This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cerna, J. F.
Right arrow Articles by Estrada-Garcia, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cerna, J. F.
Right arrow Articles by Estrada-Garcia, T.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, May 2003, p. 2138-2140, Vol. 41, No. 5
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.5.2138-2140.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Multiplex PCR for Detection of Three Plasmid-Borne Genes of Enteroaggregative Escherichia coli Strains

Jorge F. Cerna,1 James P. Nataro,2 and Teresa Estrada-Garcia1*

Department of Molecular Biomedicine, CINVESTAV-IPN, Zacatenco, Mexico DF 07360, Mexico,1 Center for Vaccine Development, Department of Pediatrics and Medicine, University of Maryland, School of Medicine, Baltimore, Maryland2

Received 9 December 2002/ Returned for modification 29 January 2003/ Accepted 13 February 2003


arrow
ABSTRACT
 
We developed a novel multiplex PCR assay for enteroaggregative Escherichia coli (EAEC) detection, by using three plasmid-borne genes (the aggregative adherence [AA] probe, aap, and aggR). One or more of the loci were detected in 24 (86%) of 28 patient isolates analyzed. The multiplex PCR assay is a fast, convenient, and sensitive molecular test to detect EAEC.


arrow
TEXT
 
The importance of enteroaggregative Escherichia coli (EAEC) strains in public health around the world is becoming increasingly clear. The pathogenicity of these strains has been proven in adult volunteers (12, 18) and in several studies from around the world (3, 4, 5, 9, 14). EAEC strains have been associated classically with persistent diarrhea (>=14 days), which represents a disproportionate share of diarrheal mortality. EAEC strains have been shown to elicit damage to the intestinal mucosa (C. Eslava, J. Villaseca, R. Morales, A. Navarro, and A. Cravioto, Abstr. 93rd Gen. Meet. Am. Soc. Microbiol. 1993, abstr. B-105, p. 44, 1993), and recent data link EAEC with growth retardation in infants (19, 24). Thus, detection of EAEC strains can make a significant contribution to public health in many areas.

EAEC diagnosis has long been problematic. The pathogen was initially defined by the presence of a characteristic stacked brick pattern, designated aggregative adherence (AA) in the HEp-2 cell adherence assay (14). Although this pattern provided the first EAEC epidemiologic associations with diarrhea, there has long been a desire to base detection on the presence of requisite virulence factors.

The pathogenesis of EAEC infection is only partly understood. EAEC adheres to the intestinal mucosa by means of aggregative adherence fimbriae (15) and then elicits damage to the mucosal epithelium, which is manifested as exfoliation of epithelial cells (19). A cytotoxin and several enterotoxins have been described (19), each of which may contribute to secretory diarrhea. EAEC diarrhea exhibits some features of an inflammatory enteritis, and the flagellin protein has been implicated in the release of interleukin-8 (IL-8) (24).

Many of the EAEC virulence factors have been localized to the 60- to 65-MDa AA plasmid, including the aggregative adherence fimbrial adhesin (16), the Pet (8) and EAST (19) enterotoxins, the transcriptional activator AggR (17), and a novel antiaggregation protein (dispersin) encoded by the aap gene (formerly known as aspU) (23). Furthermore, several other loci on the plasmid appear to be specific for EAEC, although they have not yet been implicated in virulence. The importance of the AA plasmid is underscored by the fact that an empirically derived gene probe, designated the AA or CVD432 probe, has been reported to be sensitive and specific for EAEC detection (2). In the initial publication, the AA probe hybridized with 89% of EAEC strains, although this number can vary dramatically from study to study (4, 20, 21).

It has also become apparent that strains carrying the AA plasmid represent an important subgroup of EAEC strains and that many strains which do not hybridize with the AA probe nevertheless share plasmid-borne and chromosomal virulence determinants. Indeed, it has recently been shown that adult travelers carrying EAEC strains which harbored one of more of the AA plasmid loci were significantly more likely to shed fecal IL-8 and IL-1 than if their EAEC strains lacked all plasmid-borne factors (10). Thus, developing improved methods for detecting AA plasmid-positive EAEC strains is a priority. Here, we have developed a multiplex PCR assay to detect three AA plasmid-borne genes and show that these loci are commonly but not inevitably linked.

For the sake of comparison, all EAEC reference and patients strains were first characterized by the HEp-2 assay, which was performed as described previously (6). Strains were then further characterized by the standard colony blot method (1) for the identification of the plasmid-borne genes aap and aggR and for the AA probe (7). We next developed a multiplex PCR with previously described primers for the AA probe (22). For aggR detection, we used a previously described aggR forward primer (7) and a newly chosen reverse primer (GenBank accession no. 232523). Aap primers were chosen (GenBank accession no. 232523) and optimized specifically for the present study (Table 1). For standardization purposes, we used three previously described EAEC reference strains: JM221, 042, 17-2, and 12 additional diarrheagenic E. coli reference strains (11) (three enterotoxigenic, three enteroinvasive, three Shiga-like toxin producing, and three enteropathogenic), plus two E. coli negative controls strains: 3030 and K-12. Bacterial lysates were prepared by resuspending a single colony in 1 ml of deionized water (MilliQ) in a polypropylene tube, followed by boiling for 1 min. Each PCR tube contained 23 µl of reaction mix comprised of (final concentrations): Tris-HCl (pH 8.3), 10 mM; KCl, 50 mM; MgCl2, 2 mM; gelatin, 100 µg/ml; glycerol, 5% (vol/vol); dATP, dCTP, dGTP, and dTTP (Roche), 200 µM each; AmpliTaq polymerase (Gibco-BRL), 1 U/25 µl. Also included were a mixture of the six primers (Table 1) and a 2-µl portion of the bacterial lysates. The solutions were then subjected to the following cycling conditions: 50°C (2 min, 1 cycle); 95°C (5 min, 1 cycle); 95, 55, and 72°C (45 s at each temperature, 40 cycles); and a final extension step (10 min, 72°C) in a thermal cycler (Bio-Rad). Then, 4 µl of the PCR mixture was visualized by ethidium bromide staining after electrophoresis in a 2.5% agarose gel in Tris-acetate-EDTA buffer (Gibco-BRL); the amplicon sizes are shown in Table 1 and Fig. 1. Only the presence of the correct sized gene product(s) was interpreted as a positive test.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Primers, concentrations in the PCR mix, and amplicon sizes



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 1. PCR products. Lanes 1 to 4, PCR products (in base pairs) obtained with DNA of reference strains mix 17-2, 042, JM221, and 3030, respectively, along with the primer mix; lanes 5 to 7, PCR products obtained with the 17-2 strain and primers for the AA probe, aggR, and aap genes, respectively; lanes 8 to 11, PCR products obtained with DNA of patient isolates; lane 12, 1-kb molecular weight marker in base pairs.

All three reference strains—JM221, 042, and 17-2—were found to be positive for the AA probe and aap and aggR genes (Table 2). In contrast, none of the diarrheagenic E. coli or negative controls were positive for any of the analyzed genes. The minimal concentrations of CFU detected by this assay were 322 CFU (AA probe), 471 CFU (aggR gene), and 70 CFU (aap gene)/ml of saline buffer.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Results of control, reference, and patient EAEC strains as analyzed by HEp-2 assay, colony blot, and multiplex PCR techniques

The multiplex assay was further evaluated against 28 EAEC strains isolated from patients and two other E. coli-negative control strains (from the collections of the Center of Vaccine Development, University of Maryland School of Medicine) (Table 2). All strains had been tested for HEp-2 adherence and dot blot for the EAEC genes tested here. It was observed that 24 (86%) of 28 strains were positive for at least one gene, 23 (82%) of 28 strains were positive for the three genes, none of the strains were positive for just two genes, and 4 (14%) of 28 strains were negative for the three genes (Table 2). In the 28 strains analyzed, the PCR method identified 70 genes (23 AA probe, 24 aap, and 23 aggR), and the colony blot identified only 65 genes (22 AA probe, 23 aap, and 20 aggR) revealing that the PCR method is more sensitive. This PCR sensitivity was confirmed by the fact that 23 (82%) of 28 strains were positive for the three loci by PCR compared to 20 (71%) of 28 strains found to be positive by colony blot (Table 2).

There is a need for a convenient assay to detect EAEC strains based on molecular characteristics. The HEp-2 assay remains the "gold standard" for identification of EAEC when these strains exhibit the AA pattern. However, this technique requires special expertise and facilities and is time-consuming; thus, it is only performed in a few laboratories around the world, limiting both EAEC diagnosis and the epidemiological studies. Although a simplified version of the HEp-2 assay have been reported (13), the main problem remains that the strains exhibiting the AA pattern almost certainly encompass both pathogenic and nonpathogenic clones (20). Thus, we have developed a multiplex PCR that specifically and sensitively detects loci on the AA plasmid. The test appears to be more sensitive than the colony blot technique (for the same three genes) and is also less time-consuming than the colony blot and HEp-2 assay.

Our multiplex PCR has demonstrated that most of the EAEC strains isolated from patients with diarrhea 23 (82%) of 28 were positive for the three loci, i.e., aggR, aap, and the AA probe, revealing that these plasmid-borne genes may be more frequently linked than was previously demonstrated by colony blot. These three loci have been reported in a majority of strains (7), and it has been suggested that they may be phylogenetically or pathogenetically linked (7, 23). Of course, it is possible that all three genes are markers for truly virulent EAEC clones, and this hypothesis remains to be tested. Importantly, however, the use of the multiplex assay increases both the sensitivity and the specificity of EAEC detection, which may permit earlier diagnosis of this infection and improve epidemiologic understanding of this emerging pathogen.


arrow
ACKNOWLEDGMENTS
 
Jorge F. Cerna was supported by the a CONACyT scholarship 71484. Work in the Nataro lab is supported by U.S. Public Health Service grant AI33096.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Biomedicine, CINVESTAV-IPN, Av. Instituto Politecnico Nacional 2508, Zacatenco, Mexico DF 07360, Mexico. Phone: (5255) 5-747-3327. Fax: (5255) 5-747-7134. E-mail: testrada{at}mail.cinvestav.mx. Back


arrow
REFERENCES
 
    1
  1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman, and K. Struhl (ed.). 1989. Current protocols in molecular biology. John Wiley & Sons, Inc., New York, N.Y.
    2. 2
  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. 3
  3. 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]
    4. 4
  4. Bouzari, A., A. Jafari, A. Azizi, M. Oloomi, and J. P. Nataro. 2001. Short report: characterization of enteroaggregative Escherichia coli isolates from Iranian children. Am. J. Trop. Med. Hyg. 65:13-14.[Abstract]
    5. 5
  5. 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 337:262-264.[CrossRef][Medline]
    6. 6
  6. Cravioto, A., R. J. Gross, S. Scotland, and B. Rowe. 1979. An adhesive factor found in strains of Escherichia coli belonging to the traditional enteropathogenic serotypes. Curr. Microbiol. 3:95-99.[CrossRef]
    7. 7
  7. Czeczulin, J. R., T. S. Whittam, I. R. Henderson, F. Navarro-Garcia, and J. P. Nataro. 1999. Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli. Infect. Immun. 67:2692-2699.[Abstract/Free Full Text]
    8. 8
  8. Eslava, C., F. Navarro-Garcia, J. Czeczulin, I. A. Henderson, A. Cravioto, and J. P. Nataro. 1998. Pet, an autotransporter enterotoxin from enteroaggregative Escherichia coli. Infect. Immun. 66:3155-3163.[Abstract/Free Full Text]
    9. 9
  9. 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]
    10. 10
  10. Jiang, Z.-D., D. Greenberg, J. P. Nataro, R. Steffen, and H. L. DuPont. 2002. Rate of occurrence and pathogenic effect of enteroaggregative Escherichia coli virulence factors in international travelers. J. Clin. Microbiol. 40:4185-4190.[Abstract/Free Full Text]
    11. 11
  11. López-Saucedo, C., J. F. Cerna, N. Villegas-Sepulveda, R. Thompson, F. R. Velazquez, J. Torres, P. I. Tarr, and T. Estrada-García. 2003. A single multiplex polymerase chain reaction to detect diverse loci associated with diarrheagenic Escherichia coli. Emerg. Infect. Dis. 9:127-131.[Medline]
    12. 12
  12. Mathewson, J. J., P. C. Johnson, and H. L. DuPont. 1986. Pathogenicity of enteroadherent Escherichia coli in adult volunteers. J. Infect. Dis. 154:524-527.[Medline]
    13. 13
  13. Miqdady, M. S., Zhi-Dong Jiang, J. P. Nataro, and H. L. DuPont. 2002. Detection of enteroaggregative Escherichia coli with formalin-preserved HEp-2 cells. J. Clin. Microbiol. 40:3066-3067.[Abstract/Free Full Text]
    14. 14
  14. 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]
    15. 15
  15. Nataro, J. P., Y. Deng, D. R. Maneval, A. L. German. W. C. Martin, and M. M. Levine. 1992. Aggregative adherence fimbriae I of enteroaggregative Escherichia coli mediate adherence to HEp-2 cells and hemagglutination of human erythrocytes. Infect. Immun. 60:2297-2304.[Abstract/Free Full Text]
    16. 16
  16. Nataro, J. P., D. Yikang, J. A. Giron, S. J. Savarino, M. H. Kothary, and R. Hall. 1993. Aggregative adherence fimbria I expression in enteroaggregative Escherichia coli requires two unliked plasmid regions. Infect. Immun. 61:1126-1131.[Abstract/Free Full Text]
    17. 17
  17. Nataro, J. P., D. Yikang, D. Yingkang, and K. Walter. 1994. AggR a transcriptional activator of aggregative adherence fimbria I expression in enteroaggregative Escherichia coli. J. Bacteriol. 176:4691-4699.[Abstract/Free Full Text]
    18. 18
  18. Nataro, J. P., D. Yikang, S. Cookson, A. Cravioto, S. J. Savarino, L. D. Guers, M. M. Levine, and C. O. Tacket. 1995. Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers. J. Infect. Dis. 171:465-468.[Medline]
    19. 19
  19. Nataro, J. P., and J. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142-201.[Abstract/Free Full Text]
    20. 20
  20. Okeke, I. N., A. Lamikanra, H. Steinrûck, and J. B. Kaper. 2000. Characterization of Escherichia coli strains from cases of childhood diarrhea in provincial southwestern Nigeria. J. Clin. Microbiol. 38:7-12.[Abstract/Free Full Text]
    21. 21
  21. Scaletsky, I. C. A., S. H. Fabbricotti, K. R. Aranda, M. B. Morais, and U. Fagundes-Neto. 2002. Comparison of DNA hybridization and PCR assays for detection of putative pathogenic enteroadherent Escherichia coli. J. Clin. Microbiol. 40:1254-1258.[Abstract/Free Full Text]
    22. 22
  22. Schmidt, H., C. Knop, S. Franke, S. Aleksic, J. Heesemann, and H. Karch. 1995. Development of PCR for screening of enteroaggregative Escherichia coli. J. Clin. Microbiol. 33:700-705.
    23. 23
  23. Sheikh, J., J. R. Czeczulin, S. Harrington, S. Hicks, I. R. Henderson, C. Le Bouguénec, P. Guonon, A. Phillips, and J. P. Nataro. 2002. A novel dispersin in enteroaggregative Escherichia coli. J. Clin. Investig. 110:1329-1337.[CrossRef][Medline]
    24. 24
  24. Steiner, T. S., A. M. Lima, J. P. Nataro, and R. L. Guerrant. 1998. Enteroaggregative Escherichia coli produce intestinal inflammation and growth impairment and cause interleukin-8 release from intestinal epithelial cells. J. Infect. Dis. 177:88-96.[Medline]

Journal of Clinical Microbiology, May 2003, p. 2138-2140, Vol. 41, No. 5
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.5.2138-2140.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Cordeiro, F., da Silva Gomes Pereira, D., Rocha, M., Asensi, M. D., Elias, W. P., Campos, L. C. (2008). Evaluation of a Multiplex PCR for Identification of Enteroaggregative Escherichia coli. J. Clin. Microbiol. 46: 828-829 [Full Text]  
  • Huang, D. B., Mohamed, J. A., Nataro, J. P., DuPont, H. L., Jiang, Z.-D., Okhuysen, P. C. (2007). Virulence characteristics and the molecular epidemiology of enteroaggregative Escherichia coli isolates from travellers to developing countries. J Med Microbiol 56: 1386-1392 [Abstract] [Full Text]  
  • Samie, A., Obi, C. L., Dillingham, R., Pinkerton, R. C., Guerrant, R. L. (2007). Enteroaggregative Escherichia coli in Venda, South Africa: Distribution of Virulence-Related Genes by Multiplex Polymerase Chain Reaction in Stool Samples of Human Immunodeficiency Virus (HIV)-Positive and HIV-Negative Individuals and Primary School Children. Am J Trop Med Hyg 77: 142-150 [Abstract] [Full Text]  
  • Muller, D., Greune, L., Heusipp, G., Karch, H., Fruth, A., Tschape, H., Schmidt, M. A. (2007). Identification of Unconventional Intestinal Pathogenic Escherichia coli Isolates Expressing Intermediate Virulence Factor Profiles by Using a Novel Single-Step Multiplex PCR. Appl. Environ. Microbiol. 73: 3380-3390 [Abstract] [Full Text]  
  • Weintraub, A. (2007). Enteroaggregative Escherichia coli: epidemiology, virulence and detection. J Med Microbiol 56: 4-8 [Abstract] [Full Text]  
  • Jenkins, C., Tembo, M., Chart, H., Cheasty, T., Willshaw, G. A., Phillips, A. D., Tompkins, D., Smith, H. (2006). Detection of enteroaggregative Escherichia coli in faecal samples from patients in the community with diarrhoea.. J Med Microbiol 55: 1493-1497 [Abstract] [Full Text]  
  • Huang, D. B., Mohanty, A., DuPont, H. L., Okhuysen, P. C., Chiang, T. (2006). A review of an emerging enteric pathogen: enteroaggregative Escherichia coli.. J Med Microbiol 55: 1303-1311 [Abstract] [Full Text]  
  • Reisner, A., Krogfelt, K. A., Klein, B. M., Zechner, E. L., Molin, S. (2006). In Vitro Biofilm Formation of Commensal and Pathogenic Escherichia coli Strains: Impact of Environmental and Genetic Factors.. J. Bacteriol. 188: 3572-3581 [Abstract] [Full Text]  
  • WAKIMOTO, N., NISHI, J., SHEIKH, J., NATARO, J. P., SARANTUYA, J., IWASHITA, M., MANAGO, K., TOKUDA, K., YOSHINAGA, M., KAWANO, Y. (2004). QUANTITATIVE BIOFILM ASSAY USING A MICROTITER PLATE TO SCREEN FOR ENTEROAGGREGATIVE ESCHERICHIA COLI. Am J Trop Med Hyg 71: 687-690 [Abstract] [Full Text]  
  • Afset, J. E, Bevanger, L., Romundstad, P., Bergh, K. (2004). Association of atypical enteropathogenic Escherichia coli (EPEC) with prolonged diarrhoea. J Med Microbiol 53: 1137-1144 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cerna, J. F.
Right arrow Articles by Estrada-Garcia, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cerna, J. F.
Right arrow Articles by Estrada-Garcia, T.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»