Genotyping of Campylobacter jejuni using seven single-nucleotide polymorphisms in combination with flaA short variable region sequencing

Price, Erin P.; Thiruvenkataswamy, Venugopal; Mickan, Lance; Unicomb, Leanne; Rios, Rosa E.; Huygens, Flavia; Giffard, Philip M.

doi:10.1099/jmm.0.46460-0

Cited by 35 publications

(55 citation statements)

References 40 publications

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“…However, among the flaA SVR types represented by two or more isolates, 10% of the isolates were discordant with respect to the CC, suggesting that the flaA SVR type does not consistently correlate with the chromosomal genotype. Other studies have also suggested that flaA SVR typing alone is poorly suited for investigation of the molecular epidemiology of C. jejuni isolates (8,9,11,12,35).…”

Section: Discussionmentioning

confidence: 99%

Multilocus Sequence Typing of Campylobacter jejuni Isolates from Humans, Chickens, Raw Milk, and Environmental Water in Quebec, Canada

et al. 2008

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Molecular strain typing is essential for deciphering the epidemiology of Campylobacter jejuni infections. We applied two different methods, multilocus sequence typing (MLST) and analysis of the flaA short variable repeat (SVR), to 289 isolates (163 human, 56 chicken, 34 raw milk, and 36 environmental water isolates) collected in the province of Québec, Canada, over 3 years; in addition, the analysis included the pulsed-field gel electrophoresis (PFGE) typing results for a subset of 131 isolates studied previously. MLST defined 96 sequence types (STs) and 20 clonal complexes (CCs), including 49 STs (73 isolates, 25%) and 39 alleles not previously documented in an international database. The frequency of new STs was significantly higher among water isolates than among isolates from other sources (18/36 [50%] and 55/253 [22%], respectively; P < 0.001). Nine of the 10 most prevalent CCs included isolates from humans and at least one other source; five CCs comprised exclusively or mostly human and chicken isolates. However, water and milk were the predominant nonhuman sources among the remaining CCs, suggesting that sporadic C. jejuni infections in humans may frequently arise from sources other than chickens. All three typing systems were discriminatory (discriminatory index > 0.9). Among 131 isolates analyzed by PFGE, each of the 20 types represented by two or more isolates corresponded to a single CC. In contrast, among the 14 most prevalent types detected by analysis of the flaA SVR (5 to 27 isolates each), 8 (57%) included isolates that represented multiple different CCs. The basis for these discordant results was uncertain. Antimicrobial resistance was randomly distributed among the CCs and appeared to be more closely related to the source of an isolate than its genotype. Although MLST is labor-intensive and expensive, it remains the single best method for the genotyping of C. jejuni isolates and deciphering the epidemiologic relationships among isolates.

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Section: Discussionmentioning

confidence: 99%

Multilocus Sequence Typing of Campylobacter jejuni Isolates from Humans, Chickens, Raw Milk, and Environmental Water in Quebec, Canada

et al. 2008

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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%

“…The isolates included in this study have been subjected to typing based on variation in resolution-optimized MLST database-derived SNPs and resolution-optimized binary markers (17) as well as to the methods addressed in the present study. The SNP-, binary marker-, and HRM-based methods can all be performed on the real-time PCR platform, and they all yield digitizable results (17,(23)(24)(25), thus providing a readily accessible choice of methods or combinations of methods that can be adapted to different tasks and questions. It has previously been shown that the resolution-optimized SNP-defined genotypes correlate well with the MLST-defined population structure; i.e., there was a strong tendency for isolates with identical SNP types to have identical or very similar MLSTs (25).…”

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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“…The addition of more variable targets, such as flagellin-encoding genes, increases the discriminatory power of sequence-based typing. The most frequently used gene for this purpose is flaA (2,5,7,17,20,26,29), although flaB is also used, and as a more stable gene, flaB might become more important (21). Other important factors to consider are the time and effort needed to perform the appropriate data analysis, especially in the context of internationally standardized approaches and the use of publicly available typing tools, such as http://pubmlst.org.…”

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confidence: 99%

Multiplex Strategy for Multilocus Sequence Typing, fla Typing, and Genetic Determination of Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli Isolates Collected in Switzerland

et al. 2009

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We present an optimized multilocus sequence typing (MLST) scheme with universal primer sets for amplifying and sequencing the seven target genes of Campylobacter jejuni and Campylobacter coli. Typing was expanded by sequence determination of the genes flaA and flaB using optimized primer sets. This approach is compatible with the MLST and flaA schemes used in the PubMLST database and results in an additional typing method using the flaB gene sequence. An identification module based on the 16S rRNA and rpoB genes was included, as well as the genetic determination of macrolide and quinolone resistances based on mutations in the 23S rRNA and gyrA genes. Experimental procedures were simplified by multiplex PCR of the 13 target genes. This comprehensive approach was evaluated with C. jejuni and C. coli isolates collected in Switzerland. MLST of 329 strains resulted in 72 sequence types (STs) among the 186 C. jejuni strains and 39 STs for the 143 C. coli isolates. Fourteen (19%) of the C. jejuni and 20 (51%) of the C. coli STs had not been found previously. In total, 35% of the C. coli strains collected in Switzerland contained mutations conferring antibiotic resistance only to quinolone, 15% contained mutations conferring resistance only to macrolides, and 6% contained mutations conferring resistance to both classes of antibiotics. In C. jejuni, these values were 31% and 0% for quinolone and macrolide resistance, respectively. The rpoB sequence allowed phylogenetic differentiation between C. coli and C. jejuni, which was not possible by 16S rRNA gene analysis. An online Integrated Database Network System (SmartGene, Zug, Switzerland)-based platform for MLST data analysis specific to Campylobacter was implemented. This Web-based platform allowed automated allele and ST designation, as well as epidemiological analysis of data, thus streamlining and facilitating the analysis workflow. Data networking facilitates the exchange of information between collaborating centers. The described approach simplifies and improves the genotyping of Campylobacter, allowing cost-and time-efficient routine monitoring.

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Genotyping of Campylobacter jejuni using seven single-nucleotide polymorphisms in combination with flaA short variable region sequencing

Cited by 35 publications

References 40 publications

Multilocus Sequence Typing of Campylobacter jejuni Isolates from Humans, Chickens, Raw Milk, and Environmental Water in Quebec, Canada

Multilocus Sequence Typing of Campylobacter jejuni Isolates from Humans, Chickens, Raw Milk, and Environmental Water in Quebec, Canada

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

Multiplex Strategy for Multilocus Sequence Typing, fla Typing, and Genetic Determination of Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli Isolates Collected in Switzerland

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