Subspecies characterization of urease-positive thermophilic Campylobacter (UPTC) isolated from shellfish employing modified flagellin (flaA) restriction fragment length polymorphism (RFLP) typing

John E. Moore, Motoo Matsuda, Tsuyoshi Sekizuka, Seamus Fanning, Ohoshi Murayama, Taeko Yokoi, Shizuko Kagawa, Kentaro Nagano, Hiromi Shimura, Miyuki Watabe, Yuriko Nagano, Kaori Usui, Rie Imamaki, Takao Aritomi, Chisato Harada, Haruna Iida, Naomi Mitsuhashi, Takeshi Miyatake, Makoto Shigematsu, Juluri R. Rao & 4 others Colm Lowery, B. Cherie Millar, James Dooley, Paul J. Rooney

Research output: Contribution to journalArticle

Abstract

Shellfish including oysters (Crassostrea gigas), cockles (Cerastoderma edule) and mussels (Mytilus edulis), have previously been described as an important source of thermophilic campylobacters, with the potential of causing acute bacterial gastroenteritis in humans. Previous genotyping studies employing the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) typing, based on the flagellin (flaA) gene have been unable to generate an amplicon for the urease-positive thermophilic Campylobacter (UPTC), which are the predominant taxa associated with shellfish, largely caused by sequence diversity between the UPTC group and C. jejuni. Hence the aim of this study was to develop a modified PCR-RFLP genotyping assay, employing polymorphisms within the flagellin (flaA) gene of UPTC organisms, which would now allow the successful amplification and typing of previously nontypable UPTC isolates obtained from natural marine environments. A novel primer pair (UPTC flaF/UPTC flaR) was designed based on conserved regions within the flaA gene locus of UPTC organisms to generate a 1,358 bp amplicon for all UPTC organisms tested. RFLP analysis with DdeI in combination with computational analysis of genetic relatedness using BioNumerics software demonstrated the presence of four distinct flaA genotypes, among the seven UPTC isolates. In conclusion, this study describes a PCR-RFLP method, based on modified primers from UPTC flaA gene sequences that may be successfully applied to examine subspecies relatedness of UPTC organisms from natural environments, including shellfish.
LanguageEnglish
Pages625-629
JournalJournal of Shellfish Research
Volume25
Issue number2
Publication statusPublished - Aug 2006

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flagellin
urease
Campylobacter
shellfish
restriction fragment length polymorphism
polymerase chain reaction
organisms
genotyping
Cerastoderma edule
genes
Crassostrea gigas
gastroenteritis
Campylobacter jejuni
Mytilus edulis
marine environment
oysters
genetic relationships
mussels

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Moore, John E. ; Matsuda, Motoo ; Sekizuka, Tsuyoshi ; Fanning, Seamus ; Murayama, Ohoshi ; Yokoi, Taeko ; Kagawa, Shizuko ; Nagano, Kentaro ; Shimura, Hiromi ; Watabe, Miyuki ; Nagano, Yuriko ; Usui, Kaori ; Imamaki, Rie ; Aritomi, Takao ; Harada, Chisato ; Iida, Haruna ; Mitsuhashi, Naomi ; Miyatake, Takeshi ; Shigematsu, Makoto ; Rao, Juluri R. ; Lowery, Colm ; Millar, B. Cherie ; Dooley, James ; Rooney, Paul J. / Subspecies characterization of urease-positive thermophilic Campylobacter (UPTC) isolated from shellfish employing modified flagellin (flaA) restriction fragment length polymorphism (RFLP) typing. 2006 ; Vol. 25, No. 2. pp. 625-629.
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title = "Subspecies characterization of urease-positive thermophilic Campylobacter (UPTC) isolated from shellfish employing modified flagellin (flaA) restriction fragment length polymorphism (RFLP) typing",
abstract = "Shellfish including oysters (Crassostrea gigas), cockles (Cerastoderma edule) and mussels (Mytilus edulis), have previously been described as an important source of thermophilic campylobacters, with the potential of causing acute bacterial gastroenteritis in humans. Previous genotyping studies employing the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) typing, based on the flagellin (flaA) gene have been unable to generate an amplicon for the urease-positive thermophilic Campylobacter (UPTC), which are the predominant taxa associated with shellfish, largely caused by sequence diversity between the UPTC group and C. jejuni. Hence the aim of this study was to develop a modified PCR-RFLP genotyping assay, employing polymorphisms within the flagellin (flaA) gene of UPTC organisms, which would now allow the successful amplification and typing of previously nontypable UPTC isolates obtained from natural marine environments. A novel primer pair (UPTC flaF/UPTC flaR) was designed based on conserved regions within the flaA gene locus of UPTC organisms to generate a 1,358 bp amplicon for all UPTC organisms tested. RFLP analysis with DdeI in combination with computational analysis of genetic relatedness using BioNumerics software demonstrated the presence of four distinct flaA genotypes, among the seven UPTC isolates. In conclusion, this study describes a PCR-RFLP method, based on modified primers from UPTC flaA gene sequences that may be successfully applied to examine subspecies relatedness of UPTC organisms from natural environments, including shellfish.",
author = "Moore, {John E.} and Motoo Matsuda and Tsuyoshi Sekizuka and Seamus Fanning and Ohoshi Murayama and Taeko Yokoi and Shizuko Kagawa and Kentaro Nagano and Hiromi Shimura and Miyuki Watabe and Yuriko Nagano and Kaori Usui and Rie Imamaki and Takao Aritomi and Chisato Harada and Haruna Iida and Naomi Mitsuhashi and Takeshi Miyatake and Makoto Shigematsu and Rao, {Juluri R.} and Colm Lowery and Millar, {B. Cherie} and James Dooley and Rooney, {Paul J.}",
year = "2006",
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Moore, JE, Matsuda, M, Sekizuka, T, Fanning, S, Murayama, O, Yokoi, T, Kagawa, S, Nagano, K, Shimura, H, Watabe, M, Nagano, Y, Usui, K, Imamaki, R, Aritomi, T, Harada, C, Iida, H, Mitsuhashi, N, Miyatake, T, Shigematsu, M, Rao, JR, Lowery, C, Millar, BC, Dooley, J & Rooney, PJ 2006, 'Subspecies characterization of urease-positive thermophilic Campylobacter (UPTC) isolated from shellfish employing modified flagellin (flaA) restriction fragment length polymorphism (RFLP) typing', vol. 25, no. 2, pp. 625-629.

Subspecies characterization of urease-positive thermophilic Campylobacter (UPTC) isolated from shellfish employing modified flagellin (flaA) restriction fragment length polymorphism (RFLP) typing. / Moore, John E.; Matsuda, Motoo; Sekizuka, Tsuyoshi; Fanning, Seamus; Murayama, Ohoshi; Yokoi, Taeko; Kagawa, Shizuko; Nagano, Kentaro; Shimura, Hiromi; Watabe, Miyuki; Nagano, Yuriko; Usui, Kaori; Imamaki, Rie; Aritomi, Takao; Harada, Chisato; Iida, Haruna; Mitsuhashi, Naomi; Miyatake, Takeshi; Shigematsu, Makoto; Rao, Juluri R.; Lowery, Colm; Millar, B. Cherie; Dooley, James; Rooney, Paul J.

Vol. 25, No. 2, 08.2006, p. 625-629.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Subspecies characterization of urease-positive thermophilic Campylobacter (UPTC) isolated from shellfish employing modified flagellin (flaA) restriction fragment length polymorphism (RFLP) typing

AU - Moore, John E.

AU - Matsuda, Motoo

AU - Sekizuka, Tsuyoshi

AU - Fanning, Seamus

AU - Murayama, Ohoshi

AU - Yokoi, Taeko

AU - Kagawa, Shizuko

AU - Nagano, Kentaro

AU - Shimura, Hiromi

AU - Watabe, Miyuki

AU - Nagano, Yuriko

AU - Usui, Kaori

AU - Imamaki, Rie

AU - Aritomi, Takao

AU - Harada, Chisato

AU - Iida, Haruna

AU - Mitsuhashi, Naomi

AU - Miyatake, Takeshi

AU - Shigematsu, Makoto

AU - Rao, Juluri R.

AU - Lowery, Colm

AU - Millar, B. Cherie

AU - Dooley, James

AU - Rooney, Paul J.

PY - 2006/8

Y1 - 2006/8

N2 - Shellfish including oysters (Crassostrea gigas), cockles (Cerastoderma edule) and mussels (Mytilus edulis), have previously been described as an important source of thermophilic campylobacters, with the potential of causing acute bacterial gastroenteritis in humans. Previous genotyping studies employing the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) typing, based on the flagellin (flaA) gene have been unable to generate an amplicon for the urease-positive thermophilic Campylobacter (UPTC), which are the predominant taxa associated with shellfish, largely caused by sequence diversity between the UPTC group and C. jejuni. Hence the aim of this study was to develop a modified PCR-RFLP genotyping assay, employing polymorphisms within the flagellin (flaA) gene of UPTC organisms, which would now allow the successful amplification and typing of previously nontypable UPTC isolates obtained from natural marine environments. A novel primer pair (UPTC flaF/UPTC flaR) was designed based on conserved regions within the flaA gene locus of UPTC organisms to generate a 1,358 bp amplicon for all UPTC organisms tested. RFLP analysis with DdeI in combination with computational analysis of genetic relatedness using BioNumerics software demonstrated the presence of four distinct flaA genotypes, among the seven UPTC isolates. In conclusion, this study describes a PCR-RFLP method, based on modified primers from UPTC flaA gene sequences that may be successfully applied to examine subspecies relatedness of UPTC organisms from natural environments, including shellfish.

AB - Shellfish including oysters (Crassostrea gigas), cockles (Cerastoderma edule) and mussels (Mytilus edulis), have previously been described as an important source of thermophilic campylobacters, with the potential of causing acute bacterial gastroenteritis in humans. Previous genotyping studies employing the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) typing, based on the flagellin (flaA) gene have been unable to generate an amplicon for the urease-positive thermophilic Campylobacter (UPTC), which are the predominant taxa associated with shellfish, largely caused by sequence diversity between the UPTC group and C. jejuni. Hence the aim of this study was to develop a modified PCR-RFLP genotyping assay, employing polymorphisms within the flagellin (flaA) gene of UPTC organisms, which would now allow the successful amplification and typing of previously nontypable UPTC isolates obtained from natural marine environments. A novel primer pair (UPTC flaF/UPTC flaR) was designed based on conserved regions within the flaA gene locus of UPTC organisms to generate a 1,358 bp amplicon for all UPTC organisms tested. RFLP analysis with DdeI in combination with computational analysis of genetic relatedness using BioNumerics software demonstrated the presence of four distinct flaA genotypes, among the seven UPTC isolates. In conclusion, this study describes a PCR-RFLP method, based on modified primers from UPTC flaA gene sequences that may be successfully applied to examine subspecies relatedness of UPTC organisms from natural environments, including shellfish.

M3 - Article

VL - 25

SP - 625

EP - 629

IS - 2

ER -