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

Abstract

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 ...

Comparative Genotypic and Antimicrobial Susceptibility Analysis of Zoonotic Campylobacter Species Isolated from Broilers in a Nationwide Survey, Portugal

“…This methodology has been used for the past two decades with a high level of acceptance. Although genetic material may recombine at the flagellin locus (30), the methodology is suitable for short-term and geographically limited epidemiological studies (5,17,42,44,47,50,62), as was the case in the present study. In some studies, RFLP plus pulsed-field gel electrophoresis and multilocus sequence typing have had higher discriminatory power (5) for Campylobacter isolates, whereas in others, flaA-RFLP methodology had an equivalent or superior discriminatory power (40, 48).…”

Technique d’analyse des courbes de fusion haute résolution (FHR) ou High Resolution Melting analysis (HRM) en infectiologie

“…[18,19]. Cette technique permet aussi de différencier les espèces de Brucella [20] et de génotyper les souches de Campylobacter [21] ou de Mycobacterium avium subspecies paratuberculosis [22] ou encore de Mycoplasma [23]. Enfin, cette technique peut être utilisée pour la différenciation des souches de S. aureus [24].…”

The Potential of High Resolution Melting Analysis (Hrma) to Streamline, Facilitate and Enrich Routine Diagnostics in Medical Microbiology

“…Namely, Price et al 37 first developed a HRMA assay for interrogating the hypervariable CRISPR (cluster, regularly interspaced short palindromic repeats) locus of Campylobacter jejuni. To improve the power of resolution and extend the spectrum to C. coli, the same group elaborated an HRMA approach to detection of sequence variants of the flaA flagellin gene 38 . Because of its easy and rapid performance and potential for full automation, melting analysis of RAPD products may also be useful for epidemiological surveillance in the future.…”