Development and Validation of a Comparative Genomic Fingerprinting Method for High-Resolution Genotyping of Campylobacter jejuni

Abstract

e Campylobacter spp. are a leading cause of bacterial gastroenteritis worldwide. The need for molecular subtyping methods with enhanced discrimination in the context of surveillance-and outbreak-based epidemiologic investigations of Campylobacter spp. is critical to our understanding of sources and routes of transmission and the development of mitigation strategies to reduce the incidence of campylobacteriosis. We describe the development and validation of a rapid and high-resolution comparative genomic fingerprinting (CGF) method for C. jejuni. A total of 412 isolates from agricultural, environmental, retail, and human clinical sources obtained from the Canadian national integrated enteric pathogen surveillance program (C-EnterNet) were analyzed using a 40-gene assay (CGF40) and multilocus sequence typing (MLST). The significantly higher Simpson's index of diversity (ID) obtained with CGF40 (ID ‫؍‬ 0.994) suggests that it has a higher discriminatory power than MLST at both the level of clonal complex (ID ‫؍‬ 0.873) and sequence type (ID ‫؍‬ 0.935). High Wallace coefficients obtained when CGF40 was used as the primary typing method suggest that CGF and MLST are highly concordant, and we show that isolates with identical MLST profiles are comprised of isolates with distinct but highly similar CGF profiles. The high concordance with MLST coupled with the ability to discriminate between closely related isolates suggests that CFG40 is useful in differentiating highly prevalent sequence types, such as ST21 and ST45. CGF40 is a high-resolution comparative genomics-based method for C. jejuni subtyping with high discriminatory power that is also rapid, low cost, and easily deployable for routine epidemiologic surveillance and outbreak investigations. Campylobacter spp. are a leading cause of bacterial gastroenteritis worldwide (20), and most cases are thought to be the direct result of infection by C. jejuni or C. coli (20,43). Risk factors for campylobacteriosis include exposure to contaminated water, milk, and various food products, such as poultry (1,3,6,21). The development and implementation of effective control measures for these pathogens hinge on the identification of sources of infection. Although the ingestion of contaminated food or water and animal contact play a significant role in the epidemiology of campylobacteriosis, efforts to track sources of Campylobacter infection are hampered by the sporadic nature of campylobacteriosis (25), the infrequent association with outbreaks of disease, and widespread reservoirs that include water, livestock, domestic animals, and wildlife (8,17,46,65,67).A number of different molecular subtyping methods, such as pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphism analysis of the flagellin gene (flaA RFLP), and the DNA sequencing of the flagellin gene short variable region (flaA SVR), have been used to identify genotypic clusters of Campylobacter in the context of molecular epidemiology (18, 33). More recently, a multilocus sequence typing (ML...

Prevalence and Characteristics of <i>Salmonella</i> and <i>Campylobacter</i> in Retail Poultry Meat in Japan

“…These findings suggest that the CGF40 assay is a useful tool for the determination of genetic relatedness of C. jejuni isolates in combination with PFGE. This new method can be utilized for molecular typing of C. jejuni isolates: it is highly concordant with multi-locus sequence typing results, but offers better discriminatory power (Taboada et al, [4], 2012).…”

“…CGF method: The CGF40 method was applied to C. jejuni and C. coli isolates as described by Taboada et al (4). The PCR mixture consisted of 1 U Takara Ex Taq DNA polymerase (Takara Bio), 1 × PCR buffer (Takara Bio), 2.5 mM MgCl 2 , 0.2 mM each deoxynucleoside triphosphate (dNTP), 0.4 M each of the 10 primers, and 1 μL DNA template in a 25-μL reaction mixture.…”

“…The CGF method was developed by Canadian researchers as a high-resolution comparative genomics-based method specific for C. jejuni subtyping with a high discriminatory power. It offers a rapid, low-cost, and easily deployable approach of routine epidemiologic surveillance and outbreak investigations (4).…”

Comprehensive Detection and Discrimination of Campylobacter Species by Use of Confocal Micro-Raman Spectroscopy and Multilocus Sequence Typing

“…To date, a variety of methods have been used to identify Campylobacter isolates to the species level. These include genotyping methods (76) such as traditional PCR (6,35,63,75), multilocus sequence typing (MLST) (5,13,38,52,67,71), amplified fragment length polymorphism (4,8,15,40), pulsedfield gel electrophoresis (PFGE) (24,62), loop-mediated isother-mal amplification (LAMP) (80,81), and microarray-based methods (73), as well as serotyping methods (21,56) and mass spectrometry (17,18,25,47,78). Typically, genotyping methods are time-consuming and require highly trained personnel.…”

Public Health Genomics and the New Molecular Epidemiology of Bacterial Pathogens

“…To be most beneficial, genome sequencing infrastructure must be readily available - requiring capital and support. For some, it may remain most economically feasible to perform whole-genome sequencing on only a small subpopulation and then to port genome-identified traits to higher throughput detection and typing technologies [21] in order to conduct routine screening for the distribution of these traits in wider isolate panels (i.e. those not yet sequenced).…”