INFECTION AND IMMUNITY, JUlY 1985, p. 46-510019-9567/85/070046-06$02.00/0Copyright X 1985, American Society for Microbiology

Detection of Genes for Heat-Stable Enterotoxin I in Escherichia coliStrains Isolated in Brazil

RENATA MAAS,' ROSA M. SILVA,' TANIA A. T. GOMES,2 LUIZ R. TRABULSI,2 AND WERNER K. MAAS'*Department of Microbiology, New York University School of Medicine, New York, New York 10016,' and Department of

Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, Sao Paulo, Brazil2Received 22 January 1985/Accepted 2 April 1985

Heat-stable enterotoxin I (STI) can be assayed in intestinal loops of pigs and rabbits and in the gut of infantmice. To produce a simpler and more discriminating assay procedure, we used three gene probes correspond-ing to three forms of STI called STIa, STIb, and STIc. We tested 159 Brazilian isolates, of which 40 werepositive in the infant mouse assay. The STIb and STIc probes are similar (93% DNA homology) and are bothdifferent from the STIa probe (70% DNA homology). Of 33 strains that were still active for STI 3 years aftertheir isolation, 25 reacted with both the STIb and STIc probes, 4 reacted with the STIc probe only, and 7reacted strongly with the STIa probe and weakly or not at all with the other probes. Two strains reacted withall three probes. Further analysis showed that each of these two strains contains a small plasmid that reactswith the STIa probe and a large plasmid that reacts with the STIc probe in one strain and weakly with boththe STIa and STIc probes in the other strain. It was also shown that the STIa probe reacts with the cloningvehicle pACYC184 used for the cloning of STIc. We conclude that the gene probes used can identify mostSTI-producing strains and that in cases of positive responses with several probes careful scrutiny is necessaryfor analysis.

The use of gene probes for diagnostic purposes is usefulwhen the pertinent characteristics of a pathogen are difficultto determine by other methods. For example, the productionof heat-stable enterotoxin I (STI, also referred to as STa) byEscherichia coli strains is usually determined in animalassays involving infant mice or intestinal loops of rabbits orpigs (4). For this toxin, two gene probes that correspond totwo forms of the toxin, STIa (or STIp) and STIb (or STIh),have been described (11). The differences between STIa andSTIb were analyzed at the level of the amino acid sequences(1) and the nucleotide sequences (11) and were found to beconsiderable. Thus, STI denotes a family of enterotoxinswith the same biological activities but with variations in thechemical structures. The STIa and STIb probes do notcross-react in colony hybridization tests, and thus the use ofthese two probes enables one to determine the type of STIproduced.Three years ago we began a study of enterotoxigenic E.

coli strains isolated in Brazil by using gene probes for STIaand STIb as well as a gene probe for heat-labile enterotoxin(LT) (10). For LT-producing strains, we found good agree-ment between gene probing and biological activity (Y-1adrenal cell assay), but for STI-producing strains, the agree-ment was not as good. Although strains that were negative inthe biological assay (infant mouse test) were also negativewith the two gene probes, only 40 to 50% of the infant mousetest-positive strains were positive with a gene probe. Subse-quently, we modified the published hybridization procedureby introducing a steaming step during lysis of the cells andwere able to increase considerably the sensitivity for thedetection of STI-positive colonies (7). We also obtainedanother STI probe similar to STIb that was easier to prepareand label than the STIb probe (5). We refer to it as the STIcprobe. By using the three probes and the modified procedurefor lysis, we were able to detect most of the strains that were

* Corresponding author.

positive in the infant mouse assay. In the present paper, wedescribe recent results obtained with the three probes. A fewof the strains that were positive three years ago in thebiological assay and with one of the probes had becomenegative by the time these experiments were done. In ourstudy, as in the previously reported studies from Thailand(10) and Bangladesh (11), there was a preponderance ofstrains that were positive for the STIb probe but not for theSTIa probe.

MATERIALS AND METHODS

Materials. Restriction endonucleases were purchased fromNew England BioLabs, Inc. DNA polymerase used in nicktranslation reactions was purchased from Boehringer Mann-heim Biochemicals. Labeled deoxynucleotide triphosphateprecursors (c- 32p) were purchased or were a gift from NewEngland Nuclear Corp.

Bacterial strains. The 40 strains were part of a collectionfrom Sao Paulo, Brazil. Most of them that were originallypositive for STI were clinical isolates from patients withdiarrhea, and a few were isolated from environmentalsources. Most of the ones for which complete serologicaland biochemical identification was possible were found tobelong to typical enterotoxigenic serogroups, such as 06,063, and 0128ac (12). As negative controls we used labora-tory K-12 strains and some clinical isolates that were nega-tive in the infant mouse test.

Enterotoxin probes. The three probes for STIa, STIb, andSTIc were obtained as follows. The bacterial strain contain-ing plasmid pNG10 with the STIc gene was obtained fromNigel Harford of the Smith Kline-RIT Co. via Dean Taylor.The STIc gene was originally isolated and cloned from aplasmid present in strain CRL25090 (5). In pNG10, a 510-base-pair segment containing the STIc gene is inserted in thevector pACYC184. The STIc gene has been sequenced (3),and the DNA sequence is very similar (93% homology) tothat of STIb (Nigel Harford, personal communication). The

46

Vol. 49, No. 1

GENE PROBING FOR STI IN BRAZILIAN E. COLI STRAINS

other two strains were gifts from Steve Moseley. One straincontains plasmid pRIT10036, which comprises an insert intopBR322 of a fragment containing a porcine STI gene (STIa)(10, 11). The other strain harbors plasmid pSLM004, whichwas made by inserting a fragment containing a human STIgene (STIb) (11) into pBR322.

Plasmid DNA was prepared by cesium chloride gradientcentrifugation of the cleared bacterial lysates. The probeDNA fragments were prepared as follows. For pNG10, theplasmid DNA was digested with EcoRI, and the resulting510-base-pair fragment was separated from the linearizedcloning vector on a 1% agarose gel. It was then eluted byadsorption on glass powder (15). For pRIT10036 andpSLM004, the appropriate fragments (157 base pairs forpRIT10036 and 240 base pairs for pSLM004) were preparedas described by Moseley et al. (11).The fragments were labeled with 32P by nick translation

with any one of the four possible precursors (specific activ-ity, 3,000 Ci/mmol) to a specific activity of ca. S x 107cpm/ujg of probe (13).

Immobilization of DNA from bacterial colonies on filterpaper or nylon membranes. An improved method for increas-ing the yield of immobilized DNA from bacterial colonieshas been described (7). This method involves the steaming inalkali of filter paper replicas of colony arrays. The method isparticularly useful when the genes in question, such as thegenes for STI, occur in large single-copy plasmids. We wereable to study the 40 STI+ Brazilian strains with each of thethree probes with relative ease. In a few cases, the resultswere somewhat equivocal. These strains were then testedagain with either colonies grown on GeneScreen Plus (NewEngland Nuclear) or minilysates that were filtered ontoGeneScreen Plus with a spot-blotting manifold (Schleicher &Schuell, Inc.). GeneScreen Plus membranes gave cleanerresults than Whatman no. 541 filter paper because thenegative controls tended to give lower signals.

Disks of GeneScreen Plus were suitably marked for ori-entation and identification with a marker pen, sandwichedbetween two disks of Whatman 3MM filter paper, individu-ally wrapped in tinfoil, and autoclaved. Each disk was heldwith sterile forceps over 2% agar medium in a petri dish (150by 25 mm) with the convex side facing down. The disk wasgently lowered so that contact was made in the center firstand spread spontaneously outward. When the membranewas uniformily wet, the strains were transferred onto it witheither a multipronged device or toothpicks. The bacteriawere allowed to grow overnight at 37°C, and the membraneswere processed as described previously (7).The procedure for spot blotting was as follows. Cultures (3

ml) of each strain were grown in tryptone-yeast extractmedium overnight. The cells were pelleted and suspended in1 ml of 0.5 N NaOH-1.5 N NaCl. The tubes were placed ina boiling water bath for 3 min and then in watery ice; thecells were then neutralized with 1 ml of 1 M Tris (pH 7.0)-2M NaCl. A suitably cut piece of GeneScreen Plus waswetted with 7x SSC (lx SSC is 0.15 M NaCl plus 0.015 Msodium citrate), and 200 ,ul of each neutralized bacteriallysate was filtered onto it with the help of a spot blotter. Themembrane was then ready for hybridization.

Preparation of agarose gels for hybridization. When strainswere positive with two different probes, we separated theirplasmids by agarose gel electrophoresis by the method ofKado and Liu (6) for rapid plasmid DNA preparations. Wefirst attempted to transfer the plasmid DNA after acidnicking onto nitrocellulose or GeneScreen Plus membranesbut found that large-plasmid DNA did not transfer ef-

ficiently, resulting in a very low signal after hybridization.The direct use of agarose gels for hybridization has beendescribed previously (2), but we found the unprotected driedgels difficult to manipulate. We used the following method toprotect them. Gels were stained with ethidium bromide andphotographed with UV light. They were incubated in 0.4 NNaOH-0.6 NaCl for 30 min at room temperature to denaturethe DNA and were then neutralized by incubation in 0.5 Macidic Tris-hydrochloride-1.5 M NaCl for 30 min at roomtemperature. They were mounted sandwiched between twopieces of Whatman no. 541 filter paper cut so that a 1-cm rimwas left all around the gel. It was convenient to mark thepapers before mounting the gel for identification and orienta-tion. The paper-gel-paper sandwich was dried in a gel dryer(Hoefer Scientific Instruments), and then a seam was madeall around the sandwich with a sewing machine. The prehy-bridization, hybridization, and washings could then be car-ried out on this paper-enclosed gel. The latter could also beused as such for exposure to X-ray film provided that theexposure time was relatively short (no more than overnight).If longer exposure times were required, it was desirable toremove the no. 541 paper because of its background radio-activity. This was done by cutting away the seam while thegel was still wet and remounting the gel on a fresh piece ofWhatman 3MM filter paper, which could then be dried in thegel dryer in 5 to 10 min. This method was also usedsuccessfully for hybridizing fragments of the probe plasmidsthat had been digested with restriction endonucleases.

Hybridization and washing. The hybridizations were donein 50%o formamide-5 x SSC under conditions that have beendescribed previously (7). In general, an approximate 10-foldmolar excess of probe to complementary immobilized DNAwas used (although in one experiment with STIa, the excesswas 10-fold higher still), and the washings were done asdescribed previously (7). We used relatively large hybridiza-tion volumes (15 to 20 ml/96 cm2 of membrane or paper).

RESULTSFor interpretation of the results of probing experiments

with the Brazilian strains, it is desirable to know the extentof homology between the probes. To this end, the strainscontaining the cloned probes were hybridized with the threeprobes. As shown in Table 1 and Fig. 1, pSLM004 andpNG10 hybridized with both STIb and STIc. These probesdid not hybridize with the STIa-carrying strain. The STIaprobe did not hybridize with pSLM004 (pBR322 vector) butdid hybridize with pNG10 (pACYC184 vector). This one-way hybridization was unexpected and suggested the pos-sibility that there is homology between the STIa probe andthe pACYC184 vector. This was tested by hybridizing du-plicate EcoRI digests of pNG10 DNA that had beenelectrophoresed on an agarose gel with the STIa and STIcprobes. The STIa probe was found to hybridize with thecloning vehicle, and the STIc probe was found to hybridizewith the STIc insert (Fig. 2). Although the possibility thatour STIa probe preparation was contaminated withextragenic sequences cannot be ruled out, this is veryunlikely in view of the strong response in our STIa radio-gram (45-min exposure).Of the 159 Brazilian E. coli isolates that were tested for

STI in the infant mouse assay, 40 were originally positive.During the ensuing 3-year period, probing for STI was doneseveral times (with and without the steaming step) at twolocations, Sao Paulo and New York. With steaming, STIcprobing of the 40 infant mouse assay-positive strains wasdone five times; STIb probing and STIa probing were each

VOL. 49, 1985 47

48 MAAS ET AL.

TABLE 1. Properties of Brazilian STI-producing Si

Strain Position in Serotype' Toxin andStrainFig. 1 Serotypea(colonizationfactor' STIc(July 1983)

0128AC:H120128AC:H120128AC:H120128AC:H120128AC:H120128AC:H120128AC:H70128AC:H7063:H-063:H-063:H-062:H-R:H-0128AC:H210128AC:H210128AC:H210128AC:H210128AC:H270128AC:H-0128AC:H-06:H1606:H1606:H1606:H1606:H1606:H1608:H4NT:H4NT:H-ND:H1ONT:H-NT:H4 or NT:H17NT:H28NDND:H7034:ND0128:H21023:H-ND06:H16

STI, CFA/ISTI, CFA/ISTI, CFA/ISTI, CFA/ISTI, CFA/ISTI, CFA/ISTI, CFA/ISTI, CFA/ILT, STI, CFA/ILT, STI, CFA/ILT, STI, CFA/ILT, STI, CFA/ILT, STI, CFA/ISTISTISTISTISTISTISTILT, STI, CFA/IILT, STILT, STI, CFA/IILT, STI, CFA/IILT, STILT, STI, CFA/IILT, STILT, STILT, STILT, STILT, STILT, STILT, STILT, STISTISTISTILT, STISTILT, STI

+++++++++++++++++

++++++++++++++++++

+++++++++++

+

trains

STIC(January

++++++++++++++

+++

++++

+++

+++++

++

Reaction" with:

STIb1984) (January 1984)

++

+

+++++++

+l.

+-F

ControlsC600(pRIT10036)HB101(pSLM004)MM294(pNG10)

a H-, Nonmotile; R, rough; NT, negative with available 0 sera; ND, not determined.b CFA/I, Colonization factor antigen I; CFA/II, colonization factor antigen II.c + +, Strongly reactive; +, fairly reactive; ±, weakly reactive; -, nonreactive.

done twice. The results reported in this paper for the infantmouse assay-positive strains were recently obtained in NewYork, where we do not carry out infant mouse assays. Asshown in Table 1, as of July 1983, 3 of the 40 strains (strains33, 38, and 40) had become negative with the three probes.We think that the most likely explanation for this change isa loss of the STI gene during storage in stab cultures. This isborne out by the finding that in January 1984, four strainsthat were positive for STIc only in July 1983 had becomenegative with this probe (Table 1, strains 21, 24, 25, and 31).Of the 33 strains that were positive with at least one probe

in January 1984, 25 gave clear positive results with bothSTIb and STIc (Table 1 and Fig. 1). Four strains (strains 8,10, 26, and 27) were positive with STIc and negative withSTIb, but their reaction with STIc was weak. The difference

in the response to these two probes is most likely due to thegenerally lower sensitivity obtained with the STIb probethan with the STIc probe (Fig. 1). One probable cause forthis difference is that during nick translation, the 510-base-pair STIc probe was always labeled to twice the specificactivity of the 240-base-pair STIb probe. In addition, accord-ing to Meinkoth and Wahl (8), 500-base-pair segments or

longer segments, but not shorter segments, hybridize witheach other in overlapping complementary regions to form"networks" or "hyperpolymers." This phenomenon couldalso contribute to the greater effectiveness of the STIc probethan of the STIb probe.Seven strains reacted strongly with the STIa probe and

weakly or not at all with the other probes. One of thesestrains (Table 1, strain 27) at first reacted moderately with

12345678910111213141516171819202122232425262728293031323334353637383940

A2A3A4ASA6A7A8A9A10AllB1B2B3B4BSB6B7B8B9B10Bl1B12C1C2C3C4CSC6C7 and E4C8C9C1oC1lC12DlD2D3D4DSD6

STIa(January 1984)

++++

++++

++

++

++

++

++

++

++

++

++

++

++

INFECT. IMMUN.

GENE PROBING FOR STI IN BRAZILIAN E. COLI STRAINS

1 2

-,

pop.

.e ,

Eco Rl digest of pNG 0

IO-- Inear pAcyc 184

j_- 500 bp probe

..STITb

....f... .

.w..lI

9.- + w: :.s.:: .. : ^¢: :¢>:

#: * T

FIG. 2. Duplicate EcoRI digests of pNG10 DNA. DuplicateEcoRI digests of pNG10 DNA were separated into the two resultingfragments by agarose gel electrophoresis (0.8% agarose, Tris-boratebuffer). The gels were pretreated for hybridization as described inthe text, hybridized with the labeled probes as shown, and autoradi-ographed on X-Omat AR film as described in the text. Lanes: 1,Hybridization with the STIc probe; 2, hybridization with the STIaprobe. bp, Base pairs.

the STIc probe as well. When the test was repeated withGeneScreen Plus, colony hybridization with the STIc probewas weaker for this strain, and the spot blot test produced anintensity almost as low as that of the K-12 negative control(Fig. 3, spot 7). We conclude that this strain is STIcnegative, especially since the plasmid gel hybridization testgave no reaction with the STIc probe but gave a positivereaction with the STIa probe (Fig. 4, lane 7). The serogroupsof the strains that were positive with the pRIT10036 probeonly could not be identified (Table 1).Two strains (Table 1, strains 4 and 5) reacted strongly with

the STIc probe and fairly well with the STIa probe. Probingof these strains was repeated with GeneScreen Plus and spotblotting (Fig. 3, strains 3 and 4). In plasmid gels of thesestrains (Fig. 4), it can be seen that one of the strains (lane 4)

A/(A 3

I,9

1 2 3 4 s 6 7 8 9 la It *2

c r>R1T10036 LPSLMOO4 pRIT1OO36

E iL I

FIG. 1. Colony hybridization with three different STI probes. Acolony array was printed three times from nutrient agar ontoWhatman no. 541 filter paper. Each paper was processed andhybridized as indicated with the probe for STIa (pRIT10036), STIb(pSLM004), or STIc (pNG10). The diagram is included for orienta-tion. The specific activity of the probes varied from 1 x 10i to 5 x10i cpm/Iug, and exposure was on Kodak X-Omat AR film with oneintensifying screen for 1 to 5 h. Strains containing the plasmids thatwere used for preparing the probes are indicated on the diagram. E2is a K-12 negative control. E3 is a K-12 strain containing the sameplasmid as E4 and its duplicate C7 (clinical isolate). D7, D9, D10,Dll, and D12 are clinical isolates that were STI negative in theinfant mouse test.

IIt.

i.j.h..

3 4 7 2

FIG. 3. Probing with STIc using colony hybridization (A) andspot blotting (B) of minilysates with GeneScreen Plus. DNA wasimmobilized on GeneScreen Plus. Spots 2, 3, 4, and 7 correspond tolanes of the same number in Fig. 4. Spot 2 is the control strainharboring plasmid pNG10, spots 3, 4, and 7 are clinical isolatesundergoing repeat testing, and spot 0 is a K-12 negative control.

v .L.

w .4. .. *. iX *~~~~~A~4?

...'? .4

0

--.-j

VOL. 49, 1985 49

::,

50 MAAS ET AL.

A2 3 4 5 6 7 8 9 10

B c4., a; 8 , si

am

STIa

STIcFIG. 4. Plasmid hybridization with two STI probes. (A) Gel

showing the plasmid content of various strains (strain numbers are

from Table 1). Lanes: 1, C600(pRIT10036); 2, MM294(pNG10); 3,

clinical isolate 4; 4, clinical isolate 5; 5, clinical isolate 7; 6, clinical

isolate 8; 7, clinical isolate 27; 8, clinical isolate 29; 9, clinical isolate

30; 10, E. coli K-12 harboring the STIa plasmid shown in lane 8. This

K-12 strain is in position E3 in the colony arrays (see Fig. 1).

Duplicate gels were hybridized with the STIa probe (B) and the STIc

probe (C). Lanes are as in panel A.

has a large plasmid that hybridizes with the STIc probe and

therefore presumably produces the corresponding toxin. The

other strain (lane 3) has two large plasmids, one of which

reacts weakly with both the STIc probe and the STIa probe.The kind of toxin produced by the gene in this plasmid,if any, remains unknown. In both strains there are

small plasmids that hybridize with the STIa probe from

pRIT10036. Although so far genes for STI in natural isolateshave been observed only in large plasmids, in view of thelocation of the STIa gene in a transposon (14), it is conceiv-able that the STIa gene might also occur in small plasmids.We think that this is unlikely in the present case, since thereare usually more copies of a small plasmid than of a largeplasmid in a cell and yet in colony hybridizations with theSTIa probe strains 4 and 5 gave weaker responses thanstrains responding only to the STIa probe (Table 1). In theplasmid gel hybridization (Fig. 4), the intensity of the smallplasmid in lanes 3 and 4 is probably related to the relativelyhigh yield of small-plasmid DNA in the Kado-Liu procedure(6). It should be noted that strains with two STI genes havebeen reported, each gene being located in a large plasmid(16). As for the other lanes in Fig. 4, 1 and 2 are plasmidcontrols for STIa and STIc, respectively, 6 is representativeof a clinical STIc-positive strain, and 7, 8, and 9 arerepresentative of clinical STIa-positive strains.

DISCUSSIONColony hybridization with clinical isolates presents prob-

lems not ordinarily encountered with laboratory K-12strains, since the latter are uniform and the genes to betested are often cloned into multicopy vectors. However,with our modified lysis procedure and the three probes, wewere able to detect all STI-producing Brazilian isolates asdefined by a biological assay and observed no false-positivecolony hybridizations among the STI-negative strains. Actu-ally, two probes, STIa and STIc, would have sufficed. STIband STIc are very similar (Nigel Harford, personal com-munications), and both show only 70% homology with theSTIa probe (3, 11). The advantages of the STIc probe overthe STIb probe are that the STIc probe is easier to prepareand somewhat more efficient in hybridization reactions.Otherwise, there is not much difference between the STIbprobe and the STIc probe.The type of STI whose gene hybridizes with the STIc and

STIb probes is more frequent among the Brazilian clinicalisolates than the type whose gene hybridizes with the STIaprobe. The gene probes for STIc and STIb were originallyobtained from human isolates (3, 11), whereas the STIa geneprobe was derived from a porcine isolate (10). In previousstudies with the STIa and STIb probes (10, 11), a similarpreponderance of clinical isolates reacting with the STIbprobe was observed.

It is clear from our results that care has to be taken in theinterpretation of hybridization results. Although we did notsee any false-positives among the Brazilian isolates thatwere negative in the infant mouse assay, this possibility isindicated by our finding of hybridization of the STIa probewith the cloning vehicle pACYC184. The 157-base-pair STIaprobe contains 141 base pairs of the STI gene (14), so that itis unlikely that the hybridization with pACYC184 is due tothe remaining 16 base pairs. It suggests rather that there issome homology between the STIa gene and a region of thepACYC184 plasmid.Two strains that reacted with all three probes in colony

hybridizations were investigated further by spot blotting ofextracts and Southern hybridization of plasmid DNA. Thefinding of positive reactions with the STIa probe of smallplasmids of these strains raises two possible explanations,both unexpected. Either a gene for STIa toxin is present inthese small plasmids, or the small plasmids contain DNAsequences that do not code for STI toxin but are homologouswith STIa. So far we have not distinguished experimentallybetween the two possibilities. Another unexpected finding

INFECT. IMMUN.

GENE PROBING FOR STI IN BRAZILIAN E. COLI STRAINS

was the weak hybridization of a large plasmid with both theSTIa and STIc probes. A possible explanation for thisfinding is the presence of a type of STI gene that isintermediary in its sequence between STIa and STIc.

ACKNOWLEDGMENTSWe thank Steve Moseley and Dean Taylor for supplying us with

the gene probes and Nigel Harford for unpublished informationabout plasmid pNG10.

This work was supported by grant PCM-8118494 from the Na-tional Science Foundation, by a joint grant from the NationalScience Foundation (Int-8020261) and the Brazilian National Re-search Council under the Cooperative Science Program for LatinAmerica, and by a grant from the Fundacao de Amparo a Pesquisade Sao Paulo. W.K.M. is the holder of Public Health ServiceResearch Career Award GM15129 from the National Institute ofGeneral Medical Sciences. a

LITERATURE CITED

1. Aimoto, S., T. Takao, Y. Shimonishi, S. Hara, T. Takeda, Y.Takeda, and T. Miwatani. 1982. Amino-acid sequence of aheat-stable enterotoxin produced by human enterotoxigenicEscherichia coli. Eur. J. Biochem. 129:257-263.

2. Dalbadie-McFarland, G., L. W. Cohen, A. D. Riggs, C. Horin,K. Itakura, and J. H. Richards. 1982. Oligonucleotide-directedmutagenesis as a general and powerful method for studies ofprotein function. Proc. Natl. Acad. Sci. U.S.A. 79:6409-6413.

3. De Wilde, M., M. Ysebaert, and N. Harford. 1981. DNAsequence of the STA2 enterotoxin gene from an E. coli strain ofhuman origin, p. 596. In S. B. Levy, R. C. Clowes, and E. L.Koenig (ed.), Molecular biology, pathogenicity, and ecology ofbacterial plasmids. Plenum Publishing Corp., New York.

4. Gyles, C. L. 1979. Limitations of the infant mouse test forEscherichia coli heat-stable enterotoxin. Can. J. Comp. Med.43:371-379.

5. Harford, N., M. De Wilde, and T. Cabezon. 1981. Cloning of twodistinct but related ST enterotoxin genes from porcine andhuman strains of E. coli, p. 611. In S. B. Levy, R. C. Clowes,and E. L. Koenig (ed.), Molecular biology, pathogenicity, and

ecology of bacterial plasmids. Plenum Publishing Corp., NewYork.

6. Kado, C. I., and S. T. Liu. 1981. Rapid procedure for detectionand isolation of large and small plasmids. J. Bacteriol.145:1365-1373.

7. Maas, R. 1983. An improved colony hybridization method withsignificantly increased sensitivity for detection of single genes.Plasmid 10:296-298.

8. Meinkoth, J., and G. Wahl. 1984. Hybridization of nucleic acidsimmobilized on solid supports. Anal. Biochem. 138:267-284.

9. Moseley, S., I. Huq, A. R. M. A. Alim, M. So, M. Samadpour-Motalebi, and S. Falkow. 1980. Detection of enterotoxigenicEscherichia coli by DNA colony hybridization. J. Infect. Dis.142:892-898.

10. Moseley, S. L., P. Echeverria, J. Seriwatana, C. Tirapat, W.Chaicumpa, T. Sakuldaipeara, and S. Falkow. 1982. Identifica-tion of enterotoxigenic Escherichia coli by colony hybridizationusing three enterotoxin gene probes. J. Infect. Dis. 145:863-869.

11. Moseley, S. L., J. W. Hardy, M. I. Huq, P. Echeverria, and S.Falkow. 1983. Isolation and nucleotide sequence determinationof a gene encoding a heat-stable enterotoxin of Escherichia coli.Infect. Immun. 39:1167-1174.

12. Reis, M. H. L., D. P. Matos, A. F. P. DeCastro, M. R. F. Toledo,and L. R. Trabulsi. 1980. Relationship among enterotoxigenicphenotypes, serotypes, and sources of strains in enterotoxigenicEscherichia coli. Infect. Immun. 28:24-27.

13. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1977.Labeling deoxyribonucleic acid to high specific activity in vitroby nick translation with DNA polymerase I. J. Mol. Biol.113:237-251.

14. So, M., and B. J. McCarthy. 1980. Nucleotide sequence ofbacterial transposon Tn1681 encoding a heat stable toxin (ST)and its identification in enterotoxigenic Escherichia coli strains.Proc. Natl. Acad. Sci. U.S.A. 77:4011-4015.

15. Vogelstein, B., and D. Gillespie. 1979. Preparative and analyticalpurification of DNA from agarose. Proc. Natl. Acad. Sci.U.S.A. 76:615-619.

16. Yamamoto, T., and T. Yokota. 1983. Plasmids of enterotoxi-genic Escherichia coli H10407: evidence for two heat-stableenterotoxin genes and a conjugal transfer system. J. Bacteriol.153:1352-1360.

VOL. 49, 1985 51