Genetic relationships between clinical and environmental Vibrio cholerae isolates based on multilocus enzyme electrophoresis

Abstract

A total of 107 isolates of Vibrio cholerae, including 29 strains belonging to serogroup O139, were studied using multilocus enzyme electrophoresis (MLEE) to determine allelic variation in 15 housekeeping enzyme loci. All loci were polymorphic and 99 electrophoretic types (ETs) were identified from the total sample. No significant clustering of isolates was detected in the dendrogram generated from a matrix of coefficients of distances with respect to serogroup, biotype or country of isolation. The mean genetic diversity of this V. cholerae population (H:=0.50) was higher than reported previously. Linkage disequilibrium analysis of the MLEE data showed a clonal structure for the entire population, but not in some of the population subgroups studied. This suggests an epidemic population structure. The results showed that the O139 strains were not clustered in a unique ET, in contrast to previous MLEE studies. This higher genetic variation of the O139 serogroup is concordant with ribotyping studies. The results also confirm that the O139 and O1 ElTor isolates are genetically more closely related to each other than to all the other subpopulations of V. cholerae studied.

Full text

Microbiology (2000), 146, 2613–2626 Printed in Great Britain
Genetic relationships between clinical and
environmental Vibrio cholerae isolates based
on multilocus enzyme electrophoresis
M. Farfa
!
n, D. Min
4
ana, M. C. Fuste
!
and J. G. Lore
!
n
Author for correspondence: J. G. Lore
!
n. Tel: j34 93 402 44 97. Fax: j34 93 402 18 96.
e-mail: loren!farmacia.far.ub.es
Departament de
Microbiologia i Parasitologia
Sanita
'
ries, Divisio
!
de
Cie
'
ncies de la Salut, Facultat
de Farma
'
cia, Universitat de
Barcelona, Avda Joan XXIII
s/n, 08028 Barcelona, Spain
A total of 107 isolates of Vibrio cholerae, including 29 strains belonging to
serogroup O139, were studied using multilocus enzyme electrophoresis (MLEE)
to determine allelic variation in 15 housekeeping enzyme loci. All loci were
polymorphic and 99 electrophoretic types (ETs) were identified from the total
sample. No significant clustering of isolates was detected in the dendrogram
generated from a matrix of coefficients of distances with respect to serogroup,
biotype or country of isolation. The mean genetic diversity of this V. cholerae
population (Hl050) was higher than reported previously. Linkage
disequilibrium analysis of the MLEE data showed a clonal structure for the
entire population, but not in some of the population subgroups studied. This
suggests an epidemic population structure. The results showed that the O139
strains were not clustered in a unique ET, in contrast to previous MLEE studies.
This higher genetic variation of the O139 serogroup is concordant with
ribotyping studies. The results also confirm that the O139 and O1 ElTor isolates
are genetically more closely related to each other than to all the other
subpopulations of V. cholerae studied.
Keywords: MLEE, linkage disequilibrium, cholera, population genetics, electrophoretic
types
INTRODUCTION
Vibrio cholerae is an autochthonous inhabitant of
marine and freshwater environments, which is often
associated with phyto- and zooplankton (Baumann et
al., 1984; Colwell & Huq, 1994). This bacterium is
classified, by the composition of its major surface
antigen (O), into serogroups, of which there are nearly
200 (Beltra
!
n et al., 1999). Important distinctions within
the species are made on the basis of serogroup,
production of cholera toxin, which is responsible for
severe diarrhoea, and potential for epidemic spread.
Only two serogroups of V. cholerae, O1 and O139, have
been considered as causative agents of cholera, but some
strains of these serogroups do not produce cholera toxin
and are not involved in epidemics. All the others
serogroups, non-O1\non-O139, are more frequently
isolated from environmental sources and are associated
with sporadic cases of gastroenteritis or extraintestinal
.................................................................................................................................................
Abbreviations: AFLP, amplified fragment length polymorphism; ET,
electrophoretic type; MLEE, multilocus enzyme electrophoresis ; RAPD,
random amplified polymorphic DNA.
infections. Although occasional strains can produce
cholera toxin or other virulence factors, none of them
has caused large epidemics (Kaper et al., 1995).
Historically, it seems most probable that cholera
emerged after the Neolithic, which began in the Middle
East some 10000 years ago, when the adoption of
agricultural practices by nomadic groups enabled higher
densities of humans to subsist (Byun et al., 1999). There
are references to deaths due to dehydrating diarrhoea
dating back to Hippocrates and Sanskrit writings
(Colwell, 1996). Cholera has been endemic on the Indian
subcontinent for centuries. The literature describes the
first pandemic spread of cholera outside Asia in 1817
(Blake, 1994). Since then seven pandemics have been
recorded. The fifth and sixth were caused by the classical
biotype of O1 strains, but the nature of the strains
causing the first four pandemics is unknown. In contrast,
in 1961 the seventh pandemic started in Indonesia and
was due to the ElTor biotype. Recently, in 1992 an
epidemic clone of a non-O1 strain with serogroup O139
Bengal caused a large cholera outbreak in Bangladesh
and neighbouring countries (Albert et al., 1993;
Ramamurthy et al., 1993). Several studies have shown
0002-3939 # 2000 SGM
2613

M. FARFA
!
N and OTHERS
that the O139 strain is phylogenetically and pheno-
typically very similar to the O1 ElTor strain, and most
probably derived from an O1 seventh-pandemic clone
strain by horizontal gene transfer (Bik et al., 1995;
Stroeher et al., 1997). At the beginning of the outbreak,
the O139 strain totally displaced the V. cholerae O1
strains, including both classical and ElTor biotypes,
which coexisted only in Bangladesh. The subsequent
emergence of a new clone of V. cholerae O1 ElTor that
transiently displaced the O139 strains during 1994 and
1995, and the reemergence in 1996 of V. cholerae O139
as the main cause of cholera in Calcutta and its
coexistence with the O1 ElTor strains demostrated
temporal changes in the epidemiology of the cholera
(Faruque et al., 1997a, b; Mukhopadhyay et al., 1998).
The factors that determine the emergence, disappear-
ance or continued presence of particular clones of
toxigenic V. cholerae are not clear.
The continual emergence of new toxigenic strains of V.
cholerae and their selective enrichment during cholera
outbreaks constitute essential mechanisms for the sur-
vival and evolution of V. cholerae and the genetic
elements that mediate the transfer of virulence genes
(Faruque et al., 1998). The molecular mechanisms of
cholera pathogenesis are currently being elucidated, as
exemplified by the description of the lysogenic fila-
mentous bacteriophage (CTX
φ), which encodes cholera
toxin (Waldor & Mekalanos, 1996), and its receptor, the
TCP (toxin-coregulated pilus), which is part of a larger
genetic element, the TCP pathogenicity island (Karaolis
et al., 1998). These findings may help us to understand
the possible origin of new toxigenic clones of V.
cholerae, and raise the possibility that all strains of V.
cholerae have the potential to become agents of epidemic
cholera (Faruque et al., 1998).
New analysis methods have permitted studies of the
genetic variability of V. cholerae on a global scale
(Faruque et al., 1998). One of them is MLEE, which
analyses the electrophoretic mobility differences in
multiple enzymes to study divergence of bacterial strains
of the same species. Previous studies were primarily
concerned with the analysis of strains belonging to the
serogroup O1, collected during outbreaks, which were
responsible for cholera epidemics and pandemics (Salles
& Momen, 1991 ; Wachsmuth et al., 1993; Evins et al.,
1995). More recently, Beltra
!
n et al. (1999) applied the
MLEE technique to the study of a collection of V.
cholerae isolates from Mexico and Guatemala ; they also
included reference strains of all serogroups (toxigenic
and non-toxigenic). Moreover, various molecular tech-
niques such as PFGE, RAPD and ribotyping have also
been applied in attempts to establish the genetic popu-
lation structure of V. cholerae. Recent work in this field
includes the comparative nucleotide sequence analysis
of pathogenic isolates (Byun et al., 1999) and the AFLP
fingerprinting of clinical and environmental isolates
(Jiang et al., 2000). However, the relationship of the
pathogenic clones with the environmental isolates re-
mains unclear. In the present study, we analysed by
MLEE a collection of V. cholerae strains isolated from
several countries, including toxigenic (O1 and O139)
and non-toxigenic serogroups from environmental and
clinical sources, to determine the population structure of
V. cholerae.
METHODS
Bacterial strains. This study was carried out on a collection of
100 isolates of Vibrio cholerae, from several countries,
including toxigenic (O1 and O139) and non-toxigenic sero-
groups, obtained from clinical and environmental sources
(Table 1). Strains were kindly provided by G. B. Nair
(National Institute of Cholera & Enteric Diseases, Calcutta,
India), M. A. R. Chowdhury (Marine Laboratory, Depart-
ment of Microbiology, University of Maryland, MD 20740,
USA) and M. Talledo (Laboratorio Microbiologı
!
a y Bio-
tecnologı
!
a Microbiana, Facultad de Ciencias Biolo
!
gicas,
Universidad Nacional Mayor de San Marcos, Lima, Peru). Six
reference strains of V. cholerae from the Spanish Type Culture
Collection (CECT 514, CECT 569, CECT 652, CECT 655,
CECT 658 and CECT 659) and a reference strain of classical
O1 V. cholerae (ATCC 14035) were also included in the study.
Preparation of lysates for electrophoresis. Each isolate was
grown overnight at 35 mC in Trypticase Soy Broth (TSB), in a
shaker. The cells were harvested by centrifugation (7000 g for
15 min at 6 mC), suspended in TE-NADP buffer (Tris\HCl 10
mM, EDTA 1 mM and NADP 0n5 mM, pH 6n8) and lysed by
freezing and thawing. The cell extracts were obtained after
three repeated freezing–thawing cycles (at k20 mC for 12–24 h
and at 36n6 mC for 5 min) and centrifugation at 110 000 g for
25 min at 6 mC. Aliquots of supernatant were transferred to
Eppendorf tubes and stored at k40 mC until use. Protein was
measured by the Lowry method, with bovine serum albumin
(Sigma) as a standard.
Electrophoresis and specific enzyme staining. Nondenaturing
vertical polyacrylamide gel electrophoresis was used for all the
enzymes. The acrylamide concentration in the gels depended
on the enzyme studied (10% continuous polyacrylamide gels
and 10 %\8% or 8%\5% discontinuous polyacrylamide
gels). Tris\HCl 0n8 M (pH 8n8) buffer was used in continuous
gels and Tris\HCl 0n125 M (pH 6n8) stacking buffer and
Tris\HCl 0n4 M (pH 8n8) resolving buffer were used in
discontinuous gels. Tris\glycine 0n19 M (pH 8n3) buffer was
used for the electrode compartments. Gels were used within
24 h of preparation and run at 7 mC. A constant voltage,
depending on the acrylamide concentration of the gel, was
applied until the bromophenol blue band reached the bottom
of the gel. All strains were run at least twice to confirm their
genotype.
The staining of the gels to reveal specific enzyme activity was
performed following Selander et al. (1986), except in the
case of catechol 2,3-oxygenase (Gibson, 1971; Kataeva &
Golovleva, 1990). The following 15 enzymes were assayed :
glucose-6-phosphate dehydrogenase (G6P), isocitrate dehy-
drogenase (IDH), alanine dehydrogenase (ALD), NAD-
dependent glyceraldehyde-phosphate dehydrogenase (GP1),
malate dehydrogenase (MDH), fumarase (FUM), aspartate
dehydrogenase (ASD), leucine aminopeptidase (LAP), malic
enzyme (ME), esterase (EST), catechol 2,3-oxygenase (C23O),
nucleoside phosphorylase (NSP), xanthine dehydrogenase
(XDH), phosphoglucose isomerase (PGI) and 6-phospho-
gluconate dehydrogenase (6PG). For each enzyme, distinct
mobility variants were designated as electromorphs and
numbered in order of increasing migration towards the anode.
2614

MLEE of Vibrio cholerae
Table 1. Characteristics of the bacterial isolates used in this study and their allele profiles at each locus
ET No. of
strains
Reference
isolates
Allele at enzyme locus Serogroup/biotype Source* Country
G6P IDH ALD GP1 MDH FUM ASD LAP ME EST C23O NSP XDH PGI 6PG
1 1 TM35123-77 431113271123141non-O1\non-O139 E Brazil
2 2 TM34162-77 431213241123141non-O1\non-O139 E Brazil
TM9024-79 431213241123141non-O1\non-O139 E Brazil
3 1 TM52479-78 431212152213146non-O1\non-O139 E Brazil
4 1 TM23256-79 331112152213121non-O1\non-O139 E Brazil
5 1 TM19225-79 311514252213423non-O1\non-O139 E Brazil
6 1 GGPB10 331212152213535non-typed U Brazil
7 1 TM1705-82 331215252223131non-O1\non-O139 E Brazil
8 3 TM48733-82 431323271123141non-O1\non-O139 E Brazil
TM11079-80 431323271123141O1ElTor E Brazil
CO484 431323271123141non-O1\non-O139 E India
9 1 TM1187-83 331214253213431non-O1\non-O139 E Brazil
10 1 CT10738-92 331216253213433non-O1\non-O139 E Brazil
11 1 CT10834-92 331214052213425non-O1\non-O139 E Brazil
12 1 CT12009-92 341234325223131non-O1\non-O139 E Brazil
13 1 GGPB29 331115253213121non-typed U Brazil
14 1 GM30-90 321233334233133non-O1\non-O139 E Brazil
15 1 GM31-90 431113273123141non-O1\non-O139 E Brazil
16 1 GM33-90 331112152213125non-O1\non-O139 E Brazil
17 1 GM34-90 331212152213121non-O1\non-O139 E Brazil
18 1 GM35-90 431213213123141non-O1\non-O139 E Brazil
19 1 GM36-90 411213273123441non-O1\non-O139 E Brazil
20 1 GM37-90 331215253213131non-O1\non-O139 E Brazil
21 1 GM38-90 231214213233141non-O1\non-O139 E Brazil
22 1 CT7606-93 631323271113441O1ElTor E Brazil
23 1 CT9995-93 431113273113441O1ElTor E Brazil
24 1 GGPB44 421113271123141non-typed U Brazil
25 2 TM207832-78 431223241123141O1ElTor E Brazil
ALVC-92-0344 431223241123141O1ElTor C Peru
26 1 CT25016-91 631323271113443O1ElTor E Brazil
27 1 CT7021-94 631223231123441O1ElTor E Brazil
28 1 TM16457-78 331114153213135O1classical E Brazil
29 1 CT7649-94 431223271123141O1ElTor E Brazil
30 1 CTMARMI2-
92
431113213123143O1ElTor E Brazil
31 1 CTMACMI1-
92
331224263123141O1ElTor E Brazil
2615

M. FARFA
!
N and OTHERS
Table 1 (cont.)
ET No. of
strains
Reference
isolates
Allele at enzyme locus Serogroup/biotype Source* Country
G6P IDH ALD GP1 MDH FUM ASD LAP ME EST C23O NSP XDH PGI 6PG
32 1 MX121 311214152213125non-O1\non-O139 F Mexico
33 1 MX129 331112152221131non-O1\non-O139 F Mexico
34 1 MX134 431113271213125non-O1\non-O139 F Mexico
35 1 MX157 431123221123141non-O1\non-O139 F Mexico
36 1 MX158 331215153213131non-O1\non-O139 F Mexico
37 1 MX159 431323221123131non-O1\non-O139 F Mexico
38 1 AT01 421213241123141non-O1\non-O139 C India
39 1 CO391 431215253213125O139 F India
40 1 CO407 331214253223131O139 F India
41 1 CO487 411213243123441O1ElTor C India
42 1 CO870 341112132213435O1ElTor E India
43 1 SG24 311213211213143O139 C India
44 1 CECT514 631227223113642O1classical F UK
45 1 CECT569 611227211113441O1classical C India
46 1 CECT652 631227223123141O1 CUnknown
47 1 CECT655 431227223123541non-O1\non-O139 E Bangladesh
48 1 CECT658 631323221123441non-O1\non-O139 E Bangladesh
49 1 CECT659 632323221113144non-O1\non-O139 E Bangladesh
50 1 1-7\31 431223221213141O1ElTor E USA
51 1 6-3\6 331116231223141O1ElTor E USA
52 1 NT330 431223221123551O139 C India
53 2 CO402 631323221113441O139 F India
SO30 631323221113441O139 C India
54 1 CO414 632323221113143O139 F Unknown
55 1 NT642 631324223123141O139 C India
56 1BO1 631323221113141O139 F India
57 1 ATCC14035 631223221213041O1classical C UK
58 1 2030 H 131213233213415non-O1\non-O139 E USA
59 1 NT329 631323221113144O139 C India
60 2 25872 631223221113541non-O1\non-O139 E Unknown
CO406 631223221113541O139 F India
61 1 BLO9 631223231113143O1ElTor C Bangladesh
62 1 MDO90 431123231113141O139 C India
63 1 SO29 631423221113441O139 C India
64 1VO6 431323221113041O139 C India
65 1BO2 431126231113441O139 F India
66 1 CO404 NT656 611323241101152O139 F India
2616

MLEE of Vibrio cholerae
Table 1 (cont.)
ET No. of
strains
Reference
isolates
Allele at enzyme locus Serogroup/biotype Source* Country
G6P IDH ALD GP1 MDH FUM ASD LAP ME EST C23O NSP XDH PGI 6PG
67 1 CO417 631323221113541O1ElTor F India
68 1 3\06\02 421113251113141non-O1\non-O139 F USA
69 1 5-6\37 421223231113143O1classical E Tanzania
70 1 1196\78 341216211212526O1classical E USA
71 1 CO403 431223231113441O139 F India
72 1 2076\29 322214231223131non-O1\non-O139 E Unknown
73 1 3-6\69 421213231203152O1ElTor E USA
74 1 MEX332 431323341123140O1classical C Mexico
75 1 NM4392 331215241223130O1ElTor C Unknown
76 1329 431223231123141O139 C Unknown
77 134-1 412233314213242non-O1\non-O139 E Unknown
78 1 1-7\69 331216231213124O1ElTor E USA
79 1 MEX445 341212112113441O1classical C Mexico
80 1 NM286 531324241223050O1ElTor C India
81 1 653\36 341214211223040O139 C India
82 1 25873 431123231113444non-O1\non-O139 E Unknown
83 1 CO396 NT646 631223211213143O139 C India
84 1 3-6\3 331214271223141non-O1\non-O139 E USA
85 1 6-3\2 321214231223141O1ElTor E USA
86 1 6-3\50 331233344223041non-O1\non-O139 E USA
87 1 NPO388 321233334203100O139 C India
88 1 CO416 321233314223231O1ElTor F India
89 1 NT648 631223271102352O139 C India
90 2 NPO390 631323231113141O139 C India
BO4 631323231113141O139 F India
91 1 SO19 331213271223136O139 C India
92 1 CO415 631223221123141O139 F India
93 1 MOD084 631223231123041O139 C India
94 1 NT638 631323221123141O139 C India
95 1 1074-78 341211133213143O1classical E USA
96 1 CO418 631123231113141O139 F India
97 2 ALVC-91-0803 431223271113141O1ElTor C Peru
ALVC-91-1019 431223271113141O1ElTor C Peru
98 1 ALVC-92-0534 631323211123141O1ElTor C Peru
99 1 ALVC-93-0024 431123241123141O1ElTor C Peru
* C, clinical; E, environmental ; F, faecal ; U, unknown.
2617

M. FARFA
!
N and OTHERS
Displacement of the electromorphs was expressed in terms of
relative electrophoretic mobility with respect to the bromo-
phenol blue band. Electromorphs of an enzyme were equated
with alleles at the corresponding structural gene locus.
Absence of enzyme activity was attributed to a null allele, and
designated as 0. Distinct combinations of alleles over the 15
loci assayed were named as electrophoretic types (ETs).
Data treatment. Genetic diversity for a locus was calculated
according to Nei (1978). The probability that two isolates
differ at the jth locus is h
j
l (1 k Σp
#
ij
) n\(n k 1), where p
ij
is
the frequency of allele i at locus j and n is the number of
isolates. The mean genetic diversity, H, is the arithmetic mean
of h
j
for m loci. Genotypic diversity was calculated as Gl 1 k
Σg
#
j
, where g
j
is the frequency of the jth genotype (ET).
Clustering of data obtained by MLEE was performed with the
 package (Felsenstein, 1993) from a matrix of coeffi-
cients of distances by the unweighted pair-group method for
arithmetic averages (UPGMA). Distance between pairs of ETs
was calculated as the proportion of loci at which dissimilar
electromorphs occurred. The cophenetic correlation coef-
ficient was calculated using -pc, version 1n80 (Rohlf,
1993). Multilocus linkage disequilibrium was estimated on the
basis of the distribution of allelic mismatches between pairs of
bacterial isolates among all the loci examined. The ratio of the
observed variance in mismatches (V
O
) to the expected variance
at linkage equilibrium (V
E
) provides a measure of multilocus
linkage disequilibrium that can be expressed as the index of
association (I
A
); I
A
l (V
O
\V
E
) k 1 (Brown et al., 1980 ;
Maynard Smith et al., 1993). For populations in linkage
equilibrium, V
O
l V
E
and I
A
is not significantly different from
zero, whereas values of I
A
greater than zero indicate that
recombination has been rare or absent. To determine whether
V
O
is significantly different from V
E
in any sample, a
Montecarlo procedure was generated by randomly sampling
alleles, without replacement, according to their respective
frequencies at each locus (Fuste
!
et al., 1996). Computer
programs written by T. S. Whittam (Selander et al., 1986) and
J. G. Lore
!
n were used to calculate V
O
and V
E
and to perform
the Montecarlo randomization.
Estimates of the genetic differentiation between sub-
populations were obtained by using Nei’s genetic distance
(Nei, 1972). Phylogenetic analyses were performed with the
 package by using the neighbour-joining method. The
reliability of the cladograms obtained (Fig. 3) was determined
by bootstrapping as follows. Gene frequencies were randomly
selected, with replacement, to produce 100 replicated data sets
of the same size as the original data sets. New Nei’s distance
matrices were then calculated and subjected to the neighbour-
joining method. Comparison of the 100 bootstrapped clado-
grams generated was made using the Consensus program of
the  package.
RESULTS
ETs and genetic diversity
From a collection of 107 isolates of V. cholerae analysed
by MLEE, 99 ETs were identified. The genotypic
diversity (G) for all samples was 0n9872. All the enzyme
loci studied were polymorphic, and the number of alleles
ranged from two (ALD and EST) to seven (FUM, LAP,
XDH and 6PG) (Table 2). The mean number of alleles
per locus was 4n8. A small number of null alleles was
observed (17 among the 99 ETs), distributed over 4 of
the 15 enzymes assayed. Genetic diversity ranged from
0n08 for the least polymorphic loci (ALD and NSP) to
0n83 for the most polymorphic locus (LAP), with a mean
genetic diversity (H)of0n50 (Table 2). (A comparison of
genetic diversity between the different sample sets is
shown in Table 5.)
Genetic relationships among multilocus genotypes
The genetic relationship among the 99 ETs is shown in
Fig. 1. The cophenetic correlation coefficient of the total
sample was Rl0n81. The shortest genetic distance
observed between ETs (0n06) corresponds to a single
locus difference. No significant clustering among isolates
was detected according to their serogroup, biotype or
country of isolation. All but six of the total ETs are
represented in two major divisions in the dendrogram,
designated as divisions I and II, which diverge at a
genetic distance of 0n6. Division II is constituted by 34
ETs, most of which are environmental non-O1\non-
O139 strains. In division I we have designated two
subgroups: Ia, which includes a large number of
environmental strains, and Ib, with a greater number of
clinical strains. The third group, division III, diverges at
a genetic distance of 0n65. This is the deepest lineage
found in the dendrogram and is clearly differentiated
from all the other strains. This lineage is represented by
six ETs (77, 12, 14, 87, 86, 88) without any apparent
relationship between them.
Table 4 shows the 30 pairs of ETs that differ at a single
enzyme locus. The loci that occur most frequently in this
analysis are GP1 (seven pairs of ETs) and LAP (six pairs
of ETs) ; none of these 30 pairs of ETs differ in the loci
EST, FUM, NSP or PGI. In addition, we identified 15
pairs of ETs which are formed by strains of the same
serogroup: seven pairs of O139 strains, five of non-
O1\non-O139 and three of O1 ElTor isolates. In four of
these cases, the pairs of ETs are represented by strains
from a different country of isolation. On the other hand,
the other pairs of ETs are formed by strains of different
serogroups, except O1 classical.
Linkage disequilibrium analysis
Allele mismatch distribution among 99 ETs is shown in
Fig. 2. Our population of V. cholerae presents a
unimodal allele mismatch distribution, which suggests
that it has a panmictic structure (Whittam, 1995). The
complete set of isolates and population subsets was
analysed for multilocus linkage disequilibrium (Table
5). The index of association (I
A
) found for the total
sample was 1n25p0n14, which suggested that this
population of V. cholerae presents a significant level of
linkage disequilibrium. Table 5 also shows the results
obtained for several population subsets studied. When
we considered divisions of the dendrogram, serogroups,
sources and geographical origin of the strains, all subsets
showed I
A
values differing significantly from zero, except
division II and III, O1 classical and USA\Mexico
subgroups, which exhibited I
A
values of 0n41, 0n08, 0n64
and 0n42, respectively.
2618

MLEE of Vibrio cholerae
Table 2. Allele frequencies and genetic diversities at 15 enzyme loci in 99 ETs of V.
cholerae
Enzyme
locus
Max. no of
alleles
Frequency of each allele h*
12345670
G6P 6 0n010 0n010 0n354 0n343 0n010 0n273 0n69
IDH 4 0n081 0n101 0n758 0n061 0n41
ALD 2 0n960 0n040 0n08
GP1 5 0n202 0n586 0n192 0n010 0n010 0n58
MDH 3 0n465 0n475 0n061 0n56
FUM 7 0n010 0n081 0n606 0n152 0n061 0n051 0n040 0n60
ASD 4 0n121 0n798 0n071 0n010 0n35
LAP 7 0n121 0n222 0n212 0n101 0n192 0n010 0n141 0n83
ME 5 0n596 0n121 0n222 0n051 0n010 0n58
EST 2 0n556 0n444 0n50
C23O 4 0n515 0n424 0n020 0n040 0n56
NSP 3 0n020 0n020 0n960 0n08
XDH 7 0n636 0n020 0n010 0n202 0n061 0n010 0n061 0n55
PGI 6 0n010 0n111 0n172 0n647 0n051 0n010 0n54
6PG 7 0n626 0n051 0n111 0n040 0n091 0n030 0n051 0n59
Mean genetic diversity, H 0n50
* h, genetic diversity.
Genetic distances within population subgroups
The clustering of strains considering serogroups and
geographical origin from values of genetic distances
between population subgroups is shown in Fig. 3. The
cladogram of the geographical subpopulations showed
that the strains from the USA, Brazil and Mexico were
more genetically related to each other than to the
isolates of V. cholerae from India and Peru. On the other
hand, the cladogram of the serogroup\biotype subpopu-
lations confirmed that the O1 ElTor and O139 strains
were genetically more related to each other than to other
serogroups.
DISCUSSION
Nowadays V. cholerae is a well-defined species based on
biochemical tests and DNA homology studies (Baumann
et al., 1984), though it is a highly heterogeneous group
with respect to its pathogenic potential: only two out of
the nearly 200 serogroups defined at present, O1 and
O139, have been associated with epidemics (Kaper et al.,
1995). Therefore many aspects of the ecology of V.
cholerae and its relationship to the pathogenesis and
epidemiology of cholera remain unknown (Faruque et
al., 1998).
Bacterial population genetics is the study of the natural
variability of bacterial populations and has led to the
formulation of theories to account for this diversity
(Maynard Smith et al., 1993). Classical and molecular
methods have continously been used to study the
population diversity of V. cholerae and have been
extensively applied to characterize different serogroups
and biotypes (Baumann et al., 1984), ribotypes (Karaolis
et al., 1994), RAPD fingerprint types (Rivera et al., 1995)
and insertion sequence fingerprint types (Bik et al.,
1996). These methods have shown a considerable
amount of variation in V. cholerae. MLEE constitutes
the classical methodology in the study of bacterial
population genetics (Selander et al., 1986). By using
MLEE data we can obtain estimates of genetic and
genotypic diversity and estimates of the frequency of
recombination in natural populations. These aspects of
V. cholerae population biology are of great importance
to molecular epidemiological studies of cholera.
Previous MLEE studies with V. cholerae have shown a
limited genetic diversity among toxigenic strains of this
bacterium. Pathogenic isolates from various sources all
showed identical electrophoretic profiles or differed in
only a few loci (Salles & Momen, 1991; Wachsmuth et
al., 1993 ; Evins et al., 1995). Some authors indicate that
the sixth-pandemic, the seventh-pandemic and the US
Gulf isolates are three independent clones (Kaper et al.,
1982; Waschmuth et al., 1993; Karaolis et al., 1994,
1995; Evins et al., 1995). In a recent study, it has been
postulated that all strains of the O139 serogroup belong
to a unique ET; similarly the toxigenic O1 ElTor
isolates cluster in only three ETs (Beltra
!
n et al., 1999).
Our results show that when MLEE methods were
applied to a large collection of isolates of diverse origin
including serogroup O1 (classical and ElTor biotypes),
O139 and non-O1\non-O139 strains, the different V.
cholerae populations showed a high degree of genetic
variation. The number of ETs found in our work does
2619

M. FARFA
!
N and OTHERS
.................................................................................................................................................................................................................................................................................................................
Fig. 1. For legend see facing page.
2620

MLEE of Vibrio cholerae
Table 3. Groups of strains with identical genotype
ET No. of strains Reference isolates Serogroup/biotype Source* Country
2 2 TM34162-77 non-O1\non-O139 E Brazil
TM9024-79 non-O1\non-O139 E Brazil
8 3 TM48733-82 non-O1\non-O139 E Brazil
TM11079-80 O1 ElTor E Brazil
CO484 non-O1\non-O139 E India
25 2 TM207832-78 O1 ElTor E Brazil
ALVC-92-0344 O1 ElTor C Peru
53 2 CO402 O139 F India
SO30 O139 C India
60 2 25872 non-O1\non-O139 E Unknown
CO406 O139 F India
90 2 NPO390 O139 C India
BO4 O139 F India
97 2 ALVC-91-0803 O1 ElTor C Peru
ALVC-91-1019 O1 ElTor C Peru
* C, clinical; E, environmental; F, faecal.
not coincide with data previously published (Salles &
Momen, 1991; Wachsmuth et al., 1993 ; Evins et al.,
1995; Beltra
!
n et al., 1999). From 107 isolates analysed,
99 ETs were found, 84 containing a single strain, six
containing two strains and only one containing three
strains. Previous studies described a more limited
diversity of V. cholerae populations, with most isolates
clustering in two or three ETs with minimal differences
between them, especially when toxigenic strains were
included (Salles & Momen, 1991; Wachsmuth et al.,
1993; Evins et al., 1995). The estimate for the mean
genetic diversity per locus of the total 99 ETs (Hl0n50)
in our study is greater than other values reported for V.
cholerae (Chen et al., 1991; Salles & Momen, 1991;
Wachsmuth et al., 1993; Evins et al., 1995 ; Beltra
!
n et al.,
1999). Likewise, the mean number of alleles per locus
(4n8), and the genotypic diversity (0n9872) were higher
than those given in similar studies, with the exception of
Beltra
!
n et al. (1999), who reported a value of 9n5 alleles
per locus as an average. Also, the work of Beltra
!
n et al.
(1999) differed in the number of strains that fell into the
same ET.
Differences in the serogroup, geographical origin and
year of isolation could explain this diversity. Another
factor that may influence the results is the methodology
used; some previous studies used starch as a matrix for
the gels, whose resolution could be distinct from that of
polyacrylamide (Wachsmuth et al., 1993; Evins et al.,
1995; Beltra
!
n et al., 1999). Finally, the number and type
of enzymes studied are different ; most of them are
monomorphic and only a few contribute to the genetic
diversity. However, in our work all the enzymes were
polymorphic with a considerable genetic diversity, thus
hampering comparisons between studies.
Fig. 1. Dendrogram constructed by the UPGMA method, showing genetic relationships among 99 ETs of V. cholerae
strains. The sources of the strains are indicated by the letters C (clinical), E (environmental), F (faecal) and U (unknown).
Asterisks (*) denote ETs that contain more than one strain (see Table 3). The scale indicates genetic distance.
The dendrogram obtained in the present study shows no
association between isolates with regard to serogroup,
biotype or geographical origin. The same serogroup is
present in several lineages and does not cluster separately
from the others (Fig. 1). Similar results were obtained by
Beltra
!
n et al. (1999), who showed that strains of the
same serogroup may belong to two or more divergent
ET lineages. Division II has more non-O1\non-O139
strains than other groups. On the other hand, most of
the O139 serogroup strains clustered in division I (24 of
29 isolates), while other serogroups seem to be randomly
distributed. With regard to the source of the strains
studied, division II seems to contain most of the
environmental strains, while clinical isolates predomi-
nate in the subgroup Ib. Although this clustering did not
coincide with previous studies, the value of the co-
phenetic correlation coefficient obtained (Rl0n81) falls
into the range (0n74–0n90) of most frequently occurring
cophenetic correlations reported by Sneath & Sokal
(1973).
Unimodal allele mismatch distribution (Fig. 2) and the
shape of the dendrogram (Fig. 1) are typical of a
panmictic population (Whittam, 1995). However, evi-
dence for clonal proliferation is provided by the multi-
locus linkage disequilibrium calculations, which reveal a
significant level of association between alleles when the
whole population sample is analysed. Values of I
A
among all ETs indicated that there is a nonrandom
distribution of alleles, which is clear evidence of a clonal
population structure, with a significant degree of linkage
disequilibrium I
A
l 1n25p0n14 (no significant differ-
ences were obtained when we considered strains or
ETs). Nevertheless, when we analysed data subsets
corresponding to divisions of the dendrogram, sero-
2621

M. FARFA
!
N and OTHERS
Table 4. ETs that differ at a single locus
ET Serogroup Country of
isolation
Locus ET Serogroup Country of
isolation
Locus
1 non-O1\non-O139 Brazil ME 29 O1 ElTor Brazil C23O
15 non-O1\non-O139 Brazil ME 97 O1 ElTor Peru C23O
1 non-O1\non-O139 Brazil IDH 35 non-O1\non-O139 Mexico LAP
24 Unknown Brazil IDH 99 O1 ElTor Peru LAP
2 non-O1\non-O139 Brazil MDH 48 non-O1\non-O139 Bangladesh C23O
25 O1 ElTor Brazil MDH 53 O139 India C23O
O1 ElTor Peru MDH
48 non-O1\non-O139 Bangladesh XDH
2 non-O1\non-O139 Brazil IDH 94 O139 India XDH
38 non-O1\non-O139 India IDH
49 non-O1\non-O139 Bangladesh 6PG
4 non-O1\non-O139 Brazil 6PG 54 O139 Unknown 6PG
16 non-O1\non-O139 Brazil 6PG
49 non-O1\non-O139 Bangladesh ALD
4 non-O1\non-O139 Brazil GP1 59 O139 India ALD
17 non-O1\non-O139 Brazil GP1
53 O139 India XDH
8 non-O1\non-O139 Brazil GP1 56 O139 India XDH
non-O1\non-O139 India GP1 67 O1 ElTor India XDH
O1 ElTor Brazil GP1
29 O1 ElTor Brazil GP1 53 O139 India GP1
63 O139 India GP1
19 non-O1\non-O139 Brazil LAP
41 O1 ElTor India LAP 56 O139 India 6PG
59 O139 India 6PG
20 non-O1\non-O139 Brazil ASD
36 non-O1\non-O139 Mexico ASD 56 O139 India LAP
90 O139 India LAP
22 O1 ElTor Brazil 6PG
26 O1 ElTor Brazil 6PG 56 O139 India C23O
94 O139 India C23O
22 O1 ElTor Brazil LAP
53 O139 India LAP 60 non-O1\non-O139 Unknown GP1
O139 India LAP O139 India GP1
67 O1 ElTor India GP1
25 O1 ElTor Brazil LAP
O1 ElTor Peru LAP 62 O139 India G6P
29 O1 ElTor Brazil LAP 96 O139 India G6P
76 O139 Unknown LAP
90 O139 India GP1
25 O1 ElTor Brazil GP1 96 O139 India GP1
O1 ElTor Peru GP1
99 O1 ElTor Peru GP1 92 O139 India GP1
94 O139 India GP1
27 O1 ElTor Brazil XDH
93 O139 India XDH 94 O139 India LAP
98 O1 ElTor Peru LAP
groups or geographical origin, some of them (divisions
II, III, O1 classical and USA\Mexico strains) showed I
A
values less than one (0n41, 0n08, 0n64 and 0n42, re-
spectively). The V
O
values of these subgroups were also
within the 95 % confidence limits of V
E
and within the
maximum and minimum values of the variance obtained
by the Montecarlo procedure. However, I
A
values
corresponding to the division III and the O1 classical
subgroup should be taken cautiously because of the low
number of ETs in these groups (six and nine ETs,
respectively). These results are partially concordant
with those obtained by Beltra
!
n et al. (1999), who found
2622

MLEE of Vibrio cholerae
0·14
0·12
0·10
0·08
0·06
0·04
0·02
0123 456789101112131415
No. of mismatches
Frequency
.....................................................................................................
Fig. 2. Allele mismatch distribution among
99 ETs of the total sample studied.
Table 5. Multilocus linkage disequilibrium analysis of V. cholerae
No. of
ETs
Mean no. of
alleles per
locus
HV
O
V
E
95 % confidence
limits of V
E
*
I
A
† Montecarlo
V minimum
Randomization
V maximum
Total 107 isolates 99 4n80 0n50 7n13 3n17 2n29–4n04 1n25p0n14 2n54 3n89
Dendrogram
Division I 59 3n47 0n35 3n91 2n62 1n67–3n56 0n49p0n18 1n87 3n77
Division II 34 3n73 0n44 3n77 2n67 1n41–3n92 0n41p0n24 1n78 3n88
Division III 6 2n33 0n40 2n03 1n88 0n24–3n99 0n08p0n56 0n27 7n52
Serogroup/biotype‡
O1 35 4n00 0n49 6n54 3n04 1n63–4n44 1n15p0n23 2n15 4n35
O1 ElTor 24 3n60 0n45 7n01 2n94 1n29–4n57 1n39p0n28 1n74 5n03
O1 classical 9 3n33 0n57 4n35 2n65 0n20–5n09 0n64p0n46 0n99 7n56
Non-O1\non-O139 37 4n00 0n51 6n72 3n20 1n75–4n64 1n10p0n23 2n28 4n68
O139 26 3n53 0n40 8n05 2n94 1n34–4n52 1n74p0n27 1n51 5n09
Country of
isolation‡
America§ 54 4n26 0n49 6n37 3n10 1n94–4n25 1n05p0n19 2n28 4n09
Brazil 31 3n53 0n49 7n82 3n01 1n53–4n49 1n59p0n24 2n14 4n25
USA\Mexico 20 3n86 0n51 4n49 3n15 1n22–5n08 0n42p0n31 1n71 5n83
India 32 4n06 0n46 8n74 3n05 1n57–4n53 1n86p0n24 1n74 4n61
Source‡
Environmental 47 4n33 0n52 6n17 3n16 1n89–4n42 0n95p0n19 2n38 4n43
F223n53 0n48 10n16 3n27 1n35–5n18 2n10p0n29 1n84 5n96
C313n67 0n42 6n17 2n96 1n50–4n42 1n08p0n25 1n75 4n63
* Calculated according to Brown et al. (1980).
† Index of associationp.
‡ The same ET could be represented in various subgroups when it includes strains of diverse origin, source, serogroup or biotype. See
Tables 1 and 3.
§ Considering isolates from Brazil, Mexico, Peru and USA together.
I
A
values quite similar to ours (1n248p0n083) for the
whole population, and I
A
values close to zero when they
analysed population subgroups. Therefore, our results
do not rule out the possibility that the population of V.
cholerae studied has a clonal structure, nor do they
exclude recombination. We believe that this population
has a clonal structure which has not been broken by
genetic recombination. Another possibility is to consider
that the population has an epidemic structure (Maynard
Smith et al., 1993), in which strains of different origin
would coexist with others of identical origin corre-
sponding to an outbreak of disease from a single person
in a specific geographical area (e.g. in July, 1994, the
massive outbreak of O1 ElTor V. cholerae among
2623

M. FARFA
!
N and OTHERS
.....................................................................................................
Fig. 3. Unrooted phylogenetic trees of
different subpopulations from the total
sample generated by the neighbour-joining
method of clustering of Nei’s distances. The
bootstrap values for nodes are presented for
only those clusters of subpopulations which
occurred more than 80% of the time. The
scale bars indicate genetic distance.
.................................................................................................................................................................................................................................................................................................................
Fig. 4. Comparison of the genetic diversity of two subpopulations of the total sample, O1 ElTor and O139. Asterisks (*)
denote ETs that contain more than one strain (see Table 3). The scale bars indicate genetic distance.
Rwandan refugees in Goma, Zaire : Sanchez & Taylor,
1997).
The analysis of the genetic distances for isolates be-
longing to the six subpopulations that we could establish
on the basis of the country of isolation (Fig. 3), showed
that strains from Peru cluster in the same group as those
isolated from India. This result is fully concordant with
the origin of Peru’s strains previously determined by
Wachsmuth et al. (1993). In the same context, we might
consider the results obtained with strains isolated in
USA and Mexico. USA strains are longer established
and remotely related with Mexico’s isolates, as
Wachsmuth et al. (1993) pointed out.
If we consider population subsets referring to serogroup\
biotype, there is also a concordance with previous
references in relation to the origin of O1 and O139
serogroups (Bik et al., 1995; Stroeher et al., 1997). From
the analysis of the genetic distances between populations
it might be deduced that V. cholerae O1 ElTor and O139
originated from a unique splitting event (Fig. 3) and
consequently, O139 and O1 ElTor might have gained a
similar diversity (Hl0n40 and Hl0n45, respectively).
Indeed, looking at Fig. 4, we can see that O139
subpopulation strains were distributed in different ETs
in the same way as those of O1 ElTor. Nonetheless, the
slight differences in the genetic diversity can easily be
explained if we consider the geographical origin of the
two sets of strains. O139 are all Indian isolates, whereas
O1 ElTor were isolated in several countries (Table 1).
There are two possible explanations for the diversity
found among O139. First, the dissociation might have
happened a long time ago and consequently both
populations have been accumulating diversity length-
wise. Second, the split might have happened recently,
but several recombination events have taken place in a
diverse genetic background. Neither of these hypotheses
agrees with the idea of a unique ET supported by
Popovic et al. (1995) and Beltra
!
n et al. (1999). However,
they would explain the diversity we observed in the
O139 population.
Other population studies based on the analysis of
restriction fragment length polymorphisms in genes for
conserved rRNA and cholera toxin (ctxA) or in DNA
sequences flanking these genes revealed four different
ribotypes and four different ctx genotypes among 93
strains of V. cholerae O139 studied (Faruque et al.,
1997b). These results agree with the idea that strains
belonging to the O139 serogroup may have emerged
from similar serotype-specific genetic changes in more
than one progenitor strain of V. cholerae. Considering
2624

MLEE of Vibrio cholerae
this background, it is hard to believe that all the O139
strains could belong to a unique ET. Results obtained in
our work are in agreement with the hypothesis of a more
diverse origin of these strains, a conclusion that can also
be reached from ribotyping analysis.
Data from the sequences of some housekeeping genes of
V. cholerae showed a high degree of similarity when the
different strains were compared, especially in clinical
isolates. Karaolis et al. (1995) determined the DNA
sequences of the asd (aspartate semialdehyde dehydro-
genase) genes from 45 isolates of V. cholerae, which
included five O139 isolates. Byun et al. (1999) sequenced
the mdh (malate dehydrogenase) and hylA (haemolysin
A) genes from environmental and pathogenic strains of
V. cholerae, including seven strains of the O139 sero-
group. Both groups of authors found a high degree of
similarity among the pathogenic isolates, suggesting
that they are very closely related. These results may
appear to disagree with the high diversity found in our
work. This contradiction, however, is only apparent.
Our results show that mdh and asd presented a low
genetic diversity within the O139 isolates studied (mdh,
hl0n32; asd, hl0n07). Moreover, the number of O139
strains studied by these authors is low (in our work we
studied 29 O139 isolates). Recently, Jiang et al. (2000)
published a study applying the AFLP method to a
collection of clinical and environmental isolates of V.
cholerae, which supports the idea that pathogenic V.
cholerae strains have evolved from multiple independent
sources like environmental O1 or non-O1 strains. We
believe that more extensive areas of the bacterial
chromosome should be analysed (as in the case of
MLEE) in order to reach a reasonable conclusion about
the likely origin of a bacterial population.
The persistence of V. cholerae in the environment, for
months and probably years, is facilitated by its ability to
enter a viable nonculturable state in which its nutrient
and oxygen requirements are much decreased (Ravel et
al., 1995). It has also been described that V. cholerae can
bind to chitin in crustacean shells, and colonize the
surfaces of algae, phytoplankton, copepods and the
roots of aquatic plants such as water hyacinth. One
scenario to explain our findings is that during inter-
epidemics V. cholerae populations might be associated
with an ecological reservoir. We can considerer that all
V. cholerae cells constitute a metapopulation integrated
by multiple ecological populations. Previous studies
have shown that bacterial recombination is too rare to
prevent neutral sequence divergence between distinct
ecological populations. Moreover, the episodes of per-
iodic selection that purge diversity operate within
populations and do not prevent their divergence (Young,
1989). In their reservoir V. cholerae clones could diverge
from each other. Because the genes for the major
virulence factors can be acquired horizontally, we can
postulate that each V. cholerae cell would have the same
probability of being transformed into a toxigenic strain
(Beltra
!
n et al., 1999). In the next epidemic episode, some
unknown environmental factors might trigger an ex-
plosive multiplication of V. cholerae populations outside
this reservoir (Epstein et al., 1993 ; Harvell et al., 1999).
This global population would be formed by bacteria
from various local populations and include a diversity of
toxigenic clones that potentially could infect humans.
ACKNOWLEDGEMENTS
We are grateful to Dr R. Montilla for providing strains for this
study and for his helpful comments. We are indebted to Dr J.
Palomar for his help in setting up the MLEE technique. We
also thank G. B. Nair, M. A. R. Chowdhury and M. Talledo
for kindly supplying isolates of V. cholerae.
M. Farfa
!
n is the recipient of the grant ‘Formacio
!
en la Recerca
i Doce
'
ncia per a alumnes de tercer cicle’ from the University
of Barcelona. This work was supported by a grant from the
Vicerectorat de Recerca of the University of Barcelona.
REFERENCES
Albert, M. J., Siddique, A. K., Islam, M. S., Faruque, A. S. G.,
Ansaruzzaman, M., Faruque, S. M. & Sack, R. B. (1993). Large
outbreak of clinical cholera due to Vibrio cholerae non-O1 in
Bangladesh. Lancet 341, 704.
Baumann, P., Furniss, A. L. & Lee, J. V. (1984). Genus I. Vibrio.In
Bergey’s Manual of Systematic Bacteriology, vol. 1, pp. 518–538.
Edited by N. R. Kreig & J. G. Holt. Baltimore: Williams &
Wilkins.
Beltra
!
n, P., Delgado, G., Navarro, A., Trujillo, F., Selander, R. K. &
Cravioto, A. (1999). Genetic diversity and population structure of
Vibrio cholerae. J Clin Microbiol 37, 581–590.
Bik, E. M., Bunschoten, A. E., Gouw, R. D. & Mooi, F. R. (1995).
Genesis of the novel epidemic Vibrio cholerae O139 strain :
evidence for horizontal transfer of genes involved in poly-
saccharide synthesis. EMBO J 14, 209–216.
Bik, E. M., Gouw, R. D. & Mooi, F. R. (1996). DNA fingerprinting
of Vibrio cholerae strains with a novel insertion sequence
element: a tool to identify epidemic strains. J Clin Microbiol 34,
1453–1461.
Blake, P. A. (1994). Historical perspectives on pandemic cholera.
In Vibrio cholerae and Cholera: Molecular to Global Perspec-
tives, pp. 293–295. Edited by I. K. Wachsmuth, P. A. Blake & O.
Olsvik. Washington, DC : American Society for Microbiology.
Brown, A. D., Feldman, M. W. & Nevo, E. (1980). Multilocus
structure of natural populations of Hordeum spontaneum.
Genetics 96, 523–536.
Byun, R., Elbourne, L. D., Lan, R. & Reeves, P. R. (1999).
Evolutionary relationships of pathogenic clones of Vibrio chol-
erae by sequence analysis of four housekeeping genes. Infect
Immun 67, 1116–1124.
Chen, F., Evins, G. M., Cook, W. L., Almeida, R., Hargrett-Bean, N.
& Wachsmuth, K. (1991). Genetic diversity among toxigenic and
nontoxigenic Vibrio cholerae O1 isolated from the Western
Hemisphere. Epidemiol Infect 107, 225–233.
Colwell, R. R. (1996). Global climate and infectious disease : the
cholera paradigm. Science 274, 2025–2031.
Colwell, R. R. & Huq, A. (1994). Vibrios in the environment : viable
but nonculturable Vibrio cholerae.InVibrio cholerae and
Cholera: Molecular to Global Perspectives, pp. 117–133. Edited
by I. K. Wachsmuth, P. A. Blake & O. Olsvik. Washington, DC:
American Society for Microbiology.
Epstein, P. R., Ford, T. E. & Colwell, R. R. (1993). Marine eco-
systems. Lancet 342, 1216–1219.
2625

M. FARFA
!
N and OTHERS
Evins, G. M., Cameron, D. N., Wells, J. G., Greene, K. D., Popovic,
T., Giono-Cerezo, S., Wachsmuth, I. K. & Tauxe, R. V. (1995). The
emerging diversity of the electrophoretic types of Vibrio cholerae
in the Western Hemisphere. J Infect Dis 172, 173–179.
Faruque, S. M., Ahmed, K. M., Abdul Alim, A. R. M., Qadri, F.,
Siddique, A. K. & Albert, M. J. (1997a). Emergence of a new clone
of toxigenic Vibrio cholerae O1 biotype ElTor displacing V.
cholerae O139 Bengal in Bangladesh. J Clin Microbiol 35,
624–630.
Faruque, S. M., Ahmed, K. M., Siddique, A. K., Zaman, K., Abdul
Alim, A. R. M. & Albert, M. J. (1997b). Molecular analysis of
toxigenic Vibrio cholerae O139 Bengal strains isolated in Bangla-
desh between 1993 and 1996 : evidence for emergence of a new
clone of the Bengal vibrios. J Clin Microbiol 35, 2299–2306.
Faruque, S. M., Albert, M. J. & Mekalanos, J. J. (1998). Epi-
demiology, genetics and ecology of toxigenic Vibrio cholerae.
Microbiol Mol Biol Rev 62, 1301–1314.
Felsenstein, J. (1993).  package, version 3n5. Seattle, WA:
University of Washington (http:\\evolution.genetics.washing-
ton.edu\phylip.html).
Fuste
!
, M. C., Pineda, M. A., Palomar, J., Vin
4
as, M. & Lore
!
n, J. G.
(1996). Clonality of multidrug-resistant nontypeable strains of
Haemophilus influenzae. J Clin Microbiol 34, 2760–2765.
Gibson, D. T. (1971). Assay of enzymes of aromatic metabolism.
Methods Microbiol 6A, 463–478.
Harvell, C. D., Kim, K., Burkholder, J. M. & 10 other authors
(1999). Emerging marine diseases – climate links and anthropo-
genic factors (review). Science 285, 1505–1510.
Jiang, S. C., Matte, M., Matte, G., Huq, A. & Colwell, R. R. (2000).
Genetic diversity of clinical and environmental isolates of Vibrio
cholerae determined by amplified fragment length polymorphism
fingerprinting. Appl Environ Microbiol 66, 148–153.
Kaper, J. B., Bradford, H. B., Roberts, N. C. & Falkow, S. (1982).
Molecular epidemiology of Vibrio cholerae in the U. S. Gulf
Coast. J Clin Microbiol 16, 129–134.
Kaper, J. B., Morris, J. G. & Levine, M. M. (1995). Cholera. Clin
Microbiol Rev 8, 48–86.
Karaolis, D. K., Lan, R. & Reeves, P. R. (1994). Molecular evolution
of the seventh-pandemic clone of Vibrio cholerae and its
relationship to other pandemic and epidemic V. cholerae isolates.
J Bacteriol 176, 6199–6206.
Karaolis, D. K., Lan, R. & Reeves, P. R. (1995). The sixth and
seventh cholera pandemics are due to independent clones
separately derived from environmental, nontoxigenic, non-O1
Vibrio cholerae. J Bacteriol 177, 3191–3198.
Karaolis, D. K., Johnson, J. A., Bailey, C. C., Boedeker, E. C.,
Kaper, J. B. & Reeves, P. R. (1998). A Vibrio cholerae pathogenicity
island associated with epidemic and pandemic strains. Proc Natl
Acad Sci U S A 95, 3134–3139.
Kataeva, I. A. & Golovleva, L. A. (1990). Catechol 2,3-dioxy-
genases from Pseudomonas aeruginosa 2x. Methods Enzymol
188, 115–121.
Maynard Smith, J., Smith, N. H., O’Rourke, M. & Spratt, B. G.
(1993). How clonal are bacteria ? Proc Natl Acad Sci U S A 90,
4384–4388.
Mukhopadhyay, A. K., Basu, A., Garg, P., Bag, P. K., Ghosh, A.,
Bhattacharya, S. K., Takeda, Y. & Nair, G. B. (1998). Molecular
epidemiology of reemergent Vibrio cholerae O139 Bengal in
India. J Clin Microbiol 36, 2149–2152.
Nei, M. (1972). Genetic distance between populations. Am Nat
106, 283–292.
Nei, M. (1978). Estimation of average heterozygosity and genetic
distance from a small sample of individuals. Genetics 89, 583–590.
Popovic, T., Fields, P. I., Olsvik, O. & 11 other authors (1995).
Molecular subtyping of toxigenic Vibrio cholerae O139 causing
epidemic cholera in India and Bangladesh, 1992–1993. J Infect Dis
171, 122–127.
Ramamurthy, T., Garg, S., Sharma, R. & 8 other authors (1993).
Emergence of a novel strain of Vibrio cholerae with epidemic
potential in Southern and Eastern India. Lancet 341, 703–704.
Ravel, J., Knight, I. T., Monahan, C. E., Hill, R. T. & Colwell, R. R.
(1995). Temperature-induced recovery of Vibrio cholerae from
the viable but nonculturable state: growth or resuscitation?
Microbiology 141, 377–383.
Rivera, I. G., Chowdhury, M. A. R., Huq, A., Jacobs, D., Martins,
M. T. & Colwell, R. R. (1995). Enterobacterial repetitive intergenic
consensus sequences and the PCR to generate fingerprints of
genomic DNAs from Vibrio cholerae O1, O139 and non-O1
strains. Appl Environ Microbiol 61, 2898–2904.
Rohlf, F. J. (1993). Numerical taxonomy and multivariate analysis
system, version 1n80. New York : Exeter Software.
Salles, C. A. & Momen, H. (1991). Identification of Vibrio cholerae
by enzyme electrophoresis. Trans R Soc Trop Med Hyg 85,
544–547.
Sanchez, J. L. & Taylor, D. N. (1997). Cholera. Lancet 349,
1825–1830.
Selander, R. K., Caugant, D. A., Ochman, H., Musser, J. M.,
Gilmour, M. N. & Whittam, T. S. (1986). Methods of multilocus
enzyme electrophoresis for bacterial population genetics and
systematics. Appl Environ Microbiol 51, 873–884.
Sneath, P. H. A. & Sokal, R. R. (1973). Numerical Taxonomy. San
Francisco: Freeman.
Stroeher, U. H., Parasivam, G., Dredge, B. K. & Manning, P. A.
(1997). Novel Vibrio cholerae O139 genes involved in lipopoly-
saccharide biosynthesis. J Bacteriol 179, 2740–2747.
Wachsmuth, I. K., Evins, G. M., Fields, P. I., Olsvik, O., Popovic, T.,
Bopp, C. A., Wells, J. G., Carrillo, C. & Blake, P. A. (1993). The
molecular epidemiology of cholera in Latin America. J Infect Dis
167, 621–626.
Waldor, M. K. & Mekalanos, J. J. (1996). Lysogenic conversion by
a filamentous phage encoding cholera toxin. Science 272, 1910–
1914.
Whittam, T. S. (1995). Genetic population structure and patho-
genicity in enteric bacteria. In Population Genetics of Bacteria,
pp. 217–245. Edited by S. Baumberg, J. P. Young, E. M. H.
Wellington & J. R. Saunders. Cambridge : Cambridge University
Press.
Young, J. P. W. (1989). The population genetics of bacteria. In
Genetics of Bacterial Diversity. Edited by D. A. Hopwood &
K. F. Chater. London : Academic Press.
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Received 20 December 1999 ; revised 22 May 2000 ; accepted 26 June
2000.
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