Applied and Environmental Microbiology, October 2000, p. 4555-4558, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Rapid and Simple Determination of the
Escherichia coli Phylogenetic Group
Olivier
Clermont,
Stéphane
Bonacorsi, and
Edouard
Bingen*
Laboratoire d'études de génétique
bactérienne dans les infections de l'enfant (EA3105),
Université Denis Diderot-Paris 7, Hôpital Robert
Debré, Paris, France
Received 15 February 2000/Accepted 7 July 2000
 |
ABSTRACT |
Phylogenetic analysis has shown that Escherichia coli
is composed of four main phylogenetic groups (A, B1, B2, and D) and that virulent extra-intestinal strains mainly belong to groups B2 and
D. Actually, phylogenetic groups can be determined by multilocus enzyme
electrophoresis or ribotyping, both of which are complex, time-consuming techniques. We describe a simple and rapid phylogenetic grouping technique based on triplex PCR. The method, which uses a
combination of two genes (chuA and yjaA) and an
anonymous DNA fragment, was tested with 230 strains and showed
excellent correlation with reference methods.
| |
TEXT |
Escherichia coli is a
normal inhabitant of the intestines of most animals, including humans.
Some E. coli strains can cause a wide variety of intestinal
and extra-intestinal diseases, such as diarrhea, urinary tract
infections, septicemia, and neonatal meningitis (18).
Phylogenetic analyses have shown that E. coli strains fall
into four main phylogenetic groups (A, B1, B2, and D) (10,
21) and that virulent extra-intestinal strains belong mainly to
group B2 and, to a lesser extent, to group D (4, 7, 12, 19),
whereas most commensal strains belong to group A. These studies have
also given us a better understanding of how pathogenic strains acquire
virulence genes (4). Actually, phylogenetic grouping can be
done by multilocus enzyme electrophoresis (10, 20) or
ribotyping (2-4, 8), but both of these reference techniques
are complex and time-consuming and also require a collection of typed strains.
Creation of a subtractive library for two E. coli strains
belonging to different phylogenetic groups (6) and
characterization of an anonymous 14.9-kb fragment strongly associated
with neonatal meningitis strains (3) suggested that certain
genes or DNA fragments might be specific phylogenetic group markers.
Three candidate markers were studied further: (i) chuA, a
gene required for heme transport in enterohemorrhagic O157:H7 E. coli (6, 16, 23, 24); (ii) yjaA, a gene
initially identified in the recent complete genome sequence of E. coli K-12, the function of which is unknown (5); and
(iii) an anonymous DNA fragment designated TSPE4.C2 from our
subtractive library (6). Here we describe a rapid technique
for determining the phylogenetic groups of E. coli strains
based on PCR detection of the chuA and yjaA genes
and DNA fragment TSPE4.C2. The method was evaluated by testing 230 strains that had already been grouped by using reference methods.
Bacterial strains and growth conditions.
The 72 strains of the
ECOR collection (17) were kindly provided by R. Selander
(Department of Biology, University of Rochester, Rochester, N.Y.).
These reference strains, isolated from a variety of hosts and
geographic locations, are representative of the range of genotypic
variation in the species. Sixty-eight of these strains belong to the
four main phylogenetic groups (A, B1, B2, and D), and four are
unclassified (10, 21). We also tested a set of 86 E. coli strains causing neonatal meningitis (ECNM strains) (4), 34 E. coli strains responsible for neonatal
septicemia without meningitis, 30 E. coli strains isolated
from feces of healthy neonates, the J96 uropathogenic E. coli strain (O4:K6) (kindly provided by J. Hacker, Institut
für Molekulare Infektionsbiologie, Würzburg, Germany)
(22), and 10 verotoxin-producing E. coli O157:H7
strains, including 1 strain obtained from A. D. O'Brien (Uniformed Services University of the Health Sciences, Bethesda, Md.)
and 9 strains isolated from different locations in France (P. Mariani,
Hôpital Robert-Debré, Paris, France). The phylogenetic group distribution of 69 of the 86 ECNM strains has been published previously (4). The other 17 ECNM strains and the 75 remaining clinical isolates were classified by ribotyping as previously described (4). The E. coli laboratory K-12 strain
MG1655, which belongs to phylogenetic group A, was also studied
(10). Bacteria were grown at 37°C in Luria-Bertani broth
or on Luria-Bertani agar. When necessary, ampicillin (100 µg per ml)
was used.
PCR amplification and Southern blotting.
As a first step, PCR
was performed with a standard protocol. Each reaction was carried out
by using a 20-µl mixture containing 2 µl of 10× buffer (supplied
with Taq polymerase), 20 pmol of each primer, each
deoxynucleoside triphosphate at a concentration of 2 µM, 2.5 U of
Taq polymerase (ATGC Biotechnologie, Noisy-le-Grand, France), and 200 ng of genomic DNA. The PCR was performed with a
Perkin-Elmer GeneAmp 9600 thermal cycler with MicroAm tubes under the
following conditions: denaturation for 5 min at 94°C; 30 cycles of
30 s at 94°C, 30 s at 55°C, and 30 s at 72°C; and a final extension step of 7 min at 72°C. The primer pairs used were ChuA.1 (5'-GACGAACCAACGGTCAGGAT-3') and ChuA.2
(5'-TGCCGCCAGTACCAAAGACA-3'), YjaA.1
(5'-TGAAGTGTCAGGAGACGCTG-3') and YjaA.2
(5'-ATGGAGAATGCGTTCCTCAAC-3'), and TspE4C2.1
(5'-GAGTAATGTCGGGGCATTCA-3') and TspE4C2.2
(5'-CGCGCCAACAAAGTATTACG-3'), which generate 279-, 211-, and
152-bp fragments, respectively. In a simplified protocol, a two-step
triplex polymerase reaction based on previously described methods
(1, 9) was assessed. The components of the reaction
mixture were the same as those in the standard protocol, except that
(i) DNA was directly provided by 3 µl of bacterial lysate or a piece
of a colony, (ii) the six above-mentioned primers were mixed, and (iii)
the PCR steps were as follows: denaturation for 4 min at 94°C, 30 cycles of 5 s at 94°C and 10 s at 59°C, and a final
extension step of 5 min at 72°C.
Southern blotting was performed by capillary transfer to positively
charged nylon membranes. Hybridization was performed at 65°C in 1%
sodium dodecyl sulfate-1 M NaCl-50 mM Tris HCl (pH 7.5)-1% blocking
reagent (Boehringer, Mannheim, Germany). The membranes were washed in
2× SSC for 15 min at room temperature, then in 2× SSC-0.1% sodium
dodecyl sulfate for 30 min at 65°C, and finally in 0.1× SSC for 5 min at room temperature (1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate). Chemiluminescence detection was performed according to the
manufacturer's instructions (DIG Luminescent Detection Kit for nucleic
acids; Boehringer). The probes were produced by PCR according to the
manufacturer's instructions (PCR DIG Probe Synthesis Kit; Boehringer)
by using the primers and amplification procedure described above for
the standard protocol.
PCR grouping results.
A total of 230 strains were analyzed.
Their phylogenetic groups, as determined by reference methods, were as
follows: 43 belonged to group A, 23 belonged to group B1, 51 belonged
to group D, and 113 belonged to group B2. Table
1 shows the PCR results for the entire
set of strains according to their phylogenetic groups. The
chuA gene was present in all strains belonging to groups B2 and D and was absent from all strains belonging to groups A and B1.
This allowed us to separate groups B2 and D from groups A and B1. In
the same way, the yjaA gene allowed perfect discrimination between group B2 (100% of the strains were positive) and group D
(100% of the strains were negative). Finally, clone TSPE4.C2 was
present in all but two of the group B1 strains and absent from all
group A strains. All the PCR results were confirmed by Southern
hybridization (data not shown). The results of these three
amplifications made it possible to establish a dichotomous decision
tree (Fig. 1) for phylogenetic grouping.
With this tree, 228 of the 230 strains tested (99%) were correctly
grouped, while only 2 strains were wrongly classified (group B1 strains
were identified as group A strains). Identical results were obtained with standard and simplified PCR protocols. Figure
2 shows the different profiles obtained
by triplex PCR for the four phylogenetic groups.
View this table:
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TABLE 1.
PCR amplification of the chuA and
yjaA genes and DNA fragment TSPE4.C2 in E. coli
strains from various collections according to phylogenetic group
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FIG. 1.
Dichotomous decision tree to determine the phylogenetic
group of an E. coli strain by using the results of PCR
amplification of the chuA and yjaA genes and DNA
fragment TSPE4.C2.
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View larger version (52K):
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FIG. 2.
Triplex PCR profiles specific for E. coli
phylogenetic groups. Each combination of chuA and
yjaA gene and DNA fragment TSPE4.C2 amplification allowed
phylogenetic group determination of a strain. Lanes 1 and 2, group A;
lane 3, group B1; lanes 4 and 5, group D; lanes 6 and 7, group B2. Lane
M contained markers.
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In this work, we developed a PCR method to rapidly determine the
phylogenetic groups of E. coli strains and obtained an
accuracy of more than 99% compared to the reference method.
Phylogenetic characterization of E. coli strains on the
basis of a very few phenotypic or genotypic features initially appeared
to be very difficult. Such genotypic traits (presence or absence of a
gene, for example) must meet different criteria for use in phylogenetic characterization. First, the gene must have been acquired or deleted when the group that it characterizes emerged. Second, the same gene
must have been "stabilized," thereby ruling out its subsequent deletion or horizontal transfer among bacteria belonging to other phylogenetic groups. Finally, recombination events in the candidate gene must be very rare. In other words, the gene product must not be
targeted by natural selection, which favors new genetic recombinations
(24). Previous attempts to identify specific phylogenetic
group characteristics based on phenotypic (21) or genotypic
features (11) were not sufficiently discriminative. For the
first time, we describe the use of two genes and an anonymous DNA
fragment in a simple phylogenetic grouping method. Too little information is available on yjaA and the DNA fragment to
speculate on their evolutionary history. In contrast, the study by
Wyckoff et al. (25) of heme transport genes, together with
our results, suggests that chuA was acquired by sister
groups B2 and D (15) soon after their emergence rather than
being present in a common ancestor and subsequently being lost by
groups B1 and A.
However, two strains (ECOR 70 and an ECNM strain) belonging to
phylogenetic group B1 were classified in group A by our method. This
discrepancy may be explained by an intermediate genetic base between
these two groups in these strains and by the fact that the region
studied with our method (chuA, yjaA, and TSPE4.C2
are located at 78.7 min [25], 90.8 min
[5], and approximately 87 min [6],
respectively, in relation to the genome of E. coli K-12) may
be more closely related to group A regions than to the regions studied
with the reference methods. Indeed, it has been demonstrated that
groups A and B1 are sister groups (15). Moreover, recent
multiple chromosomal nucleotide sequence analysis has shown that ECOR
70 may be considered a "hybrid" strain, in which some housekeeping
genes exhibit nucleotide sequences shared by group A ECOR strains and
some other genes exhibit nucleotide sequences shared by group B1 ECOR
strains (15; E. Denamur, personal communication). Thus, the phylogenetic group of ECOR 70 remains to be settled. In
addition to the rapidity of our PCR-based method, no reference collection is required, meaning that the assay can easily be used in
any laboratory. Furthermore, in contrast to other methods, group
allocation is unequivocal. Indeed, the four unclassified strains in the
ECOR collection, ECOR 31, ECOR 37, ECOR 42, and ECOR 43, were
classified by our method; the first three strains belong to group D,
and the fourth belongs to group A. It is noteworthy that all the
sequences of the housekeeping genes studied in the latter strain were
characteristic of those found in group A strains (Denamur, personal communication).
In conclusion, our simple and rapid phylogenetic grouping technique
could have several practical applications. The first is in bioclinical
practice, given the established link between phylogenetic group and
virulence (4, 7, 12, 19). The second is as a
biotechnological screening tool for eliminating potentially pathogenic
strains when candidate strains for cloning are screened. Such screening
tools have been developed, for example, to identify E. coli
K-12 strains by PCR (13) or to detect E. coli
strains with no known virulence genes by a reverse dot blot procedure described by Kuhnert et al. (14). Our method has the
advantage of being capable of identifying nonpathogenic strains other
than E. coli K-12 and suitable for large-scale strain
screening. Thus, after all strains belonging to groups B2 and D, which
are potentially pathogenic, are eliminated, the reverse dot blot
technique (14) could be applied to group A or B1 strains to
eliminate rare strains harboring virulence factors.
| |
ACKNOWLEDGMENTS |
We thank Walter Nanni and Colin Tinsley for technical help and
Xavier Nassif, Jacques Elion, and Erick Denamur for helpful discussions.
This work was supported in part by the Programme de Recherche
Fondamentale en Microbiologie, Maladies Infectieuses et Parasitaires (Appel d'offre 1998), "Recherche de déterminants
génétiques de pathogénicité chez E. coli K1 responsable de méningite néonatale," and by
the Programme Hospitalier de Recherche Clinique (grant AOM 96069).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Service de
Microbiologie, Hôpital Robert Debré, 48 blvd.
Sérurier, 75395 Paris cedex 19, France. Phone: 33 1 40 03 23 40. Fax: 33 1 40 03 24 50. E-mail: edouard.bingen{at}rdb.ap-hop-paris.fr.
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REFERENCES |
| 1.
|
Belgrader, P.,
W. Benett,
D. Hadley,
G. Long,
R. Mariella, Jr.,
F. Milanovich,
S. Nasarabadi,
W. Nelson,
J. Richards, and P. Stratton.
1998.
Rapid pathogen detection using a microchip PCR array instrument.
Clin. Chem.
44:2191-2194[Abstract/Free Full Text].
|
| 2.
|
Bingen, E.,
E. Denamur, and J. Elion.
1994.
Use of ribotyping in epidemiological surveillance of nosocomial outbreaks.
Clin. Microbiol. Rev.
7:311-317[Abstract/Free Full Text].
|
| 3.
|
Bingen, E.,
E. Denamur,
N. Brahimi, and J. Elion.
1996.
Genotyping may provide rapid identification of Escherichia coli K1 organisms that cause neonatal meningitis.
Clin. Infect. Dis.
22:152-156[Medline].
|
| 4.
|
Bingen, E.,
B. Picard,
N. Brahimi,
S. Mathy,
P. Desjardins,
J. Elion, and E. Denamur.
1998.
Phylogenetic analysis of Escherichia coli strains causing neonatal meningitis suggests horizontal gene transfer from a predominant pool of highly virulent B2 group strains.
J. Infect. Dis.
177:642-650[Medline].
|
| 5.
|
Blattner, F. R.,
G. I. Plunkett,
C. A. Bloch,
N. T. Perna,
V. Burland,
M. Riley,
J. Collado-Vides,
J. D. Glasner,
C. K. Rode,
G. F. Mayew,
J. Gregor,
N. W. Davis,
H. A. Kirkpatrick,
M. A. Goeden,
D. J. Rose,
B. Mau, and Y. Shao.
1997.
The complete genome sequence of Escherichia coli K-12.
Science
277:1453-1461[Abstract/Free Full Text].
|
| 6.
|
Bonacorsi, S. P. P.,
O. Clermont,
C. Tinsley,
I. Le Gall,
J. C. Beaudoin,
J. Elion,
X. Nassif, and E. Bingen.
2000.
Identification of regions of the Escherichia coli chromosome specific for neonatal meningitis-associated strains.
Infect. Immun.
68:2096-2101[Abstract/Free Full Text].
|
| 7.
|
Boyd, E. F., and D. L. Hartl.
1998.
Chromosomal regions specific to pathogenic isolates of Escherichia coli have a phylogenetically clustered distribution.
J. Bacteriol.
180:1159-1165[Abstract/Free Full Text].
|
| 8.
|
Desjardins, P.,
B. Picard,
B. Kaltenböck,
J. Elion, and E. Denamur.
1995.
Sex in Escherichia coli does not disrupt the clonal structure of the population: evidence from random amplified polymorphic DNA and the restriction fragment length polymorphism.
J. Mol. Evol.
41:440-448[CrossRef][Medline].
|
| 9.
|
Haedicke, W.,
H. Wolf,
W. Ehret, and U. Reischl.
1996.
Specific and sensitive two-step polymerase reaction assay for the detection of the Salmonella species.
Eur. J. Clin. Microbiol. Infect. Dis.
15:603-607[CrossRef][Medline].
|
| 10.
|
Herzer, P. J.,
S. Inouye,
M. Inouye, and T. S. Whittam.
1990.
Phylogenetic distribution of branched RNA-linked multicopy single-stranded DNA among natural isolates of Escherichia coli.
J. Bacteriol.
172:6175-6181[Abstract/Free Full Text].
|
| 11.
|
Hurtado, A., and F. Rodriguez-Valera.
1999.
Accessory DNA in the genomes of representatives of the Escherichia coli reference collection.
J. Bacteriol.
181:2548-2554[Abstract/Free Full Text].
|
| 12.
|
Johnson, J. R., and A. L. Stell.
2000.
Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise.
J. Infect. Dis.
181:261-272[CrossRef][Medline].
|
| 13.
|
Kuhnert, P.,
J. Nicolet, and J. Frey.
1995.
Rapid and accurate identification of Escherichia coli K-12 strains.
Appl. Environ. Microbiol.
61:4135-4139[Abstract].
|
| 14.
|
Kuhnert, P.,
J. Hacker,
I. Mühldorfer,
A. P. Burnens,
J. Nicolet, and J. Frey.
1997.
Detection system for Escherichia coli-specific virulence genes: absence of virulence determinants in B and C strains.
Appl. Environ. Microbiol.
63:703-709[Abstract].
|
| 15.
|
Lecointre, G.,
L. Rachdi,
P. Darlu, and E. Denamur.
1998.
Escherichia coli molecular phylogeny using the incongruence length difference test.
Mol. Biol. Evol.
15:1685-1695[Abstract].
|
| 16.
|
Mills, M., and S. Payne.
1995.
Genetics and regulation of haem iron transport in Shigella dysenteriae and detection of an analogous system in Escherichia coli O157:H7.
J. Bacteriol.
177:3004-3009[Abstract/Free Full Text].
|
| 17.
|
Ochman, H., and R. K. Selander.
1984.
Standard reference strains of Escherichia coli from natural populations.
J. Bacteriol.
157:690-692[Abstract/Free Full Text].
|
| 18.
|
Orskov, F., and I. Orskov.
1992.
Escherichia coli serotyping and disease in man and animals.
Can. J. Microbiol.
38:699-704[Medline].
|
| 19.
|
Picard, B.,
J. S. Garcia,
S. Gouriou,
P. Duriez,
N. Brahimi,
E. Bingen,
J. Elion, and E. Denamur.
1999.
The link between phylogeny and virulence in Escherichia coli extraintestinal infection.
Infect. Immun.
67:546-553[Abstract/Free Full Text].
|
| 20.
|
Selander, R. K.,
D. A. Caugant,
H. Ochman,
J. M. Mussser,
M. N. Gilmour, and T. S. Whittam.
1986.
Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics.
Appl. Environ. Microbiol.
51:873-884[Free Full Text].
|
| 21.
|
Selander, R. K.,
D. A. Caugant, and T. S. Whittam.
1987.
Genetic structure and variation in natural populations of Escherichia coli, p. 1625-1648.
In
F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C.
|
| 22.
|
Swenson, D. L.,
N. O. Bukanov,
D. E. Berg, and R. A. Welch.
1996.
Two pathogenicity islands in uropathogenic Escherichia coli J96: cosmid cloning and sample sequencing.
Infect. Immun.
64:3736-3743[Abstract].
|
| 23.
|
Torres, A., and S. Payne.
1997.
Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7.
Mol. Microbiol.
23:825-833[CrossRef][Medline].
|
| 24.
|
Whittam, T.
1996.
Genetic variation and evolutionary processes in natural populations of Escherichia coli, p. 2708-2720.
In
F. C. Neidhardt (ed.), Escherichia coli and Salmonella: cellular and molecular biology. American Society for Microbiology, Washington, D.C.
|
| 25.
|
Wyckoff, E. E.,
D. Duncan,
A. G. Torres,
M. Mills,
K. Maase, and S. M. Payne.
1998.
Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria.
Mol. Microbiol.
28:1139-1152[CrossRef][Medline].
|
Applied and Environmental Microbiology, October 2000, p. 4555-4558, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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