Emerging Infectious Diseases
[Volume 6 No.2 / March - April 2000]


Research

Vibrio cholerae O139 in Calcutta, 1992-1998: Incidence,
Antibiograms, and Genotypes

Arnab Basu,* Pallavi Garg,* Simanti Datta,* Soumen
Chakraborty,* Tanuja Bhattacharya,* Asis Khan,* T.
Ramamurthy,* S.K. Bhattacharya,* Shinji Yamasaki, Yoshifumi
Takeda, and G. Balakrish Nair*
*National Institute of Cholera and Enteric Diseases, Calcutta,
India; International Medical Center of Japan, Tokyo, Japan; and
National Institute of Infectious Diseases, Tokyo, Japan

We report results of surveillance for cholera caused by Vibrio
cholerae O139 from September 1992, when it was first identified,
to December 1998. V. cholerae O139 dominated as the causative
agent of cholera in Calcutta during 1992-93 and 1996-97, while the
O1 strains dominated during the rest of the period. Dramatic shifts
in patterns of resistance to cotrimoxazole, neomycin, and
streptomycin were observed. Molecular epidemiologic studies
showed clonal diversity among the O139 strains and continuous
emergence of new epidemic clones, reflected by changes in the
structure, organization, and location of the CTX prophages in the
V. cholerae O139 chromosome.

Vibrio cholerae, the gram-negative organism that causes cholera, is
well defined on the basis of biochemical tests and DNA homology
studies (1), but the serogroups of the species differ in their
pathogenic potential. Of the 193 recognized "O" serogroups of V.
cholerae (2), only O1 and O139 cause epidemic and pandemic
cholera. V. cholerae O139 was first identified in September 1992
in southern India (3) and rapidly spread to all cholera-endemic
areas in India (4) and neighboring countries (5). In February 1994,
a new clone of V. cholerae O1 El Tor biotype (6) replaced the
O139 serogroup as the dominant serogroup causing cholera in
Calcutta (7). After a 33-month quiescent period, a new clone of V.
cholerae O139 (8,9) appeared in August 1996 in Calcutta (10) and
was the dominant serogroup until September 1997. This new clone
has also spread to other parts of India (11). The National Institute
of Cholera and Enteric Diseases, Calcutta, conducts continuous
surveillance for cholera in Calcutta in particular, and in India in
general. During September-October 1998, we observed an increase
in the incidence of O139 cholera, prompting study of these O139
strains. We report the findings of surveillance performed from
September 1992 to December 1998 in India, which enabled us to
track and catalog changes in the O139 strains since its
identification.

The Study

Hospital Surveillance and Bacteriology

Stool specimens were obtained from patients admitted to the
Infectious Diseases Hospital, Calcutta, the only hospital that admits
cholera patients from the city and its suburbs. Since 1995, on two
randomly selected days per week, every fifth patient has been
enrolled from all patients with diarrhea or dysentery, with or
without other complaints, visiting the emergency department of the
Infectious Diseases Hospital, Calcutta. Before 1995, only patients
with diarrhea admitted to the Infectious Diseases Hospital,
Calcutta, from Monday to Friday between 9 a.m. and 1 p.m. were
enrolled. Methods of collection, transport, and bacteriologic
examination of stool samples and identification and serotyping of
V. cholerae have been described (7). The National Institute of
Cholera and Enteric Diseases, Calcutta, is India's national
reference laboratory for cholera, and strains from throughout the
country are sent here for confirmation and phage typing. Strains
received are characterized by an array of tests (4). In this study,
representative strains of V. cholerae O139 isolated from
hospitalized patients in Calcutta from 1992 to 1998 and 15 V.
cholerae O139 strains from other parts of India (eastern, central,
northern, and southern India) isolated in 1998 were randomly
selected for molecular characterization.

Table 1. Oligonucleotide primer sequences used in polymerase
chain reaction assays of Vibrio cholerae O139

Antimicrobial Susceptibility

The V. cholerae O139 strains were examined for resistance to
ampicillin (10 g), chloramphenicol (30 g), cotrimoxazole (25 g),
ciprofloxacin (5 g), furazolidone (100 g), gentamycin (10 g),
neomycin (30 g), nalidixic acid (30 g), norfloxacin (10 g),
streptomycin (10 g), and tetracycline (30 g) by using antibiotic-
impregnated commercial disks (Hi Media, Mumbai, India).
Characterization of the strains as susceptible or resistant was based
on the size of inhibition zones around each disk, according to the
manufacturer's instructions, which followed World Health
Organization recommendations (12). Strains showing an
intermediate zone of inhibition were interpreted as resistant on the
basis of previous minimum inhibitory concentration studies
conducted with V. cholerae (13).

Polymerase Chain Reaction (PCR) Assay

PCR assays (both multiple and single) were used to screen the
O139 strains for five important virulence genes. The first PCR
assay used three pairs of oligonucleotide primers for virulence
genes ctxA (301 bp), tcpA (classical; 617 bp), and tcpA (El Tor;
471 bp), and the second and third PCR assays used primer pairs for
the virulence genes zot and ace, respectively (Table 1) (15). The
composition of the reaction mix and cycling conditions for
amplification have been described (16). Amplification was done by
using an automated thermal cycler (Biometra Gottingen, Germany).
V. cholerae strain VC20 (El Tor, Ogawa), 569B (classical, Inaba)
and Escherichia coli DH5-   strains were used as positive and
negative controls. Amplified products were electrophoresed in a
2% agarose gel (SRL, Bombay, India) in 1x TAE buffer along with
standard molecular weight markers (MWM), stained with ethidium
bromide (Sigma), and recorded with a video documentation system
(Pharmacia Biotech, San Francisco, CA).

Pulsed-Field Gel Electrophoresis (PFGE)

Genomic DNA of various strains of V. cholerae was prepared in
agarose plugs (17). For complete digestion of the DNA, 50U of
NotI enzyme was used. PFGE of inserts was done by the contour-
clamped homogeneous electric field method on a CHEF-mapper
(Bio-Rad) with 0.5x TBE Buffer [44.5 mM Tris, 44.5 mM boric
acid, 1 mM EDTA (pH 8.0)] for 40.24 hours. A DNA size standard
( -ladder; Bio-Rad) was used as molecular weight standard. A
model 1,000 minichiller (Bio-Rad) was used to maintain the
temperature of buffer at 14C. Run conditions were generated by
the auto-algorithm mode of a CHEF Mapper PFGE system with a
size range of 20-300 kb. Gels were stained in distilled water
containing 1.0 g ethidium bromide per ml for 30 minutes, rinsed
several times, and photographed. After electrophoresis, ethidium
bromide staining and photography, transfer of DNA from gel to
Hybond N + membrane (Amersham International PLC,
Buckinghamshire, England) and Southern blot hybridization with
an O139-specific probe were done as described (17). The DNA
adjacent to Tn5lac insertion in MO10 (an O139 strain from the
Madras outbreak) rendered the strain O139 negative in
agglutinability with O139-specific antiserum. The O139 probe
used in this study was prepared as described earlier (18).

Restriction Fragment Length Polymorphism (RFLP) of ctxA Gene
and O139 Specific Gene

A ctxA probe consisting of a 540-bp XbaI-ClaI fragment of ctxA
cloned in pKTN901 with EcoRI 

linkers was used (19). A modification of the method of Murray and
Thompson (20) was used for DNA extraction for ctx RFLP (16).
The transfer of DNA from gel to Hybond N + membrane
(Amersham) and hybridization with probes was done (16) with the
ECL Nucleic Acid Detection System (Amersham). The membranes
were then washed and exposed to Kodak Biomax film (Eastman
Kodak Co., Rochester, NY) and developed according to
manufacturer's instructions.

Figure 1. Monthly isolation profile of the Vibrio cholerae O1 and
O139 serogroups from patients hospitalized with acute secretory
diarrhea at the Infectious Diseases Hospital, Calcutta, India, from
March 1992 to December 1998. Arrows denote the month in which
changes in V. cholerae strains were noted: A, appearance of V.
cholerae O139 in Calcutta in November 1992; B, displacement of
V. cholerae O1 by V. cholerae O139 in January 1993; C,
reappearance of V. cholerae O1; D, domination of V. cholerae O1
over V. cholerae O139; E, isolation of nontoxigenic V. cholerae
O139 in April 1995; F, appearance for the first time of
cotrimoxazole-susceptible strains of V. cholerae O139; G,
appearance of cotrimoxazole-susceptible and neomycin-resistant
strains of V. cholerae O139; H, domination of V. cholerae O139
over V. cholerae O1; I, replacement of V. cholerae O139 by V.
cholerae O1 as the major cholera causing serogroup; J, appearance
of cotrimoxazole- and streptomycin-susceptible strains of V.
cholerae O139. % of V. chollerae isolations

Figure 2. Monthly isolation profile of the percentage of V. cholerae
O1 and O139 serogroups from patients hospitalized with acute
secretory diarrhea at the Infectious Diseases Hospital, Calcutta,
India, from March 1992 to December 1998.


Results

The O139 serogroup dominated as the major cholera-causing
serogroup in 1993 and from September 1996 to September 1997,
while the O1 serogroup dominated during the remaining part of the
study period (Figures 1, 2). The rate of isolation of O139 serogroup
was lower in 1998 than in any other year since 1992, the year it
was identified (Table 2). The seasonality in incidence of the O139
serogroup showed an interesting trend: apart from 1993 and 1994,
more O139 cholera cases were recorded during the latter half of the
year, coinciding with the second peak of cholera cases (Figures 1,
2). However, the peak incidence of cholera caused by the O1
serogroup was generally observed during June-July, except for
1993 and 1997, when the O1 peak occurred during October and
September, respectively (Figures 1, 2). The O139 strains were the
dominant cholera-causing serogroup during these years (Table 2;
Figures 1, 2).

The antibiotic resistance patterns of randomly selected O139
strains isolated from Calcutta during 1992-1998 (Figure 3) showed
dramatic changes in resistance to cotrimoxazole, with all the 1992,
1993, and 1994 O139 strains being resistant, while most of the
strains isolated during 1997 and 1998 were sensitive. Likewise,
resistance to neomycin and streptomycin also increased until 1996,
then declined. O139 strains isolated throughout the study were
resistant to furazolidone and most were resistant to ampicillin. The
dominant drug-resistance patterns among O139 strains isolated in
Calcutta from 1993 to 1998 were A C Co Fz S (30%), A C Co Fz S
(48.1%), A Co Fz S (49.1%), A Fz N S (96%), A Fz N S (32%),
and A Fz (98.3%). PCR studies with 106 representative O139
strains showed that all but one strain (CO853). were positive for
the 471-bp tcpA (El Tor) amplicon, indicating that almost all have
the organelle required for intestinal colonization. The presence of
301-bp ctxA, 843-bp zot, and 282- bp ace amplicons in all strains
except CO853 indicates that all the tcpA-positive strains have an
intact CTX prophage.

Figure 3. Antibiotic resistance pattern of the V. cholerae O139
strains. Abbreviations: A, ampicillin; C, chloramphenicol; Cf,
ciprofloxacin; Co, cotrimoxazole; Fz, furazolidone; G, gentamycin;
N, neomycin; Na, nalidixic acid; Nx, norfloxacin; S, streptomycin;
T, tetracycline. % resistance

The PFGE profile of three randomly selected O139 strains, sharing
the unique CTX ImmCalcutta prophage and isolated from Calcutta
during 1996 and 1997, has a banding pattern similar to that of the
reference strain MO45 (ATCC 51394) isolated during 1992 in
Madras (Figure 4A). The band patterns exhibited by all the O139
strains differed from that of the classical and El Tor biotype
representative strains of V. cholerae O1, 569B, 2164-88, and
CO840. However, the pattern of the reference O139 strain MO45
was identical to that of the O1 strain MO1, which was described as
the progenitor strain of the O139 serogroup (21). The Southern blot
of the PFGE DNA fragments with O139-specific probe showed
that the probe hybridized at the same position in all the O139
strains. However, the control O1 strains and the progenitor strain
MO1 did not hybridize with the probe (Figure 4B).

RFLP was then conducted to determine any differences in number,
location, and arrangement of the CTX prophage in the genome of
O139 strains. As the CTX prophage of the 1992-93 and 1996 O139
strains is well characterized (8,11,16,22), we took only one
representative strain from 1992 (SG24) and 1996 (AM258)
(8,11,22). Five O139 strains (PG265, PG269, PG316, PG337, and
PG351) isolated during September 1998 (when an upsurge in O139
strains was observed) were included in the study. RFLP of the
CTX prophage with HindIII showed one band with SG24, PG265,
PG269, PG316, and PG351; AM258 and PG337 had three bands of
14, 9.2, and 7.0 kb (Table 3). As HindIII has no site in the CTX
prophage (23), four of the five 1998 O139 strains (PG265, PG269,
PG316, and PG351), like the 1992 O139 strain (SG24), have the
CTX prophage at only one site in the chromosome. RFLP with PstI
and BglII showed only one band each of 5.6 kb and 8 kb,
respectively, in four of the 1998 O139 strains (PG265, PG269,
PG316, and PG351), while the 1992 O139 strain (SG24) exhibited
two bands of sizes 7.0 and 5.6 and 8.0 and 7.0 kb, respectively
(Table 3). As PstI and BglII have only a single site in the CTX
prophage but not within the ctxA gene, these results indicate that
most of the 1998 O139 strains have only one CTX prophage,
unlike the 1992 O139 strain, which has two CTX prophages
arranged in tandem (Figure 5). Further, as both the 8- and 5.6-kb
bands are shared by the 1992 and 1998 V. cholerae O139 strains,
the downstream regions of the CTX prophage in the 1992 and 1998
V. cholerae O139 strains are 97% similar, suggesting that they may
share the identical location in the V. cholerae genome. Southern
blot of HindIII-digested genomic DNA, using a ctxA probe of the
1996 O139 strain (AM258), showed three bands, and the Southern
blots of the PstI-and BglII-digested genomic DNA, also using the
ctxA probe, showed only two bands. As discussed (8,16), the strain
has the same organization of the CTX prophage as the 1996-97
O139 strains isolated from Calcutta. It has three tandemly
duplicated CTX prophages, with the second and third CTX
prophages being new and differing from the first in having an
altered restriction endonuclease site (8,9) (Figure 5). The Southern
blots of the 1998 O139 strain PG337 exactly match those of the
1996-97 representative strain AM258 (Table 3), indicating that its
structure resembles that of the 1996-O139 strains.

Figure 5. Schematic diagram of CTX prophage (not to scale) as
deduced from Southern blot hybridization data. Arrows and boxes
correspond to the restriction sites and core regions of CTX
prophage, respectively, with hatched portions in the box
representing ctxAB genes. Restriction site abbreviations: A, AvaI;
BI, BglI; B, BglII; H, HindIII; P, PstI.

Table 2. Isolation rates of Vibrio cholerae O139 from patients with
acute secretory diarrhea admitted to the Infectious Diseases
Hospital, Calcutta, 19921998

Figure 4. Pulsed-field gel electrophoresis profiles of the V.
cholerae O139 strains (A) and Southern blot of the same gel with
O139-specific probe. (B). The numbers at the top indicate the
strain numbers. Lanes 1, MWM (bacteriophage  ladder); 2, 569B
(O1 classical Inaba); 3, 2164-88 (O1 El Tor Ogawa); 4, CO870
(O1 El Tor Ogawa); 5, MO1 (progenitor of the O139 strain); 6,
MO45 (O139 strain isolated during 1992-93); 7, AM231 (O139
strain isolated during 1996-97); 8, AM233 (O139 strain isolated
during 1996-97); 9, AS258 (O139 strain isolated during 1996-97);
10, MWM (bacteriophage   ladder). 

Table 3. Fragment sizes of restriction enzyme-digested genomic
DNA of Vibrio cholerae O139 strains, isolated by ctxA probe,
Calcutta, 1992-1998

Southern blot analysis of HindIII-digested genomic DNA of the 15
O139 strains isolated from different parts of India in 1998 showed
four variations when probed with ctxA. Of the 15 strains
examined, eight (designated group I strains) showed three bands of
size 14, 9.2, and 7.0 kb (the pattern displayed by the 1996-97 O139
strains); two strains had three fragments of sizes 14, 12, and 7.0 kb
(designated group II strains); and four had two fragments of sizes
23 and 7.0 kb (designated group III strains); only one had two
bands of 14 and 7.0 kb (designated group IV strains) (Table 4). All
group I and group II strains showed a single band of about 23 kb in
the blot when digested with BglII and probed with ctxA (Table 4),
indicating that in these strains the BglII site resides outside the
CTX prophage, as described (8). Thus, group I and group II strains
have the same unique organization of the CTX prophage as that of
the 1996-97 O139 strains (8). The group III and group IV strains
also showed a single band when digested with BglII and probed
with ctxA, indicating that in these strains the BglII site may also be
outside the CTX prophage (Table 4). Representative strains of the
four types showed a common band of approximately 7 kb when
digested with BglI, AvaI, and PstI and probed with the ctxA probe
(Table 4). Since all these enzymes have a single restriction site in
the CTX prophage but not within the ctxA gene (23) and the size
of an intact CTX prophage is approximately 7 kb, the 7-kb band in
all these enzymes indicates a tandem duplication in all strains. All
the group I strains have three CTX prophages in tandem (8,16),
with the second and third prophages having a HindIII site instead
of the BglII site in the RS region of the CTX prophage, like the
1996-97 O139 strains (Figure 5). The group II strains differ from
the group I strains by the position of the second band in the blots
(Table 4). Thus, the group II strains have exactly the same
arrangement of the CTX prophage in their genome as the group I
strains; the difference in the size of the second band could be due
to the integration of the prophage at a different site in the genome
(Figure 5). The group III and group IV strains showed two bands in
HindIII, BglI, and AvaI blots when probed with ctxA. There is no
HindIII restriction site in the CTX prophage of El Tor and classical
strains (23), while AvaI, BglII and BglI have only one site within
the CTX prophage (23). In the CTX ImmCalcutta prophage,
present in the 1996-97 O139 strains, the positions of the HindIII
and the BglII enzymes have been interchanged (8,9). The presence
of two bands in the blots with a common 7.0 kb in HindIII, AvaI,
and BglI blots with ctxA and one band in the BglII blot (Table 4)
with same probe indicates that group III and group IV have two
copies of the CTX prophage in tandem, with both the CTX
prophages in these strains being the new type of the O139 CTX
prophage, CTX ImmCalcutta prophage (Figure 5). Thus, these
O139 strains (all isolated in southern India) may derive from the
1996-97 O139 strains but lack the El Tor CTX prophage of the
three CTX prophages that they have in tandem. Alternatively, these
group III and group IV strains may be a new type of O139 that
lacks the El Tor CTX prophage. The only difference between the
group III and group IV strains is in the HindIII blot, which may be
due to polymorphism in the HindIII site (Table 4; Figure 5).

Table 4. Restriction enzyme-digested fragments of genomic DNA
of Vibrio cholerae O139 strains isolated with the ctxA probe, India,
1998

Discussion

The emergence of V. cholerae O139 is a puzzling event in the
history of cholera. The sudden appearance of the O139 serogroup
in late 1992, its rapid spread through Southeast Asia in 1993
followed by a quiescent period during 1994- 95 and its subsequent
emergence in 1996 and 1997 are inadequately understood, but
characterize the unpredictable nature of the epidemiology of
cholera. However, the reemergence of O139 in 1996 indicates that
its appearance was not a one-time event and the serogroup has the
potential to persist and spread to other continents. Comparison of
the O139 strains isolated during the 7-year study period revealed
interesting patterns of antibiotic resistance to various common
antibiotics. While the strains remained largely susceptible to
ciprofloxacin, tetracycline, and gentamycin, resistance to
ampicillin and susceptibility to cotrimoxazole (sulphamethoxazole,
trimethoprim), chloramphenicol, and streptomycin increased
during the period of the study. Resistance to cotrimoxazole
(sulphamethoxazole and trimethoprim) and streptomycin is
encoded by a 62-kb self- transmissible chromosomally integrated
transposon termed the SXT element, which is not apparently linked
to genes encoding O139 antigen (24). The SXT element was
present in 1992-93 O139 strains and is probably silent or absent in
1998 O139 strains, which were sensitive to cotrimoxazole and
streptomycin. The presence of this novel region in 1996 O139
strains is difficult to predict because these strains are susceptible to
cotrimoxazole (sulphamethoxazole and trimethoprim) but resistant
to streptomycin. However, this pattern of rapid shift is consistent
with recent reports indicating an enhanced mobility in genetic
elements, which confers resistance to antibiotics (25) and has also
been observed in V. cholerae O1 strains (7). A multiple-antibiotic
resistance plasmid belonging to incompatibility group C has been
associated with drug-resistance plasmid of V. cholerae O139 (17).
Since the multidrug-resistance plasmid is self-transmissible by
conjugation, the incidence of plasmid-carrying strains and hence
drug resistance may change, depending on the presence of these
plasmids in O139 strains. Molecular studies show continuous
change in the structure and organization of CTX prophage during
the study period, along with evolution of a new type of CTX
prophage. The 1992-93 strains have two CTX prophages connected
by an RS1 element, while the 1996 O139 strains have three CTX
prophages arranged in tandem (8,22). Most of the 1998 O139
strains from Calcutta have only one CTX prophage, while those
isolated from other parts of India have either the arrangement of
the 1996-97 O139 strains (group I and II strains) or have two CTX
prophages arranged in tandem (group III and IV strains). However,
as reported elsewhere (9), the 1996 O139 strains have two types of
CTX prophages, with the first of the three an El Tor type CTX
prophage and the second and third CTX prophages being a new
type of CTX prophage that differs primarily in the rstR gene, the
gene that codes for the repressor protein of CTX . In 1998, we
observed two new clones of O139 at two epicenters, Calcutta and
Alleppey. From the restriction profile data of the 1998 O139
strains, it can be predicted that most of the O139 strains from
Calcutta have only the El Tor type CTX prophage and not the
unique O139 CTX prophage of the 1996 O139 strains, while the
reverse is the case with the south Indian (Alleppey) strains.
Therefore, two different clones of O139 are circulating at two
locations with different types of CTX prophages, indicating that
reassortment in the genome is taking place in the O139 strains. Our
study indicates a continuous emergence of new clones of toxigenic
V. cholerae, possibly through natural selection involving
unidentified factors and immunity of the host population (25-28).
Another possibility is that the genetic reassortments observed here
are random changes in the organism. The strains that gained
advantage as a result of this rearrangement may have infected
humans and become enriched inside the gastrointestinal tract so
that they became detectable as new toxigenic strains. Molecular
analysis of V. cholerae strains isolated during epidemics from 1961
to 1996 in Bangladesh revealed similar clonal diversity among
strains isolated during different epidemics (25-29). These studies
demonstrated three different ribotypes among the V. cholerae O139
isolated from Bangladesh, with different ribotypes often showing
different CTX prophage genotypes (26,28).
This work was supported in part by the Japan International
Cooperation Agency (JICA/NICED Project 054-1061-E-0).

Mr. Basu holds a Masters in Biochemistry and is completing his
doctoral degree in Microbiology under the supervision of G.
Balakrish Nair, Deputy Director of the National Institute of
Cholera and Enteric Diseases, the National Reference Centre for
Cholera in India. Mr. Basu's doctoral thesis is an in-depth
molecular analysis of the CTX genetic element in strains of Vibrio
cholerae isolated in the last 3 decades, including recent isolates of
V. cholerae O139.

Address for correspondence: G. Balakrish Nair, National Institute
of Cholera and Enteric Diseases, P-33, CIT Road, Scheme XM,
Beliaghata, Calcutta, 700 010, India; fax: 91-33- 350-5066; e-mail:
gbnair@vsnl.com.


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Emerging Infectious Diseases
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