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1 JCM Accepts, published online ahead of print on 5 December 2012 J. Clin. Microbiol. doi: /jcm Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 Genotypic characterization of enterotoxigenic Escherichia coli strains causing traveler's diarrhea Fulton P. Rivera 1,2, Anicia M. Medina 1, Edelweiss Aldasoro 3, Anna Sangil 3,4, Joaquim Gascon 3, Theresa J. Ochoa 1,5, Jordi Vila 3, Joaquim Ruiz 3* 1 Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru; 2 Laboratorio de Fisiopatogenia, Departamento de Fisiología, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina; 3 Barcelona Centre for International Health Research (CRESIB- Hospital Clínic- Universitat de Barcelona), Barcelona, Spain; 4 Unitat de Malalties Infeccioses, Medicina Interna, Hospital Universitari Mutua Terrassa, Terrassa, Spain; 5 Center for Infectious Diseases, University of Texas School of Public Health, Houston, Texas, United States Downloaded from Running title: Traveler's diarrhea-associated ETEC * Corresponding author Joaquim Ruiz, PhD Centre de Recerca en Salut Internacional de Barcelona Hospital Clinic / Universitat de Barcelona Edificio CEK, Planta 1 C/Rosselló Barcelona, Spain Phone ext: joruiz@clinic.ub.es 1 on August 17, 2018 by guest

2 27 ABSTRACT This study aims to characterize the presence of virulence factors of enterotoxigenic E. coli causing traveler's diarrhea. Among 52 ETEC isolates, the most common toxin type was STh, and the most frequent CFs were CS21, CS6 and CS3. On the other hand, the nonclassical virulence factors EAST1 and EatA were frequently present. Key words: Enterotoxigenic Escherichia coli, colonization factor, enterotoxin, virulence genes, traveler's diarrhea. 2

3 Enterotoxigenic Escherichia coli (ETEC) is by far the most common pathogen causing travelers s diarrhea (TD) worldwide, being responsible for up to 30 to 60% of all TD cases (1,2). ETEC adhere to and colonize the human intestinal mucosa thanks to specific antigenic fimbriae called colonization factors (CFs) (2,3). Following CF-mediated mucosal adhesion, ETEC elaborates heat-labile toxin (LT), and/or heat-stable toxin (ST), causing secretion of water and electrolytes (4). Inactivated enterotoxins and some CFs have been used as vaccine candidates in clinical studies (5,6,7), but none of them has yielded results in clinical trials sufficiently robust to foster completion of clinical development. Moreover, the great variability of ETEC CFs requires the determination of prevalent CF types in different geographic locations and population types (2,7). An alternative approach for a vaccine development would be to focus on ETEC antigens other than enterotoxins. Other virulence factors (VF) have been identified from the prototype strain ETEC H10407 (O78:H11/LTI-STh- STp/CFAI). These nonclassical VF include adhesins (Tia, TibA), a cytoplasmic protein with GTPase activity (LeoA), an autotransporter (EatA), and an enteroaggregative E. coli heat-stable enterotoxin (EAST1) (8). Additionally, the E. coli common pili (ECP), common both to commensal and pathogenic E. coli, may be present, but its role in ETEC pathogenesis is not yet established (9). To our knowledge, these genes have not been systematically evaluated in epidemiological studies in ETEC strains associated with TD. In this study, we characterized VF of ETEC causing TD in Spanish travelers abroad. Fifty-two ETEC isolates causing TD obtained between January 2004 and August 2011 as part of a passive TD surveillance study in Barcelona, Spain were grown from frozen stock. From these, clinical data were recovered in 30 cases. TD was defined as the 3

4 occurrence between 12 hours after arrival in, and 5 days after departure from, the country visited; of three or more episodes of watery diarrhea within a 24-h period with or without other symptoms; or as the occurrence of unformed stools accompanied by one of the following: abdominal cramps, tenesmus, vomiting, nausea, fever, chills or prostration. Presence of E. coli and other bacterial enteropathogens was searched by conventional methods, while PCR was used to detect enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), and ETEC (10). The presence of classical (LT, STh, STp, CFAI, CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS8, CS12, CS14, CS17, CS18, CS19, CS20 and CS21) and nonclassical VFs (Tia, LeoA, TibA, EatA, EAST1 and ECP) was determined by PCR as previously described (11,12, 13,14,15,16) (Table 1). Chi-square or Fisher s exact tests were used for comparisons between groups as appropriate. ETEC were isolated from patients, mostly returning from Africa (15/30, 50%), Asia/Pacific (10/30, 33%) and Central America (4/30, 13%). The median duration of travel was 21 days (range d). ST-positive (ETEC-ST) strains (23/52, 44%, including STh and STp) were the most frequent, followed by strains with both LT and ST (ETEC-LT-ST) (14/52, 27%) and strains positive for only LT (ETEC-LT) (15/52, 29%) (Table 2). STh (esta+) was the toxin type most frequently identified. This study employs travelers returning with diarrhea. Since most ETEC infections are brief and self-limited, returning travelers will be biased to more severe and/or cases of longer duration. This may actually make the analysis more significant since it may reveal the factors common among the more severe traveler's pathogens, but validation in another study would be required to verify. The most common CFs were CS21 (58%), CS6 (27%) and CS3 (23%) (Table 2). There were no differences between the geographical area of travel and the CFs types 4

5 identified, with CS21 (Longus) being the most common CF in all areas. The presence of multiple CFs in a given isolate was frequent, these CFs being frequently associated with CS21. STh+ CS21 genotype (with or without other CFs) was the most prevalent among all strains (44%, 23/52). The ECP was detected in 81% of all strains. Meanwhile, the most common nonclassical VFs were EAST1 (65%), autotransporter EatA (48%) and adhesin tia (21%) (Table 2). TD owing to infection with ETEC is a considerable problem to travelers in high-risk regions. In several studies, the most prevalent CFs associated with the occurrence of diarrhea worldwide are CFAI, CS1, CS2, CS3 and CS6; more recently CS8, CS14 and CS21 have been detected with relatively high frequencies (6). In contrast with some studies performed in developing countries, where CFA/I has been described as the most prevalent CF accounting for up to 21% of samples (2), in our study, CFA/I was present only in 12% of isolates. Thus, the prevalence of CFs antigens may be related to geographical origin. Among nonclassical VFs, EAST1 (65%, 34/52), and EatA protein (48%, 25/52) were frequently detected in our strains. The presence of EAST1, structurally related to STI peptides, that also leads to increases in cgmp (17), in multiple strains may suggests functional redundancy of toxins with the capacity to provoke elevated levels of cgmp (8). EatA acts as a serine protease, belonging to the SPATE family proteins (Serine Protease Autotransporters of Enterobacteriaciae). Although a precise function or substrate for EatA is not clear, initial studies in ileal loops suggested that EatA could contribute to accelerated virulence of eata+ strains (18). Despite searching for the CFs most commonly described in the literature, 31% of all ETEC strains did not possess any of the tested CFs. In a prior study, Blackburn and 5

6 colleagues reported that 58% of ETEC strains were positive for the Escherichia coli common pilus (ECP), a percentage even higher than that of the most prevalent CFs and independent of the presence of CFs, suggesting an important role for ECP in the biology of ETEC, particularly in CF-negative strains and in human infections (9). In our study, the ECP gene (ecpa) was identified in 81% (42/52) of the present isolates. In recent years, a variety of different structures (other that colonization factor) encoded either on plasmids or the chromosome of ETEC has been identified as putative adhesins. Among these adhesins, Tia is an outer membrane protein that interacts with host cell surface proteoglycans (19) and by itself is sufficient to promote adherence and epithelial cell invasion when cloned into laboratory strains of E. coli. The success of an ETEC vaccine targeting travelers from industrialized countries to developing countries will depend on a combination of maximally-antigenic vaccine preparations and regimens for their delivery which will produce optimal immune responses. While vaccine development to prevent diarrheal illness due to ETEC is feasible, extensive efforts are needed to identify new conserved antigenic targets. Additional studies will be needed to determine the utility of these antigens as well as other autotransporters in ETEC vaccines. 6

7 139 ACKNOWLEDGEMENTS A.M.M. attended a 3-month training through the CRESIB-UPCH collaborative agreement, training was partially funded by Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica (CONCYTEC, FONDECYT) in Perú. This work was partially funded by: Agencia Española de Cooperación Internacional para el Desarrollo (AECID), Spain, Programa de Cooperación Interuniversitaria e Investigación Científica con Iberoamérica (D/019499/08, D/024648/09; D/030509/10 and A1/035720/11); Departament d'universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya, Spain 2009SGR685 (J.R) and 2009SGR385 (J.G., E.A). National Institutes of Health, USA, Public Health Service awards 1K01TW (T.J.O); J.R. has a fellowship from the program I3, of the ISCIII (grant number: CES11/012) There is no conflict of interest for any of the authors. 7

8 REFERENCES 1. Gascon J, Vila J, Valls ME, Ruiz L, Vidal J, Corachán M, Prats G, Jimenez de Anta MT Etiology of traveller's diarrhea in Spanish travellers to developing countries. Eur J Epidemiol. 9: Qadri F, Svennerholm AM, Faruque AS, Sack RB Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin. Microbiol. Rev. 18: Gaastra W, Svennerholm AM Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 4: Kaper JB, Nataro JP, Mobley HL Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2: Boedeker EC Vaccines for enterotoxigenic Escherichia coli: current status. Curr. Opin. Gastroenterol. 21: Svennerholm AM, Tobias J Vaccines against enterotoxigenic Escherichia coli. Expert. Rev. Vaccines. 7: Walker RI, Steele D, Aguado T; Ad Hoc ETEC Technical Expert Committee Analysis of strategies to successfully vaccinate infants in developing countries against enterotoxigenic E. coli (ETEC) disease. Vaccine. 25: Fleckenstein JM, Hardwidge PR, Munson GP, Rasko DA, Sommerfelt H, Steinsland H Molecular mechanisms of enterotoxigenic Escherichia coli infection. Microbes. Infect. 12:

9 Blackburn D, Husband A, Saldaña Z, Nada RA, Klena J, Qadri F, Girón JA Distribution of the Escherichia coli common pilus among diverse strains of human enterotoxigenic E. coli. J. Clin. Microbiol. 47: Vargas M, Gascón J, Gallardo F, Jimenez De Anta MT, Vila J Prevalence of diarrheagenic Escherichia coli strains detected by PCR in patients with travelers' diarrhea. Clin. Microbiol. Infect. 4: Bölin I, Wiklund G, Qadri F, Torres O, Bourgeois AL, Savarino S, Svennerholm AM Enterotoxigenic Escherichia coli with STh and STp genotypes is associated with diarrhea both in children in areas of endemicity and in travelers. J. Clin. Microbiol. 44: Del Canto F, Valenzuela P, Cantero L, Bronstein J, Blanco JE, Blanco J, Prado V, Levine M, Nataro J, Sommerfelt H, Vidal R Distribution of classical and nonclassical virulence genes in enterotoxigenic Escherichia coli isolates from Chilean children and trna gene screening for putative insertion sites for genomic islands. J. Clin. Microbiol. 49: Rodas C, Iniguez V, Qadri F, Wiklund G, Svennerholm AM, Sjöling A Development of multiplex PCR assays for detection of enterotoxigenic Escherichia coli colonization factors and toxins. J. Clin. Microbiol. 47: Sjöling A, Wiklund G, Savarino SJ, Cohen DI, Svennerholm AM Comparative analyses of phenotypic and genotypic methods for detection of enterotoxigenic Escherichia coli toxins and colonization factors. J. Clin. Microbiol. 45:

10 Vidal RM, Valenzuela P, Baker K, Lagos R, Esparza M, Livio S, Farfán M, Nataro JP, Levine MM, Prado V Characterization of the most prevalent colonization factor antigens present in Chilean clinical enterotoxigenic Escherichia coli strains using a new multiplex polymerase chain reaction. Diagn. Microbiol. Infect. Dis. 65: Yamamoto T, Echeverria P Detection of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene sequences in enterotoxigenic E. coli strains pathogenic for humans. Infect. Immun. 64: Savarino SJ, Fasano A, Watson J, Martin BM, Levine MM, Guandalini S, Guerry P Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin. Proc. Natl. Acad. Sci. U.S.A. 90: Patel SK, Dotson J, Allen KP, Fleckenstein JM Identification and molecular characterization of EatA, an autotransporter protein of enterotoxigenic Escherichia coli. Infect. Immun. 72: Fleckenstein JM, Holland JT, Hasty DL Interaction of an outer membrane protein of enterotoxigenic Escherichia coli with cell surface heparan sulfate proteoglycans. Infect. Immun. 70:

11 Table 1. Sequence of primers used in this study. Virulence Size Primer sequence Ref factor (bp) Forward (5' 3') Reverse (5' 3') STp 166 TCTTTCCCCTCTTTTAGTCAG ACAGGCAGGATTACAACAAAG 11 STh 120 TTCACCTTTCCCTCAGGATG CTATTCATGCTTTCAGGACCA 13 CFA/I 497 ACTATTGGTGCAATGGCTCTGAC CAGGATCCCAAAGTCATTACAAG 15 CS1 410 GAGAAGACCATTAGCGTTACGG CCCTGATATTGACCAGCTGTTAG 15 CS2 358 ACTGTAACTGCTAGCGTTGATCC TGCTTCCTGCATTAATAACGAGT 15 CS3 300 CCCACTCTAACCAAAGAACTGG CGTATTTCCAGCATTTTTATCCA 15 CS4 242 ATTGATATTTTGCAAGCTGATGG GTCACATCTGCGGTTGATAGAGT 15 CS5 558 CAACCGTATCAGGTTCTGTTTTG CAAATGTTACCGGAGCTACAAAG 15 CS6 165 AAATGTATCCCAGGTAACGGTCT TGTTGATTAGGCGTAACCTCTGT 15 CS7 203 TGCTCCCGTTACTAAAAATAC TAGATGTCGTATCACTACGT 12 CS8 166 ATCCGGATTATCAAGCTCCA GAAGATGTTATTGCACCACCAA 14 CS GCGAATAACAATGATGCAAG CCTGACTGGTTTACAAGATA 12 CS TTTGCAACCGACATCTACCA CCGGATGTAGTTGCTCCAAT 14 CS TAAACTTGATCTTCTGCAAGC GCATGAATCGTAAGCTGTTG 12 CS (paired with CS17-19 Forward) TCAGGCGCAGTTCCTTGTGTG 12 CS AACCAGCACCGGTGATAAAG CTGGCTGGCCATTTAAGGTA 14 CS AGGTATCCAAATCCGCACTG CATCAGCCAGCACATAGGAA 14 CS TCATGAGCCTGCTGGAAGTTATCA TCCGGCTACCTAAAGTAATTGAGT 13 Tia 535 GCTTCAGTTGTGATGCAGAC CAGCATCCAGATAGCGATAG 12 TibA 655 CACGACAAATCAAACGTACC CTTCCGCCGTAGAGATACAT 12 LeoA 574 CGTTTATGGACGATGAGTTG TCTGCCAGCTCAGTAACAAC 12 EatA 1943 ATGTGCTTTGGCAGGTTAAT ATATCCAGTCAGCACCCACT 12 EcpA 483 TGAAAAAAAAGGTTCTGGCAATAGC CGCTGATGATGGAGAAAGTGAA 9 EAST1 111 CCATCAACACAGTATATCCGA GGTCGCGAGTGACGGCTTTGT 16 Size: Number of base pairs of the amplified product; Ref: Reference

12 Table 2. Virulence factors in traveler s diarrhea-associated ETEC isolates (N=52). Virulence factors a n (%) Classical virulence factors b LT 15 (29) STp 2 (4) STh 17 (33) LT-STh 14 (27) STh-STp 4 (8) CFAI 6 (12) CS1 7 (13) CS2 4 (8) CS3 12 (23) CS4 2 (4) CS5 1 (2) CS6 14 (27) CS17 3 (6) CS19 1 (2) CS20 1 (2) CS21 30 (58) At least one colonization factor 36 (69) 2 colonization factors c 28 (54) Nonclassical virulence factors E. coli common pilus 42 (81) Enteroaggregative heat-stable toxin-1 (EAST1) 34 (65) Autotransporter EatA 25 (48) Adhesin tia 11 (21) Dispersin transporter (aata) 4 (8) GTP-binding protein (LeoA) 3 (6) Adhesin TibA 1 (2) a All virulence factors were detected by PCR. b Colonization factors CS7, CS8, CS12, CS14 and CS18 were not detected.

13 c Sixteen strains showed two CFs, including four CFAI and CS21, three CS3 and CS21, nine CS6 and CS21; eight strains showed three CFs, including one CFAI-CS6-CS21, four CS1-CS3-CS21, two CS2-CS3-CS21, one CS4-CS6-CS21; three strains showed four CFs, including one CFAI-CS4-CS6-CS21, one CS1-CS2-CS3-CS21, one CS1- CS3-CS6-CS21; and one strain showed five CFs CS2-CS3-CS5-CS6-CS21. Downloaded from on August 17, 2018 by guest