Detection of enterotoxigenic Escherichia coli in stool specimens by polymerase chain reaction

BACTERIOLOGY

Detection of Enterotoxigenic Escherichia coli in Stool Specimens by Polymerase Chain Reaction M. Yavzori, N. Porath, O. Ochana, R. Dagan, R. Orni-Wasserlauf, and D. Cohen

A polymerase chain reaction (PCR) protocol for rapid (7 h) detection of enterotoxigenic Escherichia coli (ETEC) is described. This protocol has been validated on 57 stool samples from young children by comparing it with the colony hybridization technique. A good agreement was found between the two methods with Cohen’s kappa statistics of 0.87 and 0.79 for the detection of the heat-stable toxin (ST) and heat-labile toxin (LT), respectively. Of 26 samples positive for LT and 15 samples positive for ST by colony hybridization, 21 (81%) and 15 (100%) were also found to be positive for LT and ST by PCR,

respectively. Only one sample identified as LT-negative by colony hybridization was found to be positive by PCR. However, 3 of 42 samples of ST-negative by colony hybridization were detected as positive by PCR. A reconstruction experiment revealed that PCR could detect LT-producing and STproducing ETEC at minimal concentrations of 2.5 3 103 cfu and 2.5 3 102 cfu per gram of feces, respectively. These data indicate the possible use of this method for rapid identification of ETEC-associated diarrhea in clinical and epidemiological settings. © 1998 Elsevier Science Inc.

INTRODUCTION

either alone or together with LT, were strongly associated with symptomatic infection being isolated at significantly higher rates among cases of diarrhea as compared to asymptomatic controls. No such association was found for ETEC expressing LT alone (Albert et al. 1995; Levine et al. 1993). Lopez-Vidal et al. (1990) showed that ST-only and ST/LT-ETEC strains are more frequently associated with colonization factor antigens (CFAs) expression, in contrast to LT-only ETEC strains. ETEC causes a spectrum of disease symptoms ranging from mild diarrhea to severe cholera-like watery diarrhea (Merson et al. 1976; Sack et al. 1971). The evidence on the relative involvement of ETEC in the etiology of diarrheal diseases in various regions throughout the world is limited due to difficulties in its detection. The correlation between the presence of specific O and H antigens and production of LT and/or ST in the same E. coli strains led to the use of serotyping as a means of ETEC identification (DuPont and Mathewson 1991). Although relatively cumbersome, detection of LT and ST production by enzyme-linked immu-

It has been estimated that in developing countries enterotoxigenic Escherichia coli (ETEC) is the cause of 650 million cases of diarrhea and 800,000 deaths in children under 5 years of age (Black 1986a). In addition, ETEC strains producing a heat-labile (LT) or a heat-stable (ST) enterotoxin, or both, are detected in one-third to one-half of the cases of traveler’s diarrhea (Black 1986b; Merson et al. 1976; Steffen et al. 1988; Taylor et al. 1985). Controlled studies carried out in pediatric populations living in ETEC-endemic regions showed that ETEC organisms expressing ST, From the Medical Corps, Israel Defence Force (M.Y., D.C.); Medical School, Ben-Gurion University, Israel (N.P., O.O., R.D.); Tel Aviv Medical Center, Tel Aviv, Israel (R.O.-W.); and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel (D.C.). Address reprint requents to: Miri Yavzori, Military Post 02149, Israel Defence Force, Israel. Presented in part at The 35th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), San Francisco, California, 17–20 September 1995 (abstract D 84). Received 14 November 1997; revised and accepted 7 March 1998.

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M. Yavzori et al.

504 nosorbent assay or by their cytopathic effect on cell lines, yields higher sensitivity and specificity than does serotyping (Jertborn and Svennerholm 1991; Konowalchuk and Speirs 1979; Sack and Sack 1975; Svennerholm and Wiklund 1983; Svennerholm et al. 1986; Thompson et al. 1984). In recent years molecular biology methods involving the use of polynucleotide and later oligonucleotide DNA probes have been employed to detect plasmid genes encoding for the synthesis of LT and ST by E. coli isolates (Moseley et al. 1980; Sommerfelt et al. 1990, 1993). The identification of the LT- and ST-producing E. coli by the colony hybridization method employing DNA probes has the drawback of being a relatively lengthy procedure including initial isolation and growth of E. coli colonies, DNA extraction, probe labeling, and hybridization. Hybridization of stool blots instead of hybridization of colony blots led to a more rapid detection of LT and ST specific sequences but at the same time the sensitivity of the test decreased in patients excreting a relatively low number of ETEC organisms in their stools (Sommerfelt et al. 1993). More recently, LT and ST genes were detected in smears of lactose-positive cultures on solid selective media by polymerase chain reaction (PCR) at a higher sensitivity than by colony hybridization (Schultsz et al. 1994). We took this strategy a step further and developed a PCR protocol for detection of LT and ST DNA sequences directly in stool samples combining a short pre-enrichment stage with the use of previously described procedures of DNA release and detection of amplified sequences (Victor et al. 1991). The sensitivity and specificity of the test for detection of ETEC LT- and ST-specific plasmid genes have been evaluated versus the colony hybridization technique.

MATERIALS AND METHODS Stool Specimens Stool specimens were collected from children suffering from diarrhea and from asymptomatic subjects. The specimens, maintained frozen at 270°C until examined in the present study, were thawed and tested for presence of ETEC by the colony hybridization technique and PCR in parallel. A swab of thawed fecal material was suspended in 4 mL of brain heart infusion (BHI) broth for the preenrichment and subsequent PCR steps. Thawed specimens were inoculated onto MacConkey agar and incubated for 18 –24 h at 37°C. Five lactosefermenting colonies were picked, confirmed as E. coli by standard biochemical tests, and transferred to filter papers for further ETEC identification by the colony hybridization technique.

Simulated ETEC (LT1ST1)–Positive Stool Specimens Such specimens were created by mixing known concentrations of ETEC (LT1ST1) with feces obtained from a healthy donor. Three different fecal specimens were used for the construction experiments and similar results were obtained with all of them. Volumes of 0.5 mL phosphate-buffered saline containing 10-fold dilutions of an initial ETEC concentration of approximately 5 3 106 cfu per milliliter were mixed with 0.5 g feces. The bacterial concentration of the suspensions was estimated by plate counts done on blood agar base. The minimal number was expressed in terms of cfu per 1 gram of fecal material. Concomitantly, duplicate swabs taken from the same mixtures were plated on MacConkey agar to be tested by the colony hybridization technique, or preincubated in BHI broth, to be further tested by PCR. The subsequent steps for the two procedures were the same as those described above concerning stool specimens obtained from patients.

Bacterial Strains The following bacterial strains were examined in the study: E. coli, positive or negative for LT and ST as documented by the colony hybridization technique, Shigella flexneri 2a, Shigella sonnei form 1, Shigella boydii, Shigella dysenteriae type 1 (Shiga), Campylobacter jejuni, E. coli (serotypes O128:H53, O128:H2, O142:H2, O158:H2, O159:H2), enteroinvasive E. coli (serotypes O28ac:H2, O143:H2, O164:H2), enterohemorrhagic E. coli (serotype O157:H7), Proteus mirabilis, Proteus vulgaris, Salmonella spp. from blood and fecal cultures, Klebsiella spp., and Enterobacter spp. The source of the strains were the Israeli National Reference Center for Enterobacteriaceae, the Army Health Branch Research Laboratory of the Israel Defence Force, Medical Corps, and the Clinical Microbiological Laboratory of the Sheba Medical Center, Tel Hashomer, Israel.

Preparation of DNA From Stool Specimens and Bacterial Strains for Polymerase Chain Reaction Analysis This was done according to the protocol used by Victor et al. for detection of ETEC (Victor et al. 1991), with the following modifications. Briefly, fecal material from swabs was suspended in 4 mL of BHI. The suspension was incubated at 37°C with agitation (125 rpm) for 4 h, boiled for 10 min and immediately chilled on ice. A similar protocol was used for preparation of DNA from simulated ETEC (LT1ST1)– positive stool specimens and from the bacterial strains examined.

Detection of Enterotoxigenic E. Coli in stool by PCR

Oligonucleotides The oligonucleotides used for the ST primers (ST1: 59-TCT GTA TTG TCT TTT TCA CC-39, ST2: 59-TTA ATA GCA CCC GGT ACA AGC-39), and for the LT primers (LT1: 59-GGC GAC AGA TTA TAC CGT GC-39, LT2: 59-CCG AAT TCT GTT ATA TAT GTC39), were constructed according to Frankel et al. (1990).

Polymerase Chain Reaction Samples of 5 mL taken from the BHI broth, after preincubation of fecal material or bacterial suspensions, underwent the PCR procedure. PCR was performed in 0.5-mL microcentrifuge tubes, using a 100-mL reaction mixture consisting of 60 mL of H2O, 10 mL of 10 3 PCR buffer (100 mM Tris-Hcl, 500 mM KCl, 0.1% gelatin (w/v), 1% Triton X-100), 8 mL of 25 mM MgCl2, 15 mL of deoxynucleoside triphosphates (1 mM of each), 1 mL of each primer (100 mM), 5 mL of DNA template, and 0.2 U of Taq polymerase (Promega, Madison, WI, USA). The PCR procedure included preincubation at 95°C for 1 min, 30 cycles of 1 min at 95°C, 1.10 min at 45°C, 1.30 min at 72°C, and final incubation at 72°C for 5 min. The thermocycler used throughout the study was Programmable Thermal Controller (MJ Research, Inc., Watertown, MA, USA). The amplified DNA samples were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide and detected under ultraviolet light.

Colony Hybridization Pools of five colonies of the various E. coli strains were transferred to Whatman no. 541 filter papers (Whatman, Inc., Clifton, NJ) and treated by standard protocol with NaOH and heat to lyse the bacterial cells, denature, and fix the DNA to the filter (Maas 1983). Twenty test colonies of E. coli were spotted on each filter to be hybridized with a specific probe. A positive control specific for the tested probe and a negative control were spotted on each filter as well. DNA probes were prepared from recombinant plasmids containing the probe DNA fragments as insert. Plasmids were prepared, purified, and digested with restriction endonucleases and the appropriate restriction fragments were purified as described by Levine (1987). Two probes were used for ETEC detection (Lanata et al. 1985). The LT probe consisted of 1200-bp Hinc II fragment cloned on Bam HI linkers in pACYC189. The ST probe was a 216-bp Eco RI fragment cloned into pUC13. DNA fragments (approximately 100 ng) were labeled by multiprime DNAlabeling system with [a-32P]deoxycytidine triphosphate, 3000 Ci/mmol, and a random primers

505 DNA-labeling kit (Amersham International plc, Buckinghamshire, U.K.). The filters were hybridized under stringent conditions (50% formamide; washed at 65°C) with the 32P-labeled probe at a concentration of 106 cpm/10 filters and were incubated at 37°C overnight with gentle agitation. After washings filters were exposed to X-Omat-AR-X-ray film (Kodak, Rochester, NY) for 12 h at 280°C and then developed according to the manufacturer’s instructions.

Data Analysis The extent of agreement in detection of ETEC LTand ST-specific plasmid genes between PCR and colony hybridization was expressed in terms of Cohen’s kappa statistic (Fleiss 1981). In general, kappa greater than 0.75 indicates very good agreement, between 0.40 to 0.75 indicates fair to good agreement, and below 0.40 indicates poor agreement (Fleiss 1981).

RESULTS Sensitivity and Specificity of the ETEC Polymerase Chain Reaction on Bacterial Isolates Examination of 28 E. coli isolates for presence of LT genes yielded positive results in 11 by both PCR and colony hybridization while one additional isolate was positive by PCR alone. Of the same isolates, eight were found to be positive for ST by colony hybridization. Use of PCR led to detection of STspecific DNA in five additional isolates (Table 1). No cross-reactions of the ST and LT primers with DNA extracted from pure cultures of pathogenic and nonpathogenic enteric bacteria other than ETEC were detected (Table 2).

TABLE 1 Comparison Between LT and ST Detection in Pure Colonies by PCR and Colony Hybridization Methods PCR Positive

Negative

Total

11 1 12

0 16 16

11 17 28

8 5 13

0 15 15

8 20 28

a

Colony hybridization (LT) Positive Negative Total Colony hybridization (ST)b Positive Negative Total a b

Kappa statistic, 0.93. Kappa statistic, 0.63.

M. Yavzori et al.

506 TABLE 2 PCR of DNA Extracted From Pure Cultures of Various Pathogenic and Nonpathogenic Enteric Organismsa

Species Campylobacter jejuni Campylobacter coli Nonpathogenic Escherichia coli Enterohemorrhagic E. coli (O157:H7) ETEC (LT2, ST1) (LT1, ST1) (LT1, ST2) Proteus mirabilis Proteus vulgaris Salmonella spp. Klebsiella spp. Enterobacter spp. Enteroinvasive E. coli Shigella flexneri Shigella sonnei Shigella boydii Shigella dysenteriae (type 1) a

Primers

Isolates tested

LT

ST

1 5 8 3

2 2 2 2

2 2 2 2

10 13 10 1 1 12 2 1 10 10 6 5 4

2 1 1 2 2 2 2 2 2 2 2 2 2

1 1 2 2 2 2 2 2 2 2 2 2 2

After enrichment in BHI.

Reconstruction Study A reconstruction experiment in which a negative stool sample was mixed with LT- and ST-producing ETEC at serial dilutions revealed that the PCR protocol could detect LT- and ST-producing ETEC at minimal concentrations of 2.5 3 103 cfu and 2.5 3 102 cfu per gram of fecal material, respectively (Figure 1). The minimal concentration at which LT and ST could have been detected by the colony hybridization method was 2.5 3 104 cfu for both LT and ST (Figure 2, A and B).

FIGURE 1 Specific DNA fragments of LT and ST after PCR amplification of 10-fold dilutions of ETEC (LT1ST1) organisms mixed with fecal material, and 2% agarose gel electrophoresis. HindIII-digested bacteriophage 1 was used as size marker (Bac/g, number of bacteria per gram of feces; pc, positive control; nc, negative control).

FIGURE 2 Positive (dark spots) and negative (clear spots) results of colony hybridization with LT (A) and ST (B) radioactive DNA probes, of ETEC (LT1ST1) colonies grown from the same 10-fold dilutions of bacteria and fecal material shown in Figure 1. The numbers appearing on the top of the spots represent the bacterial concentrations per gram of fecal material (pc, positive control; nc, negative control).

Sensitivity and Specificity of the ETEC Polymerase Chain Reaction on Stool Specimens of Patients With Diarrhea Detection of LT and ST by PCR using the LT and ST primers after preincubation of stool samples in BHI broth was validated on 57 stool samples obtained from young children by comparing it with the colony hybridization technique. A good agreement was found between the two methods with Cohen’s kappa statistics of 0.87 and 0.79 for the detection of ST and LT, respectively. Of 26 samples positive for LT and of 15 samples positive for ST by the colony hybridization method, 21 (81%) and 15 (100%) were also found to be positive for LT and ST by PCR, respectively (Table 3). Only one sample identified as LT-negative by colony hybridization was found to be LT-positive by PCR. However, 3 (7%) of 42 samples ST negative by colony hybridization were detected as ST positive by PCR (Table 3). The high specificity of the PCR protocol was further reflected by the negative results obtained with 15 stool specimens of asymptomatic

Detection of Enterotoxigenic E. Coli in stool by PCR TABLE 3 Comparison Between LT and ST Detection in Stools by PCR and Colony Hybridization Methods PCR Positive

Negative

Total

21 1 22

5 30 35

26 31 57

15 3 18

0 39 39

15 42 57

a

Colony hybridization (LT) Positive Negative Total Colony hybridization (ST)b Positive Negative Total a b

Kappa statistic, 0.79. Kappa statistic, 0.87.

subjects following incubation of the samples for 4 h in BHI.

DISCUSSION The ability of the PCR to detect very low quantities of an infectious agent at high specificity encouraged many investigators to apply this technique for the direct identification of pathogens in clinical specimens including stool samples (Jiang et al. 1992; Saulnier and Andremont 1992). The yield of the enteropathogens detected by PCR directly in stool specimens was lower than expected. This was probably related to various potential inhibitors present in the fecal material, such as small particles and soluble substances, blood, gastric fluid, traces of phenol and sodium dodecyl sulfate (Black 1986a; Brisson-Noel et al. 1991; Jiang et al. 1992; Saulnier and Andremont 1992). Different strategies have been employed to remove inhibitors of PCR detection of LT and ST DNA in feces (Schultsz et al. 1994; Stacy-Phipps et al. 1995; Victor et al. 1991). They include the use of PCR on extracts of smears of lactose-positive colonies grown overnight on solid selective media (Schultsz et al. 1994; Victor et al. 1991) and more recently, the use of a glass matrix and a chaotropic solution to further purify ETEC DNA from clinical fecal specimens (Stacy-Phipps et al. 1995). To circumvent the same problem, we developed a PCR protocol in which we included a short pre-enrichment stage. In this way the number of bacteria grows and inhibitors of the PCR amplification are diluted. We have previously shown, in another study, that this pre-enrichment stage increased the sensitivity of detection of Shigella by PCR (Yavzori et al. 1994). In that study, measurement of the minimal concentration of Shigella detected by PCR before and after enrichment demonstrated that the increased sensitivity of the test after

507 enrichment is only partially related to the higher number of organisms obtained after growth (Yavzori et al. 1994). We assume that a higher DNA/protein ratio and/or amplification of a higher plasmid copy number during growth in BHI may also explain the increased sensitivities of the PCR protocols for detection of Shigella and ETEC. The PCR protocol used in the present study for detection of ETEC in stools can be completed in a single working day (7 h) and still keeps the levels of sensitivity and specificity as high or even higher than the other PCR procedures previously reported (Schultsz et al. 1994; StacyPhipps et al. 1995; Victor et al. 1991). The threshold number of bacterial cells for ST detection in the reconstruction study was 10-fold lower than that required for LT. This may be a result of the different dimensions of the amplified fragments. It has been shown that there is an inverse relationship between the amplification yield and the length of the amplified fragments. In our case, following amplification with the LT primers 750-bp fragments were obtained as compared with 186-bp fragments obtained with the ST primers. We are currently exploring the possibility of amplifying different and shorter DNA fragments with the LT primers in order to increase the ability of LT detection by the present PCR protocol. The most commonly used method for detection of ETEC is colony blot hybridization with DNA probes specific for the LT and ST toxins. We defined the colony hybridization technique as the reference test for the evaluation of the PCR protocol developed in our laboratory. The sensitivity of ST DNA detection by PCR was higher than the sensitivity of the colony hybridization technique throughout all the comparisons drawn including E. coli isolates, stool samples of patients with diarrhea, and reconstructed stools. The PCR protocol could detect ST-producing ETEC at minimal concentrations of 2.5 3 102 cfu per gram of fecal material while the minimal concentration at which ST could have been detected by the colony hybridization method was 2.5 3 104. When 57 stool specimens were tested for presence of LT, 26 were positive by colony hybridization and only 21 by PCR. In contrast, examination of 28 pure lactose-positive colonies yielded positive results in 12 by PCR and 11 by colony hybridization. In the reconstruction experiment the minimal concentration detected by the colony hybridization method was 2.5 3 104 whereas the PCR protocol was much more sensitive, detecting LT DNA at a minimal concentrations of 2.5 3 103 cfu per gram of fecal material. The higher sensitivity of PCR versus colony hybridization in detecting LT in purified colonies of E. coli or in reconstructed LT-positive stool samples and in contrast, the lower sensitivity of PCR versus the colony hybridization technique in detecting LT

508 genes in stool samples obtained from patients with diarrhea, may have the following explanation. In a stool specimen obtained from a patient with ETECLT–associated diarrhea, when the overwhelming majority of the E. coli colonies growing on MacConkey agar will be LT-producing colonies, picking five random lactose-positive colonies and examining them by colony hybridization may have a higher chance of LT detection. The proportion of ETEC-LT colonies/E. coli colonies is probably lower when mixing serial dilutions of an LT-producing ETEC strain with feces obtained from a healthy donor. Under such conditions the PCR procedure has a higher chance of detecting LT DNA than colony hybridization for which at least one of the five picked-up colonies must produce LT to yield a positive result. This assumption is supported by data reported by Schultsz et al. who showed that there will be a 100% chance of detecting LT DNA by colony hybridization when the ETEC colonies/E. coli colonies proportion is higher than 2:12 and a much lower chance (about 20%) of detecting LT by colony hybridization of stool blots when this proportion will be lower than 2:12 (Schultsz et al. 1994). It seems therefore that the sensitivity of LT detection by colony hybridization depends more on the non-LT/LT–producing colo-

M. Yavzori et al. nies proportion whereas the sensitivity of PCR is associated with the absolute concentration of LT DNA in the sample. In summary, the findings of this study demonstrate the high level of sensitivity and specificity of a PCR protocol to detect ETEC. As it was described, the procedure is simple and can be completed within 7 h. In contrast to the other ETEC identification methods already mentioned, this PCR protocol is applicable to routine diagnostic laboratories, indicating its possible use for rapid determination of the etiology of ETEC-associated epidemics and sporadic cases of diarrhea. This will be of importance for prompt and focused intervention to stop the chain of transmission of ETEC.

This study was supported by grants no. DAMD17-93-V-3001 from the U.S. Army Medical Research and Materiel Command, Fort Detrick, Frederick, Maryland, USA, and 1 PO1AI-26497 from the National Institute of Allergy and Infectious Diseases—International Collaboration in Infectious Diseases Research, National Institutes of Health, Bethesda, Maryland, USA.

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