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

2015 ◽  
Vol 53 (8) ◽  
pp. 2427-2432 ◽  
Author(s):  
Atsushi Iguchi ◽  
Sunao Iyoda ◽  
Kazuko Seto ◽  
Tomoko Morita-Ishihara ◽  
Flemming Scheutz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Low Cost ◽  
Epidemiological Studies ◽  
Biosynthesis Gene Cluster ◽  
Content Type ◽  
E Coli ◽  
O Antigen ◽  
Promising Tool ◽  
Outbreak Investigations ◽  
Wild Strains

The O serogrouping of pathogenicEscherichia coliis a standard method for subtyping strains for epidemiological studies and enhancing phylogenetic studies. In particular, the identification of strains of the same O serogroup is essential in outbreak investigations and surveillance. In a previous study, we analyzed the O-antigen biosynthesis gene cluster in all knownE. coliO serogroups (A. Iguchi et al., DNA Res, 22:101–107, 2015,http://dx.doi.org/10.1093/dnares/dsu043). Based on those results, we have arranged 162 PCR primer pairs for the identification or classification of O serogroups. Of these, 147 pairs were used to identify 147 individual O serogroups with unique O-antigen biosynthesis genes, and the other 15 pairs were used to identify 15 groups of strains (Gp1 to Gp15). Each of these groups consisted of strains with identical or very similar O-antigen biosynthesis genes, and the groups represented a total of 35 individual O serogroups. We then used the 162 primer pairs to create 20 multiplex PCR sets. Each set contained six to nine primer pairs that amplify products of markedly different sizes. This genetic methodology (E. coliO-genotyping PCR) allowed for comprehensive, rapid, and low-cost typing. Validation of the PCR system using O-serogroup references and wild strains showed that the correct O serogroups were specifically and accurately identified for 100% (182/182) and 90.8% (522/575) of references and wild strains, respectively. The PCR-based system reported here might be a promising tool for the subtyping ofE. colistrains for epidemiological studies as well as for the surveillance of pathogenicE. coliduring outbreaks.

scholarly journals Escherichia coliH-Genotyping PCR: a Complete and Practical Platform for Molecular H Typing

2018 ◽  
Vol 56 (6) ◽  
Author(s):  
Masaya Banjo ◽  
Atsushi Iguchi ◽  
Kazuko Seto ◽  
Taisei Kikuchi ◽  
Tetsuya Harada ◽  
...  
Keyword(s):  
Low Cost ◽  
Epidemiological Studies ◽  
Pathogenic Potential ◽  
Content Type ◽  
E Coli ◽  
Outbreak Investigations ◽  
Pcr Products ◽  
Wild Strains ◽  
Encoding Genes ◽  
Reference Strains

ABSTRACTInEscherichia coli, more than 180 O groups and 53 H types have been recognized. The O:H serotyping ofE. colistrains is an effective method for identifying strains with pathogenic potential and classifying them into clonal groups. In particular, the serotyping of Shiga toxin-producingE. coli(STEC) strains provides valuable information to evaluate the routes, sources, and prevalence of agents in outbreak investigations and surveillance. Here, we present a complete and practical PCR-based H-typing system,E. coliH-genotyping PCR, consisting of 10 multiplex PCR kits with 51 single PCR primer pairs. Primers were designed based on a detailed comparative analysis of sequences from all H-antigen (flagellin)-encoding genes,fliCand its homologs. The specificity of this system was confirmed by using all H type reference strains. Additionally, 362 serotyped wild strains were also used to evaluate its practicality. All 277 H-type-identified isolates gave PCR products that corresponded to the results of serological H typing. Moreover, 76 nonmotile and nine untypeable strains could be successfully subtyped into any H type by the PCR system. TheE. coliH-genotyping PCR developed here allows broader, rapid, and low-cost subtyping of H types and will assist epidemiological studies as well as surveillance of pathogenicE. coli.

scholarly journals Additional Og-Typing PCR Techniques Targeting Escherichia coli-Novel and Shigella-Unique O-Antigen Biosynthesis Gene Clusters

2020 ◽  
Vol 58 (11) ◽  
Author(s):  
Atsushi Iguchi ◽  
Hironobu Nishii ◽  
Kazuko Seto ◽  
Jiro Mitobe ◽  
Kenichi Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Strong Association ◽  
Gene Clusters ◽  
Epidemiological Studies ◽  
Content Type ◽  
E Coli ◽  
O Antigen ◽  
Wide Range ◽  
Biosynthesis Gene ◽  
Almost All

ABSTRACT The O-serogrouping of pathogenic Escherichia coli is a standard method for subtyping strains for epidemiological studies and controls. O-serogroup diversification shows a strong association with the genetic diversity in some O-antigen biosynthesis gene clusters. Through genomic studies, in addition to the types of O-antigen biosynthesis gene clusters (Og-types) from conventional O-serogroup strains, a number of novel Og-types have been found in E. coli isolates. To assist outbreak investigations and surveillance of pathogenic E. coli at inspection institutes, in previous studies, we developed PCR methods that could determine almost all conventional O-serogroups and some novel Og-types. However, there are still many Og-types that may not be determined by simple genetic methods such as PCR. Thus, in the present study, we aimed to develop an additional Og-typing PCR system. Based on the novel Og-types, including OgN32, OgN33, and OgN34, presented in this study, we designed an additional 24 PCR primer pairs targeting 14 novel and 2 diversified E. coli Og-types and 8 Shigella-unique Og-types. Subsequently, we developed 5 new multiplex PCR sets consisting of 33 primers, including the aforementioned 24 primers and 9 primers reported in previous studies. The accuracy and specificity of the PCR system was validated using approximately 260 E. coli and Shigella O-serogroup and Og-type reference strains. The Og-typing PCR system reported here can determine a wide range of Og-types of E. coli and may help epidemiological studies, in addition to the surveillance of pathogenic E. coli.

scholarly journals Rapid Identification of Different Escherichia coli Sequence Type 131 Clades

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Yasufumi Matsumura ◽  
Johann D. D. Pitout ◽  
Gisele Peirano ◽  
Rebekah DeVinney ◽  
Taro Noguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epidemiological Studies ◽  
Rapid Identification ◽  
Sequence Type ◽  
Fluoroquinolone Resistance ◽  
Pcr Assay ◽  
Content Type ◽  
E Coli ◽  
Multiplex Pcr Assay ◽  
Clade C

ABSTRACT Escherichia coli sequence type 131 (ST131) is a pandemic clonal lineage that is responsible for the global increase in fluoroquinolone resistance and extended-spectrum-β-lactamase (ESBL) producers. The members of ST131 clade C, especially subclades C2 and C1-M27, are associated with ESBLs. We developed a multiplex conventional PCR assay with the ability to detect all ST131 clades (A, B, and C), as well as C subclades (C1-M27, C1-nM27 [C1-non-M27], and C2). To validate the assay, we used 80 ST131 global isolates that had been fully sequenced. We then used the assay to define the prevalence of each clade in two Japanese collections consisting of 460 ESBL-producing E. coli ST131 (2001-12) and 329 E. coli isolates from extraintestinal sites (ExPEC) (2014). The assay correctly identified the different clades in all 80 global isolates: clades A (n = 12), B (n = 12), and C, including subclades C1-M27 (n = 16), C1-nM27 (n = 20), C2 (n = 17), and other C (n = 3). The assay also detected all 565 ST131 isolates in both collections without any false positives. Isolates from clades A (n = 54), B (n = 23), and C (n = 483) corresponded to the O serotypes and the fimH types of O16-H41, O25b-H22, and O25b-H30, respectively. Of the 483 clade C isolates, C1-M27 was the most common subclade (36%), followed by C1-nM27 (32%) and C2 (15%). The C1-M27 subclade with bla CTX-M-27 became especially prominent after 2009. Our novel multiplex PCR assay revealed the predominance of the C1-M27 subclade in recent Japanese ESBL-producing E. coli isolates and is a promising tool for epidemiological studies of ST131.

scholarly journals Variations in O-Antigen Biosynthesis and O-Acetylation Associated with Altered Phage Sensitivity in Escherichia coli 4s

2014 ◽  
Vol 197 (5) ◽  
pp. 905-912 ◽  
Author(s):  
Yuriy A. Knirel ◽  
Nikolai S. Prokhorov ◽  
Alexander S. Shashkov ◽  
Olga G. Ovchinnikova ◽  
Evelina L. Zdorovenko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Cells ◽  
Side Chain ◽  
Gram Negative Bacteria ◽  
Gene Encoding ◽  
Content Type ◽  
Surface Receptors ◽  
E Coli ◽  
O Antigen ◽  
Phage Sensitivity

The O polysaccharide of the lipopolysaccharide (O antigen) of Gram-negative bacteria often serves as a receptor for bacteriophages that can make the phage dependent on a given O-antigen type, thus supporting the concept of the adaptive significance of the O-antigen variability in bacteria. The O-antigen layer also modulates interactions of many bacteriophages with their hosts, limiting the access of the viruses to other cell surface receptors. Here we report variations of O-antigen synthesis and structure in an environmentalEscherichia coliisolate, 4s, obtained from horse feces, and its mutants selected for resistance to bacteriophage G7C, isolated from the same fecal sample. The 4s O antigen was found to be serologically, structurally, and genetically related to the O antigen ofE. coliO22, differing only in side-chain α-d-glucosylation in the former, mediated by agtrlocus on the chromosome. Spontaneous mutations ofE. coli4s occurring with an unusually high frequency affected either O-antigen synthesis or O-acetylation due to the inactivation of the gene encoding the putative glycosyltransferase WclH or the putative acetyltransferase WclK, respectively, by the insertion of IS1-like elements. These mutations induced resistance to bacteriophage G7C and also modified interactions ofE. coli4s with several other bacteriophages conferring either resistance or sensitivity to the host. These findings suggest that O-antigen synthesis and O-acetylation can both ensure the specific recognition of the O-antigen receptor following infection by some phages and provide protection of the host cells against attack by other phages.

scholarly journals O-Antigen-Dependent Colicin Insensitivity of UropathogenicEscherichia coli

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Connor Sharp ◽  
Christine Boinett ◽  
Amy Cain ◽  
Nicholas G. Housden ◽  
Sandip Kumar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Growth Conditions ◽  
Membrane Receptors ◽  
Cell Surface Receptor ◽  
Dominant Factor ◽  
Content Type ◽  
E Coli ◽  
O Antigen ◽  
K 12

ABSTRACTThe outer membrane of Gram-negative bacteria presents a significant barrier for molecules entering the cell. Nevertheless, colicins, which are antimicrobial proteins secreted byEscherichia coli, can target otherE. colicells by binding to cell surface receptor proteins and activating their import, resulting in cell death. Previous studies have documented high rates of nonspecific resistance (insensitivity) of variousE. colistrains toward colicins that is independent of colicin-specific immunity and is instead associated with lipopolysaccharide (LPS) in the outer membrane. This observation poses a contradiction: why doE. colistrains have colicin-expressing plasmids, which are energetically costly to retain, if cells around them are likely to be naturally insensitive to the colicin they produce? Here, using a combination of transposon sequencing and phenotypic microarrays, we show that colicin insensitivity of uropathogenicE. colisequence type 131 (ST131) is dependent on the production of its O-antigen but that minor changes in growth conditions render the organism sensitive toward colicins. The reintroduction of O-antigen intoE. coliK-12 demonstrated that it is the density of O-antigen that is the dominant factor governing colicin insensitivity. We also show, by microscopy of fluorescently labelled colicins, that growth conditions affect the degree of occlusion by O-antigen of outer membrane receptors but not the clustered organization of receptors. The result of our study demonstrate that environmental conditions play a critical role in sensitizingE. colitoward colicins and that O-antigen in LPS is central to this role.IMPORTANCEEscherichia coliinfections can be a major health burden, especially with the organism becoming increasingly resistant to “last-resort” antibiotics such as carbapenems. Although colicins are potent narrow-spectrum antimicrobials with potential as future antibiotics, high levels of naturally occurring colicin insensitivity have been documented which could limit their efficacy. We identify O-antigen-dependent colicin insensitivity in a clinically relevant uropathogenicE. colistrain and show that this insensitivity can be circumvented by minor changes to growth conditions. The results of our study suggest that colicin insensitivity amongE. coliorganisms has been greatly overestimated, and as a consequence, colicins could in fact be effective species-specific antimicrobials targeting pathogenicE. colisuch as uropathogenicE. coli(UPEC).

scholarly journals Distribution of novel Og-types in Shiga toxin-producing Escherichia coli isolated from healthy cattle

2020 ◽  
pp. JCM.02624-20
Author(s):  
Nguyen Thi Thu Huong ◽  
Atsushi Iguchi ◽  
Ritsuko Ohata ◽  
Hisahiro Kawai ◽  
Tadasuke Ooka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Foodborne Pathogen ◽  
Genomic Analysis ◽  
Epidemiological Studies ◽  
Biosynthesis Gene Cluster ◽  
E Coli ◽  
Shigella Boydii ◽  
Healthy Cattle ◽  
Type Strains

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen. Although most cases of STEC infection in humans are due to O157 and non-O157 serogroups, there are also reports of infection with STEC strains that cannot be serologically classified into any O-serogroup (O-serogroup untypeable, OUT). Recently, it has become clear that even OUT strains can be subclassified based on the diversity of O-antigen biosynthesis gene cluster (O-AGC) sequences. Cattle are thought to be a major reservoir of STEC strains belonging to various serotypes; however, the internal composition of OUT STEC strains in cattle remains unknown. In this study, we screened 366 STEC strains isolated from healthy cattle by using multiplex PCR kits including primers that targeted novel O-AGC types (Og-types) found in OUT E. coli and Shigella strains in previous studies. Interestingly, 94 (25.7%) of these strains could be classified into 13 novel Og-types. Genomic analysis revealed that the results of the in silico serotyping of novel Og-type strains were perfectly consistent with those of the PCR experiment. In addition, it was revealed that a dual Og8+OgSB17-type strain carried two types of O-AGCs from E. coli O8 and Shigella boydii type 17 tandemly inserted at the locus, with both antigens expressed on the cell surface. The results of this comprehensive analysis of cattle-derived STEC strains may help improve our understanding of the strains circulating in the environment. Additionally, the DNA-based serotyping systems used in this study could be used in future epidemiological studies and risk assessments of other STEC strains.

scholarly journals Shielding Effect of Escherichia coli O-Antigen Polysaccharide on J5-Induced Cross-Reactive Antibodies

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Pascal Rainard ◽  
Maryline Repérant-Ferter ◽  
Christophe Gitton ◽  
Pierre Germon
Keyword(s):  
Escherichia Coli ◽  
Vaccine Development ◽  
Dairy Farms ◽  
Shielding Effect ◽  
Coliform Bacteria ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cell Mediated Immunity ◽  
Content Type ◽  
E Coli ◽  
O Antigen

ABSTRACT Escherichia coli is the leading cause of severe mastitis in dairy farms. As E. coli mastitis is refractory to the hygienic control measures adapted to contagious mastitis, efficient vaccines are in demand. Existing mastitis vaccines, based on the use of killed rough E. coli J5 as the antigen, aim at inducing phagocytosis by neutrophils. We assessed the binding of J5-induced antibodies to isogenic rough and smooth strains along with a panel of mastitis-associated E. coli. Analysis by enzyme-linked immunosorbent assay revealed that antibodies to OmpA or killed J5 bind readily to rough E. coli but poorly to smooth strains. Flow cytometry analysis indicated that immunization with J5 induced antibodies that cross-reacted with rough E. coli strains but with only a small subpopulation of smooth strains. We identified type 1 fimbriae as the target of most antibodies cross-reacting with the smooth strains. These results suggest that the O-polysaccharide of lipopolysaccharide shields the outer membrane antigens and that only fiber antigens protruding at the bacterial surface can elicit antibodies reacting with mastitis-associated E. coli. We evaluated J5-induced antibodies in an opsonophagocytic killing assay with bovine neutrophils. J5 immune serum was not more efficient than preimmune serum, showing that immunization did not improve on the already high efficiency of naturally acquired antibodies to E. coli. In conclusion, it is unlikely that the efficiency of J5 vaccines is related to the induction of opsonic antibodies. Consequently, other research directions, such as cell-mediated immunity, should be explored to improve E. coli mastitis vaccines. IMPORTANCE Despite intensive research, mastitis remains an important disease in dairy cattle with a significant impact on animal welfare, use of antibiotics, and, in the end, the economy of dairy farms. Although vaccines available so far have shown limited efficacy against coliform mastitis, vaccination is considered one of the measures that could limit the consequences of mastitis. One reason for the lack of efficiency of current vaccines likely stems from the current evaluation of vaccines that relies mostly on measuring antibody production against vaccine antigens. This report clearly shows that vaccine-induced antibodies fail to bind to most mastitis-associated E. coli strains because of the presence of an O-antigen and, thus, do not allow for improved phagocytosis of pathogens. As a consequence, this report calls for revised criteria for the evaluation of vaccines and suggests that cell-mediated immunity should be targeted by new vaccinal strategies. More generally, these results could be extended to other vaccine development strategies targeting coliform bacteria.

scholarly journals Rapid Identification of Major Escherichia coli Sequence Types Causing Urinary Tract and Bloodstream Infections

2014 ◽  
Vol 53 (1) ◽  
pp. 160-166 ◽  
Author(s):  
M. Doumith ◽  
M. Day ◽  
H. Ciesielczuk ◽  
R. Hope ◽  
A. Underwood ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Bloodstream Infections ◽  
Epidemiological Studies ◽  
Rapid Identification ◽  
Content Type ◽  
E Coli ◽  
Genes Encoding ◽  
Signature Sequences ◽  
Sequence Types

Escherichia colisequence types (STs) 69, 73, 95, and 131 are collectively responsible for a large proportion ofE. coliurinary tract and bloodstream infections, and they differ markedly in their antibiotic susceptibilities. Here, we describe a novel PCR method to rapidly detect and distinguish these lineages. Three hundred eighteen publishedE. coligenomes were compared in order to identify signature sequences unique to each of the four major STs. The specificities of these sequences were assessedin silicoby seeking them in an additional 98 genomes. A PCR assay was designed to amplify size-distinguishable fragments unique to the four lineages and was validated using 515E. coliisolates of known STs. Genome comparisons identified 22 regions ranging in size from 335 bp to 26.5 kb that are unique to one or more of the four predominantE. coliSTs, with two to 10 specific regions per ST. These regions predominantly harbor genes encoding hypothetical proteins and are within or adjacent to prophage sequences. Most (13/22) were highly conserved (>96.5% identity) in the genomes of their respective ST. The new assay correctly identified all 142 representatives of the four major STs in the validation set (n= 515), with only two ST12 isolates misidentified as ST95. Compared with MLST, the assay has 100% sensitivity and 99.5% specificity. The rapid identification of major extraintestinalE. coliSTs will benefit future epidemiological studies and could be developed to tailor antibiotic therapy to the different susceptibilities of these dominant lineages.

scholarly journals Diagnostic Potential of Monoclonal Antibodies Specific to the Unique O-Antigen of Multidrug-Resistant Epidemic Escherichia coli Clone ST131-O25b:H4

2014 ◽  
Vol 21 (7) ◽  
pp. 930-939 ◽  
Author(s):  
Valéria Szijártó ◽  
Jolanta Lukasiewicz ◽  
Tomasz K. Gozdziewicz ◽  
Zoltán Magyarics ◽  
Eszter Nagy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Multidrug Resistant ◽  
Content Type ◽  
E Coli ◽  
Medical Needs ◽  
O Antigen ◽  
Novel Structure ◽  
Diagnostic Potential ◽  
Murine Monoclonal Antibodies

ABSTRACTTheEscherichia colilineage sequence type 131 (ST131)-O25b:H4 is a globally spread multidrug-resistant clone responsible for a great proportion of extraintestinal infections. Driven by the significant medical needs associated with this successful pathogenic lineage, we generated murine monoclonal antibodies (MAbs) against its lipopolysaccharide (LPS) O25b antigen in order to develop quick diagnostic tests. Murine monoclonal antibodies were generated by immunizing mice with whole killed nonencapsulated ST131-O25bE. colicells and screening hybridoma supernatants for binding to purified LPS molecules obtained from anE. coliST131-O25b clinical isolate. The MAbs selected for further study bound to the surface of liveE. coliO25b strains irrespective of the capsular type expressed, while they did not bind to bacteria or purified LPS from other serotypes, including the related classical O25 antigen (O25a). Using these specific MAbs, we developed a latex bead-based agglutination assay that has greater specificity and is quicker and simpler than the currently available typing methods. The high specificities of these MAbs can be explained by the novel structure of the O25b repeating unit elucidated in this article. Based on comparative analysis by nuclear magnetic resonance (NMR) and mass spectrometry, theN-acetyl-fucose in the O25a O-antigen had been replaced byO-acetyl-rhamnose in the O25b repeating unit. The genetic determinants responsible for this structural variation were identified by aligning the corresponding genetic loci and were confirmed bytrans-complementation of a rough mutant by the subserotype-specific fragments of therfboperons.

scholarly journals Rapid and EasyIn SilicoSerotyping of Escherichia coli Isolates by Use of Whole-Genome Sequencing Data

2015 ◽  
Vol 53 (8) ◽  
pp. 2410-2426 ◽  
Author(s):  
Katrine G. Joensen ◽  
Anna M. M. Tetzschner ◽  
Atsushi Iguchi ◽  
Frank M. Aarestrup ◽  
Flemming Scheutz
Keyword(s):  
Escherichia Coli ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Antigen Processing ◽  
Processing System ◽  
Outbreak Detection ◽  
Whole Genome ◽  
Content Type ◽  
E Coli ◽  
O Antigen

Accurate and rapid typing of pathogens is essential for effective surveillance and outbreak detection. Conventional serotyping ofEscherichia coliis a delicate, laborious, time-consuming, and expensive procedure. With whole-genome sequencing (WGS) becoming cheaper, it has vast potential in routine typing and surveillance. The aim of this study was to establish a valid and publicly available tool for WGS-basedin silicoserotyping ofE. coliapplicable for routine typing and surveillance. A FASTA database of specific O-antigen processing system genes for O typing and flagellin genes for H typing was created as a component of the publicly available Web tools hosted by the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org). AllE. coliisolates available with WGS data and conventional serotype information were subjected to WGS-based serotyping employing this specific SerotypeFinder CGE tool. SerotypeFinder was evaluated on 682E. coligenomes, 108 of which were sequenced for this study, where both the whole genome and the serotype were available. In total, 601 and 509 isolates were included for O and H typing, respectively. The O-antigen geneswzx,wzy,wzm, andwztand the flagellin genesfliC,flkA,fllA,flmA, andflnAwere detected in 569 and 508 genome sequences, respectively. SerotypeFinder for WGS-based O and H typing predicted 560 of 569 O types and 504 of 508 H types, consistent with conventional serotyping. In combination with other available WGS typing tools,E. coliserotyping can be performed solely from WGS data, providing faster and cheaper typing than current routine procedures and making WGS typing a superior alternative to conventional typing strategies.

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