Sequence of the
 Escherichia coli
 O121 O-Antigen Gene Cluster and Detection of Enterohemorrhagic
 E. coli
 O121 by PCR Amplification of the
 wzx
 and
 wzy
 Genes

Fratamico, Pina M.; Briggs, Connie; Needle, D.; Chen, Chin Yi; DebRoy, Chitrita

doi:10.1128/jcm.41.7.3379-3383.2003

Cited by 73 publications

(59 citation statements)

References 23 publications

(18 reference statements)

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“…They are then secreted into the outer membrane and finally polymerized. Many proteins are involved in these complex steps, but two of them have proved to be highly determinative for the appropriate O-antigen serotype, namely, the O-antigen polymerase which is encoded by the gene wzy and which polymerizes the specific trisaccharide repeat units, and the O-antigen flippase, which is encoded by the gene wzx and which is involved in the secretion process (5,7,8). The use of wzx or wzy sequences depended on the availability of the respective sequences in public sequence databases (GenBank, EMBL Bank).…”

Section: Resultsmentioning

confidence: 99%

Fast DNA Serotyping of Escherichia coli by Use of an Oligonucleotide Microarray

et al. 2007

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Classical antibody-based serotyping of Escherichia coli is an important method in diagnostic microbiology for epidemiological purposes, as well as for a rough virulence assessment. However, serotyping is so tedious that its use is restricted to a few reference laboratories. To improve this situation we developed and validated a genetic approach for serotyping based on the microarray technology. The genes encoding the O-antigen flippase (wzx) and the O-antigen polymerase (wzy) were selected as target sequences for the O antigen, whereas fliC and related genes, which code for the flagellar monomer, were chosen as representatives for the H phenotype. Starting with a detailed bioinformatic analysis and oligonucleotide design, an ArrayTube-based assay was established: a fast and robust DNA extraction method was coupled with a site-specific, linear multiplex labeling procedure and hybridization analysis of the biotinylated amplicons. . Evaluation of the microarray with a set of defined strains representing all O and H serotypes covered revealed that it has a high sensitivity and a high specificity. All of the conventionally typed 24 O groups and all of the 47 H serotypes were correctly identified. Moreover, strains which were nonmotile or nontypeable by previous serotyping assays yielded unequivocal results with the novel ArrayTube assay, which proved to be a valuable alternative to classical serotyping, allowing processing of single colonies within a single working day.Escherichia coli is a commensal bacterium of the intestinal tract of humans and various animal species. Among the many harmless strains, pathogenic isolates exist; and such strains can be harmful, especially for children but even for fully immunocompetent adults as well as animals (9). Disease-causing, virulent E. coli strains can be divided into two major categories, intestinal and extraintestinal pathogens, with the latter comprising mainly uropathogenic variants and those causing neonatal meningitis. The first category comprises various pathotypes, including enterotoxigenic E. coli, enteropathogenic E. coli, enteroinvasive E. coli, enteroaggregative E. coli, and Shigatoxin producing E. coli (STEC), with the last group including the subgroup of enterohemorrhagic E. coli. In particular, strains in the STEC group have the potential to inflict major damage on different organs, depending on the presence of several virulence factors, and STEC strains are therefore of interest for epidemiological surveillance (3, 6). Some STEC strains have a zoonotic potential, with a reservoir mainly in ruminants (2,15,16).Methods for the diagnosis of E. coli infections include biochemical methods as well as methods that detect specific virulence genes by PCR or hybridization, allowing determination of the pathotype. However, serotyping takes a central place in the differentiation of various pathogenic and nonpathogenic E. coli types, because specific serogroups are consistently associated with certain clinical syndromes (9). Serotyping with the O antigen, a polymerized s...

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Section: Resultsmentioning

confidence: 99%

Fast DNA Serotyping of Escherichia coli by Use of an Oligonucleotide Microarray

et al. 2007

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“…Traditional serotyping requires the use of a large panel of antisera; moreover, it is subjective and cross-reactive (16). In recent years, PCR assays based on O-serotype-specific genes have been proposed by us and others for molecular typing of many Shigella and E. coli O serotypes (2, 3, 7, 8, 10, 11, 12, 15,18,31,34,36).…”

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confidence: 99%

Development of a Serotype-Specific DNA Microarray for Identification of Some Shigella and Pathogenic Escherichia coli Strains

Liú²,

Cao

et al. 2006

J Clin Microbiol

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Shigella and pathogenic Escherichia coli are major causes of human infectious diseases and are responsible for millions of cases of diarrhea worldwide every year. A convenient and rapid method to identify highly pathogenic serotypes of Shigella and E. coli is needed for large-scale epidemiologic study, timely clinical diagnosis, and reliable quarantine of the pathogens. In this study, a DNA microarray targeting O-serotypespecific genes was developed to detect 15 serotypes of Shigella and E. coli, including Shigella sonnei; Shigella flexneri type 2a; Shigella boydii types 7, 9, 13, 16, and 18; Shigella dysenteriae types 4, 8, and 10; and E. coli O55, O111, O114, O128, and O157. The microarray was tested against 186 representative strains of all Shigella and E. coli O serotypes, 38 clinical isolates, and 9 strains of other bacterial species that are commonly present in stool samples and was shown to be specific and reproducible. The detection sensitivity was 50 ng genomic DNA or 10 4 CFU per ml in mock stool specimens. This is the first report of a microarray for serotyping Shigella and pathogenic E. coli. The method has a number of advantages over traditional bacterial culture and antiserum agglutination methods and is promising for applications in basic microbiological research, clinical diagnosis, food safety, and epidemiological surveillance.

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“…The sequences of sugar transferase genes and O-unitprocessing genes are normally specific to a particular O antigen. Specific PCR methods based on O-antigen-specific genes have been proposed for molecular typing of many E. coli and Shigella O serogroups (21,25,26,28,54,(57)(58)(59).…”

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confidence: 99%

Structural and Genetic Characterization of Enterohemorrhagic Escherichia coli O145 O Antigen and Development of an O145 Serogroup-Specific PCR Assay

et al. 2005

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Enterohemorrhagic Escherichia coli O145 strains are emerging as causes of hemorrhagic colitis and hemolytic uremic syndrome. In this study, we present the structure of the E. coli O145 O antigen and the sequence of its gene cluster. The O145 antigen has repeat units containing three monosaccharide residues: 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamidoylamino-2,6-dideoxy-L-galactose, and N-acetylneuraminic acid. It is very closely related to Salmonella enterica serovar Touera and S. enterica subsp. arizonae O21 antigen. The E. coli O145 gene cluster is located between the JUMPStart sequence and the gnd gene and consists of 15 open reading frames. Putative genes for the synthesis of the O-antigen constituents, for sugar transferase, and for O-antigen processing were annotated based on sequence similarities and the presence of conserved regions. The putative genes located in the E. coli O145 O-antigen gene cluster accounted for all functions expected for synthesis of the structure. An E. coli O145 serogroup-specific PCR assay based on the genes wzx and wzy was also developed by screening E. coli and Shigella isolates of different serotypes.

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Sequence of the Escherichia coli O121 O-Antigen Gene Cluster and Detection of Enterohemorrhagic E. coli O121 by PCR Amplification of the wzx and wzy Genes

Cited by 73 publications

References 23 publications

Fast DNA Serotyping of Escherichia coli by Use of an Oligonucleotide Microarray

Fast DNA Serotyping of Escherichia coli by Use of an Oligonucleotide Microarray

Development of a Serotype-Specific DNA Microarray for Identification of Some Shigella and Pathogenic Escherichia coli Strains

Structural and Genetic Characterization of Enterohemorrhagic Escherichia coli O145 O Antigen and Development of an O145 Serogroup-Specific PCR Assay

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