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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Gomes, T. A. T. Articles by Ramos, S. R. T. S. Search for Related Content PubMed PubMed Citation Articles by Gomes, T. A. T. Articles by Ramos, S. R. T. S.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, December 1998, p. 3609-3613, Vol. 36, No. 12
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Adherence Patterns and Adherence-Related DNA Sequences in Escherichia coli Isolates from Children with and without Diarrhea in São Paulo City, Brazil

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, S.P., Brazil, CEP 04023-062¹; Instituto da Criança, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, S.P., Brazil, CEP 05403-900²; and Foodborne and Diarrheal Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia 30333³

Received 8 June 1998/Returned for modification 23 July 1998/Accepted 21 September 1998

	ABSTRACT

Top Abstract Introduction Materials & Methods Results Discussion References

The correlation between various adherence patterns and adherence-related DNA sequences in Escherichia coli isolates from 1- to 4-year-old children with and without diarrhea in São Paulo, Brazil, was evaluated. A total of 1,801 isolates obtained from 200 patients and 200 age-matched controls were studied. The adherence patterns found were classified as diffuse, aggregative, aggregative in a 6-h assay, aggregative predominantly in coverslips, localized, localized-like, and noncharacteristic. In general, the DNA sequences used as probes showed excellent specificities (>93%), but their sensitivities varied. Thus, the results of bioassays and assays with DNA probes normally used to search for adherent E. coli did not correlate well, and the best method for the identification of these organisms in the clinical research setting remains controversial. Isolates presenting diffuse adherence or hybridizing with the related daaC probe, or both, were by far the most frequent in patients (31.5, 26.0, and 23.0%, respectively), followed by isolates presenting aggregative adherence or hybridizing with the related EAEC probe, or both (21.5, 13.0, and 10.5%, respectively). None of the different combinations of adherence patterns and adherence-related DNA sequences found were associated with acute diarrhea.

	INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

The first step in the establishment of the diarrheal diseases caused by the various categories of diarrheagenic Escherichia coli is adherence to epithelial cells of the intestinal mucosa. In vitro assays with eukaryotic cell lines (HeLa and HEp-2 cells) have identified three distinct adherence patterns among fecal isolates of E. coli: localized, diffuse, and aggregative (37, 38, 41). Localized adherence (LA) is characterized by formation of bacterial microcolonies on a restricted area(s) of the cell surface, while diffuse adherence (DA) is the scattered attachment of bacteria over the whole surface of the cell (41). The pattern of aggregative adherence (AA) consists of bacterial attachment to the cells and the intervening cell growth surface in a stacked brick-like lattice (37).

The LA pattern was first detected in strains classified as enteropathogenic E. coli (EPEC) among serogroups associated with outbreaks of infantile diarrhea (41). Although E. coli strains exhibiting DA (DAEC) have been isolated at similar frequencies from feces of infants and young children with acute diarrhea and nondiarrheic controls in some populations (3, 10, 11, 14, 18), they were significantly associated with diarrhea in other settings (1, 17, 24, 29, 33). E. coli strains showing AA, termed enteroaggregative E. coli (EAEC), have been linked to sporadic persistent diarrhea (3, 4, 7, 10, 13, 26, 27, 44) and to outbreaks of diarrhea in both developing and developed countries (8, 12, 28, 43). However, the role of EAEC in acute diarrhea is still controversial: some studies have shown a correlation (7, 23, 25, 27, 34, 37), but others (1, 3, 6, 10, 11, 13-15, 17, 18, 24, 26, 29, 33, 44) have not.

DNA probes derived from adherence-related sequences have been constructed (2, 5, 16, 31, 36) and used in hybridization assays for the detection of the different established and putative categories of diarrheagenic E. coli in many epidemiological studies.

We evaluated the relationship between the LA, DA, and AA patterns and hybridization with adherence-related DNA sequences and tested children 1 to 4 years old with and without acute diarrhea for the presence of adherent E. coli strains.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Patients. Rectal swab specimens were collected from 200 children with acute (7 days) diarrhea (patients) and 200 children who had not had any gastrointestinal signs or symptoms during the 30 days prior to collection (controls). These children were selected by a computer-generated random-number table from among 505 patient-control pairs obtained for a study on the etiology of acute diarrheal diseases (19). The population analyzed consisted of children visiting the emergency room of the Hospital Infantil Menino Jesus, São Paulo, Brazil, for medical attention between April 1989 and March 1990; most children were of low socioeconomic status. Controls were matched with case patients in the following age groups: 12 to 23, 24 to 35, and 36 to 59 months.

Microbiologic methods. Adenovirus, Aeromonas spp., thermophilic Campylobacter, Salmonella spp., Shigella spp., Yersinia enterocolitica, and rotavirus were searched for by standard methods (20, 30). Five lactose-fermenting and any non-lactose-fermenting colonies typical of E. coli were selected from the isolation plates. Biochemically confirmed E. coli isolates were stored in 15.0% glycerol at 70°C. All E. coli isolates were tested with specific DNA probes designed to detect enterotoxigenic E. coli (ETEC) (LT-I, LT-II, ST-Ip, ST-Ih, and ST-II probes), enteroinvasive E. coli (EIEC) (Inv probe), and Shiga-toxin-producing E. coli (STEC) (Stx1 and Stx2 probes), as described previously (22).

HeLa cell adherence assays. The HeLa cell adherence assays were performed as described by Cravioto et al. (9) with HeLa cells grown to 60% confluence and a single infection period of 3 h. Weakly adherent and nonadherent isolates were retested by the same method with an additional incubation period of 3 h (6-h assay) (9). The following E. coli strains were included as controls: E2348/69 (LA) (36), C1845 (DA) (5), and 0431-4/85 (AA) (18). Strains were tested in duplicate and were examined blindly by immersion microscopy.

Search for adherence-related DNA sequences in E. coli. The following specific DNA probes were used: daaC (associated with the biogenesis of F1845, a fimbrial adhesin involved in DA), a 350-bp PstI fragment of pSLM852 (5); EAEC (EAEC adherence plasmid), a 1-kb EcoRI-PstI fragment of pCVD432 (2); EAF (EPEC adherence factor), a 1-kb BamHI-SalI fragment derived from plasmid pMAR2 (36); bfpA (encoding the subunits of a fimbrial adhesin of EPEC strains, termed the bundle-forming pilus), an 852-bp EcoRI fragment of pMSD207 (16); and eaeA (encoding intimin, an outer membrane protein involved in the attaching-effacing lesions promoted by EPEC and some STEC strains), a 1-kb SalI-KpnI fragment from plasmid from pCVD434 (31). The fragment probes were labeled with [-³²P]dATP and used in colony hybridization assays performed under stringent conditions as described by Maas (35).

Statistical analysis. Analysis of patients and controls maintained the matched pairs and used the Pike-Morrow adaptation of the Mantel-Haenszel test to calculate P values (42). Epi-Info 6.02 was used to perform the calculations.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

E. coli adherence to HeLa cells. The adherence patterns of 273 isolates could not be determined because they promoted complete detachment of the HeLa cell monolayers in the 3-h assays. Among the remaining 1,801 E. coli isolates tested (879 from patients and 922 from controls), 47.0% were nonadherent (NA; i.e., sparse bacteria adhering to <1% of the cells in the 6-h assay). The adherence patterns of the other isolates are presented in Table 1.

View this table: [in this window] [in a new window]   TABLE 1.   Relationship between hybridization with DNA probes for adherence-related sequences and patterns of adherence to HeLa cells of E. coli isolated from children with and without diarrhea

The degree and frequency of DA varied, but this pattern could always be detected in the 3-h assay. Regarding the AA pattern, 3 types were detected: typical AA, AA that could be clearly discerned only in the 6-h assay (AA6h), and AA predominantly in coverslips (AAcs). The typical LA adherence pattern was characterized by one to three tight bacterial clusters per cell on 75 to 100% of the cells. An LA-like (LAL) pattern of adherence (40), characterized by loose and compact clusters of bacteria on 1 to 45% of the cells, was detected in the 6-h assay for some isolates that presented an undefined pattern in the 3-h assay. Moreover, some isolates presented adherence patterns distinct from those described in the literature; the adherence patterns of these isolates were classified as noncharacteristic (NC).

Correlation between adherence patterns and hybridization with adherence-related DNA probes. The relationship between the distinct adherence patterns detected and hybridization with specific DNA probes found in this study is presented in Table 1. The daaC probe reacted with 202 of 321 isolates with DA (sensitivity, 62.9%) and with 99 isolates that were NA or that presented adherence patterns distinct from DA (specificity, 93.3%). The EAEC probe detected 190 of the 256 isolates with AA and 5 of the 132 isolates with AAcs (sensitivity, 47.0%) and reacted with only 29 isolates showing none of the different AA types (specificity, 97.9%). The eaeA probe reacted with all isolates with LA and LAL (sensitivity, 100.0%) and with 60 non-LA- and non-LAL-producing isolates (specificity for the detection of LA- and LAL-producing E. coli, 96%). All isolates with LA hybridized with the EAF and bfpA probes, whereas none of the isolates with LAL reacted with the EAF probe, and only five (35.7%) isolates reacted with the bfpA probe. Most (60.3%) of the 273 cytodetaching isolates did not hybridize with any of the probes used.

Prevalence of adherence patterns and adherence-related DNA sequences in patients and control children. The distribution of the E. coli isolates showing different patterns of adherence or related DNA sequences in patients and controls is presented in Table 2. Approximately 50% of the children studied carried more than one type of isolate. The statistical analysis of the association with diarrhea was performed for those children in whose stools none of the other pathogens were identified (adenovirus, Aeromonas, Campylobacter, EIEC, ETEC, rotavirus, Salmonella, Shigella, and STEC).

View this table: [in this window] [in a new window]   TABLE 2.   Association between the different patterns of adherence and the adherence-related DNA sequences in E. coli and acute diarrhea in 200 patients and 200 age-matched controls

None of the adherence patterns detected was associated with acute diarrhea. DA was the most frequent pattern among isolates from both patients and controls (31.5 versus 28.5%), followed by AA (21.5 versus 19.0%) and AAcs (12.5 versus 15.0%). Likewise, none of the adherence-related DNA sequences were associated with acute diarrhea. Isolates carrying daaC were the most frequent among the patients and controls (26.0 versus 21.0%). Similarly, none of the different combinations of adherence patterns and related DNA sequences were associated with diarrhea (Table 3). Isolates carrying daaC and producing DA (23.0%) and EAEC-positive isolates producing AA (10.5%) were the most frequent in patients.

View this table: [in this window] [in a new window]   TABLE 3.   Prevalence of different patterns of adherence combined with adherence-related DNA sequences in E. coli isolated from 200 children with acute diarrhea and 200 age-matched controlsa

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

In this study we analyzed the correlation between different adherence patterns and the presence of adherence-related DNA sequences in E. coli isolates from diarrheic and control children. The adherence-related DNA probes used showed excellent specificities (>93.0%), but their sensitivities varied. The low sensitivities of the daaC and EAEC probes found in this study (62.9 and 47.0%, respectively) are in accordance with those found in other field studies and confirms the heterogeneous nature of the DAEC and EAEC categories (2, 11, 13, 17, 29). It is interesting that none of the isolates that showed AA6h reacted with the EAEC probe; whether these isolates comprise a homogeneous population remains to be established. Furthermore, all 56 isolates that were both EAEC and daaC positive produced AA but not DA, and the significance of daaC in these isolates is under investigation. All isolates with LA carried eaeA, bfpA, and EAF, whereas all isolates with LAL carried eaeA (100.0%) and occasionally bfpA (35.7%). So far, the distribution of bfpA has not been extensively analyzed, but this probe is considered to be more sensitive than the EAF probe in detecting LA-producing E. coli (16). However, our data suggest that both probes are equally sensitive in detecting LA.

The different combinations of adherence patterns and adherence-related sequences found in this study demonstrated that the results obtained with the DNA probes and by the bioassays normally used to search for DAEC and EAEC do not correlate well. Moreover, although adherence to HEp-2 and HeLa cells has been used as the "gold standard" assay for the detection of these categories of isolates, variations in the AA (29, 32) and LA (22, 40) patterns are occasionally reported, and thus, the best method of identifying these organisms in the clinical research setting remains controversial.

None of the different adherence patterns or adherence-related DNA sequences, or a combination of both, were associated with acute diarrhea in children 1 to 4 years old in São Paulo. This lack of association was observed even when the data for any of the three age groups studied (12 to 23, 24 to 35, and 36-59 months) were analyzed (data not shown).

The children studied here were selected from a larger population in which Shigella and rotavirus were the most frequent pathogens found (19). Although DAEC and EAEC were very frequently found in these children, they were not associated with acute diarrhea, suggesting that both categories are heterogeneous and each comprises pathogenic and nonpathogenic strains or that the high level of asymptomatic carriage of DAEC and EAEC, often observed with other well-established enteropathogens (20), masks an association with the disease in our population. The low incidence of E. coli with LA found in this study is probably due to differences in the risk factors that these children have (39), since LA is mainly detected among strains of EPEC serotypes, which prevail in the first year of life (21).

	ACKNOWLEDGMENTS

We are indebted to J. B. Kaper, M. M. Levine, J. A. Girón, and S. L. Moseley for providing the probes used in this study, to B. E. C. Guth for critical reading of the manuscript, and L. R. M. Marques for helpful suggestions.

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Financiadora de Estudos e Projetos/Ministério da Ciência e Tecnologia/Programa de Apoio a Núcleos de Excelência (FINEP/MCT/PRONEX).

	FOOTNOTES

^* Corresponding author. Mailing address: Departamento de Microbiologia, Immunologia e Parasitologia, Universidade Federal de São Paulo, Escola Paulista de Medecina, Rua Botucatu 862, 3° Andar, São Paulo, S.P., Brazil, CEP 04023-062. Phone: 055-011-5084-3213. Fax: 055-011-571-6504. E-mail: tatgomes.dmip{at}epm.br.

	REFERENCES

Top Abstract Introduction Materials & Methods Results Discussion References

1.	Baqui, A. H., R. B. Sack, R. E. Black, K. Haider, A. Hossain, A. R. M. A. Alim, M. Yunus, H. R. Chowdhury, and A. K. Siddique. 1992. Enteropathogens associated with acute and persistent diarrhea in Bangladeshi children <5 years of age. J. Infect. Dis. 166:792-796[Medline].
2.	Baudry, B., S. J. Savarino, P. Vial, J. B. Kaper, and M. M. Levine. 1990. A sensitive and specific DNA probe to identify enteroaggregative Escherichia coli, a recently discovered diarrheal pathogen. J. Infect. Dis. 161:1249-1251[Medline].
3.	Bhan, M. K., P. Raj, M. M. Levine, J. B. Kaper, N. Bhandari, R. Srivastava, R. Kumar, and S. Sazawal. 1989. Enteroaggregative Escherichia coli associated with persistent diarrhea in a cohort of rural children in India. J. Infect. Dis. 159:1061-1064[Medline].
4.	Bhan, M. K., V. Khoshoo, H. Sommerfelt, P. Raj, S. Sazawal, and R. Srivastava. 1989. Enteroaggregative Escherichia coli and Salmonella associated with nondysenteric persistent diarrhea. Pediatr. Infect. Dis. J. 8:499-502[Medline].
5.	Bilge, S. S., C. R. Clausen, W. Lau, and S. L. Moseley. 1989. Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coli to HEp-2 cell. J. Bacteriol. 171:4281-4289[Abstract/Free Full Text].
6.	Biswas, R., E. A. S. Nelson, P. J. Lewindon, D. J. Lyon, P. B. Sullivan, and P. Echeverria. 1996. Molecular epidemiology of Escherichia coli diarrhea in children in Hong Kong. J. Clin. Microbiol. 34:3233-3234[Abstract].
7.	Chan, K. N., A. D. Philips, S. Knutton, H. R. Smith, and J. A. Walker-Smith. 1994. Enteroaggregative Escherichia coli: another cause of acute and chronic diarrhea in England? J. Pediatr. Gastroenterol. Nutr. 18:87-91[Medline].
8.	Cobeljic, M., B. Miljkovic-Selimovic, D. Paunovic-Todosijevic, Z. Velickovic, Z. Lepsanovic, N. Zec, D. Savic, R. Ilic, S. Konstantinovic, B. Jovanovic, and V. Kostic. 1996. Enteroaggregative Escherichia coli associated with an outbreak of diarrhea in a neonatal nursery ward. Epidemiol. Infect. 117:11-16[Medline].
9.	Cravioto, A., R. J. Gross, S. M. Scotland, and B. Rowe. 1979. An adhesive factor found in strains of Escherichia coli belonging to the traditional infantile enteropathogenic serotypes. Curr. Microbiol. 3:95-99.
10.	Cravioto, A., A. Tello, A. Navarro, J. Ruiz, H. Villafan, F. Uribe, and C. Eslava. 1991. Association of Escherichia coli HEp-2 adherence patterns with type and duration of diarrhoea. Lancet i:262-264.
11.	Echeverria, P., O. Serichantalerg, S. Changehawalit, B. Baudry, M. M. Levine, F. Orskov, and I. Orskov. 1992. Tissue culture-adherent Escherichia coli in infantile diarrhea. J. Infect. Dis. 165:141-143[Medline].
12.	Eslava, C., J. Villaseca, R. Morales, A. Navarro, and A. Cravioto. 1993. Identification of a protein with toxigenic activity produced by enteroaggregative Escherichia coli, abstr. B-105, p. 44. In Abstracts of the 93rd General Meeting of the American Society for Microbiology 1993. American Society for Microbiology, Washington, DC.
13.	Fang, G. D., A. A. M. Lima, C. V. Martins, J. P. Nataro, and R. L. Guerrant. 1995. Etiology and epidemiology of persistent diarrhea in northeastern Brazil: a hospital-based, prospective, case-control study. J. Pediatr. Gastroenterol. Nutr. 21:137-144[Medline].
14.	Forestier, C., M. Meyer, S. Favre-Bonte, C. Rich, G. Malpuech, C. Le Bougenec, J. Sirot, B. Joly, and C. de Champs. 1996. Enteroadherent Escherichia coli and diarrhea in children: a prospective case-control study. J. Clin. Microbiol. 34:2897-2903[Abstract].
15.	Germani, Y., E. Bégaud, P. Duval, and C. Le Bouguenec. 1996. Prevalence of enteropathogenic, enteroaggregative, and diffusely adherent Escherichia coli among isolates from children with diarrhea in New Caledonia. J. Infect. Dis. 174:1124-1126[Medline].
16.	Girón, J. A., M. S. Donnenberg, W. C. Martin, K. G. Jarvis, and J. B. Kaper. 1993. Distribution of the bundle-forming pilus structural gene (bfpA) among enteropathogenic Escherichia coli. J. Infect. Dis. 168:1037-1041[Medline].
17.	Girón, J. A., T. Jones, F. Millan-Velasco, E. Castro-Munoz, L. Zarate, J. Fry, G. Frankel, S. L. Moseley, B. Baudry, J. B. Kaper, G. K. Schoolnik, and L. W. Riley. 1991. Diffuse-adhering Escherichia coli (DAEC) as a putative cause of diarrhea in Mayan children in Mexico. J. Infect. Dis. 163:507-513[Medline].
18.	Gomes, T. A. T., P. A. Blake, and L. R. Trabulsi. 1989. Prevalence of Escherichia coli strains with localized, diffuse, and aggregative adherence to HeLa cells in infants with diarrhea and matched controls. J. Clin. Microbiol. 27:266-269[Abstract/Free Full Text].
19.	Gomes, T. A. T., S. R. T. S. Ramos, D. Rodrigues, D. Rodrigue', V. Rassi, M. R. F. Toledo, M. A. M. Vieira, S. V. Gatti, P. Holck, and P. M. Griffin. 1994. Etiology of acute diarrhea in children 1 to 5 years old in São Paulo, Brazil, abstr. C-299, p. 543. In Abstracts of the 94th General Meeting of the American Society for Microbiology, 1994. American Society for Microbiology, Washington, D.C.
20.	Gomes, T. A. T., V. Rassi, K. L. MacDonald, S. R. T. S. Ramos, L. R. Trabulsi, M. A. M. Vieira, B. E. C. Guth, J. A. N. Candeias, C. Ivey, M. R. F. Toledo, and P. A. Blake. 1991. Enteropathogens associated with acute diarrheal disease in urban infants in São Paulo, Brazil. J. Infect. Dis. 164:331-337[Medline].
21.	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 diarrhea in São Paulo, Brazil. J. Infect. Dis. 160:131-135[Medline].
22.	Gonçalves, A. G., L. C. Campos, T. A. T. Gomes, J. Rodrigues, V. Sperandio, T. S. Whittam, and L. R. Trabulsi. 1997. Virulence properties and clonal structures of strains of Escherichia coli O119 serotypes. Infect. Immun. 65:2034-2040[Abstract].
23.	González, R., C. Díaz, M. Mariño, R. Cloralt, M. Pequeneze, and I. Pérez-Schael. 1997. Age-specific prevalence of Escherichia coli with localized and aggregative adherence in Venezuelan infants with acute diarrhea. J. Clin. Microbiol. 35:1103-1107[Abstract].
24.	Gunzburg, S. T., B. J. Chang, S. J. Elliott, V. Burke, and M. Gracey. 1993. Diffuse and enteroaggregative patterns of adherence of enteric Escherichia coli isolated from aboriginal children from the Kimberley region of Western Australia. J. Infect. Dis. 167:755-758[Medline].
25.	Haider, K., S. M. Faruque, N. S. Shahid, M. J. Albert, S. Nahar, A. Malek, S. Tzipori, and A. N. Alam. 1991. Enteroaggregative Escherichia coli infections in Bangladeshi children: clinical and microbiological features. J. Diarrheal Dis. 9:318-322.
26.	Henry, F. J., A. S. Udoy, C. A. Wanke, and K. M. A. Aziz. 1992. Epidemiology of persistent diarrhea and etiologic agents in Mirzapur, Bangladesh. Acta Paediatr. Suppl. 381:27-31[Medline].
27.	Huppertz, H. I., S. Rutkowski, S. Aleksic, and H. Karch. 1997. Acute and chronic diarrhoea and abdominal colic associated with enteroaggregative Escherichia coli in young children living in western Europe. Lancet 349:1660-1662[Medline].
28.	Itoh, Y., I. Nagano, M. Kunishima, and T. Ezaki. 1997. Laboratory investigation of enteoaggregative Escherichia coli O untypable:H10 associated with a massive outbreak of gastrointestinal illness. J. Clin. Microbiol. 35:2546-2550[Abstract].
29.	Jallat, C., V. Livrelli, A. Darfeuille-Michaud, C. Rich, and B. Joly. 1993. Escherichia coli strains involved in diarrhea in France: high prevalence and heterogeneity of diffusely adhering strains. J. Clin. Microbiol. 31:2031-2037[Abstract/Free Full Text].
30.	Janda, J. M., and P. S. Duffey. 1988. Mesophilic Aeromonas in human disease: current taxonomy, laboratory identification, and infectious disease spectrum. Rev. Infect. Dis. 10:980-995[Medline].
31.	Jerse, A. E., J. Yu, B. Tall, and J. B. Kaper. 1990. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc. Natl. Acad. Sci. USA 87:7839-7843[Abstract/Free Full Text].
32.	Knutton, S., K. S. Robert, K. B. Maharaj, H. R. Smith, M. M. McConnel, T. Cheasty, P. H. William, and T. J. Baldwin. 1992. Ability of enteroaggregative Escherichia coli strains to adhere in vitro to human intestinal mucosa. Infect. Immun. 60:2083-2091[Abstract/Free Full Text].
33.	Levine, M. M., C. Ferreccio, V. Prado, M. Cayazzo, P. Abrego, J. Martinez, L. Maggi, M. M. Baldini, W. Martin, D. Maneval, B. Kay, L. Guers, H. Lior, S. S. Wasserman, and J. P. Nataro. 1993. Epidemiologic studies of Escherichia coli diarrheal infections in a low socio-economic level peri-urban community in Santiago, Chile. Am. J. Epidemiol. 138:849-869[Abstract/Free Full Text].
34.	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 diarrheagenic Escherichia coli. J. Infect. Dis. 158:224-228[Medline].
35.	Maas, R. 1983. An improved colony hybridization method with significantly increased sensitivity for detection of signal genes. Plasmid 10:296-298[Medline].
36.	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 with a DNA probe. J. Infect. Dis. 152:560-565[Medline].
37.	Nataro, J. P., J. B. Kaper, R. Robins-Browne, V. Prado, P. Vial, and M. M. Levine. 1987. Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells. Pediatr. Infect. Dis. J. 6:829-831[Medline].
38.	Nataro, J. P., I. C. A. Scaletsky, J. B. Kaper, M. M. Levine, and L. R. Trabulsi. 1985. Plasmid-mediated factors conferring diffuse and localized adherence of enteropathogenic Escherichia coli. Infect. Immun. 48:378-383[Abstract/Free Full Text].
39.	Ramos, S. R. T. S. 1996. Risk factors for EPEC infections. Rev. Microbiol. São Paulo 27(Suppl. 1):34-39.
40.	Rodrigues, J., I. C. A. Scaletsky, L. C. Campos, T. A. T. Gomes, T. S. Whittam, and L. R. Trabulsi. 1996. Clonal structure and virulence factors in strains of Escherichia coli of the classic serogroup O55. Infect. Immun. 64:2680-2686[Abstract].
41.	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[Abstract/Free Full Text].
42.	Schlesselman, J. J. 1982. Basic methods of analysis, p. 171-220. In J. J. Schlesselman (ed.), Case-control studies: design, conduct, analysis. Oxford University Press, Inc., New York, N.Y.
43.	Scotland, S. M., H. R. Smith, B. Said, G. A. Willshaw, T. Cheasty, and B. Rowe. 1991. Identification of enteropathogenic Escherichia coli isolated in Britain as enteroaggregative or as members of a subclass of attaching-and-effacing E. coli not hybridising with the EPEC adherence-factor probe. J. Med. Microbiol. 35:278-283[Abstract].
44.	Wanke, C. A., J. B. Schorling, L. J. Barret, M. A. Desouza, and R. L. Guerrant. 1991. Potential role of adherence traits of Escherichia coli in persistent diarrhea in an urban Brazilian slum. Pediatr. Infect. Dis. J. 10:746-751[Medline].

This article has been cited by other articles:

• Scavia, G., Staffolani, M., Fisichella, S., Striano, G., Colletta, S., Ferri, G., Escher, M., Minelli, F., Caprioli, A. (2008). Enteroaggregative Escherichia coli associated with a foodborne outbreak of gastroenteritis. J Med Microbiol 57: 1141-1146 [Abstract] [Full Text]  
• Moreira, F. C., Vieira, M. A. M., Ferreira, A. J. P., Girao, D. M., Vaz, T. M. I., Rosa, A. C. P., Knobl, T., Irino, K., Freymuller, E., Gomes, T. A. T. (2008). Escherichia coli Strains of Serotype O51:H40 Comprise Typical and Atypical Enteropathogenic E. coli Strains and Are Potentially Diarrheagenic. J. Clin. Microbiol. 46: 1462-1465 [Abstract] [Full Text]  
• Servin, A. L. (2005). Pathogenesis of Afa/Dr Diffusely Adhering Escherichia coli. Clin. Microbiol. Rev. 18: 264-292 [Abstract] [Full Text]  
• Piva, I. C., Pereira, A. L., Ferraz, L. R., Silva, R. S. N., Vieira, A. C., Blanco, J. E., Blanco, M., Blanco, J., Giugliano, L. G. (2003). Virulence Markers of Enteroaggregative Escherichia coli Isolated from Children and Adults with Diarrhea in Brasilia, Brazil. J. Clin. Microbiol. 41: 1827-1832 [Abstract] [Full Text]  
• Blanc-Potard, A.-B., Tinsley, C., Scaletsky, I., Le Bouguenec, C., Guignot, J., Servin, A. L., Nassif, X., Bernet-Camard, M.-F. (2002). Representational Difference Analysis between Afa/Dr Diffusely Adhering Escherichia coli and Nonpathogenic E. coli K-12. Infect. Immun. 70: 5503-5511 [Abstract] [Full Text]  
• Scaletsky, I. C. A., Fabbricotti, S. H., Aranda, K. R., Morais, M. B., Fagundes-Neto, U. (2002). Comparison of DNA Hybridization and PCR Assays for Detection of Putative Pathogenic Enteroadherent Escherichia coli. J. Clin. Microbiol. 40: 1254-1258 [Abstract] [Full Text]  

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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Gomes, T. A. T. Articles by Ramos, S. R. T. S. Search for Related Content PubMed PubMed Citation Articles by Gomes, T. A. T. Articles by Ramos, S. R. T. S.