IDENTIFICATION OF THE asta GENE IN ENTEROTOXIGENIC ESCHERICHIA COLI STRAINS RESPONSIBLE FOR DIARRHEA IN PIGS

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1 Bull. Vet. Inst. Pulawy 47, 9-15, 2003 IDENTIFICATION OF THE asta GENE IN ENTEROTOXIGENIC ESCHERICHIA COLI STRAINS RESPONSIBLE FOR DIARRHEA IN PIGS JACEK OSEK Department of Microbiology, National Veterinary Research Institute, Pulawy, Poland Received for publication August 12, The asta gene responsible for production of enteroaggregative Escherichia coli heatstable enterotoxin 1 (EAST1) was identified in E. coli strains isolated from pigs with postweaning diarrhea. Two hundred and twenty-six isolates were tested using PCR for the asta marker as well as for heat-labile I (LTI), heat-stable I (STI), and heat-stable II (STII) enterotoxin genes characteristic for enterotoxigenic E. coli (ETEC). The asta-positive isolates were also analysed for fimbrial adhesins using the slide agglutination test and PCR (F18 fimbriae). It was shown that 115 (50.9 per cent) of the isolates possessed the asta gene encoding the EAST1 toxin. A close correlation between the presence of F4 fimbriae and the EAST1 gene was observed: 106 of 115 (92.2%) asta + isolates expressed the F4 antigen. The results of the present study indicate that EAST1 enterotoxin may represent an additional virulence marker playing a role in the pathogenesis of porcine colibacillosis. Key words: E. coli, pigs, diarrhea, virulence markers, asta gene, PCR. Escherichia coli is an important enteric pathogen, causing diarrhea in humans and animals (7, 12). E. coli isolates associated with diarrhea have been classified into five major groups, on the base of their distinct virulence properties: enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohaemorrhagic (EHEC), enteroinvasive (EIEC) and enteroaggregative (EAEC) (12). Among them, ETEC is a predominant bacterial agent of postweaning diarrhea in piglets (9). Bacteria of this group are defined as E. coli that elaborate at least one enterotoxin: heat-labile I (LTI) or heat-stable I (STI) or heat-stable II (STII) (1, 10, 22, 23). Most porcine ETEC possess one of the pilus adherence factors - F4, F5, F6, F18, or F41, responsible for the colonization of the small intestine (5). Strains of enteroaggregative E. coli (EAEC), the most recently recognized category of diarrheagenic E. coli, adhere to HeLa cells in vitro in an aggregative adherence pattern and are associated with watery diarrhea in young children in the developing world (12, 21). The pathogenesis of EAEC infection is not fully understood; however, a characteristic histopathological lesion and several candidate virulence factors have been described (12, 21). One of them is a 38-amino-acid protein

2 10 called enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1), encoded by the asta gene, located on plasmids, on the chromosome, or on both of them (20, 28). The role of EAST1 in induction of diarrhea has not been clearly determined, however, the production of this toxin with several human ETEC strains has been demonstrated (19, 28). Recently, Choi et al. (3) and Frydendahl (4) reported that EAST1 enterotoxin was present not only in EAEC strains but also in other groups of human and animal (especially pig) diarrheagenic E. coli, including ETEC, EPEC, and EHEC. Although enteric colibacillosis is common in weaned piglets, there is very little information concerning the prevalence of EAST1-positive E. coli among bacteria isolated from diarrheal cases in these animals. In the present study, the presence of the asta gene encoding EAST1 enterotoxin in ETEC strains isolated from piglets with diarrhea was investigated by polymerase chain reaction (PCR) and its relationship with fimbrial and other enterotoxin markers was determined. Material and Methods Bacterial strains. E. coli strains (n = 226) were isolated from weaned pigs (four- to six-weeks old) with diarrhea and characterized previously (13, 15). The strains were obtained from eight geographically separated pig farms in the western part of Poland. Rectal swabs were taken from 226 piglets, plated on MacConkey s agar (Oxoid) and from each sample five bacterial colonies were picked and identified as E. coli by the PCR test with primers specific for the E. coli universal stress protein gene (uspa) as described previously (2, 16). One bacterial colony, determined to be E. coli (designated as strain), was selected from each piglet for further analyses. After isolation, E. coli were stored in agar stabs at room temperature and were not subcultured before examination. Virulence factors. F4, F5, F6, F17, and F41 fimbrial antigens were determined using the slide agglutination test as described previously (13, 18). PCR was used to detect the presence of the feda gene encoding the major fimbrial subunit of F18 fimbriae (8). The heat-labile I (LTI), heat-stable I and II (STI and STII) enterotoxin genes were detected by PCR as described previously (17). The presence of Shiga toxin 1 and 2, including the 2e variant genes was analysed using the 3-step PCR procedure as described previously (14). Identification of the asta gene. All 226 E. coli isolates were tested for the presence of the asta gene, responsible for production of enteroaggregative E. coli heatstable enterotoxin 1 (EAST1) using PCR with primers EAST1-1 (5 - CCATCAACACAGTATATC-3 ) and EAST1-2 (5 -GTCGCGAGTGACGGCTTTGT- 3 ) which amplified a 111 base pair (bp) fragment located within the asta genetic marker (26). Thirty amplification cycles at 94 C for one minute, 53 C for one minute, and 72 C for one minute were performed in a thermal cycler (PTC-100; MJResearch), followed by one final elongation step at 72 C for five minutes. The E. coli astapositive H10407 (O78:K80) strain (kindly supplied by Dr Y. Bertin, France) and C600 (E. coli K-12; kindly obtained from Dr P. Gallien, Germany) were used in each PCR run as the reference positive and negative controls, respectively. The asta PCR products were visualized by gel electrophoresis in 4.5 per cent Micropor Nu agarose

3 11 (Prona; Spain) in Tris-Acetate-EDTA (TAE) buffer at 100 V. A 100 bp DNA ladder ( bp range; DNA Gdansk) was included in each agarose run. Results PCR identification of the asta gene revealed that among 226 E. coli strains analysed, 115 (50.9 per cent) isolates were positive as determined by the presence of the 111 bp amplified product (Fig. 1). The remaining 111 (49.1 per cent) strains tested with the PCR method for the asta marker with primers EAST1-1 and EAST1-2 did not generate a PCR amplicon of 111 bp or any other size. M bp Fig. 1. Agarose gel electrophoresis presenting the PCR results obtained with EAST1-1 and EAST1-2 primers. Lane M, molecular mass marker ( bp range); lane 1, asta-positive, F4-positive E. coli; lane 2, asta-positive, F4-negative E. coli; lane 3, asta-positive reference E. coli (H10407); lane 4, asta-negative E. coli (C600); lane 5, water control. Enterotoxin profiles and the presence of F4 fimbrial antigen in ETEC strains examined in the study are shown in Table 1. Among the asta-positive isolates, the most prevalent strains had LTI and STII toxin genes and expressed F4 fimbriae (100 of 115 strains; 87.0%). On the other hand, the EAST1-negative bacteria were mostly able to produce LTI enterotoxin (103 out of 111 strains; 92.8%); such isolates were not detected among asta-positive E. coli strains tested. The only fimbrial antigen detected among EAST1-positive E. coli isolates recovered from piglets with diarrhea was F4 adhesive marker, although F5 and F6 fimbriae or F18 fimbrial gene were found among asta-negative strains tested (data not shown). There was a close correlation between the presence of F4 fimbriae and the EAST1 gene: all 106 F4-positive isolates harboured the asta genetic marker whereas only 9 strains without F4 adhesin possessed the EAST1 virulence determinant. The relationship between the presence of the asta gene, fimbriae and enterotoxin genes among EAST1-positive E. coli isolates was determined (Table 1). The majority of the strains (112 out of 115 isolates; 97.4 per cent) that carried the EAST1 genes possessed the genetic marker for heat-labile enterotoxin I (LTI), either with heat-stable enterotoxin II (93 isolates) or with both STI and STII toxins (8 strains). Only one asta + E. coli strain was LTI/STI-positive and additionally one strain had the

4 12 STI genetic determinant only as tested by PCR. Among the 3 EAST1-positive and LTInegative isolates, one of these strains produced only STI and two had genes for STII enterotoxin. EAST1 + isolates, that expressed the F4 fimbrial antigen, were mainly LTI/STII-positive (100 out of 106 F4 + /asta + strains) or either possessed genetic markers for all three enterotoxins tested (4 strains) or for STII toxin only (2 strains). None of the 226 E. coli strains tested possessed the genetic markers for production of Shiga toxins. Table 1 Prevalence of F4 fimbriae and enterotoxin genes among asta-positive and astanegative E. coli strains isolated from weaned pigs with diarrhea Enterotoxin gene F4 fimbriae asta-positive asta-negative No of strains LTI LTI LTI+STI LTI+STI LTI+STII LTI+STII LTI+STI+STII LTI+STI+STII STI STII Total Discussion In the present study, the prevalence of the EAST1 gene and its relationship with fimbriae and enterotoxin genes in E. coli isolated from piglets with postweaning diarrhea was examined. It was clearly showed that most E. coli strains possessing genes for at least one enterotoxin type (classified as ETEC) harboured the additional marker encoding the production of EAST1 toxin. Moreover, a close association of the asta gene with the presence of porcine fimbrial colonization factor F4 was demonstrated: many F4-positive E. coli strains recovered from piglets with diarrhea were EAST1- positive. The exceptions were strains possessing the elti (LTI) enterotoxin gene only (total 103 isolates) which were always EAST1-negative (Table 1). As presented in several previous studies (5, 6, 9, 11, 13, 24, 25), F4 is the major adhesin of ETEC of porcine origin. Therefore, the association of the EAST1 gene with F4-positive enterotoxigenic strains recovered from piglets with enteric disorders suggests that the enteroaggregative E. coli heat-stable enterotoxin 1 may be a virulence marker of isolates pathogenic for these animals. The EAST1 gene was also detected in human ETEC isolated from patients with diarrhea and was mainly found among strains possessing major adherence factors such as colonization factor antigens (CFA) I and II (26). These authors have also

5 13 demonstrated that the genes for EAST1 and CFA are located on the same plasmid whereas in porcine ETEC strains the asta and F4 virulence factors are carried on separate plasmids (27). Moreover, the EAST1 gene sequence of porcine F4-positive ETEC isolates was different from that of human CFA + ETEC strains (28). On the other hand, the pig origin EAST1 gene sequence is identical to that of strain O42 pathogenic for human volunteers (26). Therefore, EAST1 toxin expressed by E. coli strains recovered from pigs may play a role in pathogenicity of diarrhea. Recently, Frydendahl (4) analyzed 563 E. coli isolates from 503 pigs with postweaning diarrhea and showed a high frequency (65.8%) of strains possessing the asta gene. The author also observed a close correlation between the presence of the EAST1 toxin marker and F4 fimbrial as well as LTI and STII enterotoxin genes. Choi et al. (3) analysed 720 E. coli strains isolated from piglets with enteric colibacillosis for the presence of the asta gene and again a close association of the EAST1 gene with the F4 fimbrial marker was found. Moreover, many EAST1-positive isolates possessed the genes encoding heat-stable enterotoxin I (STI), either alone or in combination with STII enterotoxin. As demonstrated in the present study, there was a high frequency of the LTI gene and a low frequency of the STI gene in the EAST1- positive E. coli isolates. A similar relationship between the EAST1 and LTI genes in porcine ETEC strains was observed by Yamamoto and Nakazawa (27). Moreover, these authors showed that STI-positive ETEC were mainly EAST1-negative. Several studies demonstrated that porcine F4 + E. coli are usually LTI-positive, either alone or with STI or STII (6, 9, 13, 24). Therefore, it is not surprising that most of the asta + strains detected in the present study and in the study of Yamamoto and Nakazawa (27) were able to produce heat-labile enterotoxin I (LTI). In conclusion, the results of the present work indicate that the EAST1 gene is widely distributed among E. coli strains isolated from piglets with postweaning diarrhea. The data show that this toxin is mainly produced by F4-positive ETEC that mainly express LTI or LTI together with STII enterotoxins. Therefore, EAST1 toxin may represent an additional determinant playing a role in the pathogenesis of E. coli postweaning diarrhea in pigs. Moreover, the presence of the asta gene makes the porcine E. coli strains similar to human EAST1-positive isolates that are potentially able to induce diarrhea. Acknowledgments: This paper was granted by the Open Society Institute ZUG Foundation; Project Code: (Log-in ID: ). References 1. Celemin C., Anguita J., Naharro G., Suarez S.: Evidence that E. coli isolated from healthy pigs intestine hybridize with LT-II, ST-Ib and SLTII DNA probes. Microb. Pathog., 1994, 16, Chen J., Griffiths N.W.: PCR differentiation of Escherichia coli from other gramnegative bacteria using primers derived from the nucleotide sequences flanking the gene encoding the universal stress protein. Lett. Appl. Microbiol., 1998, 27,

6 14 3. Choi C., Kwon D., Chae C.: Prevalence of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene and its relationship with fimbrial and toxin genes in E. coli isolated from diarrheic piglets. J. Vet. Diagn. Invest., 2001, 13, Frydendahl K.: Prevalence of serogroups and virulence genes in Escherichia coli associated with postweaning diarrhoea and edema disease in pigs and a comparison of diagnostic approaches. Vet. Microbiol., 2002, 85, Gaastra W., De Graaf F.K.: Host-specific fimbrial adhesins of noninvasive enterotoxigenic Escherichia coli strains. Microbiol. Rev., 1982, 46, Harel J., Lapointe H., Fallara A., Lortie L.A., Bigras-Poulin M., Lariviere S., Fairbrother J.M.: Detection of genes for fimbrial antigens and enterotoxins associated with Escherichia coli serogroups isolated from pigs with diarrhea. J. Clin. Microbiol., 1991, 29, Holland R.E.: Some infectious cases of diarrhea in young farm animals. Clin. Microbiol. Rev., 1990, 3, Imberechts H., Wild P., Charlier G., De Greve H., Lintermans P., Pohl P.: Characterization of F18 fimbrial genes fede and fedf involved in adhesion and length of enterotoxemic Escherichia coli strain 107/86. Microb. Pathog., 1996, 21, Nagy B., Fekete P.Z.: Enterotoxigenic Escherichia coli (ETEC) in farm animals. Vet. Res., 1999, 30, Nair G.B., Takeda Y.: The heat-stable enterotoxins. Microb. Pathog., 1998, 24, Nakazawa M., Sugimoto C., Isayama Y., Kashiwazaki M.: Virulence factors in Escherichia coli isolated from pigs with neonatal and post-weaning diarrhea in Japan. Vet. Microbiol., 1987, 13, Nataro J.P., Kaper J.B.: Diarrheagenic Escherichia coli. Clin. Microbiol. Rev., 1998, 11, Osek J.: Prevalence of virulence factors of Escherichia coli strains isolated from diarrheic and healthy piglets after weaning. Vet. Microbiol., 1999, 68, Osek J.: Prevalence of shiga toxin genes among Escherichia coli strains isolated from pigs. Vet. Rec., 1999b, 145, Osek J.: Virulence factors and genetic relatedness of Escherichia coli strains isolated from pigs with post-weaning diarrhea. Vet. Microbiol., 2000, 71, Osek J.: Multiplex polymerase chain reaction assay for identification of enterotoxigenic Escherichia coli strains. J. Vet. Diagn. Invest., 2001, 13, Osek J., Gallien P., Truszczynski M., Protz D.: The use of polymerase chain reaction for determination of virulence factors of Escherichia coli strains isolated from pigs in Poland. Comp. Immunol. Microbiol. Infect. Dis., 1999, 22, Osek J., Svennerholm A.M.: Determination of K88 antigens and enterotoxins of Escherichia coli strains isolated from Polish piglets with diarrhea by the use of enzyme-linked immunosorbent assays. Vet. Microbiol., 1991, 29, Savarino S.J., Fasano A., Robertson D.C., Levine M.M.: Enteroaggregative Escherichia coli elaborate a heat-stable enterotoxin demonstrable in an in vitro rabbit intestinal model. J. Clin. Invest., 1991, 87, Savarino S.J., Fasano A., Watson J., Martin B.M. Levine M.M., Guandalini S., Guerry P.: Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin. Proc. Natl. Acad. Sci. USA, 1993, 90,

7 Scaletsky I.C.A., Silva M.L.M., Trabulsi L.R.: Distinctive patterns of adherence of enteropathogenic Escherichia coli to HeLa cells. Infect. Immun., 1984, 45, Sears C.L., Kaper J.B.: Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion. Microbiol. Rev., 1996, 60, Seriwatana J., Brown J.E., Echeverria P., Taylor D.N., Suthienkul O., Newland J.: DNA probes to identify Shiga-like-toxins I and II producing bacterial pathogens isolated from patients with diarrhea in Thailand. J. Clin. Microbiol., 1988, 26, Wilson R.A., Francis D.H.: Fimbriae and enterotoxins associated with Escherichia coli serogroups isolated from pigs with colibacillosis. Am. J. Vet. Res., 1986, 47, Wray C., McLaren I.M., Carroll P.J.: Escherichia coli isolated from farm animals in England and Wales between 1986 and Vet. Rec., 1993, 133, 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., 1996, 64, Yamamoto T., Nakazawa M.: Detection and sequences of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene in enterotoxigenic E. coli strains isolated from piglets and calves with diarrhea. J. Clin. Microbiol., 1997, 35, Yamamoto T., Wakisaka N., Sato F., Kato A.: Comparison of the nucleotide sequence of enteroaggregative Escherichia coli heat-stable enterotoxin 1 genes among diarrhea-associated Escherichia coli. FEMS Microbiol. Lett., 1997, 147,