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Journal of Clinical Microbiology, September 2007, p. 3068-3071, Vol. 45, No. 9
0095-1137/07/$08.00+0     doi:10.1128/JCM.00646-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

PCR-Based Identification of Common Colonization Factor Antigens of Enterotoxigenic Escherichia coli

Abhisek Ghosal, Rudra Bhowmick, Ranjan Kumar Nandy, T. Ramamurthy, and Nabendu Sekhar Chatterjee^*

National Institute of Cholera and Enteric Diseases, Kolkata, India

Received 23 March 2007/ Returned for modification 21 May 2007/ Accepted 20 June 2007

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Colonization factor antigens (CFAs) of enterotoxigenic Escherichia coli (ETEC) have been classified into several groups based on their distinct antigenicity. We describe here a PCR-based method to detect common CFAs of ETEC, which were characterized using conventional serology. This PCR assay is simple and sensitive for the detection of expressed CFA genes.

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Enterotoxigenic Escherichia coli (ETEC) infection is a major cause of infantile and traveler's diarrhea, causing substantial effects in terms of morbidity and economic consequences (1, 16, 17). Apart from heat-labile and heat-stable enterotoxins, ETEC pathogenesis is mediated by specific antigenically distinct colonization factor antigens (CFAs). Several CFAs have been identified; among these, CFA/I, CFA/II, and CFA/IV are the most common (5). CFA/II is composed of coli surface (CS) antigens CS1, CS2 and CS3, whereas CFA/IV is composed of CS4, CS5, and CS6 (10, 11, 13). In addition, several other CFAs have been identified, namely, CS12, CS14, CS17, and CS21.

CFA typing of ETEC strains is done by serology using specific monoclonal antibodies raised against these antigens. Such typing is limited to the reference laboratories and could not be used in the routine surveillance. In this study, we have developed a simple PCR-based method for the detection of CFAs and evaluated the specificity and sensitivity of the method by comparing the results with the conventional serology-based methods.

Thirty-three ETEC strains with established CFA types were included in this study. These strains were isolated from diarrheal patients in different parts of India, and the CFA typing was done at the International Centre for Diarrheal Diseases Research, Bangladesh (14). Diarrheagenic E. coli other than ETEC, along with the K-12 derivative strain DH5, served as negative controls. All the strains were maintained at –70°C in Luria broth containing 15% glycerol. The cultures were grown on CFA agar plates (4) at 37°C for PCR experiments.

Nucleotide sequences from different CFAs were aligned to identify different CFA specific regions and were utilized to develop CFA-specific PCR primers (Table 1). Boiled bacterial cell lysate in water served as the genomic DNA template for the PCR assay. Total RNA was prepared from the ETEC strains grown on CFA agar plates (4), and the cDNA was prepared by a reverse transcription (RT) reaction as described previously (2). The PCR products were electrophoresed onto 2% agarose gels stained with ethidium bromide and digitally recorded (UVP Biospectrum AC Imaging Systems). To check the PCR specificity, amplicons were sequenced using an automated DNA sequencer (ABI Prism 3200; Applied Biosystems). PCR results revealed that most of the ETEC strains harbored genes encoding CS6 alone or in combination with other CFA types (Fig. 1 and Table 2). This prompted us to further screen the strains with an anti-CS6 antibody to correlate the phenotypes with reverse transcription-PCR (RT-PCR) results. For this, we procured an anti-CS6 antibody commercially (GenScript Corp., Piscataway, NJ), which was developed against the most antigenic region of the CssA subunit of CS6. The most antigenic region was predicted by hydropathy plot (Kyte-Doolittle plot) using the amino acid sequence of CS6 (GenBank accession no. U04844). The region from amino acid residues 131 to 144 of the CssA subunit of CS6 (YTSGDKEIPPGIYN) was selected to raise the antibody in rabbits.

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TABLE 1. Primers used in this studya

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FIG. 1. PCR results obtained with representative ETEC strains. Specific CFAs based on the amplicon size on the agarose gel are shown: lane 1, CFA/I; lane 2, CS1; lane 3, CS2; lane 4, CS3; lane 5, CFA/III; lane 6, CS4; lane 7, CS5; lane 8, CS6; lane 9, CS14; and lane 10, CS17. M, 100-bp size marker. Representative strains used are shown here with respective strain numbers on the right side of the panel. The top eight panels are from RT-PCR analyses; the bottom panel (strain 933) is from a genomic PCR analysis.

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TABLE 2. PCR assay and serological results with ETEC and non-ETEC strains

To check its recognition specificity, this antibody was evaluated by dot blot enzyme-linked immunosorbent assay (ELISA) using heat-saline extract (HS) prepared from strains that gave CS6-specific amplicons in the RT-PCR assay (11). In the dot blot assay, equal concentrations of HS extracts, measured by the Bradford method, were blotted onto a nitrocellulose membrane (Bio-Rad). CS6 was detected by anti-CssA antibody, followed by alkaline phosphatase-conjugated secondary antibody, and developed according to a standard protocol. As a negative control, HS extract from six randomly chosen CS6-positive ETEC strains grown at 17°C was used, since CS6 was not expressed at this temperature (8, 11).

Initially, 20 serologically CFA-typed ETEC strains were used to validate the data generated by the newly developed PCR-based CFA detection assay. Comparative analysis revealed that five of them (1571, 2654, 3539, 6547, and 881) gave amplicons for additional CFAs, along with those detected by serological assay. Such an observation can be explained by the fact that the expression of CFAs is dependent on the culture condition (3), and PCR-based detection using genomic DNA depends on the presence of the gene per se but not its functional expression in vitro. Therefore, the analysis was extended by repeating the PCR assay using RNA converted to cDNA instead of genomic DNA since it relied on the expressed CFA genes, which might mimic the serological CFA typing. In this study, RT-PCR and the serological typing results matched perfectly for 17 of the 20 strains, except for strains 4266, 1453, and 5349 (Table 2). For strains 4266 and 5349, CS5 was detected by serology but not by RT-PCR. Strain 4266 was also negative for CS5 in the genomic PCR. Similarly, strain 1453 showed the presence of CS1 and CFA/III by PCR instead of CS3 as detected by serology. Screening of CFA types by serological assay has been reported to give false-positive results in certain cases (5), and this may be true in the case of two ETEC strains (4266 and 1453). Strain 5349, though produced CS6-specific amplicon along with CS5 in the genomic PCR assay, failed to generate CS5-specific amplicon in the RT-PCR assay. The authenticity of the amplicons was confirmed by DNA sequencing (data not shown). These data indicate that the tested culture condition may not be favorable for the expression of CS5 by the strain 5349 and raise the possibility that such differential expression of CFA in vitro may be strain specific.

In order to expand the scope of this study, 13 clinical ETEC strains identified as untypeable by serological CFA typing were analyzed. CFA encoding genes were detected in all of them, but two strains (E-7 and E-13) did not express CFA genes as detected by RT-PCR. This can be attributed either to antibody specificity (15) or to the lack of corresponding CFA transcripts under the tested laboratory condition. The dot blot ELISA results obtained with anti-CssA antiserum are presented in Table 2. We found that of 28 strains positive for the CS6-encoding gene, 24, 14, and 19 were positive by RT-PCR, serology, and dot blot assay, respectively. We assume that different culture conditions could give a better correlation with the phenotypic assay. Ten of the ETEC strains were untypeable by serology, and six were untypeable by dot blot, but all were determined to be CS6 positive by RT-PCR. Of 33 ETEC strains analyzed, 28 were positive by RT-PCR, whereas 20 were detected by the available antibody-based method. Two strains remained untypeable by both methods. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of RT-PCR compared to the serological results were 85, 15.4, 60.7, and 40%, respectively (Table 3). The likelihood ratio suggested that the RT-PCR positive result is almost onefold more likely to occur than the serology. The RT-PCR-based detection of CFAs was also statistically more sensitive (P < 0.001, MacNemer chi-square test) than serology. Thus, the PCR-based detection of CFAs in ETEC strains is more sensitive and as specific as the existing serology method. A comparative analysis of RT-PCR and serology results for 28 strains that were positive for CS6-encoding gene was also performed. The sensitivity, specificity, PPV, and NPV were 100, 28.6, 58.3, and 100%, respectively (Table 3). The newly developed antibody in the dot blot assay gave slightly better specificities and PPV values compared to serology (Table 3).

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TABLE 3. Comparison between RT-PCR and antibody-based detection

In conclusion, it appears that an antibody typing method alone may not be sufficient to classify ETEC strains because many factors, such as the antigenic variation of colonization factor antigens (18), the differential expression of CFAs (3, 6, 12), and gene silencing due to transcriptional control, may influence the conventional assay results. Considering these factors, the PCR assay seems to be simple and perform well with several advantages compared to the conventional methods for the detection of CFAs. Moreover, the RT-PCR method developed here is based on CFA gene expression under established culture conditions. The results presented here suggest that this method is as sensitive as serological assays and very handy when an isolate is untypeable with the available antibodies. The DNA hybridization assay reveals the existence of genes in the genome but does not give any information about phenotypes (7). The other disadvantage of using a probe hybridization assay is the cross-reactivity among the genes encoding CFAs. Antibody-based typing in slide agglutination and detection of antibody in patients' immune systems also has not proved very successful due to cross-reactivity and sequence variations (9, 15, 18). Our PCR assays can detect the presence of common CFA genes, as well as their expression in the same strain. To expedite the identification of ETEC CFAs and its epidemiology, a multiplex PCR system can be developed in the future with specific combinations of primers and PCR conditions.

 
This study was supported in part by grant from the Ministry of Health, Labor, and Family Welfare of Japan (project H17-Shinkou-3) and Japan International Cooperation Agency project.

We thank S. R. Choudhury, E. Ghosh, P. Bagchi, and R. Tapadar for technical assistance. We are particularly grateful to F. Quadri, International Center for Diarrheal Diseases, Bangladesh, for screening the ETEC strains for CFAs.

 
* Corresponding author. Mailing address: Division of Biochemistry, National Institute of Cholera and Enteric Diseases, P33 CIT Road, Scheme XM, Beliaghata, Kolkata 700 010, India. Phone: 91(33) 2363-3372. Fax: 91(33) 2350-5066. E-mail: nschatterjee{at}rediffmail.com

Published ahead of print on 27 June 2007.

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1
1. Black, R. E. 1993. Epidemiology of diarrheal disease: implications for control by vaccine. Vaccine 11:100-106.[CrossRef][Medline]
2
2. Bhowmick, R., A. Ghosal, and N. S. Chatterjee. 2007. Effect of environmental factors on expression and activity of chitinase genes of vibrios with special reference to Vibrio cholerae. J. Appl. Microbiol. 103:97-108.[CrossRef][Medline]
3
3. Chakraborty, S., J. S. Deokule, P. Garg, S. K. Bhattaccharya, R. K. Nandi, G. B. Nair, S. Yamasaki, Y. Takeda, and T. Ramamurthy. 2001. Concomitant infection of enterotoxigenic Escherichia coli in an outbreak of cholera caused by Vibrio cholerae O1 and O139 in Ahmedabad, India. J. Clin. Microbiol. 39:3241-3246.[Abstract/Free Full Text]
4
4. Evans, D. G., D. J. Evans, Jr., S. Clegg, and J. A. Pauley. 1979. Purification and characterization of the CFA/I antigen of enterotoxigenic Escherichia coli. Infect. Immun. 25:738-748.[Abstract/Free Full Text]
5
5. Gaastra, W., and A. M. Svennerholm. 1996. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 4:444-452.[CrossRef][Medline]
6
6. Grewal, H. M. S., A. Helander, A. M. Svennerholm, M. K. Bhan, W. Gaastra, and H. Sommerfelt. 1994. Genotypic and phenotypic identification of coli surface antigen 6-positive enterotoxigenic Escherichia coli. J. Clin. Microbiol. 32:1295-1301.[Abstract/Free Full Text]
7
7. Hans Steinsland, P. Valentiner-Branth, H. M. S. Grewal, W. Gaastra, K. Mølbak, and H. Sommerfelt. 2003. Development and evaluation of genotypic assays for the detection and characterization of enterotoxigenic Escherichia coli. Diagn. Microbiol. Infect. Dis. 45:97-105.[CrossRef][Medline]
8
8. Helandera, A., C. H. Gunnar, and A. M. Svennerholm. 1997. Binding of enterotoxigenic Escherichia coli to isolated enterocytes and intestinal mucus. Microb. Pathog. 23:335-346.[CrossRef][Medline]
9
9. Helander, A., H. M. S. Grewal, W. Gaastra, and A. M. Svennerholm. 1997. Detection and characterization of the coli surface antigen 6 of enterotoxigenic Escherichia coli strains by using monoclonal antibodies. J. Clin. Microbiol. 35:867-872.[Abstract]
10
10. Kaper, J. B., J. P. Nataro, and H. L. Mobley. 2004. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2:123-140.[CrossRef][Medline]
11
11. Marcia, K., G. Wolf, P. Andrews, B. D. Tall, M. M. McConnell, M. M. Levine, and E. C. Boedeker. 1989. Characterization of CS4 and CS6 antigenic components of PCF8775, a putative colonization factor complex from enterotoxigenic Escherichia coli E8775. Infect. Immun. 57:164-173.[Abstract/Free Full Text]
12
12. McConnell, M. M., L. V. Thomas, G. A. Willshaw, H. S. Smith, and B. Rowe. 1988. Genetic control and properties of coli surface antigens of colonization factor antigen IV (PCF8775) of enterotoxigenic Escherichia coli. Infect. Immun. 56:1974-1980.[Abstract/Free Full Text]
13
13. Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142-201.[Abstract/Free Full Text]
14
14. Qadri, F., S. K. Das, A. S. G. Faruque, G. J. Fuchs, M. J. Albert, R. Sack, and A. M. Svennerholm. 2000. Prevalence of toxin types and colonization factors in enterotoxigenic Escherichia coli isolated during a 2-year period from diarrheal patients in Bangladesh. J. Clin. Microbiol. 38:27-31.[Abstract/Free Full Text]
15
15. Qadri, F., F. Ahmed, T. Ahmed, and A. M. Svennerholm. 2006. Homologous and cross-reactive immune responses to enterotoxigenic Escherichia coli colonization factors in Bangladeshi children. Infect. Immun. 74:4512-4518.[Abstract/Free Full Text]
16
16. Sack, R. B., S. L. Gorbach, J. G. Banwell, D. B. Chatterjee, and R. C. Mitra. 1971. Enterotoxigenic Escherichia coli isolated from patients with severe cholera-like disease. J. Infect. Dis. 123:378-385.[Medline]
17
17. World Health Organization. 2004. World Health Organization report. World Health Organization, Geneva, Switzerland. [Online.] http://www.who.int/whr/2004.
18
18. Wolf, M. K., L. A. De Haan, F. J. Cassels, G. A. Willshaw, R. Warren, E. C. Boedeker, and W. Gaastra. 1997. The CS6 colonization factor of human enterotoxigenic Escherichia coli contains two heterologous major subunits. FEMS Microbiol. Lett. 148:35-42.[CrossRef][Medline]

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Journal of Clinical Microbiology, September 2007, p. 3068-3071, Vol. 45, No. 9
0095-1137/07/$08.00+0     doi:10.1128/JCM.00646-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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