145, and O157. The ability to detect the STEC serogroups in ground beef samples artificially inoculated at a level of ca. 2-20 CFU/25 g and subjected to enrichment in mTSB or BPW was similar. Following enrichment, all inoculated ground beef samples showed amplification of the correct set of target genes carried by each strain. Samples inoculated with STEC serogroups O26, O45, O103, O111, O121, O145, and O157 were subjected to immunomagnetic separation, and isolation was achieved by plating onto Rainbow agar O157. Colonies were confirmed by PCR assays targeting stx1, stx2, eae, and serogroup-specific genes. Thus, this work demonstrated that GeneDisc assays are rapid, sensitive, and reliable and can be used for screening ground beef and potentially other foods for STEC serogroups that are important food-borne pathogens worldwide.

Immunomagnetic separation

10.3389/fcimb.2012.00152

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Citations (34)

Microbial competition: effect of Pseudomonas fluorescens on the growth of Listeria monocytogenes

Food Microbiology (1999)

Robert L. Buchanan Lori K. Bagi

Pseudomonas fluorescens

Brain heart infusion

Bacterial growth

Gompertz function

10.1006/fmic.1998.0264