JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1992, p. 2080-2083
Vol. 30, No. 8
0095-1137/92/082080-04$02.00/0 Copyright © 1992, American Society for Microbiology
Detection of Genes in Feces by Booster Polymerase Chain Reaction P. SAULNIER1
AND
A. ANDREMONT .2*
Laboratoire d 'Ecologie Microbienne, Institut Gustave-Roussy, Villejuif, 1 and Laboratoire de Microbiologie, Faculte de Pharmacie, Chatenay-Malabry France Received 2 January 1992/Accepted 7 May 1992
A 321-bp fragment intragenic to the gene ereA carried byEscherichia coli BM2195 was used as a model target to study the conditions under which DNA amplification by booster polymerase chain reaction can be used to detect specific bacterial DNA sequences in fecal specimens. When target E. coli cells were mixed with 41 freshly obtained fecal specimens, the polymerase chain reaction detection limit varied from 4.5 to 7.1 log CFU/g of feces, depending on the individual fecal specimen used to prepare the test sample. These variations were not statistically related to the sex or age of the subject from whom the specimen was obtained. After storage of the samples for 4 weeks at room temperature on swabs or filter papers, no loss in sensitivity was observed.
Detection of pathogenic bacteria in fecal samples is based the culture of fecal specimens on solid selective medium with or without previous enrichment growth. However, in many laboratories in developing countries, selective and sensitive media either do not exist for some pathogens (e.g., enterotoxigenic or enteroinvasive Escherichia coli) or are not available for certain pathogens (e.g., Campylobacter sp. or Clostridium difficile). In addition, the conservation of isolated clones until their final identification in reference laboratories is difficult because of the spontaneous loss of virulence plasmids or because deletion of plasmid-encoded genes can occur during in vitro growth, storage, and transon
port.
To overcome these problems, DNA-DNA hybridization techniques with specific probes either on dots of extracted DNA (8, 10, 14, 22, 27) or on bacterial colonies lysed on filters (18, 21, 27) have been proposed. Subsequently, DNA amplification by the polymerase chain reaction (PCR) combined with hybridization with a specific probe was used to detect enterotoxigenic E. coli after DNA extraction from fecal clinical specimens and partial DNA purification (19). Enterotoxigenic E. coli and Shigella sp. have also been detected in diarrheal stools by using multigene amplification of a crude fecal DNA extract (11, 12). Theoretically, these techniques can be used in epidemiological surveys even if the bacteria die during storage or transport. However, their sensitivities have not been determined under laboratory or field conditions. We report here the quantitative results obtained when PCR was performed on test samples stored under various conditions in a model in which fecal samples were mixed with a well-characterized resistance gene cloned in an E. coli strain and used as a target for DNA amplification. MATERIALS AND METHODS
The E. coli strains used in this study were BM2195 (2), which carries the low-copy-number plasmid pIP1100 (5) and contains the gene ereA that encodes for erythromycin esterase type I (20), and BM2570, which contains the gene ereB that encodes for erythromycin esterase type II. The ereB gene has been shown to have no significant homology with ereA (6). Both genes endow E. coli with a high level of *
Corresponding author.
erythromycin resistance (MIC, >512 ,g/ml). E. coli HB101 and Cla (from P. Courvalin) and five clinical members of the family Enterobacteriaceae (two E. coli, one Kiebsiella oxytoca, and two Klebsiella pneumoniae) which were not highly resistant to erythromycin were used as negative controls. Amplification was performed on DNA extracts, which either were used undiluted after extraction from pure bacterial cultures (13) or were diluted 10- to 100-fold in sterile water after extraction from fecal samples constructed in the laboratory. These samples were prepared by mixing known quantities of E. coli BM2195 with 20 mg of fecal material taken from clinical samples chosen among the nondiarrheic or dysenteric specimens sent to our laboratory. These samples did not contain highly erythromycin resistant enteric bacteria, as shown by plating on Drigalski agar containing 512 jig of erythromycin per ml (3), and as described below, they were negative when tested for the presence of gene ereA by PCR before they were mixed with E. coli BM2195. Total DNA was extracted from the pure bacterial cultures or the constructed samples either extemporaneously after culture or sample preparation or after procedures designed to simulate transport and storage conditions were performed. These simulation procedures included the transfer of 20 mg of constructed fecal samples to cotton swabs (Consortium Materiel Laboratoires, Nemours, France) and the dilution of the same amount of fecal samples in 4 ml of phosphate-buffered saline (PBS; pH 7.4), 200 RI of which was then dropped onto filter papers (10 by 15 mm; 3MM; Whatman, Clifton, N.J.). The swabs and filter papers were stored for 4 weeks at room temperature. The fecal material on the swabs was then squeezed out into 4 ml of PBS, and the filter papers were immersed in 0.4 ml of a previously described extraction medium (11). When DNA extraction was performed extemporaneously, the fecal material was first suspended in 4 ml of PBS and then centrifuged for 90 s at 2,800 x g at room temperature. Three milliliters of the supernatant was transferred into Eppendorf tubes and centrifuged at 12,000 x g for 5 min. The pellets were suspended in 75 ,ul of 50 mM Tris hydrochloride (pH 8.0) containing 20% (wt/vol) sucrose and 50 mM EDTA, and 10 pI of a 25-mg/ml lysozyme solution (Appligene, France) was then added. The resulting preparation was incubated for 30 min at 37°C. Next, 300 pu1 of 50 mM NaCl containing 1% (wt/vol) sodium dodecyl sulfate (SDS) and 80 ,ul of a solution containing 10 mg of proteinase K (Boehringer, Mannheim, France) per ml were added, and the 2080
VOL. 30, 1992
incubation was continued for 60 min. The DNA in the solution was precipitated with 800 pl of absolute ethanol; harvested by centrifugation; and resuspended in 40, 300, and 400 ,ul of sterile water for samples from papers, swabs, and fresh specimens, respectively. DNA amplification was performed on 1- to 5-,ul aliquots of DNA extracts diluted up to 50 ,ul in a medium containing 15 mM Tris hydrochloride (pH 8.4, at 20°C), 40 mM KCI, 1.5 mM MgCl2, 0.01% (wt/vol) gelatin, 2.5 U of Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, Conn.), and 10 ,uM (each) PP11 primer 5'-TGAGCGATTTTCGGATAC CC-3' and PP22 primer 5'-AGCTfCAATCTGGTTACCC C-3', which are complementary to bases 330 to 349 and 631 to 650, respectively, of the ereA gene. The samples were overlaid with 60 p.l of mineral oil, denatured at 94°C for 2 min, and subjected to 20 cycles of amplification (94°C for 30 s, 62°C for 4 min, and 72°C for 30 s for each cycle) in a thermocycler (Gene Ataq Controller; Pharmacia, Paris, France). The concentrations of the two primers were then raised to 1 mM, and 20 additional amplification cycles were performed by the same procedure, except that the annealing time was 1 min instead of 4 min and the final polymerization cycle lasted for 10 min at 72°C. This amplification procedure is known as booster PCR (23, 24). Preliminary experiments with our primers and target showed that this booster PCR was 10 to 20 times more sensitive than conventional PCR in detecting amplification products in electrophoresis gels (data not shown). The amplified DNAs were restricted with PstI and were analyzed by submarine gel electrophoresis by using 2.7% Nusieve and 0.9% Seakem GTG agaroses (FMC Bioproducts, Rockland, Maine) in 89 mM Tris-borate-2 mM EDTA (pH 8.0) buffer. Gels were blotted onto a Hybond-N+ membrane (Amersham, Les Ullis, France) as described previously (26), and the blots were hybridized with the 40-bp intragenic PS1 probe 5'-CGCGAATCAGGAAGAAAACT GCAGTTAGTCGGAATCGCCT-3', which is complementary to bases 400 to 439 of the ereA gene. This probe, which was named PS1, was chosen with the help of BISANCE software (Centre Inter Universitaire du Traitement de l'Information 2, Paris, France) and was labeled with [y-3'2P]ATP (Amersham). Hybridization was evidenced by autoradiography, which was done for 1 to 12 h. RESULTS When the booster PCR was performed on DNA extracted from E. coli BM2195, it amplified a 321-bp product (Fig. 1A, lane g) that was visible on the gel when as few as 1 to 10 cells were used as the target (Fig. 1A, lane d). No increase in sensitivity was revealed by hybridization with the PS1 probe (Fig. 1A and B, lanes d to h). After restriction with PstI (Fig. 1A, lane h), the amplified product generated two fragments whose sizes corresponded to the results of the sequence analysis of the ereA gene and which hybridized with probe PS1 (Fig. 1B, lane h). No amplification was detected when PCR was performed on DNA from E. coli BM2570, HB101, or Cla or the five clinical members of the family Enterobacteriaceae (data not shown). There was no significant difference in the sensitivity of PCR for detection of the ereA gene whether pure bacteria were harvested extemporaneously from a fresh culture or had been stored for 4 weeks on swabs or filter papers (Table 1). The limit of sensitivity on constructed fecal samples was never lower than 4.5 log E. coli cells per g of feces (Fig. 2)
DETECTION OF GENES IN FECES
2081
A .
a
.
f
...
4-
W
4-
FIG. 1. Electrophoretic separation of the products of PCR amplification performed on various DNA extracts from E. coli BM2195. (A) Ethidium bromide-stained agarose gel. (B) Autoradiograph of the transferred DNA hybridized with a PS1 probe. Lanes a and i, size standards obtained by digesting pBR322 DNA with HaeIII (marker V; Boehringer); lane b, no DNA extract; lanes c through g, potential numbers of target cells of 0 (no target cells were added to the sample used for DNA extraction), 1 to 10, 10' to 102, 102 to 10', and 10 respectively; lane h, PCR products digested with PstI. ,
because of the need to dilute the fecal specimens in buffers and to use small volumes for the amplification procedures. Large variations in sensitivity were observed when feces from different subjects were used to prepare the test samples. However, no significant difference was noted when constructed specimens prepared with fecal material from the TABLE 1. Sensitivity of PCR for detection of E. coli BM2195 from pure cultures submitted to three types of storage Type of storage
No. of expts
Absolute mean no. (range) of cells detected
Fresh cultures
16
2.5 (0.2-13)
Storage ona: Filter papers Swabs
6 8
2.3 (0.3-6.0) 3.5 (0.2-8.0)
a At room temperature for 4 weeks.
SAULNIER AND ANDREMONT
2082
J. CLIN. MICROBIOL.
7.5
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a)
7.0.
a)
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00 0
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D
M
000 00
6.0
0*@ 0 0 0 000 0
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6.-
0
E
._
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*0
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samples n=41
on
on
swabs filter papers n=1 9 n=21 Storage conditions
FIG. 2. Limit of detection of E. coli BM2195 by booster PCR amplification in constructed fecal samples stored under various conditions. Individual circles indicate the limits of detection in samples from different subjects.
subject were tested either extemporaneously after preparation or after storage for 4 weeks on swabs or filter same
papers.
We found no statistically significant variations whether the subjects from whom the feces were obtained were males or females (6.0 0.7 versus 6.1 t 0.7 log CFU/g) or whether they were older or younger than 15 years (6.0 0.6 versus 6.2 0.9 log CFU/g).
traces of phenol and SDS (7) can also inhibit Taq polymerase. Although we used SDS for DNA extraction, this apparently did not inhibit the booster PCR technique, since the detection limit on pure bacterial cultures was of the order of magnitude of a single copy of the target DNA. In any case, large sample-to-sample variations in the sensitivity of this PCR technique may increase the risk of false-negative results if the counts of the target organisms are low. In previously published studies in which PCR alone (11, 12, 19) or immunomagnetic enrichment combined with either DNA hybridization (17) or PCR (15, 25) was used to detect bacterial genes in fecal specimens, variations in sensitivity from one specimen to another were not investigated. In this study, the sensitivity of gene detection was not significantly different whether PCR was performed on extemporaneously prepared cultures, test samples, or samples that were stored in the laboratory for 4 weeks on filter papers or swabs. This is of particular interest for the detection of target DNA in fecal samples obtained during epidemiological surveys conducted in countries without modem laboratory facilities. In conclusion, we showed that the sensitivity limit of the present booster PCR technique varied greatly among individual samples containing a given number of living target cells. This may not perfectly reflect the content of clinical samples, because these contain both dead and living bacteria. However, because it proved to be possible to conserve fecal samples on inert supports, further research designed to improve the sensitivity of the PCR technique appears to be
justified. ACKNOWLEDGMENTS We thank J. A. Giron for valuable suggestions concerning DNA extraction from stool specimens, M. Arthur and P. Courvalin for the gifts of E. coli strains, and M. Dreyfus for English-language revision. This work was supported in part by l'Association pour la Recherche sur le Cancer, la Ligue Nationale Frangaise Contre le Cancer, and the European Economic Community (contract TS2-0219-F).
±
DISCUSSION When PCR was used under conditions that allowed detection in pure culture of a single cell containing a specific bacterial gene, it detected as little as 104 of these cells per g of material after the cells were mixed with freshly passed feces. We chose as a model the well-characterized resistance gene ereA, which was cloned in E. coli, because a selective and sensitive medium was available to quantify even low counts of cells carrying the gene and because the bacteria contained in most fecal specimens did not express a high level of resistance to erythromycin (4). It was therefore possible to construct in the laboratory fecal samples containing known numbers of cells carrying the target DNA. When feces from different subjects were used to construct the test samples, the variations in the sensitivity of the PCR booster technique used for the detection of our target were as large as 1,000-fold. They were not related to the age or the sex of the patients, but they may have been due to the presence in some fecal samples of nonspecific inhibitors of the PCR technique, as has been observed previously (1, 19, 28). In addition, molecules such as urea (16) and hemoglobin and heparin (7, 9), which may be present in samples, have been identified as inhibitors of Taq polymerase. Lastly,
REFERENCES 1. Allard, A., R. Girones, P. Juto, and G. Wadell. 1990. Polymerase chain reaction for detection of adenoviruses in stool samples. J. Clin. Microbiol. 28:2659-2667. 2. Andremont, A., G. Gerbaud, and C. Courvalin. 1986. Plasmidmediated high-level resistance to erythromycin in Escherichia coli. Antimicrob. Agents Chemother. 29:515-518. 3. Andremont, A., G. Gerbaud, C. Tancrede, and P. Courvalin. 1985. Plasmid-mediated susceptibility to intestinal microbial antagonisms in Escherichia coli. Infect. Immun. 49:751-755. 4. Andremont, A., H. Sancho-Garnier, and C. Tancrede. 1986. Epidemiology of intestinal colonization by members of the family Enterobacteriaceae highly resistant to erythromycin in a hematology-oncology unit. Antimicrob. Agents Chemother. 29: 1104-1107. 5. Arthur, M., A. Andremont, and P. Courvalin. 1986. Heterogeneity of genes conferring high-level resistance to erythromycin by inactivation in enterobacteria. Ann. Inst. Pasteur/Microbiol.
(Paris) 137A:125-134. 6. Arthur, M., and P. Courvalin. 1986. Contribution of two different mechanisms to erythromycin resistance in Eschenichia coli. Antimicrob. Agents Chemother. 30:694-700. 7. Bentler, E., T. Gelbart, and W. Kuhl. 1990. Interference of heparin with the polymerase chain reaction. BioTechniques 9:166. 8. Boileau, C. R., H. M. Hauteville, and P. J. Sansonetti. 1984. DNA hybridization technique to detect Shigella species and enteroinvasive Escherichia coli. J. Clin. Microbiol. 20:959-961. 9. Brisson-Noel, A., C. Aznar, C. Churean, S. Nguyen, C. Pierre,
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10.
11.
12.
13. 14.
15.
16. 17.
18.
M. Bartoli, R. Bonete, G. Pialoux, B. Gicquel, and G. Garrigue. 1991. Diagnosis of tuberculosis by DNA amplification in clinical practice evaluation. Lancet 338:364-366. Fitts, R., M. Diamond, C. Hamilton, and M. Neri. 1983. DNA hybridization assay for detection of Salmonella species in foods. Appl. Environ. Microbiol. 46:1146-1151. Frankel, G., J. A. Giron, J. Valmassoi, and G. K. Schoolnilk 1989. Multi-gene amplification: simultaneous detection of three virulence genes in diarrheal stool. Mol. Microbiol. 3:1729-1734. Frankel, G., L. Riley, J. A. Giron, J. Valmossoi, A. Friedmann, N. Strockbine, S. Falkw, and G. K. Schoolick. 1990. Detection of Shigella in feces using DNA amplification. J. Infect. Dis. 161:1252-1256. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193197. Horn, J. E., T. Quinn, M. Hammer, L. Palmer, and S. Falkow. 1986. Use of nucleic acid probes for the detection of sexually transmitted infectious agents. Diagn. Microbiol. Infect. Dis. 4:S101-S109. Hornes, E., Y. Wasteson, and 0. Olswik. 1991. Detection of Escherichia coli heat-stable enterotoxin genes in pig stool specimens by an immobilized, colorimetric, nested polymerase chain reaction. J. Clin. Microbiol. 29.2375-2379. Khan, G., H. 0. Kangro, P. J. Coates, and R B. Heath. 1991. Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J. Clin. Pathol. 44:360-365. Lund, A., Y. Wasteson, and 0. Olsvik. 1991. Immunomagnetic separation and DNA hybridization for detection of enterogenic Escherichia coli in a piglet model. J. Clin. Microbiol. 29:22592262. Moseley, S. L., I. Huq, A. L. Alim, M. So, M. SamadpourMotalebi, and S. Falkow. 1980. Detection of enterotoxigenic Escherichia coli by DNA colony hybridization. J. Infect. Dis. 142:892-898.
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19. Olive, D. M. 1989. Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase. J. Clin. Microbiol. 27:261-265. 20. Ounissi, H., and P. Courvalin. 1985. Nucleotide sequence of the gene ereA encoding the erythromycin esterase in Escherichia colt. Gene 37:271-278. 21. Pao, C.-C., S.-S. Tin, S.-Y. Wu, W.-M. Juang, C.-H. Chang, and J.-Y. Lin. 1988. The detection of mycobacterial DNA sequences in uncultured clinical specimens with cloned Mycobacterium tuberculosis DNA as probe. Tubercle 69:27-36. 22. Roberts, M. C., C. McMilliam, and M. B. Coyle. 1987. Whole chromosomal DNA probes for rapid identification of Mycobacterinum tuberculosis and Mycobacterium avium complex. J. Clin. Microbiol. 25:1239-1243. 23. Ruano, G., W. Fenton, and K. K. Kidd. 1989. Biphasic amplification of very dilute DNA samples via booster PCR. Nucleic Acids Res. 17:5407. 24. Ruano, G., K. K. Kidd, and J. C. Stephens. 1990. Haplotype of multiple polymorphisms resolved by enzymatic amplification of single DNA molecules. Proc. Natl. Acad. Sci. USA 87:62966300. 25. Shirai, H., M. Nishibuchi, T. Ramamurthy, S. K. Bhattacharya, S. C. Pal, and Y. Takeda. 1991. Polymerase chain reaction for detection of the cholera enterotoxin operon of Vibrio cholerae. J. Clin. Microbiol. 29:2517-2521. 26. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 27. Tompkins, L., and M. Krajden. 1986. Approaches to the detection of enteric pathogens including Campylobacter, using nucleic acid hybridization. Diagn. Microbiol. Infect. Dis. 4:S78S88. 28. Xu, L., D. Harbour, and M. A. McCrae. 1990. The application of polymerase chain reaction to the detection of rotaviruses in faeces. J. Virol. Methods 27:29-38.
Vol. 30, No. 8
0095-1137/92/082080-04$02.00/0 Copyright © 1992, American Society for Microbiology
Detection of Genes in Feces by Booster Polymerase Chain Reaction P. SAULNIER1
AND
A. ANDREMONT .2*
Laboratoire d 'Ecologie Microbienne, Institut Gustave-Roussy, Villejuif, 1 and Laboratoire de Microbiologie, Faculte de Pharmacie, Chatenay-Malabry France Received 2 January 1992/Accepted 7 May 1992
A 321-bp fragment intragenic to the gene ereA carried byEscherichia coli BM2195 was used as a model target to study the conditions under which DNA amplification by booster polymerase chain reaction can be used to detect specific bacterial DNA sequences in fecal specimens. When target E. coli cells were mixed with 41 freshly obtained fecal specimens, the polymerase chain reaction detection limit varied from 4.5 to 7.1 log CFU/g of feces, depending on the individual fecal specimen used to prepare the test sample. These variations were not statistically related to the sex or age of the subject from whom the specimen was obtained. After storage of the samples for 4 weeks at room temperature on swabs or filter papers, no loss in sensitivity was observed.
Detection of pathogenic bacteria in fecal samples is based the culture of fecal specimens on solid selective medium with or without previous enrichment growth. However, in many laboratories in developing countries, selective and sensitive media either do not exist for some pathogens (e.g., enterotoxigenic or enteroinvasive Escherichia coli) or are not available for certain pathogens (e.g., Campylobacter sp. or Clostridium difficile). In addition, the conservation of isolated clones until their final identification in reference laboratories is difficult because of the spontaneous loss of virulence plasmids or because deletion of plasmid-encoded genes can occur during in vitro growth, storage, and transon
port.
To overcome these problems, DNA-DNA hybridization techniques with specific probes either on dots of extracted DNA (8, 10, 14, 22, 27) or on bacterial colonies lysed on filters (18, 21, 27) have been proposed. Subsequently, DNA amplification by the polymerase chain reaction (PCR) combined with hybridization with a specific probe was used to detect enterotoxigenic E. coli after DNA extraction from fecal clinical specimens and partial DNA purification (19). Enterotoxigenic E. coli and Shigella sp. have also been detected in diarrheal stools by using multigene amplification of a crude fecal DNA extract (11, 12). Theoretically, these techniques can be used in epidemiological surveys even if the bacteria die during storage or transport. However, their sensitivities have not been determined under laboratory or field conditions. We report here the quantitative results obtained when PCR was performed on test samples stored under various conditions in a model in which fecal samples were mixed with a well-characterized resistance gene cloned in an E. coli strain and used as a target for DNA amplification. MATERIALS AND METHODS
The E. coli strains used in this study were BM2195 (2), which carries the low-copy-number plasmid pIP1100 (5) and contains the gene ereA that encodes for erythromycin esterase type I (20), and BM2570, which contains the gene ereB that encodes for erythromycin esterase type II. The ereB gene has been shown to have no significant homology with ereA (6). Both genes endow E. coli with a high level of *
Corresponding author.
erythromycin resistance (MIC, >512 ,g/ml). E. coli HB101 and Cla (from P. Courvalin) and five clinical members of the family Enterobacteriaceae (two E. coli, one Kiebsiella oxytoca, and two Klebsiella pneumoniae) which were not highly resistant to erythromycin were used as negative controls. Amplification was performed on DNA extracts, which either were used undiluted after extraction from pure bacterial cultures (13) or were diluted 10- to 100-fold in sterile water after extraction from fecal samples constructed in the laboratory. These samples were prepared by mixing known quantities of E. coli BM2195 with 20 mg of fecal material taken from clinical samples chosen among the nondiarrheic or dysenteric specimens sent to our laboratory. These samples did not contain highly erythromycin resistant enteric bacteria, as shown by plating on Drigalski agar containing 512 jig of erythromycin per ml (3), and as described below, they were negative when tested for the presence of gene ereA by PCR before they were mixed with E. coli BM2195. Total DNA was extracted from the pure bacterial cultures or the constructed samples either extemporaneously after culture or sample preparation or after procedures designed to simulate transport and storage conditions were performed. These simulation procedures included the transfer of 20 mg of constructed fecal samples to cotton swabs (Consortium Materiel Laboratoires, Nemours, France) and the dilution of the same amount of fecal samples in 4 ml of phosphate-buffered saline (PBS; pH 7.4), 200 RI of which was then dropped onto filter papers (10 by 15 mm; 3MM; Whatman, Clifton, N.J.). The swabs and filter papers were stored for 4 weeks at room temperature. The fecal material on the swabs was then squeezed out into 4 ml of PBS, and the filter papers were immersed in 0.4 ml of a previously described extraction medium (11). When DNA extraction was performed extemporaneously, the fecal material was first suspended in 4 ml of PBS and then centrifuged for 90 s at 2,800 x g at room temperature. Three milliliters of the supernatant was transferred into Eppendorf tubes and centrifuged at 12,000 x g for 5 min. The pellets were suspended in 75 ,ul of 50 mM Tris hydrochloride (pH 8.0) containing 20% (wt/vol) sucrose and 50 mM EDTA, and 10 pI of a 25-mg/ml lysozyme solution (Appligene, France) was then added. The resulting preparation was incubated for 30 min at 37°C. Next, 300 pu1 of 50 mM NaCl containing 1% (wt/vol) sodium dodecyl sulfate (SDS) and 80 ,ul of a solution containing 10 mg of proteinase K (Boehringer, Mannheim, France) per ml were added, and the 2080
VOL. 30, 1992
incubation was continued for 60 min. The DNA in the solution was precipitated with 800 pl of absolute ethanol; harvested by centrifugation; and resuspended in 40, 300, and 400 ,ul of sterile water for samples from papers, swabs, and fresh specimens, respectively. DNA amplification was performed on 1- to 5-,ul aliquots of DNA extracts diluted up to 50 ,ul in a medium containing 15 mM Tris hydrochloride (pH 8.4, at 20°C), 40 mM KCI, 1.5 mM MgCl2, 0.01% (wt/vol) gelatin, 2.5 U of Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, Conn.), and 10 ,uM (each) PP11 primer 5'-TGAGCGATTTTCGGATAC CC-3' and PP22 primer 5'-AGCTfCAATCTGGTTACCC C-3', which are complementary to bases 330 to 349 and 631 to 650, respectively, of the ereA gene. The samples were overlaid with 60 p.l of mineral oil, denatured at 94°C for 2 min, and subjected to 20 cycles of amplification (94°C for 30 s, 62°C for 4 min, and 72°C for 30 s for each cycle) in a thermocycler (Gene Ataq Controller; Pharmacia, Paris, France). The concentrations of the two primers were then raised to 1 mM, and 20 additional amplification cycles were performed by the same procedure, except that the annealing time was 1 min instead of 4 min and the final polymerization cycle lasted for 10 min at 72°C. This amplification procedure is known as booster PCR (23, 24). Preliminary experiments with our primers and target showed that this booster PCR was 10 to 20 times more sensitive than conventional PCR in detecting amplification products in electrophoresis gels (data not shown). The amplified DNAs were restricted with PstI and were analyzed by submarine gel electrophoresis by using 2.7% Nusieve and 0.9% Seakem GTG agaroses (FMC Bioproducts, Rockland, Maine) in 89 mM Tris-borate-2 mM EDTA (pH 8.0) buffer. Gels were blotted onto a Hybond-N+ membrane (Amersham, Les Ullis, France) as described previously (26), and the blots were hybridized with the 40-bp intragenic PS1 probe 5'-CGCGAATCAGGAAGAAAACT GCAGTTAGTCGGAATCGCCT-3', which is complementary to bases 400 to 439 of the ereA gene. This probe, which was named PS1, was chosen with the help of BISANCE software (Centre Inter Universitaire du Traitement de l'Information 2, Paris, France) and was labeled with [y-3'2P]ATP (Amersham). Hybridization was evidenced by autoradiography, which was done for 1 to 12 h. RESULTS When the booster PCR was performed on DNA extracted from E. coli BM2195, it amplified a 321-bp product (Fig. 1A, lane g) that was visible on the gel when as few as 1 to 10 cells were used as the target (Fig. 1A, lane d). No increase in sensitivity was revealed by hybridization with the PS1 probe (Fig. 1A and B, lanes d to h). After restriction with PstI (Fig. 1A, lane h), the amplified product generated two fragments whose sizes corresponded to the results of the sequence analysis of the ereA gene and which hybridized with probe PS1 (Fig. 1B, lane h). No amplification was detected when PCR was performed on DNA from E. coli BM2570, HB101, or Cla or the five clinical members of the family Enterobacteriaceae (data not shown). There was no significant difference in the sensitivity of PCR for detection of the ereA gene whether pure bacteria were harvested extemporaneously from a fresh culture or had been stored for 4 weeks on swabs or filter papers (Table 1). The limit of sensitivity on constructed fecal samples was never lower than 4.5 log E. coli cells per g of feces (Fig. 2)
DETECTION OF GENES IN FECES
2081
A .
a
.
f
...
4-
W
4-
FIG. 1. Electrophoretic separation of the products of PCR amplification performed on various DNA extracts from E. coli BM2195. (A) Ethidium bromide-stained agarose gel. (B) Autoradiograph of the transferred DNA hybridized with a PS1 probe. Lanes a and i, size standards obtained by digesting pBR322 DNA with HaeIII (marker V; Boehringer); lane b, no DNA extract; lanes c through g, potential numbers of target cells of 0 (no target cells were added to the sample used for DNA extraction), 1 to 10, 10' to 102, 102 to 10', and 10 respectively; lane h, PCR products digested with PstI. ,
because of the need to dilute the fecal specimens in buffers and to use small volumes for the amplification procedures. Large variations in sensitivity were observed when feces from different subjects were used to prepare the test samples. However, no significant difference was noted when constructed specimens prepared with fecal material from the TABLE 1. Sensitivity of PCR for detection of E. coli BM2195 from pure cultures submitted to three types of storage Type of storage
No. of expts
Absolute mean no. (range) of cells detected
Fresh cultures
16
2.5 (0.2-13)
Storage ona: Filter papers Swabs
6 8
2.3 (0.3-6.0) 3.5 (0.2-8.0)
a At room temperature for 4 weeks.
SAULNIER AND ANDREMONT
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J. CLIN. MICROBIOL.
7.5
CO)
a)
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a)
01)
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00 0
0) lL 0
D
M
000 00
6.0
0*@ 0 0 0 000 0
5.5.
0000 000
0
0
a)
a) -o
5.0
6.-
0
E
._
4.5.
*0
4.0 f resh
samples n=41
on
on
swabs filter papers n=1 9 n=21 Storage conditions
FIG. 2. Limit of detection of E. coli BM2195 by booster PCR amplification in constructed fecal samples stored under various conditions. Individual circles indicate the limits of detection in samples from different subjects.
subject were tested either extemporaneously after preparation or after storage for 4 weeks on swabs or filter same
papers.
We found no statistically significant variations whether the subjects from whom the feces were obtained were males or females (6.0 0.7 versus 6.1 t 0.7 log CFU/g) or whether they were older or younger than 15 years (6.0 0.6 versus 6.2 0.9 log CFU/g).
traces of phenol and SDS (7) can also inhibit Taq polymerase. Although we used SDS for DNA extraction, this apparently did not inhibit the booster PCR technique, since the detection limit on pure bacterial cultures was of the order of magnitude of a single copy of the target DNA. In any case, large sample-to-sample variations in the sensitivity of this PCR technique may increase the risk of false-negative results if the counts of the target organisms are low. In previously published studies in which PCR alone (11, 12, 19) or immunomagnetic enrichment combined with either DNA hybridization (17) or PCR (15, 25) was used to detect bacterial genes in fecal specimens, variations in sensitivity from one specimen to another were not investigated. In this study, the sensitivity of gene detection was not significantly different whether PCR was performed on extemporaneously prepared cultures, test samples, or samples that were stored in the laboratory for 4 weeks on filter papers or swabs. This is of particular interest for the detection of target DNA in fecal samples obtained during epidemiological surveys conducted in countries without modem laboratory facilities. In conclusion, we showed that the sensitivity limit of the present booster PCR technique varied greatly among individual samples containing a given number of living target cells. This may not perfectly reflect the content of clinical samples, because these contain both dead and living bacteria. However, because it proved to be possible to conserve fecal samples on inert supports, further research designed to improve the sensitivity of the PCR technique appears to be
justified. ACKNOWLEDGMENTS We thank J. A. Giron for valuable suggestions concerning DNA extraction from stool specimens, M. Arthur and P. Courvalin for the gifts of E. coli strains, and M. Dreyfus for English-language revision. This work was supported in part by l'Association pour la Recherche sur le Cancer, la Ligue Nationale Frangaise Contre le Cancer, and the European Economic Community (contract TS2-0219-F).
±
DISCUSSION When PCR was used under conditions that allowed detection in pure culture of a single cell containing a specific bacterial gene, it detected as little as 104 of these cells per g of material after the cells were mixed with freshly passed feces. We chose as a model the well-characterized resistance gene ereA, which was cloned in E. coli, because a selective and sensitive medium was available to quantify even low counts of cells carrying the gene and because the bacteria contained in most fecal specimens did not express a high level of resistance to erythromycin (4). It was therefore possible to construct in the laboratory fecal samples containing known numbers of cells carrying the target DNA. When feces from different subjects were used to construct the test samples, the variations in the sensitivity of the PCR booster technique used for the detection of our target were as large as 1,000-fold. They were not related to the age or the sex of the patients, but they may have been due to the presence in some fecal samples of nonspecific inhibitors of the PCR technique, as has been observed previously (1, 19, 28). In addition, molecules such as urea (16) and hemoglobin and heparin (7, 9), which may be present in samples, have been identified as inhibitors of Taq polymerase. Lastly,
REFERENCES 1. Allard, A., R. Girones, P. Juto, and G. Wadell. 1990. Polymerase chain reaction for detection of adenoviruses in stool samples. J. Clin. Microbiol. 28:2659-2667. 2. Andremont, A., G. Gerbaud, and C. Courvalin. 1986. Plasmidmediated high-level resistance to erythromycin in Escherichia coli. Antimicrob. Agents Chemother. 29:515-518. 3. Andremont, A., G. Gerbaud, C. Tancrede, and P. Courvalin. 1985. Plasmid-mediated susceptibility to intestinal microbial antagonisms in Escherichia coli. Infect. Immun. 49:751-755. 4. Andremont, A., H. Sancho-Garnier, and C. Tancrede. 1986. Epidemiology of intestinal colonization by members of the family Enterobacteriaceae highly resistant to erythromycin in a hematology-oncology unit. Antimicrob. Agents Chemother. 29: 1104-1107. 5. Arthur, M., A. Andremont, and P. Courvalin. 1986. Heterogeneity of genes conferring high-level resistance to erythromycin by inactivation in enterobacteria. Ann. Inst. Pasteur/Microbiol.
(Paris) 137A:125-134. 6. Arthur, M., and P. Courvalin. 1986. Contribution of two different mechanisms to erythromycin resistance in Eschenichia coli. Antimicrob. Agents Chemother. 30:694-700. 7. Bentler, E., T. Gelbart, and W. Kuhl. 1990. Interference of heparin with the polymerase chain reaction. BioTechniques 9:166. 8. Boileau, C. R., H. M. Hauteville, and P. J. Sansonetti. 1984. DNA hybridization technique to detect Shigella species and enteroinvasive Escherichia coli. J. Clin. Microbiol. 20:959-961. 9. Brisson-Noel, A., C. Aznar, C. Churean, S. Nguyen, C. Pierre,
VOL. 30, 1992
10.
11.
12.
13. 14.
15.
16. 17.
18.
M. Bartoli, R. Bonete, G. Pialoux, B. Gicquel, and G. Garrigue. 1991. Diagnosis of tuberculosis by DNA amplification in clinical practice evaluation. Lancet 338:364-366. Fitts, R., M. Diamond, C. Hamilton, and M. Neri. 1983. DNA hybridization assay for detection of Salmonella species in foods. Appl. Environ. Microbiol. 46:1146-1151. Frankel, G., J. A. Giron, J. Valmassoi, and G. K. Schoolnilk 1989. Multi-gene amplification: simultaneous detection of three virulence genes in diarrheal stool. Mol. Microbiol. 3:1729-1734. Frankel, G., L. Riley, J. A. Giron, J. Valmossoi, A. Friedmann, N. Strockbine, S. Falkw, and G. K. Schoolick. 1990. Detection of Shigella in feces using DNA amplification. J. Infect. Dis. 161:1252-1256. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193197. Horn, J. E., T. Quinn, M. Hammer, L. Palmer, and S. Falkow. 1986. Use of nucleic acid probes for the detection of sexually transmitted infectious agents. Diagn. Microbiol. Infect. Dis. 4:S101-S109. Hornes, E., Y. Wasteson, and 0. Olswik. 1991. Detection of Escherichia coli heat-stable enterotoxin genes in pig stool specimens by an immobilized, colorimetric, nested polymerase chain reaction. J. Clin. Microbiol. 29.2375-2379. Khan, G., H. 0. Kangro, P. J. Coates, and R B. Heath. 1991. Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J. Clin. Pathol. 44:360-365. Lund, A., Y. Wasteson, and 0. Olsvik. 1991. Immunomagnetic separation and DNA hybridization for detection of enterogenic Escherichia coli in a piglet model. J. Clin. Microbiol. 29:22592262. Moseley, S. L., I. Huq, A. L. Alim, M. So, M. SamadpourMotalebi, and S. Falkow. 1980. Detection of enterotoxigenic Escherichia coli by DNA colony hybridization. J. Infect. Dis. 142:892-898.
DETECTION OF GENES IN FECES
2083
19. Olive, D. M. 1989. Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase. J. Clin. Microbiol. 27:261-265. 20. Ounissi, H., and P. Courvalin. 1985. Nucleotide sequence of the gene ereA encoding the erythromycin esterase in Escherichia colt. Gene 37:271-278. 21. Pao, C.-C., S.-S. Tin, S.-Y. Wu, W.-M. Juang, C.-H. Chang, and J.-Y. Lin. 1988. The detection of mycobacterial DNA sequences in uncultured clinical specimens with cloned Mycobacterium tuberculosis DNA as probe. Tubercle 69:27-36. 22. Roberts, M. C., C. McMilliam, and M. B. Coyle. 1987. Whole chromosomal DNA probes for rapid identification of Mycobacterinum tuberculosis and Mycobacterium avium complex. J. Clin. Microbiol. 25:1239-1243. 23. Ruano, G., W. Fenton, and K. K. Kidd. 1989. Biphasic amplification of very dilute DNA samples via booster PCR. Nucleic Acids Res. 17:5407. 24. Ruano, G., K. K. Kidd, and J. C. Stephens. 1990. Haplotype of multiple polymorphisms resolved by enzymatic amplification of single DNA molecules. Proc. Natl. Acad. Sci. USA 87:62966300. 25. Shirai, H., M. Nishibuchi, T. Ramamurthy, S. K. Bhattacharya, S. C. Pal, and Y. Takeda. 1991. Polymerase chain reaction for detection of the cholera enterotoxin operon of Vibrio cholerae. J. Clin. Microbiol. 29:2517-2521. 26. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 27. Tompkins, L., and M. Krajden. 1986. Approaches to the detection of enteric pathogens including Campylobacter, using nucleic acid hybridization. Diagn. Microbiol. Infect. Dis. 4:S78S88. 28. Xu, L., D. Harbour, and M. A. McCrae. 1990. The application of polymerase chain reaction to the detection of rotaviruses in faeces. J. Virol. Methods 27:29-38.
