Department ofMedical Microbiology and Immunology, University of Goteborg, S-413 46 Goteborg,. Sweden5; and .... Dhaka, Bangladesh (11); C, Dhaka, Bangladesh (7); D, Argentina (provided ..... Fundamentals of biostatistics, 3rd ed. PWS-.
JOURNAL OF CLINICAL MICROBIOLOGY, July 1992, p. 1823-1828 0095-1137/92/071823-06$02.00/0 Copyright ©) 1992, American Society for Microbiology
Vol. 30, No. 7
Use of Nonradioactive DNA Hybridization for Identification of Enterotoxigenic Escherichia coli Harboring Genes for Colonization Factor Antigen I, Coli Surface Antigen 4, or Putative Colonization Factor 0166 HALVOR SOMMERFELT,l.2* HARLEEN M. S. GREWAL,"3 WIM GAASTRA,4 A.-M. SVENNERHOLM,s AND MAHARAJ K. BHAN2 Centre for Intenational Health and Medical Department B, University of Bergen, Haukeland Hospital, N-5021 Bergen, 1* and Center of Biotechnology, University of Bergen, N-5020 Bergen,3 Norway; Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, 3508 TD Utrecht, The Netherlands4; Department of Medical Microbiology and Immunology, University of Goteborg, S-413 46 Goteborg, Sweden5; and Department of Pediatrics, Division of Gastroenterology and Enteric Infections, All India Institute of Medical Sciences, 110029 New Delhi, India2 Received 18 November 1991/Accepted 14 April 1992
We developed an accurate nonradioactive colony hybridization assay (NCHA) using a digoxigenin-labeled polynucleotide probe and an antidigoxigenin alkaline phosphatase conjugate for the identification of enterotoxigenic Escherichia coli (ETEC) harboring genes for colonization factor antigen I (CFA/I), coli surface antigen 4 (CS4), or putative colonization factor 0166 (PCF0166). In this 2-day assay, visual registration of color intensity could be used to distinguish between CFAII-positive strains and strains with the genetic potential to express CS4 or PCF0166. A rapid NCHA was developed by which the results could be read visually 7 h and 45 min after inoculation of the bacteria. In the rapid NCHA, densitometry verified the visual discrimination between four groups of E. coli; ETEC with the CFA/I gene, ETEC with the CS4 gene, ETEC with the PCF0166 gene, and E. coli strains that lack such genes. As a confirmatory test, plasmids from ETEC with the CFA/I, CS4, or PCF0166 gene were differentiated by their characteristic restriction fragment patterns in nonradioactive Southern blot hybridization.
Enterotoxigenic Escherichia coli (ETEC) is of major importance for childhood morbidity and mortality in developing countries and is probably the main cause of diarrhea in travelers from industrialized countries who visit less developed countries (3, 4, 24). ETEC bacteria possess a variety of fimbriae and outer membrane structures that anchor the bacteria to receptors on mucosal cells (10, 17). The heatlabile and heat-stable toxins (LT and STa, respectively) are thereby delivered in close proximity to the enterocytes. Furthermore, this anchorage promotes colonization of the small bowel. In strains pathogenic to humans, a number of antigenically distinct fimbriae have been described. The best characterized are the colonization factor antigens (CFAs) CFA/I, CFA/II, and CFA/IV; CFA/II and CFA/IV comprise coli surface antigens (CSs) in various combinations (8, 17, 23, 25, 31, 38, 39, 41). Thus, strains bearing CFA/II express CS3 and may, in addition, produce CS1 or CS2 (8, 31), while CFA/IV strains express CS6 alone or express CS6 in combination with CS4 or CS5 (23). Recently, new fimbrial antigens of ETEC have been described, including CS7, CS17, CFA/III, PCF0166, and PCF0159:H4 (14-16, 21, 22, 40). The prevailing CFAs are likely to be important components in future oral vaccines against ETEC diarrhea (17, 18, 38). The prevalence of ETEC bacteria that express the different CFAs shows considerable geographic variation (2, 6, 11, 38, 42). Hence, detailed epidemiological mapping of CFAs on isolates from different regions should precede the construction of candidate ETEC vaccines. A number of accurate methods, including enzyme-linked *
immunosorbent assays (ELISAs), have been developed for the identification of CFAs (12, 19, 20, 33). Recently, a radioisotope-based colony hybridization assay that specifically identifies ETEC bacteria carrying the CFA/I gene under high-stringency (HS) conditions was described (12). Under low-stringency (LS) conditions, the polynucleotide probe designated CFA/I-P1-P2 was shown to hybridize even to CS4- and PCF0166-associated DNAs (12, 33, 34). Southern blot hybridization of PstI-digested ETEC plasmid DNA yielded characteristic CFA/I, CS4, and PCF0166 restriction fragment patterns (34). The antibodies and antigens required for the CFA ELISAs are not widely available, while radioactive labeling of gene probes and cumbersome test procedures limit the use of diagnostic hybridization, particularly in less well equipped laboratories in developing countries. We developed a nonradioactive colony hybridization assay (NCHA) for detection of ETEC bacteria that harbor the structural gene for CFA/I, CS4, or PCF0166. The assay was evaluated against established genotypic and phenotypic tests for CFAs on a large battery of ETEC strains. MATERIALS AND METHODS Bacterial strains. The characteristics of the human ETEC strains used in the study are given in Table 1. ETEC strains with the CFA/I, CS4, or PCF0166 gene were identified by radioisotope-based colony and Southern blot hybridizations (12, 33, 34), while CS1, CS2, CS3, and CS5 were detected with monoclonal antibodies in ELISAs or slide agglutination tests (20, 37). CS6 was identified by using polyclonal antisera in a double-diffusion assay (28, 39). The reference CS7-
Corresponding author. 1823
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TABLE 1. Characteristics of human ETEC strains used in evaluation of the CFA/I-P5-P2 NCHA No. of strains
Enterotoxin gene profile
Type of fimbriaa
STa
LT
+ +
+ +
CFA/I+ CFA/I+
1
-
+
-'
1 3 12 4 2 3 4 1 4 1 1 1 24 14 45 3 4 1 1 1
+
-
+ + +
+
CFA/I+ CFA/I +d CFA/I +d CS1+ CS3+ CS2+ CS3+ CS3+ CS4+ CS6+ CS5+ CS6+ CS5+ CS6+ CS5+ CS6+ CS4+ CS6+ CS5+ CS6+ CFA/I CS4- PCF0166-d CFA/I CS4- PCF0166CFA/I- CS4- PCF0166-d PCF0166+d PCF0166+d CS7+ CS17+ PCF0159:H4+
1 7
+ + + + +
+ +
+ + +
-
-
+
+ + + + -
+
-
+ + -
+ + + + +
+
+
Origin'
A B B B F F C C C C
C D D E
E F F
F F F G G H
ETEC harboring CFA/I, CS4, or PCFO166 genes were detected by colony and Southern blot hybridization assays by using the CFA/I-P1-P2 probe labeled with 32p (12, 33, 34). CS1, CS2, CS3, or CS5 was identified with monoclonal antibodies in ELISAs or slide agglutination tests (20, 37), while CS6 production was assessed in a double-diffusion assay by using polyclonal antisera (28, 39). " Origins: A, strain H10407+ (provided by D. G. Evans, Houston, Tex.); B, Dhaka, Bangladesh (11); C, Dhaka, Bangladesh (7); D, Argentina (provided by N. Binsztein, Buenos Aires, Argentina); E, provided by B. Rowe (Colindale, London, United Kingdom); F, India (1, 12, 34, 35); G, provided by M. M. McConnell (Colindale, London, United Kingdom) (14, 22); H, provided by C. 0. Tacket (Baltimore, Md.) (40). ' This strain was previously STa+ LT+ CFA/I+ but lost its STa and CFA/I genes during storage and subcultures (12). d The strains were previously characterized only for CFA/I, CS4, and PCFO166 genes (12, 33, 34).
producing (CS7+) strain E29101A (14), the CS17+ strain E20738A (22), and the PCFO159:H4+ strain 350C1 (40) were also included. To control for probe contamination by vector DNA, a strain with pBS (Stratagene, La Jolla, Calif.), the plasmid vector of the CFAII-P1-P2 probe, was examined. In addition, eight ETEC isolates from animals with diarrhea, nine nonenterotoxigenic E. coli isolates from healthy children, and three isolates of the newly described diarrheal pathogen enteroaggregative E. coli (1) were included to further evaluate the specificity of the probe. Probe preparation and labeling. The polynucleotide CFA/ I-P1-P2 probe was prepared as described previously (12), with one modification. Thus, to simplify probe isolation, after digestion of pIVB3-c17 with PstI and EcoRI and electrophoretic separation of the resulting fragments in 1.2% agarose (30), the gel piece containing the CFAII-P1-P2 probe (12, 33) was centrifuged for 10 min at 2,000 x g through siliconized glass wool, thereby trapping the agarose (13). After ethanol precipitation, the probe was dissolved in 10 mM Tris-HCl (pH 7.5)-i mM EDTA and labeled with digoxigenin in a random hexanucleotide-primed, Klenow enzyme-mediated reaction (9) by using a gene probe labeling
and detection kit (catalog no. 1093 657; Boehringer, Mannheim, Germany) according to the enclosed instructions, with minor modifications. Thus, while scaling up the reaction volumes by a factor of 2 and allowing the labeling to proceed for 20 h in order to maximize the incorporation of digoxigenin-dUTP, we added 600 ng of template DNA, thus anticipating 1,000 ng of digoxigenin-labeled probe. Colony hybridization. Three colonies from each of the human (Table 1) and animal ETEC isolates, the nonenterotoxigenic and enteroaggregative E. coli strains, and the strain harboring the probe vector were inoculated in parallel onto two sets of clean but not autoclaved BA85/20 nitrocellulose membranes (NCMs; Schleicher & Schuell, Dassel, Germany) overlying MacConkey agar, one set for the HS and one set for the LS NCHA. After incubation for 5 h, the colonies were lysed and the DNA was denatured by placing the NCMs for 3 min on filter papers soaked with 10% sodium dodecyl sulfate (SDS) and subsequently for 5 min on filter papers with 0.5 M NaOH-1.5 M NaCl; this was followed by neutralization for 5 min and then for 3 min with 0.5 M Tris-HCl (pH 7)-1.5 M NaCl. After air drying for 1 h, the DNA was immobilized on the NCMs by baking without vacuum at 65°C overnight. The NCHA was performed along previously described guidelines (32), with modifications. Thus, the NCMs were initially equilibrated in 5 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 1 min. To reduce the assay time (5) and to minimize health hazards, formamide was eliminated from the prehybridization and hybridization mixtures, while the concentration of labeled probe was kept at 10 ng per ml of hybridization solution. Prehybridization was performed for 30 min, and hybridization was performed for 2 h at 67°C. After three 10-min nonstringent washes in 3 x SSC at room temperature, two 20-min LS washes (67°C) were performed on one set of the NCMs in lx SSC-0.1% SDS. The other NCM set was washed twice for 20 min each time under HS conditions in 0.07x SSC-0.1% SDS (67°C) (12, 33). Immunological detection of the hybridized digoxigeninlabeled probe was performed as described previously (32). A 1-day rapid NCHA (RNCHA) was evaluated by using a subset of the main battery of strains. Thus, in our laboratory in India, six colonies from each of the CFA/I+ strains H10407+, 254175-2, 257200, and Bh3-10 (11, 12); two strains carrying the PCFO166 gene (H561A and 258A) (34); the CS4+ strains VM66257 and VX67356 (7, 33); E20738A (CS17+) (22); and two nonenterotoxigenic E. coli isolates were examined. In addition, the PCFO166+ reference strain E7476A (21) (kindly provided by M. M. McConnell) was probed. To examine the possible effect of laboratory-tolaboratory variation in sample processing and assay performance, six colonies from each of the CFA/I1 strains 255570-1, 222986-1, 244975-2, and 325542-1 (11, 12); four strains with the PCFO166 gene (176A, 428A, H555A, and H574B) (34); the CS4+ strains VM66252 and VX68011 (7, 33), 359C1 (PCFO159:H4+) (40); and two nonenterotoxigenic E. coli were examined by the RNCH-A in our laboratory at the Center of Biotechnology, Bergen, Norway. In both laboratories, one colony from each of the strains H10407+, VM66257, and E7476A was used as a control. After a 3.5-h of incubation of the bacterial inocula, the colony blots were prepared as described above for the NCHA except that, following denaturation and neutralization, the NCMs were air dried for 4 min at room temperature in front of a table fan and then in an incubator at 37°C for 10 min before the NCMs were baked without vacuum at 75°C for 25 min. Without prior equilibration in 5 x SSC, prehy-
NONRADIOACTIVE HYBRIDIZATION FOR CFA/I, CS4, PCF0166
VOL. 30, 1992
bridization (67°C) was performed for 10 min. To ensure complete strand separation of the digoxigenin-labeled CFA/ I-P1-P2 probe (concentration, 15 ng/ml), the entire hybridization solution was heated to 95°C for 5 min immediately before hybridization (67°C), which was allowed to proceed for only 40 min. Five nonstringent washes at room temperature in 3x SSC for 1 min each time were followed by two 15-min LS washes at 670C in lx SSC-0.1% SDS. Immunological detection of the hybridized digoxigenin-labeled probe was performed as described previously (32), except that blocking was reduced to 25 min and washing of unbound conjugate was reduced to four times for 5 min each time. Registration and interpretation of the signals were performed before breaking the reference code, as described elsewhere (32). In the NCHA, the signal intensities were recorded visually after 1, 2, 4, and 7 h of development, while the RNCHA signals were read after 30 min of development in both laboratories. The color reaction was arrested after 30 min in the RNCHA performed in India, while in Norway, it was stopped when the color intensities of the control colony lysates corresponded visually to those processed in India. To improve on the objectivity of the RNCHA, after arresting the development, the color intensities over the lysates were recorded with a Repromaster densitometer (model RM 21; Oce-Helioprint, Kvistgaard, Denmark). Statistical analyses of densitometric readings. The means of the densitometric values of the six colony lysates were used to represent each strain. If the colony lysates of a strain showed marked differences in color intensity, with some of the lysates showing signs of spontaneous loss of a structural fimbrial gene (33), only the colonies with visible coloration were included. Two-sample t tests (29) were used to examine whether the densities observed in the Indian and the Norwegian laboratories were significantly different within each group of E. coli, i.e., E. coli without a gene for CFAII, CS4, or PCFO166; ETEC with the CFA/I gene; ETEC with the CS4 gene; and ETEC with the PCFO166 gene. A one-way analysis of variance supplemented with Scheffe's method for multiple comparisons (29) was used to compare the mean densitometric values of each group of E. coli. Southern blot hybridization. Plasmid DNAs from the CFA/I+ strains H10407+ and 254175-2, a nonenterotoxigenic E. coli strain, the CS4+ strain VX67356, and the PCFO166+ strains H561A and E7476A were extracted by a small-scale alkaline lysis method (30) and were digested with PstI restriction endonuclease. The resulting fragments were visualized under UV light after separation on an ethidium bromide-stained 0.8% agarose gel and transferred to a BA85 NCM (Schleicher & Schuell) by vacuum-mediated Southern blotting (30, 36). The DNA was hybridized for 2 h with the digoxigenin-labeled CFA/I-P1-P2 probe; this was followed by LS washing and color development for 90 min. RESULTS Detection of CFA/I-, CS4-, and PCFO166-associated nucleotide sequences by NCHA and RNCHA. In the HS NCHA, after 1 h of color development, the 24 strains that were positive in the reference radioisotope-based CFAII colony hybridization assay yielded weak signals. After 2 h, the 24 strains were strongly positive, while the 5 strains with the CS4 gene and 4 of the 7 strains with the PCFO166 gene gave weakly positive signals, the remaining 3 strains with the PCFO166 gene being negative. After 4 and 7 h of color development, the 24 CFAII strains gave strong signals, while
1
3
7
5 4
2
6
11
9
8
1825
10
. #~ .
.$
4
;.
*91
*
9
FIG. 1. RNCHA for 40 min with a digoxigenin-labeled CFA/IP1-P2 probe after LS washing and color development for 30 min. The
vertical sequence of inoculation started from the top of the far left column, and 11 strains (six colonies of each strain) were examined. Signal intensity could be used to distinguish between strains carrying the CFA/I (columns 1, 7, 8, and 10), the CS4 (columns 3 and 11), and the PCF0166 (columns 4 and 5) genes, all of which gave signals that were clearly different from the negligible background observed over colony lysates of the CS17' ETEC (column 9) and the nonenterotoxigenic E. coli (columns 2 and 6) strains. The second, fourth, and sixth colonies of VM66257 (column 11) showed evidence of spontaneous loss of the CS4 gene. The hybridization solution had been used twice prior to the experiment whose results are shown here.
the 5 CS4 and all 7 PCFO166 strains gave weakly positive signals. In the LS NCHA, a distinction between strains with the CFA/I gene on the one hand and those with the CS4 or PCFO166 gene on the other was simpler in that, after 1, 2, 4, and 7 h of color development, the former yielded strong signals, while the latter yielded weakly positive signals. Although colony lysates from strains harboring the PCFO166 gene tended to yield somewhat weaker signals than those from strains with the CS4 gene, the difference was not sufficiently marked to allow for a reliable differentiation between the two fimbrial systems. In the HS and LS NCHA, the 103 human ETEC strains that carried neither of these genes, the 8 ETEC strains of animal origin, the 3 enteroaggregative E. coli strains, the 9 nonenterotoxigenic E. coli strains, and the strain harboring the plasmid vector of the probe were all negative. In the RNCHA, differentiation between the strong CFA/I signals and the weaker CS4 and PCFO166 signals could be made 30 min after initiation of the color reaction (Fig. 1) in both the Indian and the Norwegian laboratories. Moreover, CS4 signals were found to be stronger than PCFO166 signals, which again were clearly distinguishable from the negligible background (Fig. 1). These distinctions were verified in unbiased observations by two observers in both laboratories. In comparison with the reference assays, no false-positive or false-negative observations were made. All six colonies from each of the examined strains yielded uniform results, with one exception. Thus, only three colonies of strain VM66257 gave signals characteristic of CS4 DNA; the remaining three were negative (Fig. 1), a finding that corresponds with previous observations of structural CS4 gene instability in this strain (33). In the Norwegian laboratory, the color reaction was arrested after 50 min in
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A
0I I
AA
l
B 23
4 S
1;
7
1
2 3 45 6 7 _23.1 - 9.4 - 6.6 - 4.4
z
_ 2.3 - 2.0
0
PCF0166
CS4
CFA/1
FIG. 2. Densitometry after an RNCHA with the CFA/I-P1-P2 probe on the following four groups of E. coli: ETEC with the CFA/I (CFA/I), ETEC with the CS4 (CS4), and ETEC with the PCFO166 (PCFO166) genes and E. coli lacking such genes (0). Means and 95% confidence limits are indicated with thick and thin lines, respectively. A, RNCHA performed in our Indian laboratory; 0, RNCHA performed in our Norwegian laboratory. The differences between the mean values of all possible pairs of groups were statistically significant (all P values were
