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JOURNAL OF CLINICAL MICROBIOLOGY,
0095-1137/97/$04.0010Mar. 1997, p. 624–630 Vol. 35, No. 3
Copyright q1997, American Society for Microbiology
Emergence of a New Clone of Toxigenic Vibrio cholerae O1 Biotype
El Tor Displacing V. cholerae O139 Bengal in Bangladesh
SHAH M. FARUQUE,
1
* KAZI MOKIM AHMED,
1
A. R. M. ABDUL ALIM,
1
FIRDAUSI QADRI,
1
A. K. SIDDIQUE,
2
AND M. JOHN ALBERT
1
Molecular Genetics Laboratory, Laboratory Sciences Division,
1
and Epidemic Control Preparedness Programme,
Health and Population Extension Division,
2
International Centre for Diarrhoeal Disease Research,
Bangladesh, Dhaka 1000, Bangladesh
Received 3 September 1996/Returned for modification 25 November 1996/Accepted 6 December 1996
The emergence of Vibrio cholerae O139 Bengal in 1992, its rapid spread in an epidemic form, in which it
replaced existing strains of V. cholerae O1 during 1992 and 1993, and the subsequent reemergence of V. cholerae
O1 of the El Tor biotype in Bangladesh since 1994 have raised questions regarding the origin of the reemerged
El Tor vibrios. We studied 50 El Tor vibrio strains isolated in Bangladesh and four other countries in Asia and
Africa before the emergence of V. cholerae O139 and 32 strains isolated in Bangladesh during and after the
epidemic caused by V. cholerae O139 to determine whether the reemerged El Tor vibrios were genetically
different from the El Tor vibrios which existed before the emergence of V. cholerae O139. Analysis of restriction
fragment length polymorphisms in genes for conserved rRNA, cholera toxin (ctxA), and zonula occludens toxin
(zot) or in DNA sequences flanking these genes showed that the El Tor strains isolated before the emergence
of V. cholerae O139 belonged to four different ribotypes and four different ctx genotypes. Of 32 El Tor strains
isolated after the emergence of O139 vibrios, 30 strains (93.7%) including all the clinical isolates belonged to
a single new ribotype and a distinctly different ctx genotype. These results provide evidence that the reemerged
El Tor strains represent a new clone of El Tor vibrios distinctly different from the earlier clones of El Tor
vibrios which were replaced by the O139 vibrios. Further analysis showed that all the strains carried the
structural and regulatory genes for toxin-coregulated pilus (tcpA,tcpI, and toxR). All strains of the new clone
produced cholera toxin (CT) in vitro, as assayed by the G
M1
-dependent enzyme-linked immunosorbent assay,
and the level of CT production was comparable to that of previous epidemic isolates of El Tor vibrios. Further
studies are required to assess the epidemic potential of the newly emerged clone of V. cholerae O1 and to
understand the mechanism of emergence of new clones of toxigenic V. cholerae.
The emergence of Vibrio cholerae O139 Bengal in 1992, its
initial rapid spread throughout Bangladesh and neighboring
countries, and its propensity to replace the existing strains of V.
cholerae O1 led experts to suspect V. cholerae O139 as the new
pandemic strain of cholera (1, 14, 24, 25, 30). The epidemic
caused by V. cholerae O139 moved quickly and affected the
entire coastal and estuarine tidal plains of southern Bang-
ladesh. By the end of March 1993, the epidemic reached the
middle and northern parts of the country and the coastal areas
and spread eastward (1, 30). In the beginning the new strain
totally displaced the existing V. cholerae O1 strains, including
both Classical and El Tor biotypes, which coexisted only in
Bangladesh. Nearly 2 years after the initial detection of O139
vibrios, striking differences in the distribution of O139 and O1
vibrios were observed. In most northern and central areas of
Bangladesh, including the capital city, Dhaka, the O139 vibrios
have been replaced by V. cholerae O1 of the El Tor biotype (5,
28). In the southern coastal regions, however, the O139 vibrios
continue to exist (28).
The factors which determine the emergence and domination
of particular clones of toxigenic V. cholerae through natural
selection are not clear. The disappearance and reemergence of
the El Tor biotype of V. cholerae O1 in Bangladesh have raised
questions regarding the possible origin of the reemerged El
Tor strains. In the present study we have used molecular tech-
niques to analyze El Tor strains collected before and after the
emergence of V. cholerae O139 to investigate whether the El
Tor strains isolated before the emergence of O139 vibrios were
genetically different from the El Tor strains isolated after the
epidemic caused by V. cholerae O139.
MATERIALS AND METHODS
V. cholerae strains. A total of 82 V. cholerae O1 isolates, obtained from cholera
patients and environmental surface water either before or after the emergence of
V. cholerae O139, were included in the study. The strains isolated before the
emergence of V. cholerae O139 included 29 strains isolated in Bangladesh be-
tween 1969 and 1992 and 21 strains from four other countries in Asia and Africa
isolated during 1991 and 1992. Clinical isolates from Bangladesh were obtained
from patients who attended the treatment center of the International Centre for
Diarrhoeal Disease Research, Bangladesh (ICDDR,B), located in Dhaka. Other
Asian isolates consisted of five El Tor strains from Syria (courtesy of F. Harb,
Public Health Laboratory, Damascus, Syria) and three El Tor strains from India
(courtesy of G. B. Nair, National Institute for Cholera and Enteric Diseases,
Calcutta, India). The African strains consisted of seven El Tor strains from
Tanzania (courtesy of F. Mahlu, Muhimbili Medical Centre, Dar-es-Salam, Tan-
zania) and six El Tor strains from Nigeria (courtesy of H. van Vliet, World
Health Organization, Lagos, Nigeria). The El Tor strains isolated in Bangladesh
during and after the epidemic caused by V. cholerae O139 included 32 strains, of
which 27 were patient isolates and 5 were environmental isolates. Strains were
stored either in lyophilized form or in sealed deep nutrient agar at room tem-
perature in the culture collection of the ICDDR,B. Before use, the identities of
the cultures were confirmed by biochemical reaction and serology (37). Details of
the strains are presented in Table 1.
Preparation of colony and Southern blots. Colony blots were prepared by
using nylon filters (Hybond; Amersham International plc, Ayelesbury, United
Kingdom) and were processed by a standard method (18). Briefly, colonies were
lysed with denaturing solution (0.5 M NaOH, 1.5 M NaCl) and were neutralized
in neutralizing solution (0.5 M Tris-HCl [pH 8.0], 1.5 M NaCl), and the liberated
DNA was fixed to the nylon membrane by exposure to UV light for 3 min in
accordance with the supplier’s instructions.
* Corresponding author. Mailing address: Molecular Genetics Lab-
oratory, Laboratory Sciences Division, ICDDR,B, GPO Box 128,
Dhaka-1000, Bangladesh. Fax: 880 2 872529 and 880 2 883116. E-mail:
faruque@cholera.bangla.net.
624
For preparation of DNA blots, total cellular DNA was isolated from overnight
cultures as described previously (32). Five-microgram aliquots of the DNA were
digested with appropriate restriction enzymes (Bethesda Research Laboratories,
Gaithersburg, Md.), electrophoresed in 0.8% agarose gels, and blotted onto
nylon membranes (Hybond; Amersham) by Southern blotting (31).
Probes and hybridization. The rRNA gene probe was a 7.5-kb BamHI frag-
ment of pKK3535 (4), which is a pBR322-derived plasmid containing an Esch-
erichia coli rRNA operon consisting of one copy each of the genes coding for 5S
rRNA, 16S rRNA, 23S rRNA, and tRNA
Glu
. The gene probe for cholera toxin
(CT) was a 0.5-kb EcoRI fragment of pCVD27 (15), which is a pBR325-derived
plasmid containing an XbaI-ClaI fragment representing 94% of the gene encod-
ing the A subunit of CT (ctxA) cloned with EcoRI linkers. The probe for zonula
occludens toxin (Zot) was an 850-bp region internal to the zot gene and was
amplified from the recombinant plasmid pBB241 (3) as we have described
previously (8). The toxR gene probe was a 2.4-kb BamHI fragment of pVM7 (20),
which is a pBR322-derived plasmid carrying the entire toxR sequence.
The probes used for hybridization were labelled by random priming (10) by
using a random primer DNA labelling kit (Bethesda Research Laboratories) and
[a-
32
P]dCTP (3,000 Ci/mmol; Amersham). Southern blots and colony blots were
prehybridized and hybridized with the labelled probes at 688C as described
previously (9). Hybridized blots were washed once in 23SSC (13SSC is 0.15 M
NaCl plus 0.15 M sodium citrate) for 5 min at room temperature, two times in
23SSC–0.1% sodium dodecyl sulfate for 10 min at 688C, and once in 0.13
SSC–0.1% sodium dodecyl sulfate for 15 min at 688C. Autoradiographs were
developed from the hybridized filters by using either Kodak X-Omat AR film
(Kodak, Rochester, N.Y.) or Fuji X-ray film at 2708C as described previously
(9).
PCR assays. The presence of tcpA genes specific for the Classical and El Tor
biotypes was determined by a multiplex PCR assay as described previously (14).
The tcpI gene was detected by a PCR assay based on the published sequence of
tcpI (12), as follows. Two primers, 59-AACGATAAAGCTGATTTTCAC and
59-CCAAGGCTTAGAGCCTTTTAT, were synthesized commercially by Oswel
DNA Service (University of Edinburgh, Edinburgh, United Kingdom). Thermo-
cycle parameters for the PCR assay consisted of denaturation at 948C for 2 min,
annealing of primers at 508C for 2 min, and primer extension at 728C for 3 min.
Amplification was performed for 25 cycles, and the expected size of the amplicon
(2.1 kb) was ascertained by electrophoresis in 1.5% agarose gels. The identity of
the PCR product was further verified by digesting the PCR product with the
restriction enzymes BclI, HaeIII, or XbaI and determining the sizes of the
digestion fragments by agarose gel electrophoresis and comparing the sizes of the
fragments with expected sizes based on the published sequence of the tcpI gene
(12).
Assay for CT production. The ability of the strains to produce CT in vitro was
determined by the G
M1
-ganglioside-dependent enzyme-linked immunosorbent
assay (G
M1
-ELISA) as described previously (26, 33). For each round of the CT
assay, 5 ml of AKI medium (1.5% Bacto Peptone, 0.4% yeast extract, 0.5% NaCl,
0.3% NaHCO
3
[pH 7.4]) was inoculated with approximately 10
3
bacterial cells,
and the cells were grown for 16 h at 308C with shaking. The culture was centri-
fuged at 4,000 3gfor 5 min, and the supernatant was collected. Aliquots of the
undiluted supernatant, 10-fold and 100-fold dilutions of the supernatant, and
dilutions of purified CT (Sigma Chemical Company, St. Louis, Mo.) were used
for the toxin assay. Briefly, 100 ml of the samples was added to each well of
microtiter plates precoated with G
M1
, and the plates were incubated at room
temperature for 90 min. After washing the plates with phosphate-buffered saline
containing 0.5% Tween 20, the G
M1
-bound CT was reacted with rabbit anti-CT
monoclonal antibody (Sigma). Antibody binding to CT was detected by reaction
with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G
(whole molecule) antibody (Sigma) and the substrates o-phenyldiamine and
hydrogen peroxide. Quantitation of CT production was done by using a standard
curve prepared for each batch of the assay mixture. The amount of CT produced
by each strain was the mean value of three different assays with the same strain
and culture conditions. Statistical comparison of CT production between two
groups of strains was carried out by the Mann-Whitney test, and comparison of
that between more than two groups was done by the Kruskal-Wallis test. Dif-
ferences were considered to be significant when Pwas #0.05. Data analysis was
carried out by using statistical software (Sigmastat, version 1.0, for Windows;
Jandel Scientific, San Rafael, Calif.).
RESULTS
rRNA gene restriction patterns. Analysis of rRNA genes
with BglI produced reproducible restriction patterns, and the
82 strains could be differentiated into five different ribotypes.
The restriction patterns (Fig. 1) consisted of 7 to 10 bands
between 12 and 1.6 kb in size. Of the 50 strains isolated before
the emergence of V. cholerae O139, 23 (46%) belonged to
ribotype I, 17 (34%) belonged to ribotype II, 3 (6%) belonged
to ribotype III, and 7 (14%) belonged to ribotype IV. Of the 32
El Tor strains isolated after the emergence of V. cholerae
O139, 30 strains (93.7%), including all 27 clinical isolates,
belonged to ribotype V, and the remaining 2 environmental
isolates belonged to ribotype II (Table 1). The restriction pat-
tern representing ribotype V contained a unique band of 8.7 kb
which was not present in any of the other restriction patterns
(Fig. 1).
Restriction patterns of ctxA and zot genes. Restriction frag-
ment length polymorphism analysis of the ctxA and zot genes
with the enzyme BglI revealed five different restriction patterns
for each of the ctxA (patterns A through E) and zot (patterns
TABLE 1. Restriction endonuclease cleavage patterns of cholera toxin (ctxA), zonula occludens toxin (zot), and rRNA genes
among 82 V. cholerae O1 strains of the El Tor biotype isolated before or after the emergence of V. cholerae O139
a
Strains and country of origin Yr of
isolation Source No. of
isolates
BglI cleavage pattern of genes Designated ctx
genotype
b
ctxA zot rRNA
Strains isolated before the emergence
of V. cholerae O139
Bangladesh 1970–1976 Patient 5 A a I 1
Bangladesh 1969–1976 Patient 7 B b I 2
Bangladesh 1991–1992 S. water
c
5BbI 2
Bangladesh 1991–1992 Patient 6 C c II 3
Bangladesh 1991–1992 S. water 3 C c II 3
Bangladesh 1990 Patient 3 D d III 4
India 1992 Patient 3 B b I 2
Syria 1992 Patient 5 C c II 3
Nigeria 1992 Patient 6 B b I 2
Tanzania 1991–1992 Patient 7 B b IV 2
Strains isolated after the emergence
of V. cholerae O139
Bangladesh 1993–1994 S. water 2 C c II 3
Bangladesh 1994–1995 S. water 3 E e V 5
Bangladesh 1995 Patient 16 E e V 5
Bangladesh 1996 Patient 11 E e V 5
a
The presence of tcp genes was detected by PCR assays. All strains carried tcpA (characteristic of the El Tor biotype), tcpI, and toxR.
b
Genotype based on restriction patterns of ctxA and zot genes.
c
S. water, surface water.
VOL. 35, 1997 NEW CLONE OF V. CHOLERAE O1 625
a through e) genes. The ctxA patterns consisted of one to two
bands between 8.1 and 3.0 kb, and the zot patterns consisted of
two to three bands between 9.2 and 3.0 kb (Fig. 2A and B). The
number of bands comprising a zot gene restriction pattern was
always one band more than the number of bands comprising
the ctxA restriction pattern produced by the same strain. Four
of the ctxA restriction patterns (patterns A through D) and the
corresponding zot restriction patterns (patterns a through d)
were shared by El Tor strains isolated before the emergence of
V. cholerae O139, whereas 30 of the 32 El Tor strains isolated
after the emergence of O139 vibrios produced restriction pat-
terns E and e with the ctxA and the zot gene probes, respec-
tively (Table 1). The 82 strains were differentiated into five
different ctx genotypes on the basis of the BglI restriction
patterns of their ctxA and zot genes.
Analysis of tcp and toxR genes. All strains were positive for
the tcpA,tcpI, and toxR genes. PCR assay for tcpA amplified a
0.47-kb portion of the tcpA gene in all the strains (Fig. 3). This
was characteristic of the El Tor biotype tcpA gene. PCR assay
for the tcpI gene produced an amplicon of 2.1 kb. Subsequent
restriction analysis of the amplicon with BclI, HaeIII, or XbaI
produced sets of fragments whose sizes agreed with the ex-
pected sizes based on the published sequence of tcpI. Digestion
with BclI produced two fragments of 1.5 and 0.6 kb, digestion
with XbaI produced two fragments of 1.7 and 0.4 kb, and
digestion with HaeIII produced four fragments of 0.85, 0.75,
0.27, and 0.19 kb (Fig. 4).
Colony blot hybridization revealed that all the strains in the
present study carried the sequence for ToxR. Subsequent
Southern blot hybridization of HindIII-digested total DNA
with the toxR probe produced identical band patterns for all
the strains (Fig. 5). The patterns consisted of two bands of 9.1
and 2.3 kb.
Production of CT. All the El Tor strains isolated after the
emergence of O139 vibrios produced CT in vitro when they
were cultured in AKI medium at 308C. The level of CT pro-
duction varied between 0.25 and 12.75 ng/ml among the dif-
ferent strains. There was no significant difference (P50.464)
between the levels of CT produced by the El Tor strains iso-
lated before and after the emergence of O139 vibrios (Table
2).
DISCUSSION
Several previous studies have demonstrated the appearance
and disappearance of different clones of toxigenic V. cholerae
in Bangladesh (6, 9, 11, 27, 29, 30). These include the transient
appearance of multiple-drug-resistant strains (11), the disap-
pearance and reemergence of Classical V. cholerae in Bang-
FIG. 1. Southern hybridization analysis of genomic DNA from V. cholerae
digested with BglI and probed with a 7.5-kb BamHI fragment of the E. coli rRNA
clone pKK3535. Lanes 1 and 2, BglI restriction pattern I; lanes 3 through 5,
restriction patterns II through IV, respectively, produced by El Tor vibrios
isolated before the emergence of V. cholerae O139; lanes 6 through 11, BglI
restriction pattern V produced by El Tor strains isolated after the emergence of
V. cholerae O139. Numbers indicate the molecular sizes of bands corresponding
to a 1-kb DNA ladder (Bethesda Research Laboratories), used as a molecular
size marker.
FIG. 2. Southern hybridization analysis of genomic DNA from V. cholerae
digested with BglI and probed with a 550-bp fragment of the A subunit of the CT
gene (A) and with a 850-bp PCR-generated probe for the zonula occludens toxin
gene (B). BglI restriction pattern I (lanes 1 and 5 in Fig. 1A and B) and patterns
II through IV (lanes 2 through 4, respectively, in Fig. 1A and B) correspond to
ctx genotypes 1 and 2 through 4, respectively, demonstrated by El Tor strains
isolated before the emergence of V. cholerae O139. Restriction pattern V (lanes
6 through 11 in Fig. 1A and B) corresponds to ctx genotype 5 demonstrated by
El Tor vibrios isolated after the emergence of V. cholerae O139. Numbers
indicate the molecular sizes of bands corresponding to a 1-kb DNA ladder
(Bethesda Research Laboratories).
626 FARUQUE ET AL. J. CLIN.MICROBIOL.
ladesh (6, 27, 29), and the emergence of a non-O1 V. cholerae
(O139) strain as the predominant epidemic strain (1, 30). Soon
after the emergence of V. cholerae O139, the existing strains of
V. cholerae O1 (mostly of the El Tor biotype) were almost
completely displaced, possibly through a competitive mecha-
nism which might have involved unidentified environmental
factors as well as preexisting immunity in the host population.
However, the gradual reemergence of El Tor strains of V.
cholerae O1 since 1994 and the decline of the O139 strains
need to be explained. The present study was designed to in-
quire whether the reemergence of El Tor vibrios was a result of
the domination of preexisting clones of El Tor vibrios over
O139 vibrios due to possible changes in environmental circum-
stances or whether the reemerged El Tor strains represent a
new clone of toxigenic V. cholerae O1 which was able to com-
pete better than the previous clones. A clone refers to bacterial
isolates which share so many identical phenotypic and genetic
traits that the most likely explanation is a common origin. We
have previously examined the restriction patterns of conserved
rRNA genes (ribotypes) and CT genes or DNA flanking these
genes to differentiate among clones of toxigenic V. cholerae
which are otherwise phenotypically identical (6, 7, 9). These
studies have demonstrated that the restriction patterns are
reproducible and may be considered fairly stable markers for
identifying different clones.
Clonal diversity of El Tor strains. Clonal diversity among El
Tor strains has been documented previously (9, 17, 23), and
those studies suggested that toxigenic El Tor strains might
have evolved from several parental strains or clones. In the
present study, the 82 strains could be differentiated and were
found to belong to five different ribotypes on the basis of the
BglI cleavage patterns of their rRNA genes (Fig. 1). The El Tor
vibrios isolated before the emergence of O139 vibrios com-
prised four ribotypes (ribotypes I through IV), whereas 93.75%
(30 of 32) of the isolates obtained after the emergence of the
O139 vibrios belonged to a single ribotype (ribotype V). We
have also previously reported (9) the rRNA gene restriction
patterns corresponding to ribotypes I through IV found in the
present study. However, cleavage pattern V produced by the
post-O139 strains in this study have not been reported previ-
ously by us or other investigators who have analyzed a large
number of El Tor, Classical, and O139 strains from different
countries (7, 17, 23). This suggests that the post-O139 El Tor
strains represent a new clone of El Tor vibrios. Although all
the El Tor vibrios isolated from patients and environmental
surface water from 1994 to 1996 belonged to this new ribotype,
two strains isolated from environmental surface water during
1993 and 1994 belonged to ribotype II. These two isolates may
have been remnants of the El Tor strains which were being
replaced by V. cholerae O139.
Probing of the BglI restriction fragments of the chromosome
for the ctxA and zot genes also revealed differences among the
El Tor vibrios isolated before and after the emergence of the
O139 vibrios. In V. cholerae, the genes encoding cholera toxin
(ctxAB) and zonula occludens toxin (zot) are part of a larger
FIG. 3. Agarose gel electrophoresis of PCR products derived from multiplex
PCR assays with El Tor and Classical biotype-specific primers for the tcpA gene.
Lanes B and C, products from two El Tor strains isolated before the emergence
of V. cholerae O139; lanes D and E, PCR products from two strains isolated after
the emergence of V. cholerae O139; lanes F and G, control El Tor and Classical
strains, respectively. Numbers indicate the molecular sizes of bands correspond-
ing to low-molecular-weight fragments of a 1-kb DNA ladder (Bethesda Re-
search Laboratories) (lanes A and H).
FIG. 4. Restriction analysis of a 2.1-kb PCR-generated fragment of tcpI gene
from El Tor strains isolated before (lanes B, C, G, H, L, and M) and after (lanes
D through F, H through J, and N through P) the emergence of V. cholerae O139.
Cleavage patterns with BclI (lanes B through F), XbaI (lanes G through K), and
HaeIII (lanes L through P) are shown. Numbers indicate the molecular sizes of
bands corresponding to low-molecular-weight fragments of a 1-kb DNA ladder
(Bethesda Research Laboratories) (lanes A and Q).
FIG. 5. Southern hybridization analysis of genomic DNA from V. cholerae
digested with HindIII and probed with the toxR gene probe. Lanes 1 through 5,
strains isolated before the emergence of V. cholerae O139; lanes 6 through 11,
strains isolated after the emergence of V. cholerae O139. Numbers indicate the
molecular sizes of bands corresponding to a 1-kb DNA ladder (Bethesda Re-
search Laboratories).
VOL. 35, 1997 NEW CLONE OF V. CHOLERAE O1 627
genetic element (ctx genetic element) consisting of at least five
genes (comprising the core region) that is flanked by two or
more copies of a repeated sequence (22, 35). Although there is
very little variation among the structural sequences of CTs
from different strains, restriction fragment length polymor-
phism in ctx is observed due to variation in the number of
copies of the ctx genetic element carried by different strains as
well as variation in the chromosomal sequence flanking the ctx
element. In the present study the restriction endonuclease
used was BglI, which does not have any recognition sequence
within the ctxA gene, but it has a single cleavage site within the
zot gene located upstream and adjacent to the ctxA gene (3,
19). Consequently, the number of bands comprising each ctxA
restriction pattern represented the possible number of copies
of the ctx element carried by the strain. The number of bands
comprising the zot restriction patterns was one band more than
the number of bands comprising the ctx restriction pattern in
the same strain (Fig. 2A and B), suggesting that strains carry-
ing more than one copy of the ctx element possibly had the
copies located adjacent to each other in the chromosome. On
the basis of the ctxA and zot restriction patterns, the strains
were grouped into five different ctx genotypes (Table 1). While
ctx genotypes 1 through 4 were shared by the 50 El Tor strains
isolated before the emergence of O139 vibrios, 30 of the 32 El
Tor strains from the post-O139 period belonged to ctx geno-
type 5. All 30 strains belonged to the new ribotype (ribotype
V). The ctxA restriction patterns of the 30 isolates belonging to
the new ribotype were also different from our previously re-
ported restriction patterns of ctxA or its flanking DNA se-
quences in Classical, El Tor, or O139 vibrios (7). Hence, the
ribotype data and the ctx genotype data agreed, providing
further evidence that the post-O139 El Tor vibrios isolated in
Bangladesh represent a new clone.
Analysis of tcp and toxR genes. Colonization of brush bor-
ders in the small intestine, a crucial component of the infection
strategy of V. cholerae, is assumed to be mediated by a rigid
pilus colonization factor designated toxin-coregulated pilus
(TCP), since it is under the same genetic control as CT, and
involves the ToxR-ToxT regulatory cascade (12, 21, 34). Mo-
lecular analysis has revealed that although the major subunit of
TCP is TcpA, the formation and function of the pilus assembly
require the products of a number of other genes located on the
chromosome adjacent to the tcpA gene, and these constitute a
tcp gene cluster (21). The tcpH and tcpI genes are two ToxR-
regulated genes that affect TcpA synthesis. It has been sug-
gested that regulators such as TcpI that act downstream of
ToxR and ToxT may function to fine-tune the expression of the
TCP virulence determinant throughout the pathogenic cycle of
V. cholerae (12).
In the present study all the El Tor strains carried the tcpA,
tcpI, and toxR genes. Although the post-O139 El Tor vibrios
were different from the El Tor vibrios isolated before the
emergence of V. cholerae O139 in terms of rRNA, ctxA, and zot
restriction patterns, restriction analysis of the PCR-amplified
tcpI gene and Southern blot hybridization of the toxR gene
showed that both of these regulatory genes are highly con-
served among the 82 El Tor strains studied. A recent report by
Waldor and Mekalanos (36) suggested that lysogenic conver-
sion by a bacteriophage designated ctxFencoding CT can give
rise to toxigenic strains from nontoxigenic V. cholerae strains
and that the phage conversion requires expression of TCP,
which is used as a receptor by the bacteriophage. Hence, the
possibility that the new clone of El Tor vibrios arose as a
consequence of bacteriophage conversion of a nontoxigenic
strain of V. cholerae O1 cannot be ruled out. Furthermore,
integration of the phage genome (the ctx genetic element) into
the host chromosome at particular sites is specified by the
presence of a 17-bp sequence called attRS1 (22, 36). The dis-
tinctly different ctxA restriction pattern produced by the new
clone of El Tor vibrios suggests that integration of the ctx
genetic element in these strains might have occurred at chro-
mosomal sites different from those for the other El Tor vibrios,
possibly due to the presence of the attRS1 sequence in these
sites of the nontoxigenic parental strain. However, further
TABLE 2. CT production by 50 El Tor vibrio strains isolated before the emergence of O139 vibrios and 32 El Tor strains
isolated after the emergence of V. cholerae O139
Strain and country of origin Yr of
isolation Source No. of
isolates ctx
genotype No. of ctx
copies
CT production
(concn [ng/ml])
a
Median Range
Strains isolated before the emergence
of V. cholerae O139
Bangladesh 1970–1976 Patient 5 1 1 4.84 2.39–6.37
Bangladesh 1969–1976 Patient 7 2 2 3.96 1.32–8.67
Bangladesh 1991–1992 S. water
b
5 2 2 2.84 1.36–3.52
Bangladesh 1991–1992 Patient 6 3 2 2.72 1.12–3.82
Bangladesh 1991–1992 S. water 3 3 2 2.12 1.51–3.17
Bangladesh 1990 Patient 3 4 2 2.45 1.75–3.75
India 1992 Patient 3 2 2 3.61 2.73–5.58
Syria 1992 Patient 5 3 2 3.23 1.50–4.34
Nigeria 1992 Patient 6 2 2 3.93 3.52–7.87
Tanzania 1991–1992 Patient 7 2 1 5.81 3.99–9.64
Strains isolated after the emergence
of V. cholerae O139
Bangladesh 1993–1994 S. water 2 3 2 4.72 2.95–6.49
Bangladesh 1994–1995 S. water 3 5 2 2.95 1.92–3.49
Bangladesh 1995–1996 Patient 27 5 2 3.95 0.25–12.57
a
Differences in the median values of CT produced by different groups of strains were not statistically significant (P50.063). The difference in the median
concentrations of CT produced by the 50 El Tor strains isolated before and the 32 El Tor strains isolated after the emergence of V. cholerae O139 was also statistically
insignificant (P50.464).
b
S. water, surface water.
628 FARUQUE ET AL. J. CLIN.MICROBIOL.
studies and identification of the possible nontoxigenic parental
strain is essential to confirm these assumptions. An alternative
explanation could, however, be that strains belonging to the
new clone existed in the environment in very low numbers and
hence were not detected in the past, but that some unidentified
environmental changes have caused these strains to multiply
rapidly and to become dominant over existing strains of V. chol-
erae O139. It is interesting that in many parts of neighboring
India, as in Bangladesh, after the initial dominance of V. chol-
erae O139, it has been replaced by El Tor vibrios (2).
Epidemic potential of the new clone of El Tor vibrios. The
mechanism involved in the domination of a newly emerged
clone of toxigenic V. cholerae resulting in the displacement of
existing clones is not clear, although unidentified environmen-
tal factors are likely to influence the process. Previous exam-
ples of the emergence or reemergence of different clones of
toxigenic V. cholerae were often associated with epidemic out-
breaks of cholera caused by the newly emerged strain (1, 6, 11,
27). All the strains examined in the present study were isolated
from the capital city of Bangladesh, Dhaka, which is over-
crowded, and the growth in population has outstripped the
capacity to provide adequate housing and sanitation facilities.
The capital is surrounded by large semirural population cen-
ters and receives an enormous influx of people from rural
villages and hence serves as a catchment area for representa-
tive V. cholerae strains found throughout the country. The
epidemic potential of the new clone examined in this study is
still not clear, but recent surveillance results for Dhaka and
several rural districts of Bangladesh and results of molecular
analysis of strains isolated in Dhaka suggest that the El Tor
vibrios which have already replaced the O139 vibrios, at least
in the northern and central parts of Bangladesh, may also
belong to the new clone. Studies are under way to collect and
analyze strains from different rural areas in Bangladesh.
It has been suggested that CT, TCP, and the ToxR regulon
are essential for V. cholerae pathogenesis in humans (13). In
the present study, all the El Tor strains belonging to the new
clone carried the genes for tcpA,tcpI,ctxA, and zot and, pre-
sumably, the complete ctx genetic element and the tcp gene
clusters, in addition to the toxR gene. Most of these El Tor
vibrios were isolated from cholera patients, and the strains
produced CT in vitro, the level of which was comparable to
that produced by strains isolated from previous cholera epi-
demics. Hence, the possibility that the new clone can give rise
to spreading outbreaks of cholera under appropriate circum-
stances cannot be ruled out. The movement of V. cholerae
strains belonging to the new clone should therefore be care-
fully monitored through environmental and epidemiological
surveillance. Further molecular studies are also required to
understand the genetic basis of the apparent ability of the
clone to compete with V. cholerae O139 better than the previ-
ously existing clones of El Tor vibrios.
ACKNOWLEDGMENTS
This research was funded by the U.S. Agency for International
Development under grant HRN-5986-A-00-6005-00 with ICDDR,B.
ICDDR,B is supported by countries and agencies which share its
concern for the health problems of developing countries. Current
donors providing core support include the aid agencies of the govern-
ments of Australia, Bangladesh, Belgium, Canada, China, Denmark,
Japan, Saudi Arabia, Sri Lanka, Sweden, Switzerland, Thailand, the
United Kingdom, and the United States and international organiza-
tions including the Arab Gulf Fund, the Asian Development Bank, the
European Union, the United Nations Children’s Fund (UNICEF), the
United Nations Development Programme (UNDP), the United Na-
tions Population Fund (UNFPA), and the World Health Organization
(WHO).
We thank John Mekalanos, Harvard Medical School, Boston, Mass.,
for the ToxR clone, and Manzurul Haque for secretarial assistance.
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