Genotyping<i>Campylobacter jejuni</i>by Comparative Genome Indexing: An Evaluation with Pulsed-Field Gel Electrophoresis and<i>flaA</i>SVR Sequencing

Pittenger, Lauren G; Englen, Mark D.; Parker, Craig T.; Frye, Jonathan G.; Quiñones, Beatriz; Horn, Sharon T.; Son, Injoon; Fedorka–Cray, Paula J.; Harrison, Mark A.

doi:10.1089/fpd.2008.0185

Cited by 5 publications

(4 citation statements)

References 41 publications

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“…The flagellin gene has been a well-accepted marker for Campylobacter genotyping for over 2 decades (4,16,18,19,22,25,32). In this study we have developed a genotyping method based on flaA interrogation using HRM analysis.…”

Section: Discussionmentioning

confidence: 99%

Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment

Merchant-Patel

Blackall²,

Templeton³

et al. 2010

Appl Environ Microbiol

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The highly variable flagellin-encoding flaA gene has long been used for genotyping Campylobacter jejuni and Campylobacter coli. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The objective of this study was to apply HRM analysis to flaA-based genotyping. The initial aim was to identify a suitable flaA fragment. It was found that the PCR primers commonly used to amplify the flaA short variable repeat (SVR) yielded a mixed PCR product unsuitable for HRM analysis. However, a PCR primer set composed of the upstream primer used to amplify the fragment used for flaA restriction fragment length polymorphism (RFLP) analysis and the downstream primer used for flaA SVR amplification generated a very pure PCR product, and this primer set was used for the remainder of the study. Eighty-seven C. jejuni and 15 C. coli isolates were analyzed by flaA HRM and also partial flaA sequencing. There were 47 flaA sequence variants, and all were resolved by HRM analysis. The isolates used had previously also been genotyped using single-nucleotide polymorphisms (SNPs), binary markers, CRISPR HRM, and flaA RFLP. flaA HRM analysis provided resolving power multiplicative to the SNPs, binary markers, and CRISPR HRM and largely concordant with the flaA RFLP. It was concluded that HRM analysis is a promising approach to genotyping based on highly variable genes.Campylobacter jejuni and Campylobacter coli are the most common causes of human bacterial gastroenteritis in industrialized countries (21). The flagellin-encoding genes flaA and flaB share 95% sequence homology and are arranged in tandem (9,20). While flaA gene expression appears critical for motility, colonization, and pathogenesis, this is not the case for flaB, which is thought to be a largely nonfunctional reservoir of genetic variation that can increase the diversity of flaA by recombination and so assist the cell in evading host immune responses (1,7,8,28).The flaA gene is commonly used for typing C. jejuni and C. coli. Two methods have gained wide acceptance: flaA restriction fragment length polymorphism (RFLP) (19) and flaA short variable region (SVR) sequencing (16). The flaA RFLP technique involves PCR amplification of the entire flaA gene followed by RFLP of the PCR product (19). Sequencing the SVR of the flaA gene was developed as a more streamlined and portable alternative to flaA RFLP protocols (16), and sequence variants are compiled at a central website (http://pubmlst.org/campylobacter /flaA/) (10). While both of these methods are very effective, they have several disadvantages: the RFLP approach is multistep, because the PCR product must be cleaved with a restriction enzyme, and the fragments must be subsequently resolved by electrophoresis (33). Also, there are many changes in the sequence that will not alter the sizes of the restriction fragments. SVR sequencing is also multistep; the targeted region is small, which limits resolving power; and DNA sequencing requires expensive equipment ...

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Section: Discussionmentioning

confidence: 99%

Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment

Merchant-Patel

Blackall²,

Templeton³

et al. 2010

Appl Environ Microbiol

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“…Despite these limitations, CGI has been important for investigating the genomic diversity of C. jejuni (Pearson et al, 2003;Taboada et al, 2004;Parker et al, 2006). As a genotyping tool, CGI provides isolatespecific information on a genome-wide scale (Leonard et al, 2003;Pittenger et al, 2009). But as Taboada et al (2004) note, the number of variable marker genes most valuable (i.e., informative) for genotyping is quite limited.…”

Section: Introductionmentioning

confidence: 99%

Analysis of Campylobacter jejuni Whole-Genome DNA Microarrays: Significance of Prophage and Hypervariable Regions for Discriminating Isolates

Pittenger

Frye²,

McNerney³

et al. 2012

Foodborne Pathogens and Disease

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Campylobacter is a leading cause of foodborne illness in humans, and improving our understanding of the epidemiology of this organism is essential. The objective of this study was to identify the genes that discriminate isolates of C. jejuni by analysis with whole-genome DNA microarrays. Statistical analyses of whole-genome data from 95 geographically diverse cattle, chicken, and human C. jejuni isolates identified 142 most significant variable genes. Of this total, 125 (88%) belonged to genomic prophage and hypervariable regions. The significance of genomic prophage and hypervariable regions in determining C. jejuni isolate genomic diversity is emphasized by these results. These genes will be useful as biomarkers and components of genotyping systems for C. jejuni to improve our understanding of the epidemiology and population genetics of this major foodborne pathogen.

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“…However, Campylobacter has recently been shown to have a high rate of carriage in cattle (Chatre et al, 2010), and the consumption of undercooked beef has been shown to be a significant risk factor for Campylobacter infection (Gallay et al, 2008). Typing of Campylobacter from different sources has shown clustering of isolates by host source Pittenger et al, 2009). However, a recent study of Irish C. jejuni isolates from poultry, beef and clinical sources has shown clinical isolates to cluster with isolates from both chicken and beef, highlighting the potential role for beef in the transmission of this pathogen to humans (Burgess et al, 2009). 10.4 Primary production 10.4.1 Pathogens in the farm environment When pathogens are shed in cattle faeces they can persist in the underlying soil and grass, surviving for extended periods ranging from several weeks to many months (Duffy, 2003;Semenov et al, 2009).…”

Section: Campylobacter Sppmentioning

confidence: 99%

Improving microbial safety in the beef production chain

Burgess

Duffy

2011

Food Chain Integrity

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GenotypingCampylobacter jejuniby Comparative Genome Indexing: An Evaluation with Pulsed-Field Gel Electrophoresis andflaASVR Sequencing

Cited by 5 publications

References 41 publications

Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment

Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment

Analysis of Campylobacter jejuni Whole-Genome DNA Microarrays: Significance of Prophage and Hypervariable Regions for Discriminating Isolates

Improving microbial safety in the beef production chain

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