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Journal of Clinical Microbiology, March 1999, p. 581-590, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Genetic Diversity and Population Structure of
Vibrio cholerae
Pilar
Beltrán,1
Gabriela
Delgado,1
Armando
Navarro,1
Francisca
Trujillo,1
Robert K.
Selander,2,* and
Alejandro
Cravioto1
Departamento de Salud Pública de la
Facultad de Medicina, Universidad Nacional Autónoma de
México, México, D.F., México,1
and
Institute of Molecular Evolutionary Genetics, Pennsylvania
State University, University Park, Pennsylvania
168022
Received 9 September 1998/Returned for modification 17 November
1998/Accepted 8 December 1998
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ABSTRACT |
Multilocus enzyme electrophoresis (MLEE) of 397 Vibrio
cholerae isolates, including 143 serogroup reference strains and
244 strains from Mexico and Guatemala, identified 279 electrophoretic types (ETs) distributed in two major divisions (I and II). Linkage disequilibrium was demonstrated in both divisions and in subdivision Ic
of division I but not in subdivision Ia, which includes 76% of the
ETs. Despite this evidence of relatively frequent recombination, clonal
lineages may persist for periods of time measured in at least decades.
In addition to the pandemic clones of serogroups O1 and O139, which
form a tight cluster of four ETs in subdivision Ia, MLEE analysis
identified numerous apparent clonal lineages of non-O1 strains with
intercontinental distributions. A clone of serogroup O37 that
demonstrated epidemic potential in the 1960s is closely related to the
pandemic O1/O139 clones, but the nontoxigenic O1 Inaba El Tor reference
strain is not. A strain of serogroup O22, which has been identified as
the most likely donor of exogenous rfb region DNA to the O1
progenitor of the O139 clone, is distantly related to the O1/O139
clones. The close evolutionary relationships of the O1, O139, and O37
epidemic clones indicates that new cholera clones are likely to arise
by the modification of a lineage that is already epidemic or is closely
related to such a clone.
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INTRODUCTION |
For Vibrio cholerae, the
causative agent of cholera and a natural inhabitant of aquatic
environments, the conventional method of identifying and classifying
strains is a serotyping scheme in which nearly 200 serogroups (or
serovars) have been distinguished on the basis of epitopic variation in
the cell surface lipopolysaccharide (LPS) (52). From an
epidemiological standpoint, the species has been divided into serogroup
O1 and serogroup non-O1 strains, which were long believed to differ in
ability to cause epidemic cholera (4, 10). Historically, O1
strains have been responsible for all major epidemics, including seven
pandemics (19), but in 1992 an epidemic clone of serogroup
O139 (Bengal) appeared in southern India (30). It rapidly
spread throughout much of Southeast Asia (1, 9) and reached
western Africa in 1994 (37).
The emergence of the O139 clone with pandemic potential stimulated
increased interest in the molecular basis of pathogenesis in V. cholerae and the degree to which genes determining serotype and
virulence properties are subject to horizontal transfer and recombination among strains (17). Molecular genetic studies have shown that the origin of the O139 clone involved a complex rearrangement of the rfb region in a strain of O1 El Tor,
which included deletion of genes responsible for the biosynthesis and assembly of the side chains of the O1 cell surface LPS and insertion of
exogenous DNA mediating synthesis of the O139 LPS core (2, 3,
40-42) and a capsule (12, 45). The observations that strains with identical nucleotide sequences of the aspartate
semialdehyde dehydrogenase gene (asd) may express different
O antigens and that O1 isolates are heterogeneous in sequence provided
further evidence of the horizontal transfer of genes mediating
O-antigen synthesis (19). Most surprisingly, it was
discovered that the CTX element, which includes the structural genes
(ctxA and ctxB) for the subunits of cholera
toxin, is the integrated genome of a filamentous bacteriophage, CTXø,
and is transmissible (31, 46). Moreover, the bacterial
receptor for CTXø, the toxin-coregulated pilus, is encoded by an
operon (tcp) that is part of a transmissible pathogenicity
island (20, 21). These findings raise the possibility that
all strains of V. cholerae have the potential to become
agents of epidemic cholera.
Previous research on the evolutionary genetics of V. cholerae has been primarily concerned with the identification and
epidemiology of O1 and O139 strains that are responsible for cholera
epidemics and pandemics. This work includes the application of
multilocus enzyme electrophoresis (MLEE) to assess genotypic diversity
in a collection of 181 O1 and 79 non-O1 strains (33) and the
extensive use of this technique, in conjunction with ribotyping and
restriction fragment length polymorphism analysis of the
ctxA gene, to study various aspects of the molecular
epidemiology of cholera in Latin America and elsewhere (8, 15, 43,
44). Additionally, ribotyping and comparative sequence analysis
of the asd gene have been employed to reconstruct the
evolutionary history of O1 clones involved in the sixth and seventh
pandemics (18, 19).
We report here the results of an analysis of 397 isolates of V. cholerae by MLEE undertaken to determine the extent of genetic diversity in the species as a whole, the relationships of the epidemic
O1 and O139 clones to strains of other serogroups, and the genetic
population structure of the non-O1 segment of the species.
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MATERIALS AND METHODS |
Bacterial strains.
This study was based on 397 strains
received as V. cholerae. The sample included 143 strains in
the serogroup reference collection maintained at the National Institute
of Infectious Diseases in Japan (52). These strains were
isolated from worldwide sources in the period from 1932 to 1993; 117 of
them were recovered from humans, 13 from animals, 6 from river water, 3 from seawater, and 3 from unknown sources. The reference strains for
serogroups O1 through O83 were provided by T. Cheasty, and those for
serogroup O155 and serogroups O84 through O140, together with strain
CA-385 (rough), which is used in serotyping, were obtained from T. Shimada.
A collection of 191 strains from Sonora, Tabasco, and 14 other states
in Mexico was provided by the Instituto Nacional de Diagnóstico y
Referencia Epidemiológicos, the Laboratorio Estatal de Salud
Pública (Sonora), and the Laboratorio Regional de Salud Pública (Tabasco). A sample of 53 strains recovered from humans in Guatemala was obtained from the Instituto de Nutrición de Centroamérica y Panamá. Of the total of 244 strains from
Mexico and Guatemala, 172 were recovered from humans, 41 from well and sewage water, 15 from fish, 7 from other environmental sources, 2 from
food, and 7 from unspecified sources.
Five serogroup O139 strains from Thailand were furnished by P. Echeverria. From the Centers for Disease Control and Prevention
(CDC),
we received single isolates of O1 El Tor from Australia,
Romania, Peru,
and Louisiana and an O139 isolate from an imported
human case of
cholera in the United States in
1993.
The strains from Mexico and Guatemala, almost all of which were
isolated in the period from 1991 to 1995, were serotyped in
our
laboratory by the standard method of Sakazaki and Donovan
(
32). Eight of these strains were of a serotype that was not
then represented in the reference collection; for purposes of
this
paper, we have designated this serotype
OA.
All of the strains used in this study have been deposited in the
collection of the Facultad de Medicina, Universidad Nacional
Autónoma de México
(FMU).
MLEE.
MLEE was performed by the methods described by
Selander et al. (34). Seventeen enzyme loci were assayed for
allelic variation: 6PG (6-phosphogluconate dehydrogenase), G6P (glucose
6-phosphate dehydrogenase), IDH (isocitrate dehydrogenase), NSP
(nucleoside phosphorylase), ALD (alanine dehydrogenase), SHK (shikimate
dehydrogenase), CAT (catalase), LAP (leucine aminopeptidase), GOT
(glutamic-oxalacetic transaminase), ME (malic enzyme), MDH (malate
dehydrogenase), PLP (phenylalanyl-leucine peptidase), PGI
(phosphoglucose isomerase), HEX (hexokinase), PGM (phosphoglucomutase),
IPO (indophenol oxidase), and THD (threonine dehydrogenase).
Electromorphs (mobility variants) were equated with alleles, an absence
of enzyme activity was scored as a null allele, and
distinctive allele
profiles for the 17 loci were designated electrophoretic
types
(ETs).
Statistical analysis.
From the allele profiles of the ETs,
mean genetic diversity per locus (H) and pairwise genetic
distance were calculated as described by Selander et al.
(34), and dendrograms were constructed by the unweighted
pair-group method with arithmetic mean (UPGMA). As a measure of
multilocus population structure (linkage disequilibrium), we calculated
an index of association of alleles (IA) by using the equation 1 
VO/VE, where
VO is the variance of the observed distribution
of number of mismatched alleles between ET pairs and
VE is the mismatch variance expected when allele
associations are random (linkage equilibrium) (24, 49).
Calculations were made with computer programs written by T. S. Whittam (Pennsylvania State University, University Park).
Detection of the ctxA gene.
Tests for the
presence of the ctxA gene, which encodes a subunit protein
of cholera toxin, were performed by colony blot assay with a
digoxigenin-labeled oligonucleotide probe, according to the procedure
of Maniatis et al. (23), and by PCR amplification. The
nucleotide sequences of the probe and the primers were those specified
by Shirai et al. (39).
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RESULTS |
Serogroups.
Of the 244 strains from Mexico and Guatemala, 230 were of 59 serogroups and 14 were serologically nontypeable (NT). The
reference collection currently includes nearly 200 serogroups
(52), and thus about 30% of the described serotypic
diversity of the species is exhibited by the Mexico-Guatemala strains.
In this paper, individual reference strains are referred to by their
serogroup designations, marked with an asterisk (e.g.,
O155*).
Multilocus enzyme genotypes.
All 17 loci assayed were
polymorphic, with an average of 9.5 alleles per locus and a range of 4 (IPO) to 15 (CAT and LAP). Among the total sample of 397 isolates, 279 ETs, each representing a distinctive allele profile, were
distinguished. The 142 serogroup reference strains were of 133 ETs, and
the 244 Mexico-Guatemala strains were assigned to 154 ETs.
For each of the 17 enzymes assayed, an absence of activity was rare.
Among the 279 ETs, only 26 null alleles, distributed
over 12 of the
loci, were recorded. Thus, only 0.5% of a total
of 4,743 (17 × 279) alleles scored were nulls, and 10 of them
were represented in the
profiles of the two strains (ETs 276 and
277) that form evolutionary
subdivision x (see
below).
The mean genetic diversities per locus were 0.436 for all ETs and 0.430 for the ETs of the reference strains (Table
1).
Estimates of genetic distance among the 279 ETs are summarized in the
dendrograms presented in Fig.
1 and
2. All but four
of the ETs are members of
two major divisions (designated I and
II) that diverge from one another
at a distance of about 0.7,
which roughly corresponds to an 11-locus
difference (Fig.
1).
In addition, there are two lineages (labeled x and
y) that branch
from the major divisions and from one other at distances
greater
than 0.9. Each of these lineages includes two distantly related
ETs.
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FIG. 1.
Dendrogram showing genetic relationships of the ETs of
V. cholerae, based on MLEE analysis (17 loci). The
dendrogram was constructed from a matrix of pairwise genetic distances
by the UPGMA method. The lineages of subdivision Ic and of groups A, B,
and C of subdivision Ia are truncated. The relationships of the 37 ETs
in group A are shown in the dendrogram in Fig. 2.
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FIG. 2.
Dendrogram showing genetic relationships of the 37 ETs
of V. cholerae in group A of subdivision Ia of division I,
based on MLEE analysis (17 loci). The dendrogram was constructed from a
matrix of pairwise genetic distances by the UPGMA method.
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Division II consists of 12 ETs, each of which is represented by a
single serogroup reference strain. Seven of these strains
are from
India and the Far East, as follows: India, two strains,
both from
humans; China, one human isolate; the Philippines, one
human strain;
and Japan, two strains, from a crab and from river
water. Three strains
(O114*, O115*, and O116*) were cultured from
river water (1979), a
human (1980), and seawater (1978) in the
United States; one isolate
(O71*) was recovered from a bird in
Denmark (1978); and one strain
(O29*) was isolated from a human
at an unspecified locality
(1968).
The vast majority of strains are of ETs in division I, in which for
purposes of reference we have designated six subdivisions
(Ia to If),
each consisting of a single ET or a group of related
ETs.
Most of the 51 strains of the 40 ETs that form subdivision Ic are from
Mexico and Guatemala, but 8 strains (representing 8
ETs) are from the
serogroup reference collection, as follows:
O50*, O75*, O78*, O82*, and
O126* from India; O155* from Thailand;
and O107* and O92* from
Japan.
Subdivision Ia includes 82% of the reference collection strains and
78% of the Mexico-Guatemala strains. All but 10 of the
214 ETs in this
subdivision form three branches (designated A,
B, and C in Fig.
1) that
diverge from one another at a genetic
distance of about 0.4. With the
singular exception of the O1 Inaba
El Tor reference strain (ET 259, in
subdivision Ie), all O1 strains
in our sample, together with all O139
isolates, are of 4 ETs that
form a tight cluster in group A of
subdivision Ia, which consists
of a total of 37 ETs (Fig.
2).
Multilocus population structure.
Estimates of multilocus
association of alleles for all 279 ETs and for the ETs in several
segments of the dendrograms (Fig. 1 and 2) are shown in Table 1. For
the total sample of 279 ETs, the 133 ETs of the reference strains, and
the ETs of each of the divisions I and II and subdivision Ic, there is
evidence of significant nonrandom associations of alleles (linkage
disequilibrium), but allele associations are not demonstrably nonrandom
for the ETs of groups A, B, and C in subdivision Ia.
Relationships of O1 and O139 epidemic strains.
With allowance
for a difference in the panels of enzymes employed in MLEE analysis,
our findings for the epidemic O1 and O139 strains are fully consistent
with those reported by Evins et al. (15) and in earlier
studies by Wachsmuth's group at the CDC (43, 44). These
workers assayed variation in 16 enzymes, only 9 of which were included
in our panel of 17 enzymes. In the interest of consistency, we have
numbered the ETs of our O1 and O139 isolates to correspond to the ET
designations of the CDC group (Table 2; Fig. 2).
ET 4 is represented by 13 isolates of O1 Inaba El Tor. Eleven of these
strains are from Mexico (Quintana Roo, Campeche, Tabasco,
Veracruz,
Puebla, and Hidalgo), one is from Guatemala, and one
is from Peru. ET 4 marks the original, or first-wave, Latin American
epidemic clone
(
15).
ET 3 includes 2 strains of O1 Inaba El Tor, 38 strains of O1 Ogawa El
Tor, and 7 strains of O139. The two Inaba El Tor strains
(FMU strains
90501 and 90500) were recovered from humans in Tabasco
in 1991 and
1993. The sample of O1 Ogawa El Tor isolates includes
the reference
strain, 35 isolates from Mexico (Tabasco, Morelos,
and the state of
México), a strain from Australia, and an isolate
from Romania. ET
3 is the seventh-pandemic type, a clone that
in Latin America was first
identified in Mexico in 1991 and is
now widely distributed in Mexico
and Central America (
15). The
Australian isolate (CDC
2463-88) was distinguished, as ET 1, from
isolates of ET 3 by Evins et
al. (
15) on basis of its carrying
a different allele for
diaphosrase 1. ET 1 marks a distinctive
Australian toxigenic clone
(
13,
43).
Included in ET 2 are an O1 Inaba El Tor isolate (CDC 2164-78) collected
in Louisiana in 1978 and an O1 Inaba classical strain
(FMU 87295/0)
recovered from a tourist returning to the United
States from
Cancún, Quintana Roo, in 1983. ET 2 marks a toxigenic
clone that
is endemic to the Gulf Coast of Mexico and the United
States
(
15).
With our panel of 17 enzymes, the sole basis for distinguishing ETs 2, 3, and 4 is allelic variation at the LAP locus (Table
2). One O1 Ogawa
El Tor isolate (from Tabasco) is ET 3.1, which
differs from ET 3 in
having a distinctive G6P allele. This variant
genotype was not detected
in previous
studies.
Relationship of serogroup O37 strains.
The reference strains
O37* (India, 1969) and O102* (China, 1988), both of which are of ET 5 (Fig. 2), differ from strains of the O1/O139 cluster (ETs 2 to 4) at a
single locus, that for PGI, and share the LAP 4 allele with strains of
ET 2 (Table 2). A second O37 strain (from Guatemala) is of ET 6, which
differs from ET 5 in having a 4 allele rather than a 3 allele at the
GOT locus. Two other O37 isolates in the collection, both of which were
cultured from well water in Campeche, represent ETs 75 and 149, which
are in group B of subdivision Ia.
Serotypic diversity among non-O1 strains of the same ET.
In
addition to ETs 2 to 4 of the epidemic O1 and O139 clones, MLEE
identified nine ETs that are each represented by non-O1 isolates from
different continents or other major land masses (Table
3). In all cases, the strains from
different continents are of different serogroups. For example, ET 196 (a member of group C of subdivision Ia) is represented by a total of
seven isolates, including four (O15*, O47*, O51*, and O53*) recovered from humans in India between 1968 and 1974, a strain (O68*) obtained from seawater in Japan in 1978, and two isolates (both O7) cultured from well water in Campeche and a fish in Sonora in 1992 and 1993. The
inference is that ET 196 marks a widely distributed clone that had
persisted for at least 24 years, with several modifications in serotype
through mutation or recombination of genes of the rfb
region.
ETs 128 and 131 are each represented by a strain collected in India in
1968 and an isolate recovered in Campeche or Guatemala
in the early
1990s. It is noteworthy that ET 131 differs at only
a single locus
(that for LAP) from ET 132 of the O14 reference
strain, which was
recovered in India in 1964 (see Table
5). Thus,
ETs 131 and 132 are
members of a clonal lineage that was extant
for at least 28
years.
Nine ETs are represented by pairs or multiple strains from different
states in Mexico (Table
4). Five of these
ETs are represented
by strains of the same serogroup, and four of them
are represented
by strains of different serogroups. For example, ET 16 was represented
by seven O5 isolates from Hidalgo, Tabasco,
Yucatán, and an unspecified
locality in Mexico. Significantly, ET
16 differs by only one locus
(that for PLP) from ET 17 of the O5
reference strain, which was
collected in India in 1964 (see Table
5).
Serotypic diversity among strains of closely related ETs.
We
identified 19 cases in which pairs of ETs that differ at a single
enzyme locus are represented by strains collected on different
continents or major land masses (Table
5). In four of these cases, the strains
are of the same serotype; these are O5* from India (1964) and seven O5
isolates from Hidalgo and other states in Mexico (1991 and 1992), O14*
from India (1964) and O14 from Guatemala (1993), O37* from India (1969)
and O37 from Guatemala (1993), and O44* from India (1973) and O44 from
Chiapas (1991). In all other cases, the strains are of different
serotypes or one of them was NT.
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TABLE 5.
Serogroups and geographic sources of non-O1 strains
representing pairs of ETs that differ at a single locus
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Genetic diversity within serogroups.
MLEE analysis
demonstrated that strains of the same serogroup may belong to two or
more widely divergent ET lineages. Thus, for example, strains of
serogroup O29 were assigned to four ETs that occur in division I (ET 50 and ET 52 in group B and ET 173 in group C) and division II (ET 274),
and O53 strains are found in group C of division I (ET 196) and also in
lineage y (ET 278). Some estimated levels of genetic diversity between
or among the ETs of strains of the same serogroup are shown in Table
6. In several cases, the estimated
diversity is at least equivalent to that obtained for the 279 ETs
identified among all 397 isolates.
Distribution of the ctxA gene.
When tested with
the ctxA probe, 13 of the 143 reference strains were
positive, as shown in Table 7. With PCR
amplification of the gene, the same strains were positive, with the
exception of O1 Inaba El Tor*. Among 104 of 254 nonreference strains
that were randomly selected for testing, 2 isolates of O1 Ogawa El Tor,
3 isolates of O1 Inaba El Tor, an isolate of O1 Inaba classical, and an
isolate of O6 were positive for ctxA by both colony blot assay and PCR amplification.
All but 3 of the total of 21
ctxA-positive strains are
members of subdivision Ia of division I (Fig.
1 and
2); the exceptions
are the reference strains O1 Inaba El Tor*, in subdivision Ie,
and
O135* and O138*, in division
II.
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DISCUSSION |
Species limits.
Strains of ETs 276 to 279 in the deep lineages
x and y (Fig. 1) are sufficiently differentiated from all other strains
as to raise the question of whether they should be included in the species V. cholerae. It is likely that assessment of genomic
relatedness by DNA-DNA hybridization would show relative degrees of
annealing with other strains somewhat below the 70% standard adopted
for species inclusion by the CDC (6).
Genetic diversity.
The estimate of 0.436 for the mean genetic
diversity per locus among the 279 ETs of V. cholerae
represented in the present study is larger than the comparable value of
0.343 reported for the Escherichia coli reference collection
(35) but smaller than the corresponding value of 0.627 obtained for Salmonella enterica (36). In a
previous MLEE study of allelic variation at 13 enzyme loci among 260 isolates of V. cholerae (most of which were serogroup O1),
Salles and Momen (33) detected an average of 4.3 alleles per
locus, identified 73 ETs, and estimated the mean genetic diversity per
locus as 0.326.
Genetic structure of populations.
Comparisons of the observed
and expected variances of the mismatch distributions for ETs at several
hierarchical levels of dendrogram structure yielded only limited
evidence of linkage disequilibrium (Table 1). The cases in which the
observed variance exceeded the upper 95% confidence limit of the
variance expected under random association of alleles were those
involving all 279 ETs, the 275 ETs of divisions I and II combined, the
263 ETs of division I, the 12 ETs of division II, and the 40 ETs of
subdivision Ic of division I. Within each of the groups A, B, and C of
subdivision Ia, which include 204 ETs (Fig. 1), significant levels of
nonrandom association were not demonstrable. The inference is that, at
least among the strains of ETs in subdivision Ia, the rate of
horizontal transfer and recombination of housekeeping enzyme genes is
sufficiently high to prevent the development and long-term maintenance
of distinctive allele complexes. On the whole, it seems likely that the
frequency of recombination, both intragenic and assortative
(50), of housekeeping genes in V. cholerae is
somewhat higher than in either E. coli (48) or
S. enterica (36), a conclusion also reached by
Karaolis et al. (19) from a comparative sequence analysis of
the asd gene. But even within subdivision Ia, clonal
lineages may persist for periods of time measured in at least decades.
The most obvious examples are the epidemic and pandemic strains of
serogroups O1 and O139 (ETs 2 to 4), but our analysis identified
numerous clones and clonal lineages of non-O1 strains with widespread,
if not global, distributions (Tables 3 to 5).
A factor that has not been evaluated in studies of the genetic
structure of bacterial populations on the basis of MLEE data
is the
convergent evolution of electromorphs, which cannot be
equated with
isoalleles. Similarity in electrophoretic mobility
resulting from
convergence in the net electrostatic charge of
an enzyme will lessen
the likelihood that linkage disequilibrium
is detected from the
analysis of MLEE data. Studies of sequence
variation in several
housekeeping enzymes among multiple strains
of
E. coli and
S. enterica (
5,
26-28,
47) have shown that
individual electromorphs may exhibit substantial heterogeneity
in amino
acid sequence, much of which clearly stems from convergence
rather than
mutational divergence from a common ancestral
sequence.
There was already evidence for recombination of genes of the
rfb region of
V. cholerae, based on studies of
relatively small
numbers of strains and serogroups. Our observation
that strains
of the same serogroup frequently are found in divergent,
even
distantly related, lineages supports earlier evidence (
2,
19)
that the
rfb genes are subject to horizontal
transfer and further
suggests that this process occurs with relatively
high frequency.
Convergence in serotype is, of course, an alternative
explanation,
but reasoning by analogy from the lack of evidence for
convergence
in epitope structure in the serologically diverse
flagellins of
S. enterica (
22), we favor the
first hypothesis. The issue can
be settled by comparative sequencing of
the epitope-encoding segments
of the
rfb region.
The fact that strains of the same ET may express different O antigens
can be explained by recombination or by spontaneous
mutation of the
genes encoding O somatic
properties.
Epidemic non-O1 clones.
There are two examples of epidemic
V. cholerae expressing a non-O1 antigen. The first is the
serogroup O139 clone, which emerged in India and Bangladesh through
modification of the El Tor O1 pandemic strain by acquisition of genes
mediating the synthesis of the O139 LPS and a polysaccharide capsule.
The second is the O37 strain that reportedly was responsible for a
large outbreak of cholera in the Sudan in 1968 (54). By
IS1004 fingerprinting, Bik et al. (3) determined
that an O37 isolate from the Sudan is closely related to classical O1
strains. The O37 reference strain (ET 5), which was recovered from a
patient in India in 1969, presumably represents the same clone as the
O37 Sudan strain. As determined by MLEE, it is closely related to O1 El
Tor and other epidemic O1 strains (Fig. 2), thus confirming the result obtained by IS1004 fingerprinting. In fact, ET 5 is
distinguished from ETs 2 to 4 of the O1/O139 cluster solely by
possession of a 4 allele (versus a 3 allele) at the PGI locus. It
carries the ctxA gene and expresses cholera toxin. Yamamoto
et al. (53) reported that the amino acid sequence of the
cholera toxin produced by O37 strain S7 differs from that of most O1
strains in having single substitutions in both the CtxA and CtxB
segments (29), which are presumed to cause the formation of
an unusually large subunit B oligomer. Recently, Karaolis et al.
(20) reported that, almost uniquely among non-O1 strains, a
Sudan 1968 outbreak strain carries a chromosomal pathogenicity island
that is characteristic of epidemic and pandemic strains.
Honma et al. (
16) studied an O37 isolate that produces a
hemolysin (O37-Hly) that is antigenically similar to O1 El Tor
hemolysin (El Tor-Hly) but different in molecular size, hemolytic
activity, and glucose-binding capacity. The gene encoding O37-Hly
differs from that encoding O1 El Tor-Hly by the presence of a
4-bp
insertion that generates a premature stop codon in the downstream
region. Thus, the O37-Hly is a truncated derivative of O1 El Tor-Hly,
sharing 90% of the N-terminal
region.
In the Mexico-Guatemala collection, there are three O37 isolates, one
of which (ET 6) is almost identical in MLEE genotype
(it carries a GOT
4 rather than a GOT 3 allele) to the O37 reference
strain (ET 5) from
India but apparently lacks the
ctxA gene. It
was isolated
from a patient in Guatemala. The two other O37 isolates,
both of which
were cultured from well water in Campeche, are distantly
related (six-
and seven-locus differences) to both the reference
O37 and Guatemala
O37 strains, as well as to one another (four-locus
difference), and
neither one carries the
ctxA gene.
It is noteworthy that strain O102*, which was recovered from a patient
with diarrhea in China in 1988, is identical in MLEE
genotype (ET 5) to
strain O37* but apparently does not carry the
ctxA gene.
In sum, there is a clone of serogroup O37 that has epidemic potential
and was present in Africa and India in 1968 and 1969.
Because it is
closely related to O1 El Tor and the other O1 pandemic
clones, it
apparently represents a case similar to that of the
O139 clone, in
which an already-established pathogenic lineage
of serogroup O1
acquired a new serotype by horizontal DNA transfer
and rearrangement of
the
rfb region genes. The O37 strain from
Guatemala may be
an offshoot of this clone in which the CTX genetic
element has been
deleted. The two O37 strains from Campeche presumably
have independent
acquisitions of the O37 polysaccharide gene
region.
O1 Inaba El Tor reference strain.
The O1 Inaba El Tor
reference strain (ET 259), which does not produce cholera toxin
although it carries at least part of the ctxA gene, is not
closely related to the O1/O139 cluster of pandemic strains or to the
toxigenic O37 clone (ET 5). According to T. Shimada (38a),
the reference strain is the NIH 35-a-3 isolate listed by Burrows et al.
(7) as one of the strains used for vaccine preparation by
the U.S. Army in the early 1940s. It was received from the Central
Research Institute in Kasauli, India, in 1942, without indication of
collection date or source of isolation. Perhaps it is related to the O1
strain that caused a cholera-like disease in Hong Kong in the 1950s
(43).
Relationships of serogroup O1 strains.
Colwell et al.
(11) hypothesized that non-O1 cells may convert to the O1
serotype and vice versa under suitable conditions, a possible strategy
for survival in the environment. As noted earlier, O37* and O102* (both
of ET 5) may represent cases in which O1 clones have acquired new
serotypes. In our collection, the only apparent case of conversion of a
non-O1 strain to the O1 serotype (apart from O139) is the O1 Inaba El
Tor reference strain (ET 259), which occurs in subdivision Ie (Fig. 1)
and is distantly related to the O1 epidemic strains (ETs 2 to 4) in
group A of subdivision Ia (Fig. 2).
Source of rfb region DNA in the emergence of the
epidemic O139 clone.
The putative source of the exogenous
rfb region DNA that was involved in the transformation of an
O1 El Tor strain to the epidemic O139 clone has been identified as a
strain of serogroup O22, O141, or O155 on the basis of serotypic
cross-reactions with O139 (2, 38, 42). Molecular analysis
showed that, in common with O139, they have two open reading frames in
the rfaD region that are lacking in O1 strains.
Through study of the gene content and organization of the
rfb region adjacent to IS
1358 in strains of O139
and 13 other serogroups,
including O22 and O155, Dumontier and Berche
(
14) recently identified
the clone represented by strain
O22* (Shimada strain 169-68, from
India) as the most likely donor,
although the possibility of a
multistep rearrangement in the recipient
O1 strain cannot be excluded.
As determined by MLEE analysis, O22* (ET
128) falls in group B
of subdivision Ia and differs in ET from the
epidemic O1 and O139
strains at four or five
loci.
In our sample of strains, there were nine serogroup O155 isolates
belonging to seven ETs. ET 224 of the O155 reference strain
(Thailand,
1993) and five other ETs (represented by isolates from
Tabasco and
Campeche) are in subdivision Ic, where, however, they
do not form a
tight cluster. The remaining ET, which is represented
by two isolates
from Sonora, is in group B of subdivision Ia.
Thus, strains of
serogroup O155 belong to a moderately diverse
group of ETs, none of
which is closely related to the ETs of the
epidemic O1 and O139 clones.
This suggests that genes mediating
expression of the O155 LPS antigen
are transferred with relatively
high
frequency.
Genesis of epidemic clones.
Because genes for the major
virulence factors can be transferred horizontally and antigenic
conversion can be achieved by the acquisition and loss of
rfb genes, there is the formal possibility that any V. cholerae cell could be transformed into a virulent strain, even an
epidemic one (19, 25). However, the close evolutionary
relationships of the O1, O139, and O37 epidemic clones indicate that
new epidemic or other strongly virulent clones are likely to arise by
the modification of a lineage that is already epidemic or is closely
related to such a clone. Thus, O139 evolved from an El Tor O1 clone by
acquisition of a transposon carrying genes for the O139 LPS and a
polysaccharide capsule and deletion of most of the genes mediating
synthesis of the serotype O1 LPS (see the review by Rubin et al.
[31]). Also, the toxigenic O37 clone that caused
outbreaks in the Sudan and India in the late 1960s is closely related
to the cluster of O1/O139 epidemic clones. Analogously, E. coli O157:H7, which emerged as an agent of hemorrhagic colitis by
acquisition of the O157 antigen, a Shiga-like toxin, and the
enterohemorrhagic E. coli plasmid, is an evolutionary derivative of an O55:H7 clone that is associated with infantile diarrhea (51).
| |
ACKNOWLEDGMENTS |
We thank Thomas Cheasty, Peter Echeverria, Alma Rosa
González, Sergio León, Claudio Lezana, José Luis
Navarro-Heinze, and Toshio Shimada for supplying strains and Delia
Licona and José Luis Méndez for technical assistance in the laboratory.
This research was supported by grants from the Consejo Nacional de
Ciencia y Tecnología (project 2397PB); the Dirección General de Apoyo al Personal Académico, UNAM (project IN211496); and the National Institutes of Health (AI-22144).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Molecular Evolutionary Genetics, Mueller Laboratory 516, Pennsylvania State University, University Park, PA 16802. Phone: (814) 234-8997. Fax: (814) 863-4706. E-mail: rks3{at}psu.edu.
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Abstract
Introduction
