Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia sp. Cryptic Lineage 1 Strain 7v Harbors a Hybrid Plasmid

ABSTRACTHybrid isolates of Shiga toxin-producingEscherichia coli(STEC) and enterotoxigenicE. coli(ETEC) encoding heat-stable enterotoxin (ST) are being reported with increasing frequency from a variety of sources. However, information regarding the plasmids that these strains harbor is scarce. In this study, we sequence and characterize a plasmid, p7v, from the STEC/ETEC hybrid strain 7v. Whole-genome phylogenetic analyses of STEC/ETEC hybrid strains and prototypeE. coliisolates of other pathotypes placed 7v in theEscherichiasp. cryptic lineage 1 (CL1) clade. The complete plasmid, p7v, was determined to be 229,275 bp and encodes putative virulence factors that are typically carried on STEC plasmids as well as those often carried on ETEC plasmids, indicating that the hybrid nature of the strain extends beyond merely encoding the two toxins. Plasmid p7v carries two copies ofstawith identical sequences, which were discovered to be divergent from thestasequences found in the prototype human ETEC strains. Using a nomenclature scheme based on a phylogeny constructed fromstaandstbsequences, thestaencoded on p7v is designated STa4.In silicoanalysis determined that p7v also encodes the K88 fimbria, a colonization factor usually associated with porcine ETEC plasmids. The p7v sequence and the presence of plasmid-encoded virulence factors are compared to those of other STEC/ETEC CL1 hybrid genomes and reveal gene acquisition/loss at the strain level. In addition, the interrogation of 24 STEC/ETEC hybrid genomes for identification of plasmid replicons, colonization factors, Stx and ST subtypes, and other plasmid-encoded virulence genes highlights the diversity of these hybrid strains.IMPORTANCEHybrid Shiga toxin-producingEscherichia coli/enterotoxigenicEscherichia coli(STEC/ETEC) strains, which have been isolated from environmental, animal, and human clinical samples, may represent an emerging threat as food-borne pathogens. Characterization of these strains is important for assessing virulence potential, aiding in the development of pathogen detection methods, and understanding how the hybrid strains evolve to potentially have a greater impact on public health. This study represents, to our knowledge, both the first characterization of a closed plasmid sequence from a STEC/ETEC hybrid strain and the most comprehensive phylogenetic analysis of available STEC/ETEC hybrid genomes to date. The results demonstrate how the mobility of plasmid-associated virulence genes has resulted in the creation of a diverse plasmid repertoire within the STEC/ETEC hybrid strains.

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Virulence Genes in Expanded-Spectrum-Cephalosporin-Resistant and -Susceptible Escherichia coli Isolates from Treated and Untreated Chickens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01996-15 ◽

2015 ◽

Vol 60 (3) ◽

pp. 1874-1877 ◽

Cited By ~ 1

Author(s):

S. Baron ◽

S. Delannoy ◽

S. Bougeard ◽

E. Larvor ◽

E. Jouy ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Virulence Factors ◽

Shiga Toxin ◽

Virulence Genes ◽

Phylogenetic Groups ◽

Content Type ◽

E Coli ◽

Heat Stable ◽

Intestinal Infections

This study investigated antimicrobial resistance, screened for the presence of virulence genes involved in intestinal infections, and determined phylogenetic groups ofEscherichia coliisolates from untreated poultry and poultry treated with ceftiofur, an expanded-spectrum cephalosporin. Results show that none of the 76 isolates appeared to be Shiga toxin-producingE. colior enteropathogenicE. coli. All isolates were negative for the major virulence factors/toxins tested (ehxA,cdt, heat-stable enterotoxin [ST], and heat-labile enterotoxin [LT]). The few virulence genes harbored in isolates generally did not correlate with isolate antimicrobial resistance or treatment status. However, some of the virulence genes were significantly associated with certain phylogenetic groups.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.00115-15 ◽

2015 ◽

Vol 53 (7) ◽

pp. 2148-2153 ◽

Cited By ~ 28

Author(s):

Xuan Qin ◽

Eileen J. Klein ◽

Emmanouil Galanakis ◽

Anita A. Thomas ◽

Jennifer R. Stapp ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Primary Cultures ◽

Clinical Samples ◽

Pcr Assay ◽

Content Type ◽

E Coli ◽

Successful Recovery

Timely accurate diagnosis of Shiga toxin-producingEscherichia coli(STEC) infections is important. We evaluated a laboratory-developed real-time PCR (LD-PCR) assay targetingstx1,stx2, andrfbEO157with 2,386 qualifying stool samples submitted to the microbiology laboratory of a tertiary care pediatric center between July 2011 and December 2013. Broth cultures of PCR-positive samples were tested for Shiga toxins by enzyme immunoassay (EIA) (ImmunoCard STAT! enterohemorrhagicE. coli[EHEC]; Meridian Bioscience) and cultured in attempts to recover both O157 and non-O157 STEC.E. coliO157 and non-O157 STEC were detected in 35 and 18 cases, respectively. Hemolytic uremic syndrome (HUS) occurred in 12 patients (10 infected with STEC O157, one infected with STEC O125ac, and one with PCR evidence of STEC but no resulting isolate). Among the 59 PCR-positive STEC specimens from 53 patients, only 29 (54.7%) of the associated specimens were toxin positive by EIA. LD-PCR differentiated STEC O157 from non-O157 usingrfbEO157, and LD-PCR results prompted successful recovery ofE. coliO157 (n= 25) and non-O157 STEC (n= 8) isolates, although the primary cultures and toxin assays were frequently negative. A rapid “mega”-multiplex PCR (FilmArray gastrointestinal panel; BioFire Diagnostics) was used retrospectively, and results correlated with LD-PCR findings in 25 (89%) of the 28 sorbitol-MacConkey agar culture-negative STEC cases. These findings demonstrate that PCR is more sensitive than EIA and/or culture and distinguishes between O157 and non-O157 STEC in clinical samples and thatE. coliO157:H7 remains the predominant cause of HUS in our institution. PCR is highly recommended for rapid diagnosis of pediatric STEC infections.

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Genetic Characterization of Escherichia coli O104 Isolates from Different Sources in the United States

Applied and Environmental Microbiology ◽

10.1128/aem.07533-11 ◽

2011 ◽

Vol 78 (5) ◽

pp. 1615-1618 ◽

Cited By ~ 8

Author(s):

Lydia V. Rump ◽

Sonya Bodeis-Jones ◽

Jason Abbott ◽

Shaohua Zhao ◽

Julie Kase ◽

...

Keyword(s):

United States ◽

Escherichia Coli ◽

Virulence Genes ◽

Genetic Characterization ◽

The United States ◽

Content Type ◽

E Coli ◽

Virulence Markers ◽

Different Sources

ABSTRACTEscherichia coliO104 isolates collected from different sources in the United States were examined for virulence genes typical of enterohemorrhagicE. coliand those identified in the O104:H4 isolate associated with the 2011 German outbreak. The unexpected presence of virulence markers in these isolates highlights the importance of screening unusual and potentially pathogenic Shiga toxin-producingE. coliserotypes.

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Intimin Gene (eae) Subtype-Based Real-Time PCR Strategy for Specific Detection of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in Cattle Feces

Applied and Environmental Microbiology ◽

10.1128/aem.03161-13 ◽

2013 ◽

Vol 80 (3) ◽

pp. 1177-1184 ◽

Cited By ~ 16

Author(s):

Delphine Bibbal ◽

Estelle Loukiadis ◽

Monique Kérourédan ◽

Carine Peytavin de Garam ◽

Franck Ferré ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Specific Detection ◽

Content Type ◽

E Coli ◽

Cattle Feces ◽

Pcr Assays

ABSTRACTShiga toxin-producingEscherichia coli(STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targetedeaesubtypes. The simultaneous presence ofstx,eae, and one of the five O group markers was found in 58.0% of the samples, and the five targetedstxpluseaeplus O genetic combinations were detected 143 times. However, taking into consideration the association betweeneaesubtypes and O group markers, the resultingstxpluseaesubtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22E. colistrains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive forstx,eaeand an O group marker, but that were negative for the correspondingeaesubtype, were successful. Characterization of the 24E. coliisolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenicE. coli(aEPEC). Finally, the more discriminatingeaesubtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.

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Prevalence of Hemolysin Genes and Comparison ofehxASubtype Patterns in Shiga Toxin-Producing Escherichia coli (STEC) and Non-STEC Strains from Clinical, Food, and Animal Sources

Applied and Environmental Microbiology ◽

10.1128/aem.02200-13 ◽

2013 ◽

Vol 79 (20) ◽

pp. 6301-6311 ◽

Cited By ~ 24

Author(s):

Sandra C. Lorenz ◽

Insook Son ◽

Anna Maounounen-Laasri ◽

Andrew Lin ◽

Markus Fischer ◽

...

Keyword(s):

Escherichia Coli ◽

Virulence Factors ◽

Shiga Toxin ◽

Phenotypic Expression ◽

Food Sources ◽

Polymorphism Analysis ◽

Subtype C ◽

Content Type ◽

E Coli ◽

Subtype A

ABSTRACTShiga toxin-producingEscherichia coli(STEC) belonging to certain serogroups (e.g., O157 and O26) can cause serious conditions like hemolytic-uremic syndrome (HUS), but other strains might be equally pathogenic. While virulence factors, likestxandeae, have been well studied, little is known about the prevalence of theE. colihemolysin genes (hlyA,ehxA,e-hlyA, andsheA) in association with these factors. Hemolysins are potential virulence factors, andehxAandhlyAhave been associated with human illness, but the significance ofsheAis unknown. Hence, 435E. colistrains belonging to 62 different O serogroups were characterized to investigate gene presence and phenotypic expression of hemolysis. We further investigatedehxAsubtype patterns inE. coliisolates from clinical, animal, and food sources. WhilesheAandehxAwere widely distributed,e-hlyAandhlyAwere rarely found. Most strains (86.7%) were hemolytic, and significantly more hemolytic (95%) than nonhemolytic strains (49%) carriedstxand/oreae(P< 0.0001).ehxAsubtyping, as performed by using PCR in combination with restriction fragment length polymorphism analysis, resulted in six closely related subtypes (>94.2%), with subtypes A/D beingeae-negative STECs and subtypes B, C, E, and Feaepositive. Unexpectedly,ehxAsubtype patterns differed significantly between isolates collected from different sources (P< 0.0001), suggesting that simple linear models of exposure and transmission need modification; animal isolates carried mostly subtypes A/C (39.3%/42.9%), food isolates carried mainly subtype A (81.9%), and clinical isolates carried mainly subtype C (66.4%). Certain O serogroups correlated with particularehxAsubtypes: subtype A with O104, O113, and O8; B exclusively with O157; C with O26, O111, and O121.

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Characterization of extended-spectrum and CMY-2 ß-lactamases, and associated virulence genes in Escherichia coli from food of animal origin in México

British Food Journal ◽

10.1108/bfj-02-2018-0104 ◽

2018 ◽

Vol 120 (7) ◽

pp. 1457-1473 ◽

Cited By ~ 1

Author(s):

Edwin Barrios-Villa ◽

Gerardo Cortés-Cortés ◽

Patricia Lozano Zarain ◽

Sergio Romero-Romero ◽

Norarizbeth Lara Flores ◽

...

Keyword(s):

Escherichia Coli ◽

Virulence Genes ◽

Food Samples ◽

Animal Origin ◽

Content Type ◽

E Coli ◽

Extended Spectrum ◽

Food Of Animal Origin ◽

Cephalosporin Resistance

Purpose Broad-spectrum cephalosporin resistance is rapidly increasing in Escherichia coli, representing a food safety problem. The purpose of this paper is to characterize eight extended-spectrum-ß-lactamase (ESBL) and acquired AmpC ß-lactamase-producing E. coli isolates and virotypes associated, obtained from chicken and pork food samples in Puebla, Mexico. Design/methodology/approach Samples (36 from chicken and 10 from pork) were cultured on Levine agar plates supplemented with cefotaxime (2 mg/L) for isolation of cefotaxime-resistant (CTXR) E. coli. CTXR-E. coli isolates were detected in 33 of 46 samples (72 percent), and one isolate/sample was characterized (28 from chicken and 5 from pork), for ESBL production, phylogenetic group, sequence typing, resistance and virulence genes by PCR and sequencing. Findings Results showed 16 ESBL-E. coli (35 percent) (12/16 belonging to phylogroup B1) and 8 CMY-2-E. coli (17 percent). ESBL detected were as follows (number of isolates): CTX-M-2 (8); CTX-M-1 (2); CTX-M-15 (1); SHV-2a (4) and TEM-52c (1). In total, 20 different sequence types (STs) were identified among the ESBL- or CMY-2-producing E. coli strains, which included four new ones. The CTX-M-15 β-lactamase was detected in one E. coli ST617-ST10 Cplx-B1 strain that also carried ibeA gene. One CMY-2-positive strain of lineage ST224-B2 was detected and it carried the qnrA1 gene. Originality/value In this study, a ST131-based virotyping scheme for strains from food of animal origin was established since this kind of strains constitutes an important vehicle of virulent ESBL- and CMY-2-producing E. coli isolates, which could be transmitted to humans by direct contact or through the food chain.

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Prevalence and Genetic Characterization of Shiga Toxin-Producing Escherichia coli Isolates from Slaughtered Animals in Bangladesh

Applied and Environmental Microbiology ◽

10.1128/aem.00854-08 ◽

2008 ◽

Vol 74 (17) ◽

pp. 5414-5421 ◽

Cited By ~ 43

Author(s):

Mohammad A. Islam ◽

Abdus S. Mondol ◽

Enne de Boer ◽

Rijkelt R. Beumer ◽

Marcel H. Zwietering ◽

...

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Virulence Genes ◽

Genetic Characterization ◽

Clonal Diversity ◽

Pulsed Field Gel Electrophoresis ◽

E Coli ◽

Phage Types ◽

Distinct Restriction

ABSTRACT To determine the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in slaughter animals in Dhaka, Bangladesh, we collected rectal contents immediately after animals were slaughtered. Of the samples collected from buffalo (n = 174), cows (n = 139), and goats (n = 110), 82.2%, 72.7%, and 11.8% tested positive for stx 1 and/or stx 2, respectively. STEC could be isolated from 37.9%, 20.1%, and 10.0% of the buffalo, cows, and goats, respectively. STEC O157 samples were isolated from 14.4% of the buffalo, 7.2% of the cows, and 9.1% of the goats. More than 93% (n = 42) of the STEC O157 isolates were positive for the stx 2, eae, katP, etpD, and enterohemorrhagic E. coli hly (hly EHEC) virulence genes. STEC O157 isolates were characterized by seven recognized phage types, of which types 14 (24.4%) and 31 (24.4%) were predominant. Subtyping of the 45 STEC O157 isolates by pulsed-field gel electrophoresis showed 37 distinct restriction patterns, suggesting a heterogeneous clonal diversity. In addition to STEC O157, 71 STEC non-O157 strains were isolated from 60 stx-positive samples from 23.6% of the buffalo, 12.9% of the cows, and 0.9% of the goats. The STEC non-O157 isolates belonged to 36 different O groups and 52 O:H serotypes. Unlike STEC O157, most of the STEC non-O157 isolates (78.9%) were positive for stx 1. Only 7.0% (n = 5) of the isolates were positive for hly EHEC, and none was positive for eae, katP, and etpD. None of the isolates was positive for the iha, toxB, and efa1 putative adhesion genes. However, 35.2% (n = 25), 11.3% (n = 8), 12.7% (n = 9), and 12.7% (n = 9) of the isolates were positive for the lpf O113, saa, lpfA O157/01-141, and lpfA O157/OI-154 genes, respectively. The results of this study provide the first evidence that slaughtered animals like buffalo, cows, and goats in Bangladesh are reservoirs for STEC, including the potentially virulent STEC strain O157.

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Characterization of Clinical Escherichia coli Strains Producing a Novel Shiga Toxin 2 Subtype in Sweden and Denmark

Microorganisms ◽

10.3390/microorganisms9112374 ◽

2021 ◽

Vol 9 (11) ◽

pp. 2374

Author(s):

Xiangning Bai ◽

Flemming Scheutz ◽

Henrik Mellström Dahlgren ◽

Ingela Hedenström ◽

Cecilia Jernberg

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Virulence Genes ◽

Epidemiological Surveillance ◽

E Coli ◽

Shiga Toxin 2 ◽

Life Threatening ◽

Macrolide Resistance Gene

Shiga toxin (Stx) is the key virulence factor in the Shiga Toxin-Producing Escherichia coli (STEC), which can cause diarrhea and hemorrhagic colitis with potential life-threatening complications. There are two major types of Stx: Stx1 and Stx2. Several Stx1/Stx2 subtypes have been identified in E. coli, varying in sequences, toxicity and host specificity. Here, we report a novel Stx2 subtype (designated Stx2m) from three clinical E. coli strains isolated from diarrheal patients and asymptomatic carriers in Sweden and Denmark. The Stx2m toxin was functional and exhibited cytotoxicity in vitro. The two Swedish Stx2m-producing strains belonged to the same serotype O148:H39 and Multilocus Sequencing Typing (MLST) Sequence Type (ST) 5825, while the Danish strain belonged to the O96:H19 serotype and ST99 type. Whole-genome sequencing (WGS) data analysis revealed that the three Stx2m-producing strains harbored additional virulence genes and the macrolide resistance gene mdf (A). Our findings expand the pool of Stx2 subtypes and highlight the clinical significance of emerging STEC variants. Given the clinical relevance of the Stx2m-producing strains, we propose to include Stx2m in epidemiological surveillance of STEC infections and clinical diagnosis.

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Single-Cell-Based Digital PCR Detection and Association of Shiga Toxin-Producing Escherichia coli Serogroups and Major Virulence Genes

Journal of Clinical Microbiology ◽

10.1128/jcm.01684-19 ◽

2020 ◽

Vol 58 (3) ◽

Author(s):

Xuming Liu ◽

Lance Noll ◽

Xiaorong Shi ◽

Elizabeth Porter ◽

Yin Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Single Cell ◽

High Throughput ◽

Shiga Toxin ◽

Virulence Genes ◽

Ground Beef ◽

Digital Pcr ◽

Content Type ◽

E Coli ◽

Cattle Feces

ABSTRACT Escherichia coli serogroups O157, O26, O45, O103, O111, O121, and O145, when carrying major virulence genes, the Shiga toxin genes stx1 and stx2 and the intimin gene eae, are important foodborne pathogens. They are referred to as the “top 7” Shiga toxin-producing E. coli (STEC) serogroups and were declared by the USDA as adulterants to human health. Since top 7 serogroup-positive cattle feces and ground beef can also contain nonadulterant E. coli strains, regular PCR cannot confirm whether the virulence genes are carried by adulterant or nonadulterant E. coli serogroups. Thus, traditional gold-standard STEC detection requires bacterial isolation and characterization, which are not compatible with high-throughput settings and often take a week to obtain a definitive result. In this study, we demonstrated that the partition-based multichannel digital PCR (dPCR) system can be used to detect and associate the E. coli serogroup-specific gene with major virulence genes and developed a single-cell-based dPCR approach for rapid (within 1 day) and accurate detection and confirmation of major STEC serogroups in high-throughput settings. Major virulence genes carried by each of the top 7 STEC serogroups were detected by dPCR with appropriately diluted intact bacterial cells from pure cultures, culture-spiked cattle feces, and culture-spiked ground beef. Furthermore, from 100 randomly collected, naturally shed cattle fecal samples, 3 O103 strains carrying eae and 2 O45 strains carrying stx1 were identified by this dPCR assay and verified by the traditional isolation method. This novel and rapid dPCR assay is a culture-independent, high-throughput, accurate, and sensitive method for STEC detection and confirmation.

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In Silico Genotyping of Escherichia coli Isolates for Extraintestinal Virulence Genes by Use of Whole-Genome Sequencing Data

Journal of Clinical Microbiology ◽

10.1128/jcm.01269-20 ◽

2020 ◽

Vol 58 (10) ◽

Author(s):

Anna Maria Malberg Tetzschner ◽

James R. Johnson ◽

Brian D. Johnston ◽

Ole Lund ◽

Flemming Scheutz

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

In Silico ◽

Virulence Genes ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Content Type ◽

E Coli

ABSTRACT Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause in humans of urinary tract infection and bacteremia. The previously published web tool VirulenceFinder (http://cge.cbs.dtu.dk/services/VirulenceFinder/) uses whole-genome sequencing (WGS) data for in silico characterization of E. coli isolates and enables researchers and clinical health personnel to quickly extract and interpret virulence-relevant information from WGS data. In this study, 38 ExPEC-associated virulence genes were added to the existing E. coli VirulenceFinder database. In total, 14,441 alleles were downloaded. A total of 1,890 distinct alleles were added to the database after removal of redundant sequences and analysis of the remaining alleles for open reading frames (ORFs). The database now contains 139 genes—of which 44 are related to ExPEC—and 2,826 corresponding alleles. Construction of the database included validation against 27 primer pairs from previous studies, a search for serotype-specific P fimbriae papA alleles, and a BLASTn confirmation of seven genes (etsC, iucC, kpsE, neuC, sitA, tcpC, and terC) not covered by the primers. The augmented database was evaluated using (i) a panel of nine control strains and (ii) 288 human-source E. coli strains classified by PCR as ExPEC and non-ExPEC. We observed very high concordance (average, 93.4%) between PCR and WGS findings, but WGS identified more alleles. In conclusion, the addition of 38 ExPEC-associated genes and the associated alleles to the E. coli VirulenceFinder database allows for a more complete characterization of E. coli isolates based on WGS data, which has become increasingly important considering the plasticity of the E. coli genome.

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