Current methods for molecular typing of Campylobacter species Eduardo N. Taboada a , Clifford G. Clark b , Emma L. Sproston c , Catherine D. Carrillo d, ⁎ a Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, PO Box 640, Township Rd. 9-1, Lethbridge, AB T1J 3Z4, Canada b National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St., Winnipeg, Manitoba R3E 3R2, Canada c Bureau of Microbial Hazards, Health Canada, 251 Sir Frederick Banting Driveway, Ottawa, ON K1A 0C6, Canada d Canadian Food Inspection Agency, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada abstract article info Article history: Received 1 May 2013 Received in revised form 1 July 2013 Accepted 2 July 2013 Available online 17 July 2013 Keywords: Campylobacter Molecular typing flaA short variable region sequence typing Multi-locus sequence typing Comparative genomic fingerprinting Whole genome sequencing Campylobacter remains one of the most common bacterial causes of gastroenteritis worldwide. Tracking sources of this organism is challenging due to the large numbers of human cases, and the prevalence of this organism throughout the environment due to growth in a wide range of animal species. Many molecular subtyping methods have been developed to characterize Campylobacter species, but only a few are commonly used in mo- lecular epidemiology studies. This review examines the applicability of these methods, as well as the role that emerging whole genome sequencing technologies will play in tracking sources of Campylobacter spp. infection. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. 1. Introduction Campylobacter spp. are among the most prevalent bacterial enteric pathogens in both developed and developing nations (ECDC, 2013; Kirkpatrick and Tribble, 2011). Most human disease has been attributed to Campylobacter jejuni, with C. coli, C. lari, and C. upsaliensis causing the majority of the remaining human cases of infection. While incidence of campylobacteriosis is high, most illnesses occur sporadically, and cases are rarely (typically less than 1%) associated with outbreaks (ECDC, 2013; Silva et al., 2011). However, it is likely that outbreaks or smaller case clusters occur far more frequently than currently detected due to a number of confounding factors (Fussing et al., 2007; Miller et al., 2004; Pebody et al., 1997). To begin with, current and past methods used to detect clusters of cases may not detect any but the most obvious clusters from large point-source outbreaks of extreme- ly limited duration and geographical extent. The use of improved molec- ular typing methods for determining the relatedness of isolates has already been shown to result in better detection of outbreaks (Fussing et al., 2007; Taylor et al., 2013). However, due to the large number of cases of campylobacteriosis, there may not be sufficient resources to per- form molecular typing on the isolated organisms in order to identify common genotypes responsible for infections. In Europe, for example, characterization of most of the clinical Campylobacter isolates does not extend beyond genus level identification (ECDC, 2013). Finally, to further complicate matters, studies have found that several genotypes of Campylobacter can be isolated from a single clinical sample (Smith et al., 1999; Gilpin et al., 2012), and point source outbreaks may comprise more than one genotype of this pathogen (Hedberg et al., 2001). Despite the fact that only a small proportion of cases appear to be outbreak-related, Campylobacter was one of the three most commonly reported causes of foodborne outbreaks in Europe in 2010 (ECDC, 2013). Outbreaks due to Campylobacter spp. are most commonly associ- ated with dairy products, poultry products and untreated water (Greig and Ravel, 2009; Ravel et al., 2009; Taylor et al., 2013). However, com- mon sources of infection cannot be determined from outbreaks alone, as routes of infection may differ between outbreaks and sporadic cases (Taylor et al., 2013). Molecular typing methodologies have been instrumental in enhanc- ing epidemiological investigations aimed at tracking sources of sporadic infections with Campylobacter spp. by providing information on the genetic subtypes in circulation. Poultry products are frequently contam- inated with Campylobacter spp. and molecular typing data has linked Campylobacter spp. on these products to human infections (Batz et al., 2012; Müllner et al., 2009; Nadeau et al., 2002), particularly in urban areas (Müllner et al., 2010b). Though Campylobacter is frequently found in fresh water and in other food animals, such as cows and pigs, Journal of Microbiological Methods 95 (2013) 24–31 Abbreviations: PFGE, pulsed field gel electrophoresis; flaA-SVR, flaA short variable region sequence typing; flaA-RFLP, flaA restriction fragment length polymorphism analysis; MLST, multi-locus sequence typing; eMLST, extended MLST; RAPD, random amplification of polymorphic DNA; WGS, whole genome sequencing; NGS, next-generation sequencing; MCGH, microarray comparative genomic hybridization; CGF, comparative genomic finger- printing; SNPs, single nucleotide polymorphisms; HRM, high-resolution melting analysis. ⁎ Corresponding author. Tel.: +1 613 759 1255. E-mail addresses: Eduardo.Taboada@phac-aspc.gc.ca (E.N. Taboada), Clifford.Clark@phac-aspc.gc.ca (C.G. Clark), Emma.Sproston@hc-sc.gc.ca (E.L. Sproston), Catherine.Carrillo@inspection.gc.ca (C.D. Carrillo). 0167-7012/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.07.007 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth