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Journal of Clinical Microbiology, February 2001, p. 782-786, Vol. 39, No. 2
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.782-786.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification of a Cluster of Strains Bearing a
New Adhesin among Genetically Diverse Enterotoxigenic Escherichia
coli Isolates of Serogroup O20
Mariana
Pichel,1
Norma
Binsztein,1
Gabriel
Gutkind,2 and
Gloria
Viboud1,*
Departamento de Bacteriología,
Instituto Nacional de Enfermedades Infecciosas
ANLIS "Carlos G. Malbrán",1 and Cátedra de
Microbiología, Facultad de Farmacia y Bioquímica,
Universidad de Buenos Aires,2 Buenos Aires,
Argentina
Received 28 July 2000/Returned for modification 22 October
2000/Accepted 18 November 2000
 |
ABSTRACT |
About one-third of the enterotoxigenic Escherichia coli
isolates lack any of the known colonization factors. Among this group, those of serogroup O20 are the most frequently isolated in Argentina. By combining analysis of adhesion to Caco-2 cells, random amplified polymorphic DNA, and pulsed-field gel electrophoresis techniques, we
were able to identify three sets of closely related strains with
different binding properties. Further analysis of the most prevalent
group revealed that all these isolates expressed the recently described
adhesin CS22.
| |
TEXT |
Enterotoxigenic Escherichia
coli (ETEC) is one of the leading causative agents of infantile
diarrhea, especially in developing countries (4). Adhesion
of the bacteria to the small intestine by means of colonization factors
(CFs) precedes the production of heat-labile and/or heat-stable (ST)
enterotoxins, which induce diarrhea (2). Several
serogroups have been most frequently associated with ETEC isolates,
including O6, O78, O8, O127, O153, O159, O20, and others (3,
18).
In Argentina, strains belonging to serogroup O20 have been recovered
from children with diarrhea in different epidemiological studies
(1, 13, 15). However, in contrast with what has been
observed in other regions (18), strains belonging to this serogroup did not produce any of the known CFs (1, 13,
15). In fact, among the CF-negative strains, which constitute
between 30 and 40% of the ETEC isolated in this region (1, 13,
15), serogroup O20 was the most frequent. Although CS18
(formerly known as PCFO20) was identified among these isolates, its
prevalence was found to be low in this area.
Certain phenotypic characteristics, i.e., O:H type, biotype, toxin, and
CF profiles, have been shown to be associated in particular combinations among ETEC isolates (4, 18). These
observations lead to the suggestion that ETEC strains sharing these
phenotypic traits owe their similarity to recent descent from a common
ancestor (8). Moreover, this notion has been confirmed by
analysis of electrophoretic patterns generated by randomly
amplified polymorphic DNA (RAPD) and by pulsed-field gel
electrophoresis (PFGE) techniques (9, 17).
As a strategy to identify prevalent "new" adhesins among a group of
O20 CF-negative ETEC strains isolated in Argentina, we determined the
genetic relationship among these strains and evaluated the adhesive
properties of the isolates belonging to different clusters. By means of
a combination of DNA typing approaches and adhesion to Caco-2 cells, we
could identify a genetically related, yet not identical, group of
strains that bear the recently described adhesin CS22
(10).
Phenotypic traits.
A total of 19 well-characterized ETEC
strains belonging to serogroup O20 were selected for this study. They
had been isolated from children with diarrhea or from healthy controls
from different regions of Argentina (Table
1).
As in other parts of the world, most of the O20 ETEC isolates examined
produced ST, as determined by ganglioside GM1-enzyme-linked immunosorbent assay (1), and most of them were nonmotile,
except for three H34 strains, one H32 strain, and two strains with
undetermined flagellar antigen (H?). Dot blot tests using specific
monoclonal antibodies or polyclonal antisera against CFA/I, CS1 to CS8,
CS12, CS14, CS15, CS17, CS18, CS19, or CS20 showed that most of the isolates lacked all these CFs, except for one strain that produced CS6
and another one expressing CS18 (Table 1).
To evaluate whether these strains expressed any unidentified adhesin,
the capacity to adhere to intestinal epithelial cell line Caco-2 was
evaluated as previously described (10). The assay was
performed at least twice for each strain, and the result was expressed
as the average number of bacteria/cell ± the standard deviation.
A strongly adherent ETEC strain (350C1A, expressing CS12)
and its corresponding CF-deficient mutant (350C1B) were included as controls, and their adhesion indexes were 9.25 ± 0.35 bacteria/cell and 0.44 ± 0.08 bacteria/cell, respectively. Most of the
O20 ETEC strains bound to the epithelial cells, with adhesion indexes
ranging from 2.75 ± 1.75 bacteria/cell to 35.00 ± 3.15 bacteria/cell (Table 1; Fig. 1). However,
three of the isolates (ARG-12, ARG-16, and ARG-17) exhibited low
adhesion indexes, comparable to those of the negative control.
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FIG. 1.
Micrographs showing adhesion of ETEC strains ARG-11 (A)
and ARG-19 (B) to intestinal Caco-2 cells.
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|
Genotypic analysis.
To evaluate the genetic diversity among
the O20 ETEC isolates, we used RAPD and PFGE as molecular typing
methods. The RAPD assay was carried out as previously described
(9) using primer 1254 (CCGCAGCCAA). Relatedness
among RAPD or PFGE patterns was estimated by applying the Jaccard
coefficient (6); data recording and calculations were
performed as described previously (9). Twelve different
RAPD types were defined among the strains studied (Table 1; Fig.
2). The degree of similarity among the
various RAPD types was calculated, and a phenogram was constructed
based on the UPGMA method (Fig. 2). Thus, three main groups of
genetically related strains could be defined. The first group (cluster
I) included almost 30% of the CF-negative strains studied, which were
all nonmotile and produced ST. All of these strains exhibited an
identical RAPD pattern, yet the group included isolates from different
geographical regions, recovered at different time periods (Table 1).
The second group (cluster II) included two O20:H34 strains along with
an O20:H
strain. These strains exhibited very similar electrophoretic
patterns, sharing more than 78% of the bands. Group III comprised two
ST O20:H? strains and one nonmotile strain that shared the same band
profile and had been isolated in a one-year period from two different
locations. The remaining eight isolates showed different patterns, with
less than 72% of the bands in common. The discriminating ability of
RAPD was determined to be 0.89 using Simpson's diversity index (DI),
as recommended by Hunter and Gaston (5).
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FIG. 2.
(A) RAPD patterns exhibited by O20 ETEC strains isolated
in Argentina; (B) phenogram representing groups of related ETEC
strains. The relatedness among the isolates was estimated based on the
proportions of shared bands, which are indicated below the figure.
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|
For PFGE analysis, agarose plugs were cast by mixing equal volumes of
bacterial suspensions and 1.6% Pulse Field Certified Agarose (Bio-Rad)
and incubated overnight at 50°C in lysis buffer containing 0.5 mg of
proteinase K/ml. The plugs were subsequently washed before digestion
with 20 U of XbaI. Electrophoresis was performed in a
CHEF-DR III chamber (Bio-Rad) for 21 h at 6 V/cm, 14°C, with a
linear pulse ramp of 5 to 50 s.
The XbaI-digested chromosomal DNA gave rise to 16 different
restriction patterns (Table 1; Fig. 3), with fragments of about 40 to
450 kb. Analysis of the shared bands revealed a higher degree of
heterogeneity (DI = 0.976) than that shown by RAPD. However, in
accordance with the results of the RAPD analysis, the same groups of
closely related isolates could be defined (Fig.
3).
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FIG. 3.
(A) PFGE profiles obtained with the XbaI
enzyme; (B) phenogram representing groups of related ETEC strains. The
relatedness among the isolates was estimated based on the proportions
of shared bands, which are indicated below the figure.
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|
The six isolates belonging to group I that were shown to be identical
by RAPD gave rise to five electrophoretic patterns in PFGE.
Nevertheless, these strains appeared in the same branch of the
phenogram, sharing on average 75.5% of the bands (minimum value, 53%;
maximum value, 100%). Recent guidelines for interpreting DNA
restriction patterns produced by PFGE (12) suggest that two isolates could be considered unrelated if they share less than 50%
of the fragments present in their patterns (typically fewer than seven
bands). Accordingly, we considered the O20:H strains included in
group I, which shared at least 53% of their bands, to be genetically related.
Strains in group II showed three different patterns, which shared more
than 72% of the bands (minimum value, 72.6%; maximum value, 79%),
and the isolates included in group III exhibited a common band profile.
The rest of the strains exhibited single electrophoretic profiles, with
less than 33.4% common bands.
Relation between adhesion capacity and genetic relatedness.
Strains within the same genetic cluster appeared to exhibit similar
adhesion indexes, those of group II being the highest (between
15.75 ± 3.15 and 35.00 ± 3.15 bacteria/cell) and those of
group III being the lowest ones (0.58 ± 0.10 to 6.75 ± 1.05 bacteria/cell). The isolates comprised in group I showed quite homogeneous adhesion indexes, ranging from 5.50 ± 0.70 to
12.50 ± 2.10 bacteria/cell. Further characterization of this
group of strains led us to the identification of the recently reported CS22 adhesin (10). This adhesin, originally identified and
characterized in strain ARG-3, was later found in all of the strains
belonging to group I, as determined by dot blot and Western blot assays using specific anti-CS22 antisera as described previously
(10).
Several studies have shown that O:H serotypes represent good indicators
of the clonal relatedness among ETEC strains (7, 9).
However, in the case of H-negative types, genotypic methods are
necessary to evaluate genetic diversity. In this work, we show that
despite sharing several phenotypic traits (toxin profile and
serogroup), the Argentinean O20 ETEC isolates studied herein exhibited
a high degree of heterogeneity by both RAPD and PFGE. It has been
postulated that the nonmotile strains derive from the motile ones by
mutational loss of the flagella (11). Since most of the
strains analyzed herein are nonmotile, these results suggest that these
isolates probably derive from ancestors belonging to different H types.
Thus, strain ARG-18, included in group II with two H34 isolates,
probably derives from an H34 ancestor. On the other hand, the origin of
the strains O20:H pertaining to the main cluster could not be
assessed, since they were not related to isolates expressing the
flagellar antigens H32 or H34.
The majority of the O20 ETEC strains tested did not bear any of the
known CFs. However, most of them were able to adhere to human
intestinal epithelium cells with a certain binding profile (weak,
moderate, or strong), suggesting that they might produce previously
undescribed adhesins. Although strains from genotype II showed the
strongest adhesion indexes, the fact that the group was comprised of
only a few isolates makes it not so relevant as a candidate for
identification of a new CF. On the other hand, strains from genotype I,
which was the most frequently isolated type, presented a moderate
adhesion index, and they all were found to express the recently
identified CS22 (10). In fact, this new adhesin was found
in almost 60% of the CF-negative O20:H ST-ETEC strains isolated in
Argentina. Since this group included strains isolated at different time
periods from very distant locations, it is unlikely that they were
epidemiologically related.
The fact that adhesion is the first step in ETEC infection makes CFs
good candidates for vaccine antigens. However, since the occurrence of
the various ETEC adhesins varies significantly within the different
geographical areas, it is important to identify the more abundant CFs
in regions where ETEC vaccines need to be implemented. We found the
approach presented in this work valuable as a start point for
identifying new CFs within a population of ETEC strains showing similar
phenotypic traits.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from Centro
Argentino-Brasileño de Biotecnología (CABBIO), and
Fundación "Alberto J. Roemmers."
We thank L. C. Souza Ferreira and A. B. F. Pacheco for
very helpful discussions; A.-M. Svennerholm for providing monoclonal antibodies against ETEC CFs; A. Darfeuille-Michaud and H. Sommerfelt for reference strains and antisera against CS15, CS19, and CS20; I. Ørskov and F. Ørskov for serotyping the ETEC strains; A. Lewis for
culturing the Caco-2 cells; and G. Lafuente Devier, A. Garbini, and N. Martinez for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Molecular Genetics and Microbiology, School of Medicine, State
University of New York at Stony Brook, Stony Brook, NY 11794-5222. Phone: (631) 632-8783. Fax: (631) 632-9797. E-mail:
gviboud{at}ms.cc.sunysb.edu.
| |
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Journal of Clinical Microbiology, February 2001, p. 782-786, Vol. 39, No. 2
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.782-786.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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