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Previous Article | Next Article 
Journal of Clinical Microbiology, January 2000, p. 44-49, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Development and Evaluation of a Phage Typing Scheme
for Vibrio cholerae O139
A. K.
Chakrabarti,
A. N.
Ghosh,
G. Balakrish
Nair,
S. K.
Niyogi,
S.
K.
Bhattacharya, and
B. L.
Sarkar*
National Institute of Cholera and Enteric
Diseases, Beliaghata, Calcutta-700 010, India
Received 7 May 1999/Returned for modification 22 July 1999/Accepted 27 September 1999
 |
ABSTRACT |
The scenario of cholera that existed previously changed in 1992 and
1993 with the emergence of toxigenic Vibrio cholerae O139 in India. The genesis of the new serogroup formed the impetus to search
for O139 phages in and around the country. A total of five newly
isolated phages lytic to V. cholerae O139 strains were used
for the development of this phage typing scheme. These phages differed
from each other and also differed from the existing O1 phages in their
lytic patterns, morphologies, restriction endonuclease digestion
profiles, and immunological criteria. With this scheme, 500 V. cholerae O139 strains were evaluated for their phage types, and
almost all strains were found to be typeable. The strains clustered
into 10 different phage types, of which type 1 (38.2%) was the
dominant type, followed by type 2 (22.4%) and type 3 (18%). Additionally, a comparative study of phage types in 1993 and 1994 versus those from 1996 to 1998 for O139 strains showed a higher percentage of phage type 1 (40.5%), followed by type 3 (18.8%) during
the period between 1993 and 1994, whereas phage type 2 (32.1%) was the
next major type during the period from 1996 to 1998. This scheme
comprising five newly isolated phages would be another useful tool in
the study of the epidemiology of cholera caused by V. cholerae O139.
| |
INTRODUCTION |
Despite more than a century of
study, cholera remains an important cause of morbidity and mortality
and still presents a devastating global problem. Until 1992, Vibrio cholerae O1 was considered the sole causative agent
of the disease cholera. In October 1992 the scenario of cholera that
had existed changed because of the emergence of a new etiologic
serogroup of cholera, which is now known as O139 Bengal
(21). These strains emerged abruptly in the south Indian
coastal city of Madras and rapidly spread to other areas of India and
neighboring countries where cholera is endemic (15, 16).
Strategies for the prevention and control of an infectious disease like
cholera depend on understanding the origin, transmission, and other
characteristics associated with the epidemic and its spread. Among the
several typing methods, phage typing is one of the important and useful
methods for the identification and differentiation of V. cholerae strains. The use of bacteriophages as a method of strain
differentiation has contributed greatly to the understanding of the
epidemiology of the disease cholera. Basu and Mukerjee (4)
proposed a phage typing scheme for V. cholerae O1 biotype E1
Tor which was efficiently used to study the spread of the E1 Tor
biotype of V. cholerae O1. A new phage typing scheme was
subsequently developed at the National Institute of Cholera and Enteric
Diseases for V. cholerae O1 (7, 18). The recent
emergence of toxigenic V. cholerae O139 has prompted us to
conduct a search for bacteriophages for V. cholerae O139 and
to develop an effective phage typing scheme for this organism.
| |
MATERIALS AND METHODS |
Bacterial strains.
V. cholerae O139 NPR-4
(6), isolated from a patient in the city of Nagpur, India,
in 1993, was used as the host for propagation of V. cholerae
O139 phages as it was lysed by all the phages. A total of 500 V. cholerae O139 strains isolated from different areas in India where
cholera is endemic between 1993 and 1998 were included in the phage
typing study. Additionally, V. cholerae O1 biotype E1 Tor
strain MAK 757 (ATCC 51352), 10 E1 Tor strains (ATCC 51610 to ATCC
51619) that had been phage typed (19), and classical biotype
strains V. cholerae 154 and 569B were also used in this
study. Other enteropathogens included in the present study were
Salmonella spp., enteropathogenic Escherichia
coli, and Shigella spp. (five strains each).
Isolation and purification of bacteriophages.
In the present
study the bacteriophages were isolated from raw sewage water samples
collected from different areas in India where cholera is endemic.
Samples were collected from selected points where the sewage water
connects with the main canal, which receives both domestic and
industrial effluents. Apart from V. cholerae O139 phages,
V. cholerae O1 biotype E1 Tor typing phage D-10 (ATCC 51352)
and classical phage 
149 were also included in the present study.
All the phages isolated were purified by successive single-plaque
isolation until homogeneous plaques against the standard propagating
strain NPR-4 were obtained. The broth lysis procedure was used to
prepare phage lysates. Nutrient broth was inoculated with a 1%
(vol/vol) cell suspension from the culture of NPR-4 grown overnight.
Cultures were grown at 37°C for 3 to 4 h with continuous shaking
in a gyratory shaker. Mid-logarithmic-phase cells were infected with
phage at a multiplicity of infection (MOI) of 0.1, and the cells were
incubated with shaking at 37°C until complete lysis occurred. A few
drops of chloroform containing 1% ethanol was added, and the mixture
was centrifuged (10,000 rpm at 4°C) for 20 min. The phage suspension
was pelleted at 30,000 rpm for 2 h at 4°C with a Beckman 50.2 Ti
rotor. The pellet was suspended in 0.05 M Tris (pH 7.5) with 0.02 M
MgCl2 and was maintained at 4°C (9).
A plate lysis procedure was used for phage propagation to get a higher
titer of phages. A young broth culture of V. cholerae NPR-4
was infected with phage at an MOI of 0.01, and adsorption was allowed
to occur at room temperature. The mixture was then plated by the soft
agar overlay method and was incubated at 37°C until complete lysis
occurred. The soft agar layer was scraped off, suspended in 2 ml of
Tris-MgCl2 buffer, and centrifuged at 10,000 rpm for 20 min
at 4°C (Beckman J2-21M/E).
The bacteriophages used in the present study were purified by density
gradient centrifugation. Three of the phages, phages VE-2, CAL-3, and
MS-3, were purified by use of a cesium chloride density (1.3 to 1.7 g/ml) gradient. Phages MAD-5 and S-2 were unstable in cesium chloride;
hence, they were purified by a neutral sucrose step gradient (20 to
60%) procedure. An electron microscopic study was performed by the
method described by Ghosh et al. (9) to determine the
morphologies of the phages.
SDS-polyacrylamide gel electrophoresis (PAGE).
A phage
lysate containing 1010 PFU/ml was solubilized in 0.065 M
Tris-HCl (pH 6.8) containing 1% sodium dodecyl sulfate (SDS), 1%
mercaptoethanol, 15% glycerol, and bromophenol blue. Samples were
prepared for electrophoresis by boiling for 3 min prior to loading.
Electrophoresis was performed by the standard method (11).
The bands were detected by staining the gel with Coomassie brilliant blue.
Preparation and titration of antiphage serum.
Antiphage
serum was prepared by immunizing New Zealand White outbred rabbits of
either sex (weight, between 1.3 and 1.5 kg) as described previously
(14). One milliliter each of the high-titer purified phage
lysate was injected once a week. A booster dose was given intravenously
after 4 weeks. Test bleeding was performed a week after the last
injection. After aseptic separation, the serum was kept in a
refrigerator in a sealed ampoule without any preservatives. Preimmune
serum from the same rabbit was used as a control for the neutralization test.
The antiphage serum neutralization test was performed by the
methodology of Mukerjee (14). For the cross-neutralization test, phage stocks were diluted to 107 PFU/ml, and the
antiphage sera were diluted to 1 in 50 in broth. One volume of each of
the phage groups was mixed with 99 volumes of serum, and the mixture
was kept at 37°C in a water bath for 10 min. An aliquot of the
mixture from all the tubes was then diluted 1 in 100 in broth to stop
the further action of the serum. A total of 100 µl of these dilutions
was then plated by the soft agar overlay method. The immunological
cross-reactivities among the five phages were determined by a standard
enzyme-linked immunosorbent assay (ELISA) method (10).
Phage characterization.
Adsorption of phage MAD-5 on its
host, strain NPR-4, was studied as elaborated by Stent (22)
for bacteriophage T4. Exponentially grown V. cholerae O139
NPR-4 (2 × 108 cells/ml) was infected with phage
MAD-5 at an MOI of 0.1 at 37°C. After infection, at every 2-min
interval over a period of 15 min, the concentration of nonadsorbed
phages was determined by diluting the supernatant and plating it before
lysis. Thermal and pH inactivation of MAD-5 phage was determined by the
procedure described by Mukerjee (14).
The interaction between V. cholerae O139 and phages in a
ligated ileal loop of a rabbit was studied by the procedure described by Sarkar et al. (19).
Isolation of phage DNA.
Phage DNA was isolated by the
procedure of Sambrook et al. (17). A high-titer phage
(1011 to 1012 PFU/ml) stock was treated with
phenol-chloroform-isoamyl alcohol, and the DNA that was extracted was
precipitated with chilled ethanol. Restriction endonuclease digestion
of phage DNA was done by the procedure recommended by the manufacturer
(Genei, Bangalore, India).
Phage typing.
The plaque morphologies of the five newly
isolated phages were studied on nutrient agar plates by the soft agar
overlay method after serial decimal dilution. Initially, 100 V. cholerae strains were included for screening of the phages. The
phages were selected on the basis of the sensitivity pattern for
development of the phage typing scheme. The routine test dilutions
(RTDs) of the phages were determined by the method described by
Chattopadhyay et al. (7). High-titer phage stocks were
serially diluted, and the dilutions were spotted onto the lawn of
standard propagating strain NPR-4. The RTD used for the phage typing
study was the highest dilution that just failed to lyse the standard
propagating strain clearly or confluently. The phage typing study was
performed on the basis of the standard methodology adopted in our
laboratory (6, 7, 18). The set of phages used for typing,
namely, phages MAD-5, VE-2, CAL-3, S-2, and MS-3, were checked for RTD prior to phage typing of each batch of strains. A batch of V. cholerae O139 strains was streaked onto a nutrient agar plate and
the plate was incubated for 18 h at 37°C. On the following morning, a single colony from the nutrient agar plate was inoculated into nutrient broth (2 to 3 ml) and was incubated under stationary conditions for 3 to 4 h. A young broth culture mixed with 3.5 ml
of molten soft agar (maintained at 42°C in a water bath) was poured
into a nutrient agar plate to prepare a uniform lawn of growth of the
bacterial strain on the surface of the agar to provide an adequate
substrate for phage action. The plates were then allowed to dry at room
temperature, preferably for 20 to 30 min. After the plates were dried,
drops of phage lysate at the RTD were applied onto the plates with a
Pasteur pipette without touching the agar. The plates were kept at room
temperature so that the drops could dry and were then incubated at
37°C for 14 to 18 h. On the following day, the plates were
observed to evaluate the degree of lysis. Strong lysis, semiconfluent
lysis, or the appearance of at least five plaques were considered
positive results. Lesser degrees of lysis or reactions of inhibition
were disregarded.
| |
RESULTS |
Bacteriophage.
A total of 79 sewage water samples were
collected from different areas in India where cholera is endemic for
isolation of V. cholerae O139 phages. Of these, 40 phages
sensitive to V. cholerae O139 strains were isolated by using
NPR-4 as the host.
Host range.
V. cholerae O1 biotype E1 Tor strain MAK757
(ATCC 51352), 10 E1 Tor strains (strains ATCC 51610 to ATCC 51619) that
had been phage typed, V. cholerae O1 Classical biotype
strain 569B, and V. cholerae 154 were resistant to these
five O139 phages and caused lysis of V. cholerae O139
strains. Strains of common enteropathogens such as
Salmonella, Shigella, and enteropathogenic
E. coli were not sensitive to these phages, indicating that
they were true cholera phages.
Morphology and characteristics of MAD-5.
Electron micrographs
of the five newly isolated phages are shown in Fig.
1. The diameters of the heads (distance
between opposite apices) of MAD-5, VE-2, CAL-3, S-2, and MS-3 were
58 ± 2.7, 112.5 ± 1.8, 67.2 ± 2.9, 60.8 ± 1.8,
and 73.9 ± 2.3 nm, respectively, while the lengths of the tails
were 141.2 ± 4.8, 204 ± 2.8, 11.5 ± 2.8, 16.5 ±
1.2, and 10.8 ± 1.8, respectively. From the dimensions, the
phages could be categorized into two distinct groups. Phages CAL-3,
MS-3, and S-2 have hexagonal heads with short noncontractile tails and
belong to group C in the classification proposed by Bradley
(5). According to the International Committee on Taxonomy of
Viruses, they belong to the family Podoviridae
(1). Phages MAD-5 and VE-2 have hexagonal heads and long
contractile tails and belong to group A or the family
Myoviridae.
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FIG. 1.
Electron micrograph of five newly isolated V. cholerae O139 phages. (a) MAD-5; (b) VE-2; (c) CAL-3; (d) S-2; (e)
MS-3. Bars, 100 nm.
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|
One of the phages (MAD-5) was characterized by its physicochemical
parameters. Phage MAD-5 showed a typical biphasic pattern of adsorption
on its host, strain NPR-4. Adsorption of phage MAD-5 was dependent on
the divalent cation Mg2+, but with replacement of
Mg2+ by Ca2+, Na+, or
K+, the adsorption was almost identical in nature to that
with Mg2+. This phage was found to be stable at pHs ranging
from 4 to 11. Below pH 4 and above pH 11, phage MAD-5 was found to be
unstable. Like serology, morphology, and host range pattern, the
thermal death point is one of the parameters used to identify a phage. Phage MAD-5 was found to be most stable at 4°C, and its thermal death
point ranged between 67 and 70°C.
Protein profile.
The protein profiles of the five V. cholerae O139 phages are shown in Fig.
2. SDS-PAGE pattern analysis of the CAL-3
and MS-3 virions showed that they had more or less similar profiles but that the profiles differed from those exhibited by MAD-5, VE-2, and
S-2, which were again different among themselves. The newly isolated
phages showed distinctly different protein profiles compared to the
profile of O1 biotype E1 Tor typing phage D-10 (ATCC 51352).
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FIG. 2.
SDS-PAGE patterns of structural proteins of five newly
isolated V. cholerae O139 phages stained with Coomassie
brilliant blue. Lanes 1 and 8, molecular size marker (in kilobases);
lane 2, MAD-5; lane 3, VE-2; lane 4, CAL-3; lane 5, S-2; lane 6, MS-3;
lane 7, D-10.
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|
Immunological analysis.
The five phages were tested to
determine their relatedness in terms of inhibition of infectivity by
using antiphage sera raised against the individual phages. Antisera
against phages MAD-5, CAL-3, and MS-3 inhibited these three phages but
did not inhibit VE-2 and S-2, while antisera against VE-2 and S-2
inhibited these two phages but did not inhibit MAD-5, CAL-3, and MS-3.
Thus, on the basis of the inhibition of infectivity of the phage
particles (cross-neutralization test) they could be categorized into
two groups, with phages MAD-5, CAL-3, and MS-3 belonging to one group and phages VE-2 and S-2 belonging to another group. Analysis of the
results of ELISA (Fig. 3) revealed that
the five phages cross-reacted immunologically but showed no
immunological relatedness to O1 typing phage D-10 (ATCC 51352).
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FIG. 3.
(a) Determination of antigenic relatedness among the
five newly isolated V. cholerae O139 phages by ELISA.
Anti-MAD-5 phage antiserum was used as the antibody, and five V. cholerae O139 phages along with E1 Tor phage D-10 were used as
antigens ( , MAD-5; ×, VE-2;  , CAL-3;  , S-2;  , MS-3;  ,
D-10). (b) Determination of antigenic relatedness among the five newly
isolated V. cholerae O139 phages by ELISA. Anti-VE-2
antisera was used as the antibody, and five V. cholerae O139
phages along with E1 Tor phage D-10 were used as antigens (, VE-2;
×, MAD-5; , CAL-3; , S-2; , MS-3; , D-10).
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|
Restriction endonuclease digestion profile.
DNAs from the five
phages were extracted and digested with the restriction endonuclease
HindIII. The restriction endonuclease digestion patterns
(Fig. 4) of the DNAs of the five phages
showed polymorphism, indicating that they were different from each
other as well as from E1 Tor phage D-10 (ATCC 51352). The restriction digestion patterns showed that phages VE-2 and S-2 and phages CAL-3 and
MS-3 were similar but not identical.
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FIG. 4.
Restriction endonuclease (HindIII)
digestion patterns of DNA isolated from five newly isolated V. cholerae O139 phages. Lane 1, lambda phage DNA digested with
HindIII; lane 2, MAD-5; lane 3, VE-2; lane 4, CAL-3;
lane 5, S-2; lane 6, MS-3; lane 7, D-10. Numbers on the left are in
kilobases.
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|
Phage typing.
A total of 500 V. cholerae O139
strains isolated from different areas in India where cholera is endemic
were used for phage typing with the set of typing phages. It was
observed that almost all the strains were typeable and clustered into
10 distinct phage types (Table 1). Only
four strains could not be typed. Phage type 1 (38.2%) was the major
phage type, followed by phage type 2 (22.4%) and phage type 3 (18%)
(Table 2). Strains belonging to phage
type 1 were widely distributed and were found in all 12 areas where the
O139 strains were isolated. It was interesting that O139 strains from
Madras exhibited the maximum number of phage types (8 of 10 types).
Incidentally, the outbreak of V. cholerae O139 originated in
this southern city of India (16).
Variations in phage typing patterns of V. cholerae O139
strains isolated in 1993 and 1994 and from 1996 to 1998.
Of the
500 strains of V. cholerae O139 examined, 304 strains were
isolated during the period between 1993 and 1994, while 196 were
isolated from 1996 to 1998 when a resurgence of V. cholerae O139 was observed in Calcutta (3, 13). A comparative
analysis of the phage typing patterns of the O139 strains isolated in
1993 and 1994 versus those of the O139 strains isolated from 1996 to 1998 showed that during both periods phage types 1, 2, 3, and 4 were
the major phage types (Table 2). Phage type 1 was found as the
predominant type in the strains recovered during both periods. However,
a higher percentage of phage type 1 (40.46%) O139 strains was isolated
in 1993 and 1994 than from 1996 to 1998 (34.69%). Strains were
available from Calcutta and Nagpur during both periods. Strains
obtained from Calcutta during both periods were divided into five
different phage types. Although strains isolated in Nagpur in 1993 and
1994 were of six different types, phage types 6 and 7 were missing from
O139 strains recovered in Nagpur from 1996 to 1998. For all other
cities, only strains isolated in 1993 and 1994 or from 1996 to 1998 were available. It was observed that strains of all 10 phage types were
detected among the O139 strains recovered in 1993 and 1994, but strains
of only 5 phage types were detected among the O139 strains isolated
from 1996 to 1998. It was also observed that phage type 3 was the
second major type among the O139 strains recovered in 1993 and 1994, whereas phage type 2 was found as the second major type among the O139
isolates recovered from 1996 to 1998.
| |
DISCUSSION |
Phages can be isolated from the environment in which host
bacterial strains generally survive and can be found in sewage, feces,
soil, and water (2). In the present study, a total of 40 bacteriophages were isolated from 79 sewage water samples collected from different areas in India where cholera is endemic. A set of five
phages were selected from the total of 40 isolates. The lytic patterns
of these phages were found to vary among themselves as well as from
those of the existing O1 E1 Tor and classical phages. Strains of
V. cholerae O1 E1 Tor and classical biotypes were resistant
to these phages. These five phages were characterized on the basis of
electron microscopy and immunological and physicochemical parameters,
and their utility for use in the development of a phage typing scheme
for V. cholerae O139 was also evaluated.
The plaques formed by a particular phage are one of the important
parameters for characterization of the phage. In this study, the
diameters of the plaques formed by the five phages differed. According
to the International Committee on Taxonomy of Viruses, phages MAD-5 and
VE-2 belong to the family Myoviridae and phages CAL-3, S-2,
and MS-3 belong to the family Podoviridae. The structural proteins of the five phages selected for the present study showed marked differences from those of the E1 Tor O1 phages, as revealed by
SDS-PAGE (Fig. 2). The restriction endonuclease digestion profiles of
the five phages were different from each other as well as from that of
E1 Tor O1 phage D-10 (ATCC 51352), indicating heterogeneity in the DNAs
of O139 phages.
The use of an immunological technique is an important approach to the
examination of a phage particle and its components. The antiserum
neutralization test was performed with the phages and their homologous
antisera, and marked inhibition of the infectivities of the phages were
observed. The cross-neutralization test with the antiserum of a
particular phage with the five groups of phages showed that three of
the five phages, phages MAD-5, CAL-3, and MS-3, were related and
differed from phages VE-2 and S-2, which were related to each other.
Thus, the five phages fall into two groups in terms of inhibition of
infectivity. To correlate with immunological analysis, the antiphage
serum of one of the three related phages (anti-MAD-5 phage antisera)
and that of one of the two other related phages (anti-VE-2 phage
antisera) were further tested. The interaction of the phage protein as
the antigen with anti-MAD-5 phage antisera and anti-VE-2 phage antisera
by ELISA proves that all of the O139 phages cross-react antigenically. Antisera raised against phage MAD-5 can probably recognize the proteins
of CAL-3 and MS-3, including the tail protein (receptor recognition
site), which is responsible for phage adsorption and which thus
inactivates lytic propagation, whereas anti-MAD-5 phage antisera can
recognize proteins of phages VE-2 and S-2 other than their receptor
recognition sites and thus could not inhibit lytic propagation by
inhibiting their infectivities. Similarly, anti-VE-2 phage antibody can
recognize proteins other than the receptor recognition sites of phages
MAD-5, CAL-3, and MS-3. Interestingly, O139 phages do not cross-react
with O1 E1 Tor phages. It should be mentioned that V. cholerae O1 biotype E1 Tor phages have no prophylactic value
against V. cholerae infection (19). Likewise, V. cholerae O139 phages were also tested in a ligated rabbit
ileal loop, and it was found that O139 phages are also unable to lyse the host cell inside the intestine. It is presumed that cholera phages
have no prophylactic value against V. cholerae infection.
All of the five phages were used at the RTD to develop the phage typing
scheme. Five hundred V. cholerae O139 strains were differentiated into 10 distinct phage types. Most of these strains grouped into phage type 1 (38.2%), followed by phage type 2 (22.4%) and phage type 3 (18.0%). Eight of the phage types were present among
the O139 strains received from Madras, where the O139 serogroup originated and rapidly spread to other areas in India where cholera is
endemic. The phage sensitivities of the V. cholerae O139
strains received from different places in India showed variable
patterns. Phage types 5 and 6 were present only in strains isolated
from Ahmedabad, Madurai, and Calcutta. This variation in the
distribution of phage types in different areas indicates the presence
of different clones of V. cholerae O139 strains. Molecular
studies have shown the existence of different ribotypes and CTX
(cholera toxin genetic element) genotypes of V. cholerae
O139 (3, 8, 20). In this study, it was interesting that all
10 phage types were detected among the O139 strains isolated in 1992 and 1993, while only 5 phage types were detected among those isolated
from 1996 to 1998. A comparative study of the phage types of the O139
strains isolated in 1992 and 1993 and from 1996 to 1998 showed that
during both periods phage type 1 was the predominant type. However, the
percentage of the O139 strains that were isolated in 1992 and 1993 and
that belonged to phage type 1 (40.46%) was much higher than the
percentage of O139 strains of that type recovered from 1996 to 1998 (34.69%). Again, molecular studies have shown substantial changes in
the organization of the CTX phage module of O139 strains isolated from
1996 to 1998 compared to the organization of the CTX phage module of
O139 strains isolated in 1993 and 1994 (3, 20).
Phages absorb to specific receptor sites on the bacterial cell wall. In
gram-negative bacteria the receptors have been identified as protein
and lipopolysaccharide components of the outer membrane layer
surrounding the peptidoglycan. A particular phage or group of phages
will absorb to a specific site, and different phages will absorb to
different sites. Thus, on the surface of a given bacterial cell a
variety of different receptors are present, each type being present in
a large number of copies. Phage typing methods can test large numbers
of strains rapidly. It is a cost-effective and a simple laboratory
method and does not require sophisticated instrumentation. Besides, it
has been shown that the presence of Vibrio phages in sewage
water is a potential tool for the prediction of cholera outbreaks
(12). The most important finding of our study was the almost
complete typeabilities of the O139 strains with the set of five phages,
and the 500 O139 strains could be differentiated into 10 distinct phage
types. The V. cholerae O139-specific phages isolated and
characterized in the present study were unique in their ability to
identify and discriminate V. cholerae O139 strains. The
proposed phage typing scheme consists of a set of five newly isolated
phages, and the phage typing scheme developed by using the five phages
would be useful in the study of the epidemiology of cholera caused by
V. cholerae O139.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: National
Institute of Cholera and Enteric Diseases, P-33, CIT Rd., Scheme XM,
Beliaghata, Calcutta-700 010, India. Phone: 91-33-350-1176. Fax:
91-33-350-5066. E-mail: icmrnicd{at}ren.nic.in.
| |
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Abstract
Introduction
