Foodborne enterotoxigenic Escherichia coli: Identification and enumeration on nitrocellulose membrane by enzyme immunoassay

International Journal of Food Microbiology, 3 (1986) 79- 87 Elsevier

79

JFM 00088

Foodborne enterotoxigenic Escherichia coli: Identification and enumeration on nitrocellulose membrane by enzyme immunoassay D.B. Shah * and U.S. Rhea Division of Microbiology, Food and Drug Administration, 1090 Tusculum Avenue, Cincinnati, OH 45226, U.S.A.

(Received 7 June 1985; accepted 16 November 1985)

A solid-phase enzyme immunoassay capable of direct enumeration of Escherichia coli that produce heat-labile enterotoxin (LT) has been developed. Pure or mixed cultures of bacteria or artificially contaminated foods were plated on nitrocellulose membranes placed on semisynthetic agar containing lincomycin. After overnight growth, the colonies were lysed in situ and were reacted with rabbit antiserum to LT. The presence of rabbit IgG in a colony was detected by goat antirabbit IgG peroxidase conjugate. Results of 71 strains tested by this method were in complete accord with LT assays performed by the mouse Y-1 adrenal cells. Three foods, namely, oysters, raw milk and Brie cheese, containing 2 × 10 6, 1.3 × 105 and 3.9 × 106 total plate count, respectively, were artificially contaminated with known levels of toxigenic E. coli. Upon analysis of membrane filters on which a range of 1 x l 0 3 to 10 4 cells was deposited, 1 to 10 LT + E. coli could be enumerated with a recovery greater than 85%. The test on foods can be completed in approximately 30 h. The test is sensitive enough to visualize 1 ng of pure LT contained in 1 mm 2 area of the membrane. Key words: E. coli, enterotoxigenic; Colony immunoassay; Enumeration; Foods

Introduction E n t e r o t o x i g e n i c E s c h e r i c h i a coli ( E T E C ) c a u s e d i a r r h e a l disease in h u m a n s o f all age g r o u p s f r o m d i v e r s e g e o g r a p h i c a l l o c a t i o n s (Sack, 1975). T h e s e E T E C e x p r e s s s p e c i e s - s p e c i f i c c o l o n i z a t i o n f a c t o r s ( E v a n s et al., 1975) a n d t h u s a t t a c h a n d m u l t i p l y in t h e s m a l l bowel. T h e y i n d u c e d i a r r h e a l illness b y the e l a b o r a t i o n of e i t h e r a h e a t - l a b i l e ( L T ) o r a h e a t - s t a b l e (ST) e n t e r o t o x i n o r b o t h (Gyles, 1971). A l t h o u g h it is c l e a r t h a t i n f e c t i o n o c c u r s a f t e r i n g e s t i o n of c o n t a m i n a t e d f o o d or w a t e r ( M a r i e r et al., 1973; M e r s o n et al., 1976; Sack et al., 1977; R o s e n b e r g et al., 1977), n o e x t e n s i v e surveys h a v e b e e n m a d e to i d e n t i f y a n d e n u m e r a t e E T E C in i t e m s of food. T h i s is p r i m a r i l y d u e to the difficulties in d i f f e r e n t i a t i n g E T E C f r o m n o r m a l E. coli in r o u t i n e m i c r o b i o l o g i c a l analysis. T h u s , e p i d e m o l o g i c a l d a t a o n the

* To whom correspondence should be addressed. 0168-1605/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

8o environmental sources of these organisms and on the minimum concentration of ETEC necessary to produce the disease are not available. The true incidence of ETEC infections, therefore, remains unknown. Methods that can differentiate and enumerate ETEC colonies from normal E. coli colonies on primary isolation media have recently been developed. One such method utilizes radiolabeled DNA encoding LT or ST gene sequences for enumeration of ETEC in foods (Hill et al., 1983; Hill, manuscript in preparation). Another method, the solid-phase radioimmunodetection method, uses radiolabeled antibodies to LT toxin to identify LT-producing colonies (Shah et al., 1982). In view of some of the disadvantages of the use of radioisopes for large scale surveys in field laboratories, we have sought nonisotopic methods for the enumeration of ETEC. The procedure described in this report takes advantage of high efficacy binding of proteins to nitrocellulose. Proteins released from bacterial colonies grown on nitrocellulose membrane filters are reacted with antiserum to LT toxin. The localization of antibody to LT positive colonies is detected by enzyme-labeled anti-antibody.

Materials and Methods

Bacterial strains and characterization of toxins

More than 100 clinical isolates of ETEC strains representing wide geographic distribution were obtained from the collection of W. Spira and R.B. Sack, Johns Hopkins University, Baltimore, MD, U.S.A.; D.J. Evans, University of Texas Health Sciences Center, Houston, TX, U.S.A.; D.C. Robertson, University of Kansas, Lawrence, KS, U.S.A.; and H. Moon, U.S.D.A., Ames, IA, U.S.A. Cultures were stored at - 20°C in trypticase soy broth (TSB) containing 40% glycerol. The cultures were tested for LT by the Y-1 adrenal cell assay (Donta et al., 1974) and for ST by the infant-mouse assay (Dean et al., 1972). Media and reagents

Nitrocellulose membranes (BA85, pore size 0.45 ~m) were obtained from Schleicher & Schuell, Keene, NH, U.S.A. Affinity purified goat anti-rabbit IgGhorseradish peroxidase (GAR-HRP), and horseradish peroxidase color development reagent were obtained from Bio-Rad Laboratories, Richmond, CA, U.S.A. Normal goat serum was purchased from Green Hectares, Oregon, WI, U.S.A. All other reagents were analytical grade and were obtained from commercial sources. The agar medium (M9CA) used for colony immunobinding assay was prepared as follows: Autoclaved (121°C for 20 min) Solution A [NazHPO4, 6 g; K H z P O 4, 3 g; NaC1, 5 g; NH4C1 , 1 g per 500 ml water] and Solution B [Noble Agar 15 g in 470 ml water] were tempered to 55°C and mixed. This agar base was further supplemented with 1.0 ml of sterile 1 M Mg SO4, 10 ml of 0.01 M CaC12, 10 ml of 10% Casamino acids (Difco), 10 ml of 20% glucose, and 2 ml of 15 m g / m l filter-sterilized lincomycin (Sigma Chemical, St. Louis, MO, U.S.A.) solution in water. Plates (10 cm) contain-

81 ing approximately 25 ml of medium were used after overnight incubation at room temperature. The plates can be used up to one month if stored in a sealed bag at 4°C.

Preparation of antiserum against LT Rabbit antiserum to electrophoretically pure LT (Clements and Finkelstein, 1979) was prepared as described before (Shah, et al., 1982). The immunoglobulin G (IgG) fraction was obtained by Protein A Sepharose chromatography (Goding, 1976).

Colony immunoassay Sterile nitrocellulose membranes were soaked in sterile Tris-buffered saline (TBS/0.02 M Tris/0.5 M NaC1, p H 7.5) for 10 min to remove any detergent, and were placed on M9CA Agar by rolling the membrane to prevent entrapment of air bubbles. Sterile toothpicks were used to transfer growth from colonies, or 30/~1 of appropriate dilution of culture or food test portion was plated with the aid of a spiral plater (Spiral Systems Instruments, Inc., Bethesda, MD, U.S.A.). Plates with cells transferred with toothpick were incubated until visible growth appeared (6-8 h) and spread-plates were incubated for 16 h at 30°C or until colonies were about 1 m m in diameter. The membranes were then placed, colony side up, on a Whatman No. 3 filter soaked in TBS containing 0.05 M ethylenediaminetetraacetic acid (EDTA)/0.1% sodium dodecyl sulfate (SDS), and were exposed to chloroform vapor for 1 h at 37°C to induce cell lysis. All subsequent steps were carried out at room temperature, and all washing steps used 20 ml of TBS for 20 min on a rocking platform. The membranes were washed once, placed in 10 ml of 'blocking solution' (TBS + 5% goat normal serum) for 1 h and then transferred to 10 ml of primary antibody solution (10/~1 of rabbit IgG in TBS/0.1% normal goat serum) for 3 h with gentle agitation. After one wash in TBS containing 0.05% Tween 20 and 3 additional washings in TBS to remove excess rabbit IgG, the membranes were incubated for 2 h in G A R - H R P conjugate (1:3000 dilution in TBS/0.5% normal goat serum). The membranes were again washed 3 times and placed in color development reagent (prepared according to manufacturer's instructions) for up to 30 min. After a brief rinse in distilled water, enterotoxin-producing colonies were enumerated by counting purple spots.

Microbiological analysis of food 100 g lot of oyster samples were processed in a stomacher (Tekmar Co., Cincinnati, OH, U.S.A.) for 30 s and were filtered through sterile cheese cloth. Brie cheese samples (25 g) were homogenized with 225 ml of TBS in a blender for 3 rain. Appropriate dilutions of these food samples were plated on aerobic plate count agar. Experimentally contaminated food was prepared by the addition of E. coli to cheese cloth-filtered portion of oyster, homogenized Brie Cheese, or to raw milk.

82

Results

Limit of determination and specificity of immunoassay To examine whether antiserum generated against native LT recognizes the denatured L T on nitrocellulose and to determine the limit of determination of the immunoassay, 1 /~1 aliquots containing k n o w n amounts of LT were spotted on the membrane. The filters were processed by the standard assay procedure. Fig. 1 shows that at least 2.75 ng of L T contained in a circle approximately 2 m m in diameter can be easily determined. A perceptible signal was not observed at positions where only buffer was spotted. To test the specificity of the assay, clinical E T E C isolates were first grown on trypticase soy agar and cells from a single colony were spotted, 1.5 cm apart, on the m e m b r a n e with sterile toothpicks. After incubation for 6 - 8 h at 37°C, the colonies on the membranes were tested for the presence of L T by the immunoassay. A representative photograph (Fig. 2) of the signal obtained with 20 cultures demonstrates that seven cultures were LT-positive. Results of 71 E T E C isolates tested by this method and summarized in Table I were in complete accord with L T assays performed by the mouse Y-1 adrenal cells. Although no false positive results were

Fig. 1. Sensitivity of the the immunoassay for LT. 1 #1 samples containing the following amounts of LT in TBS/0.1% BSA were spotted and the membrane was processed by the standard immunoassay procedure. From left to right; (Row A) 55 ng, 22 ng, 11 ng; (Row B) 5.5 ng, 2.75 ng, 1.37 ng/(Row C) 275 pg, 0 pg, 0 pg.

83

Fig. 2. Immunoassay for LT identification on nitrocellulose membrane inoculated with pure E. coli strains: l-A, V517; I-B, JC1569/PSC101; l-C, TD 514C1; l-D, TD462C1; 2-A, E-38; 2-B, WS-1; 2-C, WS-2; 2-D, WS-3, 3-A, WS-4; 3-B, H10407; 3-C, K334C2; 3-D, TD225C4; 4-A, 1408; 4-B, 286C 2, 4-C, 1362; 4-D 339.

o b s e r v e d w i t h L T n e g a t i v e isolates, d i f f e r e n c e s w e r e o b s e r v e d in the i n t e n s i t y of signal g e n e r a t e d b y v a r i o u s L T p o s i t i v e - i s o l a t e s (Fig. 2).

Experimentally contaminated foods O y s t e r s a m p l e s filtered t h r o u g h sterile c h e e s e c l o t h h a d an a e r o b i c p l a t e c o u n t of 2.8 x 10 6 cells p e r g. A l i q u o t s of this p r e p a r e d s u s p e n s i o n , c o n t a m i n a t e d w i t h LT-" E. coli, w e r e p l a t e d (in d u p l i c a t e ) , so t h a t o n e a c h m e m b r a n e was d e p o s i t e d either

TABLE I Comparison of Y-1 and immunoassay for LT identification Strain designation

1. LT , ST2. LT-, ST + 3. LT ÷, ST4. LT ÷, ST +

No. of strains tested

No. of strains + by: Y-1 Assay

Immunoassay

19 35 13 4

0 0 13 4

0 0 13 4

84

Fig. 3. lmmunoassay on membranes inoculated with oysters contaminated with the following numbers of LT + E. coli cells (1) 450 cells, (2) 90 cells, (3) 18 cells, and (4) 0 cells.

450, 90, 18, or 0 LT + E. coli in addition to approximately 6000 cells representing the normal flora of the oysters. After overnight incubation, immunoassays performed on the membranes containing the contaminated aliquots proved positive for LT + E. coli, while membranes containing the uncontaminated aliquots were negative for LT + E. coli (Fig 3). Data summarized in Table II, which compares the expected and observed numbers of LT + colonies for various aliquots, shows greater than 85% recovery. Raw milk and Brie cheese on which similar experiments were performed had aerobic plate counts of 1.3 x 105 and 3.9 x 106 cells/ml, respectively. Portions of these two foods were also contaminated with known numbers of LT + E. coli. Membranes on which approximately 1200 cells from raw milk and 3500 cells from Brie cheese had been deposited, were analyzed by the immunoassay. Data sum-

85 TABLE II Recovery of LT + E. coli from artificially contaminated foods Food

Total colonies plated

Expected LT + E~ coli

Observed LT + E. coli

% Recovery

1 2 3 4

6070 5710 5638 5620

450 90 18 0

412 77 23 0

92.0 85.0 127.0 -

Raw milk aliquot 1 aliquot 2 aliquot 3

1542 1268 1200

342 68 0

332 76 0

97.0 112.0 -

Brie cheese aliquot 1 aliquot 2 aliquot 3 aliquot 4

3760 3560 3520 3510

250 50 10 0

228 48 11 0

91.2 96.0 110.0 -

Oysters aliquot aliquot aliquot aliquot

marized in Table II again show nearly complete recovery of LT ÷ E. coli from both those foods.

Discussion

The present study demonstrates that nitrocellulose membrane colony immunoassay is a simple and sensitive method for the direct enumeration of LT ÷ E. coll. As few as 10 LT ÷ E. coli can be enumerated in a background of 5000 to 10000 cells, representing a heterogeneous flora of foods. The spiral plater improved the numerical operating range so that a portion of food containing 5 × 105 cells per ml could be plated without dilution and still produce evenly distributed colonies on the membrane. Furthermore, all the reagents except the primary antibody are commercially available. An IgG fraction prepared from 1 ml of hyperimmune serum is sufficient to analyze approximately 100 membranes, and the analysis can be completed in about 2 days. Preliminary experiments showed that including SDS in the lysis mixture and limiting the size of the bacterial colony to about 1 mm in diameter are important and markedly improved the specificity of reaction. In their absence, variable false negative reactions were produced. This was probably due to the build-up of dense bacterial mass on the membrane and consequently poor penetration of primary antibody. Furthermore, the deposition of single cells rather than applying cells with toothpicks produce the best intensity of reaction. The primary antibody used in this study was produced against LT derived from a human strain of ETEC. Although the B subunit of human and porcine LT are

86

antigenically related, they differ enough so that only partial reaction is observed with heterologous antisera (Honda et al., 1981). Therefore, both antisera should be used for analysis of foods suspected to contain either human or porcine ETEC. It is known that ETEC producing only ST can cause human disease (Mosley et al., 1982). Currently, DNA probes for human and porcine ST and LT genes are available, and the isotopic method of DNA colony hybridization can be used for analysis of foods. With the development of monoclonal antibodies against ST (Thompson et al., 1983) it should be possible to develop an analogous nonisotopic method for the identification of ST producing ETEC. Both these procedures can serve as useful tools in establishing the epidemiology of ETEC infections.

Acknowledgement We thank Clifford Johnson for help in preliminary experiments. The assistance of Diana Redmond in the preparation of manuscript is greatly appreciated.

References Clements, J.D. and R.A. Finkelstein, 1979. Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from Escherichia coli cultures. Infect. Immun. 24, 760-769. 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 diarrhea in children in Honolulu. J. Infect. Dis. 125, 407-411. 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. Evans, D.G., R.P. Silver, D.J. Evans, D.G. Chase and S.L. Gorbach, 1975. Plasmid controlled colonization factor associated with virulence in Escherichia coli enterotoxigenic for humans. Infect. Immun. 12, 656-667. Goding, J.W., 1976. Conjugation of antibodies with fluorochromes: Modification to the standard methods. J. Immunol. Methods 13, 215-226. Gyles, C.L., 1971. Heat-labile and heat-stable forms of the enterotoxin from E. coil strains enteropathogenic for pigs. Ann. N.Y. Acad. Sci. 176, 314-322. Hill, W.E., J.M. Madden, B.A. McCardell, D.B. Shah, J.A. Jagow, W.L. Payne and B.K. Boutin, 1983. Foodborne enterotoxigenic Escherichia coli: Detection and enumeration by DNA colony hybridization. Appl. Environ. Microbiol. 45, 1324-1330. Honda, T., T. Tsuji, Y. Takeda and I. Miwatani, 1981. Immunological nonidentity of heat-labile enterotoxins from human and porcine enterotoxigenic Escherichia coli. Infect. Immun. 34, 337-340. Marier, R., J.G. Wells, R.C. Swanson, W. Callahan and I.J. Mehlman, 1973. An outbreak of enteropathogenic Escherichia coli foodborne disease traced to imported French cheese. Lancet ii, 1376-1378. Merson, M.H., G.K. Morris, D.A. Sack, J.G. Wells, W.B. Creech, J.C. Feeley, R.B. Sack, A.Z. Kapikian and E.J. Gangarosa, 1976. Traveler's diarrhea in Mexico: A prospective study of physicians and family members attending a congress. N. Engl. J. Med. 294, 1299-1305. Mosley, S.L., P. Echeverria, J. Seriwatana, C. Tirapat, W. Chaicumpa, T. Sakuldaipeara and S. Falkow, 1982. Identification of enterotoxigenic Escherichia coli by colony hybridization using three enterotoxin gene probes. J. Infect. Dis. 145, 863-869. Rosenberg, M.L., J.P. Koplan, I.K. Wachsmuth, J.G. Wells, E.J. Gangarosa, R.L. Guerrant and D.A. Sack, 1977. Epidemic diarrhea at Crater Lake from enterotoxigenic Escherichia coli. A large waterborne outbreak. Ann. Intern. Med. 86, 714-718.

87 Sack, R.B., 1975. Human diarrheal disease caused by enterotoxigenic Escherichia coli. Annu. Rev. Microbiol. 29, 333-353. Sack, R.B., D.A. Sack, I.J. Mehlman, F. Orskov and I. Orskov, 1977. Enterotoxigenic Escherichia coli isolated from food. J. Infect. Dis. 135, 313-317. Shah. D.B., P.E. Kauffman, B.K. Boutin and C.H. Johnson, 1982. Detection of heat-labile-enterotoxinproducing colonies of Escherichia coli and Vibrio cholerae by solid-phase sandwich radioimmunoassays. J. Clin. Microbiol. 16, 504-508. Thompson, M.R., R.A., Gianella, A. Deutsch and H. Brandwein, 1983. Monoclonal antibodies directed against E. coli heat-stable enterotoxins (STa). The 19th Joint Conference of the United States-Japan Cooperative Medical Science Program, Cholera Panel, Bethesda, MD, U.S.A.