Bacteriology Characterization of the most prevalent colonization factor antigens present in Chilean clinical enterotoxigenic Escherichia coli strains using a new multiplex polymerase chain reaction ☆ Roberto M. Vidal a, ⁎ , Patricio Valenzuela a , Kelly Baker c , Rosanna Lagos b , Mario Esparza a , Sofie Livio c , Mauricio Farfán a , James P. Nataro c , Myron M. Levine c , Valeria Prado a a Faculty of Medicine, University of Chile, Santiago, 8389100 Chile b Centro para Vacunas en Desarrollo, Santiago, Chile c Center for Vaccine Development, University of Maryland, Baltimore, MD 21201, USA Received 1 April 2009; accepted 7 July 2009 Abstract Current methods to detect the colonization factor antigens (CFAs) associated with enterotoxigenic Escherichia coli (ETEC) strains are cumbersome, with some methods requiring antibodies that are not readily available. To achieve a gene-based method, we designed 2 multiplex polymerase chain reaction reactions to detect genes encoding the most common ETEC fimbrial colonization factors, including CFA/I and coli surface (CS) antigens CS1, CS2, CS3, CS4, CS5, and CS6. Analysis of 183 clinical ETEC strains shows that the most prevalent colonization factors were CFA/I only, CS1 and CS3, CS2 and CS3, and CS6 only. Interestingly, we identified 3 clinical isolates expressing CS1 only without its regulator rns. The method described here proved to be rapid and robust and correlates well with phenotypic expression of the CFAs, becoming a novel molecular diagnostic and research tool for future epidemiologic studies. © 2009 Elsevier Inc. All rights reserved. Keywords: Enterotoxigenic Escherichia coli (ETEC); Colonization factor antigen (CFAs); Multiplex PCR; Molecular identification of ETEC; Molecular epidemiology of ETEC; rns regulator 1. Introduction Enterotoxigenic Escherichia coli (ETEC) strains are responsible for 650 million acute diarrhea episodes around the world and approximately 800 000 deaths every year (Turner et al., 2006). In developing and transitional countries, such as Chile, where sanitation and food hygiene practices are often compromised, ETEC is readily transmitted via fecally contaminated food or water (Levine et al., 1993), causing diarrheal disease among indigenous children and among travelers from industrialized countries. ETEC produces heat- stable enterotoxin (ST) of the human (STh) and/or porcine (STp) variety, heat-labile enterotoxin (LT), or both (Kaper et al., 2004; Levine, 1987). ETEC strains adhere to human small intestinal mucosa via colonization factor antigens (CFAs) (Kaper et al., 2004; Levine, 1987); the predominant CFAs expressed by human ETEC pathogens include CFA/I, the CFA/II family, and the CFA/IV family (Qadri et al., 2005, 2006a). Heretofore, it has been accepted that CFA/II strains express coli surface (CS) antigen 3, either alone or in conjunction with CS1 or CS2 (Levine et al., 1993; Nataro and Kaper, 1998; Wolf, 1997), and CFA/IV ETEC strains express CS6, either alone or in conjunction with CS4 or CS5 (Levine et al., 1993; Nataro and Kaper, 1998; Wolf, 1997). Antibodies, including intestinal SIgA, against CFAs are believed to mediate protection against diarrheal illness upon exposure to wild-type ETEC. Accordingly, several candidate ETEC vaccines incorporate CFA/I and CS1-6 to achieve broad protection (Barry et al., 2006; Qadri et al., 2006b). For such vaccines, the level of protection that can be achieved Available online at www.sciencedirect.com Diagnostic Microbiology and Infectious Disease 65 (2009) 217 – 223 www.elsevier.com/locate/diagmicrobio ☆ Presented in part in the Investigator Meeting for the Global Enterics Multi-Center Study (GEMS), Seattle, WA, September 10 to 12, 2008. Issues faced by GEMS reference laboratories, ETEC CFAs detected by M-PCR, speaker Roberto Vidal. ⁎ Corresponding author. Institute of Biomedical Sciences (ICBM), Faculty of Medicine, University of Chile, Santiago, Chile. Tel.: +56-2- 9786646; fax: +56-2-7355855. E-mail address: rvidal@med.uchile.cl (R.M. Vidal). 0732-8893/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2009.07.005