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. 1999 Aug;37(8):2719–2722. doi: 10.1128/jcm.37.8.2719-2722.1999

Molecular Detection and Identification of Intimin Alleles in Pathogenic Escherichia coli by Multiplex PCR

Sean D Reid 1, David J Betting 1, Thomas S Whittam 1,*
PMCID: PMC85327  PMID: 10405431

Abstract

A multiplex PCR was designed to detect the eae gene and simultaneously identify specific alleles in pathogenic Escherichia coli. The method was tested on 87 strains representing the diarrheagenic E. coli clones. The results show that the PCR assay accurately detects eae and resolves alleles encoding the α, β, and γ intimin variants.


Two groups of pathogenic Escherichia coli have evolved similar mechanisms of adhering to the intestinal epithelium that result in a characteristic attaching-and-effacing (A/E) histopathology (7). Both enteropathogenic E. coli (EPEC), a major cause of infantile diarrhea in the developing world, and enterohemorrhagic E. coli (EHEC), the agent responsible for foodborne epidemics of hemorrhagic colitis in North America, Europe, and Japan (35), can produce A/E lesions which contribute to the severity of diarrheal disease. Production of A/E lesions is associated with the expression of intimin, an outer membrane protein encoded by a gene (eae) that is part of the LEE (locus of enterocyte effacement) pathogenicity island (2, 7).

Evolutionary analysis has shown that E. coli strains with the virulence properties and serotypes of EPEC and EHEC are subdivided into four distinct groups of clones (EPEC 1, EPEC 2, EHEC 1, and EHEC 2) (810). The clonal lineages differ in the site where LEE is inserted in the genome (11), and they carry distinct intimin alleles (1, 6). Three variants of intimin—Int-α, Int-β, and Int-γ—are characteristic of EPEC 1, EPEC 2, and EHEC 1 respectively. A fourth intimin (Int-δ), found in EPEC strains of serotype O86:H34, has greater homology to the intimin homologue of Citrobacter rodentium than to Int-α of EPEC strain E2348/69 (1). Most members of EHEC 2 (e.g., O26:H11) express Int-β, with the exception of a closely related group of bacteria of serotypes O111:H8, O111:H11, and O111:H− whose intimin allele has yet to be determined.

The objective of the present study was to devise a multiplex PCR for rapid detection of eae and identification of the specific intimin alleles in E. coli strains. To accomplish this, we designed oligonucleotide primers for multiplex PCR based on the multiple sequence alignment of eae alleles by McGraw et al. (6). Primers eae P1 (5′-CTGAACGGCGATTACGCGAA-3′) and eae P2 (5′-CCAGACGATACGATCCAG-3′) were constructed in the N-terminal conserved region of the gene at positions 544 and 1461, respectively. PCR with eae P1 and eae P2 generated a 917-bp fragment, indicating the presence of the eae gene (Fig. 1). Primers designed to determine the specific eae allele were constructed in the same orientation as eae P2 on the noncoding strand in the part of the gene specifying the variable C-terminal region of the protein (Fig. 1). Ecoeaeα (5′-CTGGAGTTGTCGATGTT-3′) was located at position 2192, generating a 1,648-bp fragment indicative of the eae allele specifying Int-α. Ecoeaeβ (5′-GTAATTGTGGCACTCC-3′), positioned at bp 2470, generated a 1,926-bp fragment indicative of the allele specifying Int-β. Ecoeaeγ (5′-GCCTCTGACATTGTTAC-3′), positioned at bp 2314, produced a 1,770-bp fragment indicative of the allele specifying Int-γ. All primers were synthesized by a Beckman 1000 oligonucleotide synthesizer (Beckman, Fullerton, Calif.).

FIG. 1.

FIG. 1

Primer locations and fragment sizes for multiplex PCR with five primers: eae P1, eae P2, Ecoeaeα, Ecoeaeβ, and Ecoeaeγ. PCR results are given for three standards (DEC 12a [EPEC 2], DEC 4f [EHEC 1], and E2348/69 [EPEC 1]) representing the three intimin alleles (Int-β, Int-γ, and Int-α, respectively).

To test the allele-specific PCR assay, we examined 87 strains of the diarrheagenic E. coli (DEC) collection, which have been characterized by electrophoretic type based on multilocus enzyme electrophoresis of 20 housekeeping genes (Table 1). The DEC strains represent 15 common clones associated with diarrheal disease and were examined previously for the presence of eae and several other virulence factors (10). The eae genes of DEC strains 3a, 3f, 5d, 11a, and 12a have also been sequenced (6).

TABLE 1.

Characteristics of 86 DEC strains examined by multiplex PCR to detect Int-α, Int-β, and Int-γ alleles

DEC strain no. Original strain no. Serotype Isolation dataa
PCR fragment size (bp)
eae allele
Yr Locale Host 917 1,648 1,770 1,926
1a 572-56 O55:H6 1956 USA Human + + α
1b C54-58 O55:H6 1958 Suriname Human + + α
1c F196-51 O55:H6 1951 Germany Human + + α
1d F563-55 O55:H6 1955 Egypt Human + + α
1e AC-C21 O55:H6 1986 Mexico Human + + α
2a 3787-62 O55:H6 1962 Congo Human + + α
2b 5513-56 O55:H− 1956 USA Human + + α
2c 607-54 O55:H6 1954 USA Human + + α
2d F60-51 O55:H6 1951 France Human + + α
2e 2087-77 O55:H6 1977 USA Human + + α
3a 3299-85 O157:H7 1985 USA Human + + γ
3b 46240 O157:H7 1990 USA Human + + γ
3c 3104-88 O157:H7 1988 USA Human + + γ
3d 3009-88 O157:H7 1988 USA Human + + γ
3e 3077-88 O157:H7 1988 Canada Human + + γ
3f 493/89 O157:H− 1989 Germany Human + + γ
4a C1520-77 O157:H7 1977 Argentina Calf + + γ
4b C999-87 O157:H7 1987 Denmark Human + + γ
4c C374-83 O157:H7 1983 Egypt Buffalo + + γ
4d C681-87 O157:H7 1987 Japan Calf + + γ
4e C7-88 O157:H7 1988 Denmark Human + + γ
4f EDL-933 O157:H7 1982 USA Meat + + γ
5a 5624-50 O55:H7 1950 USA Human + + γ
5b 660-79 O55:H7 1979 USA Human + + γ
5c 5380-66 O55:H7 1966 USA Human + + γ
5d C586-65 O55:H7 1965 Sri Lanka Human + + γ
5e C997-63 O55:H7 1963 Iran Human + + γ
6a 5338-66 O111:H21 1966 USA Human
6b C142-54 O111:H12 1954 Germany Human
6c 2277-67 O111:H12 1967 Guatemala Human
6d F436-51 O111:H12 1951 Italy Human
6e 184-83 O111:H12 1983 Brazil Human
7a 750001 O157:H43 1975 USA Pig
7b 902034 O149:H− 1990 USA Pig
7c 820691 O157:H43 1982 USA Pig
7d 831015 O157:H43 1983 USA Pig
7e 861575 O157:H− 1986 USA Pig
8a 2198077 O111:H− 1977 USA Human + ?
8b 3030A-86 O111:H8 1986 USA Human + ?
8c 8610049 O111:H− 1986 USA Calf + ?
8d C130-53 O111:H11 1953 Cuba Human + ?
8e C194-65 O111:H8 1965 Denmark Human + ?
9a 3323-61 O26:H11 1961 USA Human + + β
9b 2262-79 O26:H− 1979 USA Human + + β
9c C240-52 O26:H− 1952 Switzerland Human + + β
9d C814-67 O26:H11 1967 Denmark Human + + β
9e 45 O26:H11 1986 Mexico Human + + β
10a H30 O26:H11 ND Canada Human + + β
10b 3047-86 O26:H11 1986 Australia Human + + β
10c 1557-77 O26:H11 1977 USA Human + + β
10d C12-52 O26:H11 1952 France Human + + β
10e 900105 O26:H11 1990 USA Calf + + β
10f RDEC-1 O15:H− 1970s USA Rabbit + + β
10g C309-64 O128:H8 1964 ND Human + + β
10h C186-61 O119:H11 1961 ND Human + + β
10i 87-1713 O145:H6 1987 Canada Human + + β
10j 88817 O70:H11 1988 Canada Human + + β
11a 2254-75 O128:H2 1975 USA Human + + β
11b 3733-71 O128:H2 1971 USA Human + + β
11c A9619-c2 O45:H2 1983 USA Human + + β
11d E335021 O128:H2 1989 UK Human + + β
11e WM-63 O128:H2 ND Brazil Human + + β
12a F1-50 O111:H2 1950 UK Human + + β
12b 2966-56 O111:H2 1956 USA Human + + β
12c 3942-67 O111:H− 1967 Panama Human + + β
12d 9101-83 O111:H2 1983 Peru Human + + β
12e 3291-86 O111:H− 1986 Kenya Human + + β
13a 3350-73 O128:H7 1973 USA Human
13b 5024-71 O128:H7 1971 USA Human
13c C500-74 O128:H7 1974 Tanzania Human
13d C1083-79B O128:H7 1979 Rwanda Human
13e 2384-81 O128:H47 1981 USA Human
14a C916-70 O128:H21 1970 Peru Human
14b C691-71 O128:H21 1971 India Human
14c 9088-83 O128:H21 1983 Peru Human
14d 1791-79 O128:H− 1979 USA Human
14e C639-77 O128:H21 1977 Bangladesh Human
15a 5430-66 O111:H21 1966 USA Human
15b 448-71 O111:H21 1971 USA Human
15c 2660-77 O111:H21 1977 USA Human
15d 2708-78 O111:H21 1978 USA Human
15e 2394-80 O111:H21 1980 USA Human
a

USA, United States; ND, not determined; UK, United Kingdom. 

In preparation for PCR, each E. coli strain was grown overnight at 37°C in 10 ml of nutrient broth (Difco, Detroit, Mich.) in a shaking water bath. Chromosomal DNA was isolated according to the instructions in the Puregene DNA isolation kit (Gentra Systems, Inc., Minneapolis, Minn.). Aliquots (1 μl) of DNA samples were each amplified in a 50-μl reaction mixture that contained 5.0 μl of PCR buffer (100 mM Tris-HCl [pH 8.3], 500 mM KCl, 15 mM MgCl2, 1% Triton, 0.05% gelatin), 2.5 μl of primer eae P1 at 200 ng/μl, 1.0 μl each of primers eae P2, Ecoeaeα, Ecoeaeβ, and Ecoeaeγ at 200 ng/μl, 1.25 mM deoxynucleoside triphosphate mixture, 5 units of displayTAQ (Display Systems Biotech), and distilled H2O to volume. Amplification in a Perkin-Elmer 480 DNA thermal cycler utilized an initial denaturing step at 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 53°C for 2 min, and 72°C for 3 min. Positive and negative controls were included with each set of strains tested. PCR products were visualized on ethidium bromide-stained gels by transillumination with UV light.

We tested the multiplex PCR assay with three positive controls for which the complete eae sequence is known (E2348/69, DEC 4f, and DEC 12a) and observed PCR fragments of the predicted sizes (Fig. 1). We then tested the 87 DEC strains by multiplex PCR and found that 57 strains produced the 917-bp fragment indicative of the presence of the eae sequence (Table 1). The allele-specific fragments showed that Int-α occurs in DEC 1 and 2 strains (EPEC 1 group), Int-β occurs both in DEC 11 and 12 (EPEC 2) and in DEC 9 and 10 (EHEC 2) strains, and Int-γ occurs both in DEC 3 and 4 (EHEC 1) and in DEC 5 (atypical EPEC of serotype O55:H7) strains. PCR results for representative DEC strains are shown in Fig. 2. Interestingly, DEC 8 strains have an eae gene, but it is sufficiently different in sequence that it is not amplified by the allele-specific primers (Fig. 2). The genetic basis of this difference remains to be determined.

FIG. 2.

FIG. 2

Detection of the eae gene and specific intimin alleles in strains of the DEC collection. The first two strains of each of the 15 electrophoretic types of the DEC collection (Table 1) are shown. The presence of eae is indicated by a 917-bp fragment. Identification of specific intimin alleles is indicated by fragments of characteristic sizes (α, 1,648 bp; β, 1,926 bp; γ, 1,770 bp). Three controls (Fig. 1) are presented on the left side of each gel for comparison.

The results demonstrate that the multiplex PCR can accurately detect the presence of the eae gene and simultaneously identify specific eae alleles. Because the eae alleles encoding Int-α, Int-β, and Int-γ are lineage specific, this multiplex PCR method provides a rapid way to classify suspected pathogens into the major clonal groups of EPEC and EHEC.

Acknowledgments

This study was supported by Public Health Service grant AI 42391 from the National Institutes of Health.

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