Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kobayashi, H. Articles by Kubo, M. Search for Related Content PubMed PubMed Citation Articles by Kobayashi, H. Articles by Kubo, M. Agricola Articles by Kobayashi, H. Articles by Kubo, M.

 Previous Article

Applied and Environmental Microbiology, January 2009, p. 292-295, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01534-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Prevalence and Characteristics of eae- and stx-Positive Strains of Escherichia coli from Wild Birds in the Immediate Environment of Tokyo Bay

Hideki Kobayashi,^* Mika Kanazaki, Eiji Hata, and Masanori Kubo

National Agriculture and Food Research Organization, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan

Received 7 July 2008/ Accepted 24 October 2008

Top ABSTRACT INTRODUCTION REFERENCES

 
The prevalence and characteristics of eae- and stx-positive Escherichia coli strains in wild birds in the immediate environment of Tokyo Bay, Japan, was examined using cloacal swab samples taken from 447 birds belonging to 62 species. PCR screening showed that the prevalences of stx- and eae-positive strains of Escherichia coli were 5% (23/447) and 25% (113/447), respectively. Four strains of stx_2f-positive E. coli were isolated from two feral pigeons, an oriental turtle dove and a barn swallow. In contrast, 39 eae-positive E. coli strains were isolated, and most of the strains possessed a subtype of intimin that is classified as a minor group of human intimins, such as intimin , , and µ. Moreover, these strains did not possess any of the other pathogenic genes tested, such as stxs, ehxA, bfp, or irp. Thus, wild birds were considered to be a reservoir of atypical enteropathogenic E. coli.

Top ABSTRACT INTRODUCTION REFERENCES

 
Pathogenic Escherichia coli, such as enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and attaching and effacing E. coli, are food-borne pathogens that cause diarrhea in humans (13, 16, 20). The pathogenicity of these E. coli strains is, in large part, due to the fact that they express genes for Shiga toxins (stx genes) and/or for intimin, a virulence factor that is an outer membrane protein (eae). Ruminants are considered to be the main reservoir of Stx-producing E. coli (STEC). However, other domestic animals such as goats, pigs, poultry, cats, and dogs can also harbor STEC, in addition to intimin-producing E. coli (3, 4). A recent investigation of the occurrence of STEC in wild birds (14, 18, 22, 24) indicated that the latest stx₂ variant (stx_2f) is found in pigeons (22); however, the carriage of stx- or eae-possessing E. coli strains in wild animals has not been thoroughly investigated. Some wild birds live in human habitats and others migrate between waste treatment plants, marketplaces, cattle pastures, and pig farms. Such interactions between humans and wild birds therefore make birds important vehicles for the spread of zoonotic infections.

The goal of the present study was to analyze the role of wild birds as reservoirs of stx- and intimin-producing strains of E. coli. The prevalence of these E. coli strains in wild birds was examined using PCR analysis, and further genetic characterization of the various virulence genes of the isolated strains was performed.

The E. coli strain O157:H7, used as a positive-control strain for analysis of the stx₁, stx₂, and eae genes by PCR, was obtained from the American Type Culture Collection (ATCC 35150). The E. coli strains Ch05031 and PGN28, used as positive-control strains in PCR analyses of the stx_2e and stx_2f genes, respectively, were derived from the stock culture collection of the National Institute of Animal Health, Japan. A total of 447 cloacal swab samples were collected from 62 species of wild birds that had been captured between 2003 and 2007 (Table 1). The birds were captured for various reasons, such as falling from nests or as a consequence of being hunted by carnivorous animals or humans. The collection area was in the immediate environment of Tokyo Bay, mainly in the Tokyo and Chiba prefectures, Japan. The birds were placed individually in a cardboard box or a metal tray during feces collection. Prior to further analysis the swab samples were enriched in Trypticase soy broth (Eiken, Tokyo, Japan) at 37°C for 18 to 20 h. Ten microliters of the Trypticase soy broth culture was then inoculated onto MacConkey agar (Eiken) and incubated at 37°C for 18 h. The presence of pathogenic genes in the swab samples was assayed by PCR analyses on a loopful of colonies from an area of confluent growth. PCR was used to analyze the presence of the eae gene and the stx₁, stx₂, and stx₂ variants (stx_2e and stx_2f) of the Shiga toxins as previously described (8, 11, 19, 22). Samples that tested positive for the eae and/or the stx genes by PCR analysis were then subjected to colony hybridization or to PCR of randomly isolated individual colonies in an effort to isolate positive colonies. For these analyses, appropriate probes were prepared by labeling stx or eae PCR amplicons from the positive-control E. coli strains O157:H7 ATCC 35150 (stx₁ and stx₂), Ch05031 (stx_2e) or PGN28 (stx_2f) with a DIG high prime kit (Roche Diagnostics GmbH, Mannheim, Germany). Up to 24 typical E. coli isolates (including no lactose-fermenting isolates) taken from the MacConkey agar plate were assayed by colony hybridization or individual colony PCR. All of the isolates that tested positive for stx or eae genes were confirmed to be E. coli by conventional biochemical tests, such as the API 20E enteric bacterial identification system (bioMerieux, La Palme, France). As many as three positive colonies per sample were randomly chosen and subjected to more-extensive characterization. These further tests included O-serogroup typing (5); classification of the stx gene as stx₁, stx₂, stx_2e (11), or stx_2f (22) by PCR analysis; and typing of intimins by DNA sequencing using PCR analysis of an 800-bp subtype-specific region at the 3' end of the eae gene (1). One strain per sample was then selected from isolates showing the same O-serogroup-, intimin-, and/or stx-type profile and was analyzed by PCR for the presence of the bundle-forming pili gene (bfp) borne by the EAF plasmid, the enterohemolysin gene (ehxA), and the iron-repressible high-molecular-weight protein gene (irp), which are located in the genomic region known as the high pathogenicity island (7, 12, 20, 25).

View this table: [in this window] [in a new window]

TABLE 1. Isolation of eae- and stx-possessing Escherichia coli strains from cloacal swabs of various wild birds between 2003 and 2007 in the immediate environment of Tokyo Bay in Japan

In addition to the E. coli strains analyzed above, 208 intestinal bacterial floral E. coli strains, considered positive for the presence of E. coli by specific PCR analysis as previously described (21), were also isolated from 208 wild birds. These E. coli strains, as well as the pathogenic E. coli isolates described above, were examined for their susceptibilities to antimicrobial agents. The antimicrobial agents tested were aminobenzylpenicillin, cephalexin, dihydrostreptomycin, chloramphenicol, colistin, oxytetracycline (OTC), nalidixic acid, and enrofloxacin. These agents (except for chloramphenicol and nalidixic acid) are the ones most commonly used for therapeutic treatment in animal production in Japan. Aminobenzylpenicillin and dihydrostreptomycin were purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan), and the other drugs were supplied by the National Veterinary Assay Laboratory (Tokyo, Japan). In vitro susceptibility tests were carried out by the agar dilution method using the recommendations of the National Committee for Clinical Laboratory Standards (17). In cases where a strain showed resistance to one or more of the above drugs, the strain was reconfirmed as being E. coli with the API 20E test.

In the PCR screening of the cloacal samples of wild birds for the presence of the stx gene, only nine samples were weakly positive for stx₁, stx₂, or stx_2e, and no STEC isolates which possessed these stx genes were found. In one study of wild birds in southwest Scotland, only one out of 231 composite fecal samples from wild birds was stx positive (6). Similarly, a study of cloacal samples from wild birds at cattle or pig farms reported that four out of 244 samples were found to be stx positive by PCR analysis and that the frequency of STEC-positive wild birds was low, at only 1.6% (18). In the present study, PCR screening indicated that only 2.0% of the wild birds tested were positive for the stx gene. Moreover, these stx-positive birds showed no signs of overt diseases due to Stx proteins. Based on these data, it could be concluded that wild birds are not a major reservoir of STEC strains; however, analysis of the presence of the stx_2f-possessing E. coli (STEC_2f) strain in the cloacal samples of wild birds shows a slightly different picture. A PCR screen for the presence of stx_2f gave a positive result in 14 samples; four of these strains were further classified and were isolated from two feral pigeons (group O132), an oriental turtle dove (group O20), and a barn swallow (group O147) (Table 1). Moreover, four of the stx_2f-positive strains were confirmed for their expression of class Stx2 by the verocytotoxin-producing E. coli reverse passive latex agglutination test (Seiken, Tokyo, Japan). The presence of STEC_2f strains in wild birds has only been described in a few reports, and to date, these strains have only been isolated from the bird order Columbiformes (doves and pigeons) and a human infant (10, 14, 22). The present identification of an STEC_2f isolate from a swallow is the first report in which an STEC strain was isolated from a wild bird other than a dove. According to previous reports, STEC_2f isolates from wild birds or the infant can be classified into one of two subtypes: O128 and O45 (10, 14, 22). However, in the present study STEC_2f strains classified as O132, O20, and O147 were detected. It has previously been reported that STEC_2f strains carried by pigeons might be associated with human disease (23). This idea received further support from a second study in which an STEC_2f strain was isolated from an infant suffering from severe diarrhea. In that study, no diarrhea-associated microorganisms could be detected other than the STEC_2f strain (10). Thus, STEC_2f strains should be considered zoonotic microorganisms capable of movement between wild birds and humans.

In contrast to the low number of positives obtained from the PCR screening of the stx gene, PCR screening revealed 113 birds to be positive for the eae gene. Thirty-nine E. coli strains were isolated from 39 birds (16 different species), and these strains were distributed among at least 20 different types of O serogroups (Table 1). Not only were these eae-positive E. coli strains distributed among different O serogroups, but they also showed variation in the subtype of intimin that they possessed. Most of the strains possessed minor groups of intimin types in humans, such as intimin µ, , and . Furthermore, there was no relationship between the O serogroup and the intimin type present, with the exception of a few serogroups such as O128 and O103 that were associated with intimin β and intimin , respectively. None of the 39 eae-positive strains possessed any of the other pathogenic genes tested, such as bfp, ehxA, or irp. This result suggests that most of the E. coli strains in wild birds that possess the eae gene are probably related to atypical EPEC strains found in humans that are eae positive and stx negative. Atypical EPEC strains are very diverse with regard to serotypes and intimin subtypes, and many of the eae-positive and stx-negative E. coli serogroups found in the birds studied have also been found in infected humans (2, 9, 15).

The results of antimicrobial susceptibility tests on the isolated E. coli strains are shown in Table 2. Strains resistant to each of the drugs tested, with the exception of colistin, were detected. Fifty-two (25%) of the strains were resistant to OTC. In contrast, a total of 43 pathogenic strains, possessing either the stx_2f or the eae gene, were susceptible to all of the drugs tested, except for three strains that were resistant to only OTC. The higher susceptibilities of the pathogenic E. coli strains to microbial agents suggest that the pathogenic E. coli strains have higher host specificities for wild birds. In contrast to the pathogenic strains found in cloacal samples, E. coli strains in the flora of the gut might move easily between wild birds, livestock animals, and humans. Alternatively, these strains in the intestinal flora might more easily take up antimicrobial resistance genes from other organisms.

View this table: [in this window] [in a new window]

TABLE 2. Antimicrobial susceptibility of floral E. coli strains isolated from cloacal swabs of 208 wild birds

The data accumulated in this study suggest that wild birds have a low value as a reservoir animal for EHEC. This conclusion is based on the fact that serotypes of E. coli that are typically related to human diseases, such as O157, O111, and O26, were not isolated from cloacal samples of wild birds. In addition, with the exception of the STEC_2f strains, the pathogenic strains from wild birds did not possess stx genes that are common in EHEC strains, such as stx₁ and/or stx₂. Although a few STEC_2f strains were isolated from wild birds, these were the only STEC strains isolated, whereas more than 30% of wild birds were positive for eae by PCR analysis. Thus, the eae-positive isolates are potentially atypical EPEC. Even if these eae-positive strains are unable to infect mammals, the potential transmission of these pathogenic genes to other enterobacterial strains may pose a major threat to the health of humans. Therefore, the epidemiological dynamics of E. coli strains in wild birds should be continuously monitored.

 
This study was supported in part by a grant-in-aid from the Zoonoses Control Project of the Ministry of Agriculture, Forestry and Fisheries, supported by grants-in-aid from the Food Safety Commission (no. 0706) of Japan.

 
* Corresponding author. Mailing address: Center for Animal Disease Control and Prevention, Group of Biological Products, Quality Control Division, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. Phone/fax: 81-29-838-7874. E-mail: reptile{at}affrc.go.jp

Published ahead of print on 7 November 2008.

Top ABSTRACT INTRODUCTION REFERENCES

 

1
1. Adu-Bobie, J., G. Frankel, C. Bain, A. Guedes Goncalves, L. R. Trabulsi, G. Douce, S. Knutton, and G. Dougan. 1998. Detection of intimins , β, , and , four intimin derivatives expressed by attaching and effacing microbial pathogens. J. Clin. Microbiol. 36:662-668.[Abstract/Free Full Text]
2
2. Afset, J. E., E. Anderssen, G. Bruant, J. Harel, L. Wieler, and K. Bergh. 2008. Phylogenetic backgrounds and virulence profiles of atypical enteropathogenic Escherichia coli strains from a case-control study using multilocus sequence typing and DNA microarray analysis. J. Clin. Microbiol. 46:2280-2290.[Abstract/Free Full Text]
3
3. Beutin, L., D. Geier, H. Steinruck, S. Zimmermann, and F. Scheutz. 1993. Prevalence and some properties of verotoxin (Shiga-like toxin)-producing Escherichia coli in seven different species of healthy domestic animals. J. Clin. Microbiol. 31:2483-2488.[Abstract/Free Full Text]
4
4. Beutin, L., D. Geier, S. Zimmermann, and H. Karch. 1995. Virulence markers of Shiga-like toxin-producing Escherichia coli strains originating from healthy domestic animals of different species. J. Clin. Microbiol. 33:631-635.[Abstract]
5
5. Blanco, J., M. Blanco, J. E. Blanco, M. P. Alonso, J. I. Garabal, and E. A. Gonzárez. 1993. Escherichia coli enterotoxigénicos, necrotoxigénicos, y verotoxigénicos de origin humano y bovino. Servicio Publicaciones Diputación Provincial de Lugo, Lugo, Spain.
6
6. Foster, G., J. Evans, H. I. Knight, A. W. Smith, G. J. Gunn, L. J. Allison, B. A. Synge, and T. W. Pennycott. 2006. Analysis of feces samples collected from a wild-bird garden feeding station in Scotland for the presence of verocytotoxin-producing Escherichia coli O157. Appl. Environ. Microbiol. 72:2265-2267.[Abstract/Free Full Text]
7
7. Franke, J., S. Franke, H. Schmidt, A. Schwarzkopf, L. H. Wieler, G. Baljer, L. Beutin, and H. Karch. 1994. Nucleotide sequence analysis of enteropathogenic Escherichia coli (EPEC) adherence factor probe and development of PCR for rapid detection of EPEC-harboring virulence plasmids. J. Clin. Microbiol. 32:2460-2463.[Abstract/Free Full Text]
8
8. Gannon, V. P., M. Rashed, R. K. King, and E. J. Thomas. 1993. Detection and characterization of the eae gene of Shiga-like toxin-producing Escherichia coli using polymerase chain reaction. J. Clin. Microbiol. 31:1268-1274.[Abstract/Free Full Text]
9
9. Ishii, S., K. P. Meyer, and M. J. Sadowsky. 2007. Relationship between phylogenetic groups, genotypic clusters, and virulence gene profiles of Escherichia coli strains from diverse human and animal sources. Appl. Environ. Microbiol. 73:5703-5710.[Abstract/Free Full Text]
10
10. Isobe, J., K. Kimata, M. Shimojima, S. Hosorogi, D. Tanaka, and Y. Gyobu. 2004. Isolation of Escherichia coli O128:HNM harboring stx2f gene from diarrhea patients. Kansenshogaku Zasshi 78:1000-1005. (In Japanese.)[Medline]
11
11. Johnson, W. M., D. R. Pollard, H. Lior, S. D. Tyler, and K. R. Rozee. 1990. Differentiation of genes coding for Escherichia coli verotoxin 2 and the verotoxin associated with porcine edema disease (VTe) by the polymerase chain reaction. J. Clin. Microbiol. 28:2351-2353.[Abstract/Free Full Text]
12
12. Karch, H., S. Schubert, D. Zhang, W. Zhang, H. Schmidt, T. Ölschläger, and J. Hacker. 1999. A genomic island, termed the high-pathogenicity island, is present in certain non-O157 Shiga toxin-producing Escherichia coli clonal lineages. Infect. Immun. 67:5994-6001.[Abstract/Free Full Text]
13
13. Karmali, M. A. 1989. Infection by verocytotoxin-producing Escherichia coli. Clin. Microbiol. Rev. 2:15-38.[Abstract/Free Full Text]
14
14. Kobayashi, H., T. Pohojanvirta, and S. Pelkonen. 2002. Prevalence and characteristics of intimin- and Shiga toxin-producing Escherichia coli from gulls, pigeons and broilers in Finland. J. Vet. Med. Sci. 64:1071-1073.[CrossRef][Medline]
15
15. Kozub-Witkowski, E., G. Kraus, G. Frankel, D. Kramer, B. Appel, and L. Beutin. 2007. Serotypes and virutypes of enteropathogenic and enterohaemorrhagic Escherichia coli strains from stool samples of children with diarrhea in Germany. J. Appl. Microbiol. 104:403-410.
16
16. Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142-201.[Abstract/Free Full Text]
17
17. National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial susceptibility testing for bacteria that grow aerobically. Approved standard M7-A4. National Committee for Clinical Laboratory Standards, Wayne, PA.
18
18. Nielsen, E. M., N. S. Marianne, J. J. Madsen, L. Jens, B. J. Jørgen, and L. B. Dorte. 2004. Verocytotoxin-producing Escherichia coli in wild birds and rodents in close proximity to farms. Appl. Environ. Microbiol. 70:6944-6947.[Abstract/Free Full Text]
19
19. Olsvik, O., and N. A. Strockbine. 1993. PCR detection of heat-stable, heat-labile, and Shiga-like toxin genes in Escherichia coli, p. 271-276. In D. H. Persing, T. F. Smith, F. C. Tenover, and T. J. White (ed.), Diagnostic molecular microbiology. American Society for Microbiology, Washington, DC.
20
20. Paton, A. W., and J. C. Paton. 1998. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfb_O111, and rfb_O157. J. Clin. Microbiol. 36:598-602.[Abstract/Free Full Text]
21
21. Riffon, R., S. Khampoun, K. Hayssam, D. Pascal, D. Marc, and L. Jacqueline. 2001. Development of a rapid and sensitive test for identification of major pathogens in bovine mastitis by PCR. J. Clin. Microbiol. 39:2584-2589.[Abstract/Free Full Text]
22
22. Schmidt, H., J. Scheef, S. Morabito, A. Caprioli, L. H. Wieler, and H. Karch. 2000. A new Shiga toxin 2 variant (Stx2f) from Escherichia coli isolated from pigeons. Appl. Environ. Microbiol. 66:1205-1208.[Abstract/Free Full Text]
23
23. Sonntag, A. K., E. Zenner, H. Karch, and M. Bielaszewska. 2005. Pigeons as a possible reservoir of Shiga toxin 2f-producing Escherichia coli pathogenic to humans. Berl. Munch. Tierarztl. Wochenschr. 118:464-470.[Medline]
24
24. Wallace, J. S., T. Cheasty, and K. Jones. 1997. Isolation of verocytotoxin-producing Escherichia coli O157 from wild birds. J. Appl. Microbiol. 82:399-404.[CrossRef][Medline]
25
25. Wieler, L. H., E. Vieler, C. Erpenstein, T. Schlapp, H. Steinruck, R. Bauerfeind, A. Byomi, and G. Baljer. 1996. Shiga toxin-producing Escherichia coli strains from bovines: association of adhesion with carriage of eae and other genes. J. Clin. Microbiol. 34:2980-2984.[Abstract]

---

Applied and Environmental Microbiology, January 2009, p. 292-295, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01534-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kobayashi, H. Articles by Kubo, M. Search for Related Content PubMed PubMed Citation Articles by Kobayashi, H. Articles by Kubo, M. Agricola Articles by Kobayashi, H. Articles by Kubo, M.