Development of a Multiplex PCR Assay Targeting O-Antigen Modification Genes for Molecular Serotyping of Shigella flexneri

Sun, Qiang; Lan, Ruiting; Wang, Yiting; Zhao, Ailan; Zhang, Shaomin; Wang, Jianping; Wang, Yan; Xia, Shengli; Jin, Dong; Cui, Zhigang; Zhao, Hongqing; Li, Zhenjun; Ye, Changyun; Zhang, Shuxia; Jing, Huaiqi; Xu, Jianguo

doi:10.1128/jcm.01259-11

Cited by 50 publications

(84 citation statements)

References 21 publications

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“…Traditionally, S. flexneri serotyping was performed by antigen-antibody agglutination reactions using in-house antisera to the LPS O-antigen raised in rabbits or commercially available antisera (9). Recently, a multiplex PCR approach to serotyping S. flexneri has been described (6). This PCR assay targets 10 O-antigen synthesis or modification genes: wzx1-5 and wzx6 are the wzx O-antigen synthesis genes specific to S. flexneri serotypes; gtrI, gtrII, gtrIV, gtrV, gtrX, and gtr1c encode serotype-specific glucosyltransferases; oac encodes an O-acetyltransferase that mediates the addition of an acetyl group to a specific sugar in the backbone structure; and opt encodes a phosphoethanolamine transferase involved in adding a phosphoethanolamine residue to the second L-rhamnose residue of the O-antigen polysaccharide backbone (7).…”

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confidence: 99%

“…The aims of this study were to modify the gel-based PCR assay described by Sun et al (6) to a real-time PCR format and to validate the assay by comparing phenotypic-and genotypic-serotyping results. Subsequently, a subset of the cultures used for this validation were whole-genome sequenced.…”

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confidence: 99%

“…The genes that confer O-antigen modification are carried on temperate bacteriophages (5). Bacteriophages infect S. flexneri strains, and the phage-borne genetic material is integrated into the bacterial chromosome through site-specific recombination, resulting in the addition of the O-antigen modification genes (6). Currently, there are 15 well-established S. flexneri serotypes (1a, 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, X, Xv, Y, and F6), which are composed of type (I, II, III, IV, and V)-, group (3:4, 6, and 7:8)-and 1c-specific antigenic determinants (6).…”

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confidence: 99%

“…Bacteriophages infect S. flexneri strains, and the phage-borne genetic material is integrated into the bacterial chromosome through site-specific recombination, resulting in the addition of the O-antigen modification genes (6). Currently, there are 15 well-established S. flexneri serotypes (1a, 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, X, Xv, Y, and F6), which are composed of type (I, II, III, IV, and V)-, group (3:4, 6, and 7:8)-and 1c-specific antigenic determinants (6). The O-antigen modification that results in the addition of phosphoethanolamine (PEtN) to either Rha II or Rha III, mediated by opt, is found in serotypes 4a variant (4av), X variant (Xv), and Y variant (Yv) (7,8).…”

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Evaluation of Molecular Methods for Serotyping Shigella flexneri

et al. 2016

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Shigella flexneri can be phenotypically serotyped using antisera raised to type-specific somatic antigens and group factor antigens and genotypically serotyped using PCR targeting O-antigen synthesis or modification genes. The aim of this study was to evaluate a real-time PCR for serotyping S. flexneri and to use whole-genome sequencing (WGS) to investigate the phenotypic and genotypic serotype identifications. Of the 244 cultures tested retrospectively, 226 (92.6%) had concordant results between phenotypic serotyping and PCR. Seventy of the 244 isolates (including 15 of the 18 isolates where a serotype-PCR mismatch was identified) were whole-genome sequenced, and the serotype was derived from the genome. Discrepant results between the phenotypic and genotypic tests were attributed to insertions/deletions or point mutations identified in O-antigen synthesis or modification genes, rendering them dysfunctional; inconclusive serotyping results due to nonspecific cross-reactions; or novel genotypes. Phylogenetic analysis of the WGS data indicated that the serotype, regardless of whether it was phenotypically or genotypically determined, was a weak predictor of phylogenetic relationships between strains of S. flexneri. WGS data provided both genome-derived serotyping, thus supporting backward compatibility with historical data and facilitating data exchange in the community, and more robust and discriminatory typing at the single-nucleotide-polymorphism level.

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Evaluation of Molecular Methods for Serotyping Shigella flexneri

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“…However, open data or a database on the O-patterns of non-agglutinable Shigella are still lacking. Further analysis of O-antigen gene cassette arrangements using whole-genome sequencing (WGS) could identify the serotype [14].…”

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Molecular diagnosis of non-serotypeable Shigella spp.: problems and prospects

Sethuvel

Ragupathi

Anandan

et al. 2017

Journal of Medical Microbiology

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It is not always possible to identify Shigella serogroups/serotypes by biochemical properties alone. Specific identification requires serotyping. Occasionally, isolates that resemble Shigella spp. biochemically, but are non-agglutinable with available antisera, have been observed. Several mechanisms have been reported to limit the efficiency of the serotyping assay. Serotype conversion is a major mechanism in Shigella spp. to escape protective host immune responses. This easy conversion through significant modification of the O-antigen backbone results in different serotypes, which makes laboratory identification difficult. Furthermore, members of the family Enterobacteriaceae are closely related and there is antigenic cross-over (intra-and inter-specific cross-reaction) which affects the agglutination reaction. The performance of the available methods for identification of non-serotypeable Shigella is discussed here, and reveals them to be non-reliable. This shows a need for an alternative method for identification and typing of Shigella spp. Sir,Shigellae are the primary causative agents of bacterial diarrhoea, representing a significant public health problem, especially in developing countries like India. Shigella spp. belong to the extremely diverse species Escherichia coli. Shigella spp. are still very significant pathogens, particularly where the standard of hygiene is poor. In the 1940s, Shigella strains were put in a different genus from E. coli to distinguish pathogenic forms from the non-pathogenic strains of E. coli and based on their medical significance [1]. Although Shigella was grouped as a subgenus of E. coli in the later 1990s, it is still difficult to distinguish atypical E. coli from non-serotypeable Shigella, where timely diagnosis is essential.Serological identification is the crucial step in diagnosing Shigella infection. Occasionally an isolate will be biochemically indistinguishable from Shigella spp. and non-agglutinable with the standard antisera. Several intra-and inter-specific cross-reactions, morphological transitions from smooth to rough forms and mutations in the O-antigen synthesis/modification genes of Shigella spp. have been reported to limit the efficiency of serotyping assays [2]. The commercially available antisera could not discriminate all possible epitopes of the Shigella O-antigen. In addition, among Shigella, new serotypes/sub-serotypes are not unusual and are reported from different parts of the world [3].Molecular identification of Shigella using 16S rRNA sequencing could not distinguish atypical E. coli from Shigella spp. The sequence similarities of Shigella flexneri, Shigella sonnei and Shigella boydii with E. coli were reported to be 99.8, 99.9 and 99.7 %, respectively. Although studies have been reported that a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS with specialized automated data analysis approach could replace the traditional phenotypic and biochemical methods for microbial identification [4,5], MALDI-TOF MS has severa...

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Discrimination of Escherichia coli, Shigella flexneri, and Shigella sonnei using lipid profiling by MALDI‐TOF mass spectrometry paired with machine learning

et al. 2022

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Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a staple in clinical microbiology laboratories.Protein-profiling of bacteria using this technique has accelerated the identification of pathogens in diagnostic workflows. Recently, lipid profiling has emerged as a way to complement bacterial identification where protein-based methods fail to provide accurate results. This study aimed to address the challenge of rapid discrimination between Escherichia coli and Shigella spp. using MALDI-TOF MS in the negative ion mode for lipid profiling coupled with machine learning. Both E. coli and Shigella species are closely related; they share high sequence homology, reported for 16S rRNA gene sequence similarities between E. coli and Shigella spp. exceeding 99%, and a similar protein expression pattern but are epidemiologically distinct.

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Development of a Multiplex PCR Assay Targeting O-Antigen Modification Genes for Molecular Serotyping of Shigella flexneri

Cited by 50 publications

References 21 publications

Evaluation of Molecular Methods for Serotyping Shigella flexneri

Evaluation of Molecular Methods for Serotyping Shigella flexneri

Molecular diagnosis of non-serotypeable Shigella spp.: problems and prospects

Discrimination of Escherichia coli, Shigella flexneri, and Shigella sonnei using lipid profiling by MALDI‐TOF mass spectrometry paired with machine learning

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