Localized Adherence and Attaching-Effacing Properties of ...

Vol. 59, No. 5

INFECTION AND IMMUNITY, May 1991, p. 1864-1868 0019-9567/91/051864-05$02.00/0 Copyright ©D 1991, American Society for Microbiology

Localized Adherence and Attaching-Effacing Properties of Nonenteropathogenic Serotypes of Escherichia coli M. JOHN

ALBERT,'* KHORSHED ALAM,1 M. ANSARUZZAMAN,1 JACQUELINE MONTANARO,2 MOYENUL ISLAM,' SHAH M. FARUQUE,1 KHALEDA HAIDER,' KARL BETTELHEIM,3 AND SAUL TZIPORI'

International Centre for Diarrhoeal Disease Research, Dhaka 1000, Bangladesh,' and Royal Children's Hospital, Parkville,2 and Fairfield Infectious Diseases Hospital, Fairfield,3 Victoria, Australia Received 21 December 1990/Accepted 20 February 1991

Traditional enteropathogenic Escherichia coli serotypes demonstrate a plasmid-mediated localized adherence in cultured HeLa or HEp-2 cells and induce an attaching-effacing intestinal lesion, both of which are considered pathognomonic and causes of diarrhea. This study describes three E. coli strains from infantile diarrhea which share these properties but belong to serotypes (02:H2, 02:H25 and 015:H2) not considered enteropathogenic.

The term enteropathogenic Escherichia coli (EPEC) was coined by Neter et al. (17) for a collection of epidemiologically incriminated E. coli serotypes which, in many studies throughout the world, were isolated with significantly greater frequency from infants with diarrhea than from healthy controls. The World Health Organization's definition of EPEC includes the following serogroups: 026, 055, 086, 0111, 0114, 0119, 0125, 0126, 0127, 0128, 0142, and 0158 (24). It was only recently demonstrated that the majority of these serotypes adhere to HeLa and HEp-2 cells in culture, with the majority of strains showing a localized adherence (LA) and a few showing a diffuse adherence (20). Baldini et al. (2) showed that LA is mediated by an -60-MDa plasmid, and the term EPEC adherence factor (EAF) was given to the gene product (1). A DNA probe constructed from EAF plasmid correctly identified LA' EPEC (16). Human volunteer studies suggested that EAF plasmid-associated product is an important virulence factor in the pathogenesis of EPEC diarrhea (13). Ultrastructural studies of intestinal mucosa of naturally infected humans and experimentally infected animals suggested that EPEC produce diarrhea by an attaching-effacing (A/E) type of lesion (15, 22, 23). In this lesion, bacteria destroy the brush border of enterocytes and closely adhere to the apical surface, causing cupping and pedestal formation by the cell membrane and electron-dense fibrillar modifications in the terminal web areas beneath the attached bacteria. Bacteria occasionally are seen within phagolysosomes. There is evidence of inflammatory cell response in the lamina propria of affected mucosa (22). In vitro studies by Knutton et al. (9, 11) suggested that EAF appears initially to facilitate contact with the mucosa, but chromosomally encoded factors are required to produce the A/E lesion. Subsequent in vivo studies confirmed this observation (21). Studies in Peru and Brazil found that LA' EPEC serotypes were significantly associated with diarrhea, whereas LA' non-EPEC serotypes which were also encountered were isolated in equal frequency from both patients with diarrhea and controls (8, 14). However, in a study in Thailand (5), the vast majority of LA' non-EPEC serotypes *

were isolated from a significantly higher proportion of diarrheal cases than from controls. In our studies on the role of diarrheagenic E. coli in the causation of diarrhea in Bangladeshi children, we isolated LA' non-EPEC serotypes from approximately 7% of 400 diarrheal children te- _\% % SS} . ^ .v

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FIG. 2. Electron micrograph of villus epithelial cells from a rabbit ileal loop inoculated with E. ccli 02:H2. (A) Effaced microvilli, pedestal formation, and cupping of plasma membrane and increased electron density of terminal web area associated with attached bacteria. A bacterium penetrating the cell via invagination of the plasma membrane is also seen (arrows). Note the elongated microvilli between attached bacteria. Magnification, x 10,900. (B) Effaced microvilli, pedestal formation, and cupping of plasma membrane and increased electron density of terminal web associated with attached bacteria are seen. Note (lower left) uptake of bacteria into phagolysosomes (thin arrows) and (upper left) an enterocyte that has almost lost its entire cytoplasm (thick arrows). Magnification, x7,200.

increased electron density in the terminal web just beneath the site of bacterial attachment. Microvilli in between the sites of bacterial attachment were elongated. Intracellular bacteria were seen in phagolysosomes. Some of the changes are shown in Fig. 2. Each strain produced similar results in both rabbits. Light and electron microscopic examinations of the lesions produced by the three nonenteropathogenic serotypes of E. coli in rabbit ileal loops showed that lesions were identical to those produced by traditional EPEC serotypes (15, 22, 23). Moreover, like traditional EPEC serotypes, they also produced LA in cultured HeLa cells. Studies to date have suggested that the two critical virulence factors for full pathogenicity of traditional EPEC are the abilities to pro-

duce LA and A/E lesions (9, 13, 21). Since the three nontraditional serotypes isolated from children with watery diarrhea also possessed these critical virulence factors, they should be considered as diarrheagenic as the traditional EPEC serotypes. Knutton et al. (10) have shown recently that some of the non-EPEC serotypes were positive for A/E lesions by FAS assay. Our three non-EPEC serotypes which were positive for A/E lesions in gut loop were also positive for A/E lesions in the FAS assay. This suggests that the FAS test may also be used as a substitute test for the difficult in vivo gut loop test to screen non-EPEC serotypes for the ability to produce A/E lesions. The traditional EPEC serotypes were initially identified in

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FIG. 2-Continued.

Western countries by serotyping during investigations of epidemics of diarrhea. Perhaps strains other than the traditional serotypes have been causing both endemic and epidemic diarrheas in developing countries. Since methods to study these agents were not available, their role as causative agents of diarrhea would have been ignored. Case control studies based on demonstration of virulence characteristics need to be carried out to assess the role of nontraditional serotypes. This research was supported by the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B). ICDDR,B is supported by countries and agencies which share its concern about

the impact of diarrheal diseases in the developing countries. Current major donors giving assistance to ICDDR,B are the Aga Khan Foundation, Arab Gulf Fund, Australia, Bangladesh, Belgium, Canadian International Development Agency (CIDA), Canadian International Development Research Centre (IDRC), Danish International Development Agency (DANIDA), France, the Ford Foundation, Japan, The Netherlands, Norwegian Agency for International Development (NORAD), SAREC (Sweden), Swiss Development Cooperation (SDC), United Kingdom, United Nations Development Programme (UNDP), United Nations Children's Fund (UNICEF), United Nations Capital Development Fund (UNCDF), United States Agency for International Development (USAID), World Health Organization (WHO), and World University Service of Canada (WUSC).

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INFECT. IMMUN.

We thank Prabashi Mahmood and Priyatosh Sukul for secretarial assistance.

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REFERENCES Baldini, M. M., J. B. Kaper, M. M. Levine, D. C. A. Candy, and H. W. Moon. 1983. Plasmid-mediated adhesion in enteropathogenic Escherichia coli. J. Pediatr. Gastroenterol. Nutr. 2:534538. Baldini, M. M., J. P. Nataro, and J. B. Kaper. 1986. Localization of a determinant for HEp-2 adherence by enteropathogenic Escherichia coli. Infect. Immun. 52:334-336. Bettelheim, K. A., and C. J. Thompson. 1987. New method of serotyping Escherichia coli: implementation and verification. J. Clin. Microbiol. 25:781-786. Chandler, M. E., and K. A. Bettelheim. 1974. A rapid method of identifying Escherichia coli H antigens. Zentrabl. Bakteriol. Mikrobiol. Hyg. Abt. 1 Orig. Reihe A 229:74-79. Chatkaeomorakot, A., P. Echeverria, D. N. Taylor, K. A. Bettelheim, N. R. Blacklow, 0. Sethabutr, J. Seriwatana, and J. B. Kaper. 1987. HeLa cell-adherent Escherichia coli in children with diarrhoea in Thailand. J. Infect. Dis. 156:669-672. Dean, A. G., Y. C. Ching, R. G. WiSliams, and L. B. Harden. 1972. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhoea in children in Honolulu. J. Infect. Dis. 125:407-411. Gentry, M. K., and J. M. Dalrymple. 1980. Quantitative microtiter cytotoxicity assay for Shigella toxin. J. Clin. Microbiol. 12:361-366. Gomes, T. A. T., M. A. M. Vieira, I. K. Wachsmuth, P. A. Blake, and L. R. Trabulsi. 1989. Serotype-specific prevalence of Escherichia coli strains with EPEC adherence factor genes in infants with and without diarrhoea in Sao Paulo, Brazil. J. Infect. Dis. 160:131-135. Knutton, S., M. M. Baldini, J. B. Kaper, and A. S. McNeish. 1987. Role of plasmid-encoded adherence factors in adhesion of enteropathogenic Escherichia coli to HEp-2 cells. Infect. Immun. 55:78-85. Knutton, S., T. Baldwin, P. H. Williams, and A. S. McNeish. 1989. Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect. Immun. 57: 1290-1298. Knutton, S., D. R. Lloyd, and A. S. McNeish. 1987. Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa. Infect. Immun. 55:69-77. Levine, M. M., and R. Edelman. 1984. Enteropathogenic Escherichia coli of classic serotypes associated with infant diar-

14.

15.

16.

17.

18.

19.

20. 21.

22.

23. 24.

rhoea: epidemiology and pathogenesis. Epidemiol. Rev. 6:3151. Levine, M. M., J. P. Nataro, H. Karch, M. M. Baldini, J. B. Kaper, R. E. Black, M. L. Clements, and A. D. O'Brien. 1985. The diarrhoeal response of man to some classical serotype enteropathogenic Escherichia coli is dependent on a plasmid encoding an entero-adhesiveness factor. J. Infect. Dis. 152:550559. Levine, M. M., V. Prado, R. Robins-Browne, H. Lior, J. B. Kaper, S. L. Moseley, K. Gicquelais, J. P. Nataro, P. Vial, and B. Tall. 1988. Use of DNA probes and HEp-2 cell adherence assay to detect diarrhoeagenic Escherichia coli. J. Infect. Dis. 158: 224-228. Moon, H. W., S. C. Whipp, R. A. Argenzio, M. M. Levine, and R. A. Gianella. 1983. Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect. Immun. 41:1340-1351. Nataro, J. P., M. M. Baldini, J. B. Kaper, R. E. Black, N. Bravo, and M. M. Levine. 1985. Detection of an adherence factor of enteropathogenic Escherichia coli using a DNA probe. J. Infect. Dis. 152:560-565. Neter, E., 0. Westphal, 0. Luderitz, R. M. Gino, and E. A. Gorzynski. 1955. Demonstration of antibodies against enteropathogenic Escherichia coli in sera of children of various ages. Pediatrics 16:801-808. Newland, J. W., and R. J. NeiUl. 1988. DNA probes for Shigalike toxins I and II and for toxin-converting bacteriophages. J. Clin. Microbiol. 26:958-987. Sack, D. A., and R. B. Sack. 1975. Test for enterotoxigenic Escherichia coli using Y-1 adrenal cells in miniculture. Infect. Immun. 11:334-336. Scaletsky, I. C. A., M. L. M. Silva, and L. R. Trabulsi. 1984. Distinctive patterns of adherence of enteropathogenic Escherichia coli to HeLa cells. Infect. Immun. 45:534-536. Tzipori, S., R. Gibson, and J. Montanaro. 1989. Nature and distribution of mucosal lesions associated with enteropathogenic and enterohemorrhagic Escherichia coli in piglets and the role of plasmid-mediated factors. Infect. Immun. 57:1142-1150. Tzipori, S., R. M. Robins-Browne, G. Gonis, J. Hayes, M. Whithers, and E. McCartney. 1985. Enteropathogenic Escherichia coli enteritis: evaluation of the gnotobiotic piglet as a model of human infection. Gut 26:570-578. Ulshen, M. H., and J. L. Roll. 1980. Pathogenesis of Escherichia coli gastroenteritis in man-another mechanism. N. Engl. J. Med. 302:99-101. World Health Organization. 1987. Programme for control of diarrhoeal diseases (CDD/83.3 Rev 1), p. 27. In Manual for laboratory investigations of acute enteric infections. World Health Organization, Geneva.