NOTES Identification of Enterotoxigenic Escherichia ... - Europe PMC

JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1988, p. 2173-2176 0095-1137/88/102173-04$02.00/0 Copyright © 1988, American Society for Microbiology

Vol. 26, No. 10

NOTES Identification of Enterotoxigenic Escherichia coli Isolates with Enzyme-Labeled Synthetic Oligonucleotide Probes PETER P. MEDON,' JANICE A. LANSER,2 PARRY R. MONCKTON,3 PENG LI,' AND ROBERT H. SYMONS1* Department of Biochemistry, University of Adelaide,1 and Division of Clinical Microbiology, Institute of Medical and Veterinary Science,2 Adelaide, South Australia 5000, and Regional Veterinary Laboratory, Department of Agriculture and Rural Affairs, Bendigo, Victoria 3550,3 Australia Received 15 March 1988/Accepted 20 June 1988

Commercially available kits containing alkaline phosphatase-labeled oligonucleotide probes for Escherichia coli heat-stable enterotoxins (STI-H, STI-P, and STII) and the heat-labile enterotoxin were compared with bioassays and radiolabeled recombinant DNA probes to identify enterotoxigenic E. coli from 100 clinical isolates. There was very good agreement between the three methods.

Enterotoxigenic Escherichia coli (ETEC) carries the gene(s) for enterotoxin production which may cause a diarrheal disease in humans and the young of some farm animals. At least five types of E. coli enterotoxins have been characterized (1). The heat-labile toxin (LT) containing two subunits, LT(A) and LT(B), is detected by visualizing cytopathic effects on cultured mouse adrenal cell lines (4). The human (STI-H) and porcine (STI-P) E. coli heat-stable enterotoxins are detected by the use of the suckling mouse assay (3), whereas the STII toxin is assayed in ligated jejunal loops of pigs (2). The genes encoding these enterotoxins are present on transmissible plasmids. Four of these genes have been cloned, and their nucleotide sequences have been determined (11, 12, 18, 22, 23). DNA colony hybridization was originally developed as a screening tool to detect recombinant DNA in bacterial isolates by using radiolabeled cloned DNA as probes (7). Since the late 1970s, the technique has been shown to be a promising replacement for tedious and expensive diagnostic assays, such as those used to detect ETEC (6, 9, 19-21). For routine diagnosis, however, the frequent use of radioisotopes is impractical, as well as imposing handling, storage, and disposal hazards. Moreover, not all diagnostic laboratories have easy access to radioisotopes. Synthetic oligonucleotides have become readily available commercially and shown to be suitable for use as probes for the diagnosis of a number of microbial agents (14, 15). We successfully developed a method using enzyme-labeled oligonucleotides complementary to the STI-H gene in a colony hybridization assay to detect ETEC in fecal specimens (13). In the study presented here, we validated the assay by using enzyme-labeled oligonucleotide probes specific for each of the four main types of ETEC. Four ETEC detection kits were kindly provided by Bresatec Ltd., Adelaide, South Australia, Australia, each containing a probe that is a mixture of enzyme-labeled oligonucleotides specific for one of the four enterotoxin genes (STI-P, STI-H, STII, and LT). In preparing each probe, four oligonucleotides, each 22 to 25 bases in length and complementary to the specific enterotoxin gene, were selected. After being separately coupled to *

alkaline phosphatase as described by Li et al. (13) and tested individually for specificity against each of the four ETEC by DNA colony hybridization, only those oligonucleotides exhibiting total specificity for their respective enterotoxin genes were mixed and provided as probes in the respective ETEC kit. The probes for the STI-P, STI-H, and LT enterotoxins each consist of a mixture of four enzymelabeled oligonucleotides, whereas the probe for the STII enterotoxin is a mixture of only three (Table 1). The lowerlevel sensitivity of each mixture of enzyme-labeled oligonucleotides was determined by dot blot hybridization with recombinant DNA plasmids containing each of the four enterotoxin genes as target, with the following results: STI-H probe, 20 pg of plasmid pSLM004 (18); STI-P probe, 5 pg of plasmid pRIT10036 (22); LT probe, 40 pg of plasmid pEWD299 (12); and STII probe, 5 pg of plasmid pCHL6 (11). One hundred E. coli isolates, previously tested for the presence of enterotoxins by bioassay as well as by hybridization using recombinant DNA probes labeled with 32P, were blind coded and used in this study. The isolates had been stored for 2 to 4 years in a snap-freeze broth at -70°C or on Dorset egg slopes at room temperature at the Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia. Sixty-five of the isolates were obtained from patients with diarrhea admitted to the Alice Springs Hospital, Alice Springs, Northern Territory, Australia (8). Thirtythree isolates were collected from piglets by the Regional Veterinary Laboratory, Bendigo, Victoria, Australia, and were obtained by the Institute of Medical and Veterinary Science 3 years ago (17). Two isolates were obtained from the Princess Margaret's Children Medical Research Foundation in Perth, Western Australia, Australia, through Jennifer Robinson. For this study, all isolates were subcultured and 3-,ul samples were applied directly onto the grid of cellulose acetate membrane GA-6 (catalog no. 66375; Gelman Sciences, Inc.) previously placed onto agar plates containing CLED medium (catalog no. CM301; Oxoid Ltd.). The plates were incubated inverted overnight at 37°C and assayed for ETEC as outlined below. The detection procedure followed was as outlined in the Bresatec laboratory manual accompanying the four kits. Briefly, after overnight incubation at 37°C, the cellulose

Corresponding author. 2173

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J. CLIN. MICROBIOL.

TABLE 1. Nucleotide sequences used in the preparation of the ETEC enzyme-coupled oligonucleotide probes ETEC

STI-H 5' CCTCAGGATGCTAAACCAGTAG-GAAC 5' CGTATGTACCTGTTGCTTTGTTG-GGGC 5' TTTGATTCAAATGTTCGTGGATGC-GGGC 5' AGTGGTCCTGAAAGCATGAATAGT-GAAC

(TC) (C 66 66 66 68

STI-P 5' CTTTTAGTCAGTCAACTGAATCAC-AAAC 5' TCCCGTCATGTTGTTTCACGGA-GGGC 5'ACAACATCACACTTTTTAGTCTCTA-GGAC 5' TATGTATTGGTGCTGAGAAAATCAT-GGGC

66 66 66 66

STII

5' AGCATCCTTTTGCTGCAACCATT-GGGC 5' TGTAGATGCATAGGCATTTGTAGC-GGGC 5' AATAT CGCATTTCTTCTTGCATCT-CCCC

66 68 64

LT(A) 5' CCTGAGATATATTGTGCTCAGATT-AAAC

66

LT(B) 5' TATCTGACCGAGACCAAAATTGAT-GGGC 5' CGATGGCAGGCAAAAGAGAAATG-CCCC 5' TGCCGCAATTGAATTGGGGGTTTT-CCCC

66 68 70

Unique sequence-spacer arm

type

a A four-nucleotide sequence to which the enzyme alkaline phosphatase was coupled, as described in Li et al. (13). b The melting temperature (Tm) of each oligonucleotide hybridized to its complementary strand was estimated at ionic strength of 0.9 M NaCI by using the formula Tm = 4(G+C) + 2(A+T), where G, C, A, and T indicate the number of corresponding nucleotides in the oligonucleotide (16).

acetate membranes were placed, colony side up, on Whatman 3MM filter paper saturated with lysis solution (0.5 M NaOH, 1.5 M NaCI) for 20 min and then neutralized on Whatman 3MM filter paper saturated with 1 M Tris hydrochloride (pH 7.0)-3 M NaCI for 10 min. The membranes were then placed on Whatman 3MM filter paper saturated with buffer A (50 mM Tris hydrochloride [pH 8.0], 125 mM NaCI, 10 mM EDTA, 0.5% sodium dodecyl sulfate) for a further 10 min. Individual membranes were further treated in 1 ml of buffer A containing 5 mg of lysozyme. The nucleic

acids were fixed by placing the membranes on Whatman 3MM filter paper saturated with 10% formaldehyde in 20x SSC (lx SSC is 150 mM NaCI and 15 mM sodium citrate [pH 7.0]) for 6 min and then removed and dried under a strong light source for 10 min. Each membrane was then placed into a polyethylene heat-sealable bag containing 2 ml of buffer A plus 0.4 mg of proteinase K and incubated at 50°C for 30 min. Finally, the membranes were immersed in 50 ml of 95% ethanol for 2 min and air dried. Membranes were prehybridized at 50°C for 1 h with shaking in 6x SSC, 25 x Denhardt solution (lx Denhardt solution is 0.02% bovine serum albumin fraction V [Sigma Chemical Co.], 0.02% Ficoll 400 [Pharmacia], and 0.02% polyvinylpyrrolidone [Sigma]), and 0.5% sodium dodecyl sulfate. Hybridization was done at 50°C for 30 min with shaking in 6x SSC, lx Denhardt solution, and 0.5% sodium dodecyl sulfate containing 100 ng of probe per ml (concentration of probe refers to the oligonucleotide moiety only). After hybridization, membranes were washed in 2x SSC0.5% sodium dodecyl sulfate-0.5% Tween 20 at 50°C and finally in 1 M NaCl-0.1 M Tris hydrochloride (pH 9.5)-S5 mM MgCl2 at room temperature with agitation. For color development, each membrane was incubated in the presence of substrate by the method of Leary et al. (10). Strong-positive specimens were identified within 1 h, whereas weak-positive specimens were detected after overnight color development. Results were compared with data obtained when replicate filters were hybridized with radiolabeled recombinant DNA probes specific for human (8) and porcine (17) ETEC. In addition, each isolate had been previously tested for enterotoxin production by using standard bioassays (2-4) by Jennifer Robinson, Princess Margaret's Children Medical Research Foundation. The results obtained with the 100 E. coli isolates by using bioassay, recombinant enterotoxin DNA probes, and enzyme-labeled probes (Bresatec ETEC kits) are presented in Table 2. The E. coli isolates fell into three classes on the basis of their enterotoxin gene contents. An additional class (class 4, Table 2) represents E. coli isolates with no entero-

TABLE 2. Comparison of three methods for identification of ETEC in 100 E. coli isolates Class

Host(s)b

Toxin(s) present in each E. coli isolate

No. of E. coli isolates identified by: Bresatec

oligonucleotide

Recombinant DNA probe

probe

Bioassay

1 A B C D

Pig

Human

Pig

LT LT and STI-P LT and STI-H LT and STII

6 2 0 16

6 1 1 16

A B

Human (1) and pig (1) Human

STI-P STI-H

4 31

2 33

35C

A B

Pig Pig

STI-P STI-P and STII

3 8

0 il

d

A B

Human (6) and pig (24)

None

30

30

29 Y

Human

6 2c

16

2

3

4

STI-(P+H)

a The E. coli isolates were grouped into four classes on the basis of the absence or presence of one or two enterotoxin genes. b The host(s) of isolates identified by the recombinant DNA probe (8, 17). The number of hosts is shown in parentheses. C Bioassay does not differentiate between STI-H- and STI-P-containing isolates (3). d Bioassay cannot confirm the presence of STII production by isolates which also produce STI-P (2).

NOTES

VOL. 26, 1988

toxin genes detectable by the oligonucleotide or DNA probes. There was very good agreement by the three methods in testing for LT-containing isolates (class 1, Table 2). The Bresatec kits also identified all STI isolates found positive by hybridization with the recombinant enterotoxin DNA probes (classes 1, 2, and 3, Table 2). However, the two DNA probe methods were not in total agreement as to which isolates were STI-P and which were STI-H (classes 1 and 2, Table 2). The Bresatec kits identified two LT isolates as also containing STI-P (class 1B, Table 2), whereas the recombinant DNA probes identified one, isolated from a pig, as containing STI-P and the other, isolated from a human, as containing STI-H (class 1C, Table 2). Similarly, the Bresatec kits identified an additional two human isolates as STI-P, not STI-H (class 2A, Table 2). Unfortunately, STI-P-producing isolates cannot be distinguished from STI-H isolates by using bioassay in suckling mice (3). The four STI-P oligonucleotide probes were selected to have at least eight mismatches compared with the published sequence of the gene for STI-H (see reference 18 and Table 1). Since it is recognized that there are practical difficulties in distinguishing between STI-P and STI-H using recombinant DNA probes (5, 8), we believe the results obtained with the oligonucleotide probes are more accurate. The Bresatec kit detected three fewer STII isolates of class 3B than did the recombinant DNA probe (Table 2). Two of the three isolates gave borderline color reactions but were scored as STII negative. On retesting, definite positives were obtained with these two isolates. The same three isolates were also found to be STI-P positive by all three methods. A possible explanation for the apparent lower sensitivity of the Bresatec STII probe is that the particular batch of STII probe used contained a mixture of only three enzyme-labeled oligonucleotides. One STI isolate (class 4B, Table 2), positive by bioassay (suckling mouse test), was not detected by the two DNA probe methods and may contain a toxin similar in biological activity and yet genetically divergent from STI. This isolate was twice tested by bioassay and recombinant DNA probing, with consistent results each time. Similar results have been reported by Moseley et al. (18). Several laboratories have reported the use of oligonucleotides for the detection of ETEC with variable results (6, 9, 21). Good correlation between radiolabeled synthetic probes and bioassays was reported by Hill et al. (9). Echeverria et al. (6) showed that the oligonucleotide probes detected slightly fewer ETEC than bioassays and recombinant DNA probes. Another study by Seriwatana et al. (21) compared synthetic probes labeled with alkaline phosphatase with those labeled with 32P. They found that the alkaline phosphatase-labeled probes detected 93% of STI-H, 73% of LT, and 67% of LT plus STI-H colonies identified by the radiolabeled probes. Several factors may account for the apparent improved performance of the enzyme-labeled ETEC test reported in our study. In contrast to groups in other laboratories which used a single synthetic oligonucleotide, 26 bases in length, to detect ETEC, we used toxin probes in a mixture of three or four oligonucleotides, 22 to 25 bases in length. Therefore, each probe mixture in the Bresatec kit contains between 66 and 100 bases, which represents approximately 10% of the LT [one oligonucleotide from LT(A) and three from LT(B)], 35% of the STI-P, 30% of the STI-H, and 20% of the STII gene. This should increase the sensitivity of the assay and also ensure that ETEC with silent mutations in their toxin

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genes would still be detected (13). An increase in the number of oligonucleotides in the STII kit from three to five or six should also bring the sensitivity up to that of the recombinant DNA probe. Overall, the enzyme-labeled ETEC test itself is quite straightforward with minimal opportunity for error and, unlike other DNA hybridization procedures, is completed in 1 day. We thank Jennifer Cassady and Tammy Edmonds for technical assistance. This work was supported by a joint grant under the Australian National Biotechnology Programme Research Grants Scheme to the Department of Biochemistry, University of Adelaide, and the Division of Medical Virology, Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia. LITERATURE CITED 1. Betley, M. J., V. L. Miller, and J. J. Mekalanos. 1986. Genetics of bacterial enterotoxins. Annu. Rev. Microbiol. 40:577-05. 2. Burgess, M. N., R. J. Bywater, C. M. Cowley, N. A. Mullan, and P. M. Newsomne. 1978. Biological evaluation of a methanolsoluble, heat-stable Escherichia coli enterotoxin in infant mice, pigs, rabbits, and calves. Infect. Immun. 21:526-531. 3. Dean, A. G., Y. C. Ching, R. G. Williams, 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. 4. Donta, S. T., H. W. Moon, and S. C. Whipp. 1974. Detection of heat-labile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture. Science 183:334-336. 5. Echeverria, P., D. N. Taylor, J. Seriwatana, A. Chatkaeomorakot, V. Khungvalert, T. Sakuldaipeara, and R. D. Smith. 1986. A comparative study of enterotoxin gene probes and tests for toxin production to detect enterotoxigenic Escherichia coli. J. Infect. Dis. 153:255-260. 6. Echeverria, P., D. N. Taylor, J. Seriwatana, and C. Moe. 1987. Comparative study of synthetic oligonucleotide and cloned polynucleotide enterotoxin gene probes to identify enterotoxigenic Escherichia coli. J. Clin. Microbiol. 25:106-109. 7. Grunstein, M., and D. S. Hogness. 1975. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. USA 72:3961-3965. 8. Higgins, G. D., J. A. Lanser, J. Robinson, G. P. Davidson, J. Erhlich, and P. A. Manlng. 1988. Enterotoxigenic Escherichia coli in central Australia: diagnosis using cloned and synthetic nucleic acid probes. Pathology 20:167-172. 9. Hil, W. E., W. L. Payne, G. Zon, and S. L. Moseley. 1985. Synthetic oligodeoxyribonucleotide probes for detecting heatstable enterotoxin-producing Escherichia coli by DNA colony hybridization. Appl. Environ. Microbiol. 50:1187-1191. 10. Leary, J. J., D. J. Brigati, and D. C. Ward. 1983. Rapid and sensitive colorimetric method for visualizing biotin-labelled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: bio blots. Proc. Natl. Acad. Sci. USA 80:40454049. 11. Lee, C. H., S. L. Moseley, H. W. Moon, S. C. Whipp, C. L. Gyles, and M. So. 1983. Characterization of the gene encoding heat-stable toxin II and preliminary molecular epidemiological studies of enterotoxigenic Escherichia coli heat-stable toxin Il producers. Infect. Immun. 42:264-268. 12. Leong, J., A. C. Vinal, and W. S. Dallas. 1985. Nucleotide sequence comparison between heat-labile toxin B-subunit cistrons from Escherichia coli of human and porcine origin. Infect. Immun. 48:73-77. 13. Li, P., P. P. Medon, D. C. Skiagle, J. Lanser, and R. H. Symons. 1987. Enzyme-linked synthetic oligonucleotide probes: nonradioactive detection of enterotoxigenic Escherichia coli in faecal specimens. Nucleic Acids Res. 15:5275-5287. 14. Lin, H. J., P. C. Wu, and C. L. Lai. 1987. An oligonucleotide probe for the detection of hepatitis B virus DNA in serum. J. Virol. Methods 15:139-149.

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J. CLIN. MICROBIOL. 20. Oprandy, J. J., S. A. Thornton, C. H. Gardiner, D. Burr, R. Batchelor, and A. L. Bourgeois. 1988. Alkaline phosphataseconjugated oligonucleotide probes for enterotoxigenic Escherichia coli in travelers to South America and West Africa. J. Clin. Microbiol. 26:92-95. 21. Seriwatana, J., P. Echeverria, D. N. Taylor, T. Sakuldaipeara, S. Changchawalit, and O. Chivoratanond. 1987. Identification of enterotoxigenic Escherichia coli with synthetic alkaline phosphatase-conjugated oligonucleotide DNA probes. J. Clin. Microbiol. 25:1438-1441. 22. So, M., and B. J. McCarthy. 1980. Nucleotide sequence of bacterial transposon Tnl681, encoding a heat-stable (ST) toxin, and its identification in enterotoxigenic Escherichia coli strains. Proc. Natl. Acad. Sci. USA 77:4011-4015. 23. Yamamoto, T., T. Tamura, and T. Yokata. 1984. Primary structure of heat-labile enterotoxin produced by Escherichia coli pathogenic for humans. J. Biol. Chem. 259:5037-5044.