Escherichia coli O-Genotyping PCR: a Comprehensive and Practical Platform for Molecular O Serogrouping

Iguchi, Atsushi; Iyoda, Sunao; Seto, Kazuko; Morita-Ishihara, Tomoko; Scheutz, Flemming; Ohnishi, Makoto

doi:10.1128/jcm.00321-15

Cited by 131 publications

(120 citation statements)

References 22 publications

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“…The O antigen was initially screened using the O-genotyping PCR method to identify and classify the E. coli O serogroups (Iguchi et al, 2015). The complete E. coli O antisera (O1-O188; Statens Serum Institut, Hillerød, Denmark) were used to confirm the PCR results.…”

Section: Methodsmentioning

confidence: 99%

Molecular and Phylogenetic Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli Strains in China

Bai¹,

Xu³

et al. 2016

Front. Cell. Infect. Microbiol.

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Shiga toxin-producing Escherichia coli (STEC) causes diarrhea and hemorrhagic colitis with life-threatening complications, such as hemolytic uremic syndrome. The aim of this study was to assess the molecular epidemiologic features of non-O157 STEC strains from different resources in China and illustrate the role of animal reservoirs or animal-derived foodstuffs in human STEC infections. A collection of 301 non-O157 STEC isolates from domestic and wild animals (i.e., cattle, goat, pig, yak, pika, and antelope), raw meats (i.e., beef, pork, mutton, chicken, and duck), diarrheal patients, and healthy carriers in different regions of China were selected in this study. Of the 301 analyzed STEC isolates, 67 serogroups, and 118 serotypes were identified; this included some predominant serogroups associated with human disease, such as O26, O45, O103, O111, and O121. Eighteen different combinations of stx subtypes were found. Eleven isolates carried the intimin gene eae, 93 isolates contained ehxA, and 73 isolates carried astA. The prevalence of other putative adhesion genes saa, paa, efa1, and toxB was 28.90% (87), 6.98% (21), 2.31% (7), and 1% (3), respectively. The phylogenetic distribution of isolates was analyzed by multilocus sequence typing (MLST). Ninety-four sequence types were assigned across the 301 isolates. A subset of isolates recovered from yak and pika residing in the similar wild environments, Qinghai-Tibetan plateau, showed similar genetic profiles and more tendencies to cluster together. Isolates from goat and mutton exhibited close genetic relatedness with those from human-derived isolates, providing evidence that transmission may have occurred locally within intraspecies or interspecies, and importantly, from animal reservoirs, or raw meats to humans. Comparing isolates in this study with highly virulent strains by MLST, along with serotyping and virulence profiles, it is conceivable that some of isolates from goat, yak, or raw meats may have potential to cause human diseases.

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Section: Methodsmentioning

confidence: 99%

Molecular and Phylogenetic Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli Strains in China

Bai¹,

Xu³

et al. 2016

Front. Cell. Infect. Microbiol.

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“…Between November 2008 and February 2014 a total of 233 Ehly-producing E. coli were isolated and stored in 80% glycerol at À70 C before further studies. Seven O5 AE-STEC from our collections were included in the genome sequencing study: four were isolated from diarrhoeic calves between 1967 and 1993 at the Veterinary Faculty of the Table 1 Primers and control strains used in this study for the pathotyping (a) and for the serogrouping (b) (adapted from Iguchi et al, 2015). University of Liège after growth on Gassner agar plates (Merck, Darmstadt, Germany) and were later typed by colony hybridization (Mainil et al, 1993) and three were isolated from humans with (bloody) diarrhoea in 2004 and 2009 at the Universitair Ziekenhuis of the Vrije Universiteit Brussel after culture on sorbitol-MacConkey (SMAC) and on SMAC with cefixime and tellurite (CT-SMAC) (LabM, Heywood, Lancashire, UK), and typing by PCR (Buvens et al, 2012).…”

Section: E Coli Isolatesmentioning

confidence: 99%

“…The triplex-PCR positive isolates were assayed with two pentaplex PCRs to detect the O antigen-encoding genes specific to the ten most frequent and pathogenic STEC serogroups in humans and animals (Mekata et al, 2014;Iguchi et al, 2015) (Table 1a and b). Control strains for the triplex and the pentaplex PCRs were included.…”

Section: Pcr Virulotyping and O Segroupingmentioning

confidence: 99%

Identification of Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli in diarrhoeic calves and comparative genomics of O5 bovine and human STEC

Fakih

Thiry

Duprez

et al. 2017

Veterinary Microbiology

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A B S T R A C TEscherichia coli producing Shiga toxins (Stx) and the attaching-effacing (AE) lesion (AE-STEC) are responsible for (bloody) diarrhoea in humans and calves while the enteropathogenic E. coli (EPEC) producing the AE lesion only cause non-bloody diarrhoea in all mammals. The purpose of this study was (i) to identify the pathotypes of enterohaemolysin-producing E. coli isolated between 2009 and 2013 on EHLY agar from less than 2 month-old diarrhoeic calves with a triplex PCR targeting the stx1, stx2, eae virulence genes; (ii) to serotype the positive isolates with PCR targeting the genes coding for ten most frequent and pathogenic human and calf STEC O serogroups; and (iii) to compare the MLSTypes and virulotypes of calf and human O5 AE-STEC after Whole Genome Sequencing using two server databases (www.genomicepidemiology.org). Of 233 isolates, 206 were triplex PCR-positive: 119 AE-STEC (58%), 78 EPEC (38%) and 9 STEC (4%); and the stx1+eae+ AE-STEC (49.5%) were the most frequent. Of them, 120 isolates (84% of AE-STEC, 23% of EPEC, 22% of STEC) tested positive with one O serogroup PCR: 57 for O26 (47.5%), 36 for O111 (30%), 10 for O103 (8%) and 8 for O5 (7%) serogroups. The analysis of the draft sequences of 15 O5 AE-STEC could not identify any difference correlated to the host. As a conclusion, (i) the AE-STEC associated with diarrhoea in young calves still belong to the same serogroups as previously (O5, O26, O111) but the O103 serogroup may be emerging, (ii) the O5 AE-STEC from calves and humans are genetically similar.2016 Published by Elsevier B.V.

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“…For the ESBL-producing UT strains, we determined the O serogroups by O-genotyping PCR (41,42). The H serogroup was determined by sequencing of the fliC gene (43) (see Table S2 in the supplemental material).…”

Section: Subjects and Strainsmentioning

confidence: 99%

Phylogenetic Analysis of Enteroaggregative Escherichia coli (EAEC) Isolates from Japan Reveals Emergence of CTX-M-14-Producing EAEC O25:H4 Clones Related to Sequence Type 131

et al. 2016

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f Enteroaggregative Escherichia coli (EAEC) causes acute or persistent diarrhea. The aggR gene is widely used as a marker for typical EAEC. The heterogeneity of EAEC is well known; however, there are few reports on the phylogenetic relationships of EAEC. Recently, CTX-M extended-spectrum ␤-lactamase (ESBL)-producing EAEC strains have been reported worldwide. To characterize EAEC strains in Japan, we investigated the population structure of EAEC. A total of 167 aggR-positive strains isolated from stool specimens from diarrheal patients in Kagoshima (139 strains) and Osaka (28 strains), Japan, between 1992 and 2010 were examined for the prevalence of EAEC virulence markers, the bla CTX-M gene, and the capacity to form biofilms. Multilocus sequence typing was also conducted. EAEC strains were widely distributed across four major E. coli phylogroups. Strains of O111: H21/clonal group 40 (CG40) (30 strains), O126:H27/CG200 (13 strains), and O86a:H27/CG3570 (11 strains) in phylogroup B1 are the historical EAEC clones in Japan, and they exhibited strong biofilm formation. Twenty-nine strains of EAEC O25:H4/ CG131 were identified in phylogroup B2, 79% of which produced CTX-M-14. This clone has emerged since 2003. The clone harbored plasmid-encoded EAEC virulence genes but not chromosomal virulence genes and had lower biofilm-forming capacity than historical EAEC strains. This clone most likely emerged from a pandemic uropathogenic O25:H4/sequence type 131 clone by acquiring an EAEC virulence plasmid from canonical EAEC. Surveillance of the horizontal transfer of both virulence and ESBL genes among E. coli strains is important for preventing a worldwide increase in antimicrobial drug resistance. Enteroaggregative Escherichia coli (EAEC) causes acute or persistent diarrhea in children and adults, in both developing (1) and industrialized (2, 3) countries. Several outbreaks of gastroenteritis linked to EAEC have been reported (4, 5). EAEC is also the second most common cause of travelers' diarrhea (6). EAEC is defined by its characteristic aggregative adherence (AA) to HEp-2 cells in culture (7). Although detection of the AA phenotype in the HEp-2 cell adherence assay is still the gold standard for EAEC identification, the transcriptional regulator AggR, which controls the expression of multiple EAEC virulence genes, is also used as a typical EAEC marker for PCR detection (8).The pathogenesis of EAEC infection involves the adherence of the bacterium to the intestinal mucosa, followed by the secretion of one or more enterotoxins and/or cytotoxins that induce mucosal inflammation (9). EAEC adherence to the mucosa is characterized by the formation of a thick biofilm (10), which may promote persistent infection. The majority of EAEC strains carry a large (ϳ100-kb) plasmid, which encodes most putative EAEC virulence factors, such as AggR, aggregative adherence fimbriae (AAF), the dispersin protein Aap (11), its transporter Aat (12), the plasmid-encoded toxin Pet (13), enteroaggregative heat-stable toxin 1 (EAST-1) (14), and the...

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Escherichia coli O-Genotyping PCR: a Comprehensive and Practical Platform for Molecular O Serogrouping

Cited by 131 publications

References 22 publications

Molecular and Phylogenetic Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli Strains in China

Molecular and Phylogenetic Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli Strains in China

Identification of Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli in diarrhoeic calves and comparative genomics of O5 bovine and human STEC

Phylogenetic Analysis of Enteroaggregative Escherichia coli (EAEC) Isolates from Japan Reveals Emergence of CTX-M-14-Producing EAEC O25:H4 Clones Related to Sequence Type 131

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