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Nov. 2000, p. 4249–4253 Vol. 38, No. 11

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Emergence of Vibrio cholerae O1 Biotype El Tor Serotype Inabafrom the Prevailing O1 Ogawa Serotype Strains in India

PALLAVI GARG,1 RANJAN K. NANDY,1 PAPIYA CHAUDHURY,1 NANDINI ROY CHOWDHURY,1

KEYA DE,1 T. RAMAMURTHY,1 SHINJI YAMASAKI,2 S. K. BHATTACHARYA,1

YOSHIFUMI TAKEDA,3 AND G. BALAKRISH NAIR1*

Department of Microbiology, National Institute of Cholera and Enteric Diseases, Calcutta 700 010, India,1 andResearch Institute, International Medical Center of Japan, Shinjuku-ku, Tokyo 162-8655,2 and

National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640,3 Japan

Received 4 April 2000/Returned for modification 1 July 2000/Accepted 20 August 2000

The toxigenic Inaba serotype of Vibrio cholerae O1 biotype El Tor reappeared in India in 1998 and 1999,almost 10 years after its last dominance in Calcutta in 1989. Extensive molecular characterization by ribotyp-ing, restriction fragment length polymorphism, and pulsed-field gel electrophoresis indicated that recent Inabastrains are remarkably different from the earlier Inaba strains but are very similar to the prevailing V. choleraeO1 Ogawa El Tor biotype strains. The antibiograms of the Inaba strains were also similar to those of the recentV. cholerae Ogawa strains. These V. cholerae O1 Inaba strains appear to have evolved from the currentlyprevailing Ogawa strains and are likely to dominate in the coming years.

The disease cholera, caused by toxigenic strains of Vibriocholerae belonging to the O1 or O139 serogroup, is character-ized by the passing of voluminous watery stools, which rapidlyleads to dehydration and, if left untreated, to death. V. choleraeO1 is further classified into two biotypes, classical and El Tor,and into two major serotypes, Inaba and Ogawa. The genesresponsible for O1 antigen biosynthesis have been designatedwbe (previously known as rfb) (18) and are localized on a21.6-kb SacI fragment of DNA (7, 24). This region is highlyconserved; the only changes observed between the Ogawa andInaba serotypes are related to a mutation in the wbeT region(24, 27). V. cholerae O1 strains can undergo serotype conver-sion or switching between the Inaba and Ogawa serotypes (5,8, 24). Observations of the epidemic that broke out in LatinAmerica in 1991 have supported this notion. Extensive bio-chemical analyses and rRNA restriction fragment length poly-morphism (RFLP) analysis have shown that the El Tor Inabaepidemic strains were unique to Latin America (13, 21). How-ever, Ogawa isolates that were identical to the epidemic strainin all other respects began to appear in about the seventhmonth of the epidemic, suggesting that the epidemic strainshad undergone a serotype conversion, possibly because of im-mune pressure in the population (13).

With the advent of the O139 serogroup in 1992 (17), theInaba serotype of V. cholerae O1 was displaced in Calcutta andother parts of India (15) by the O139 serogroup, and the lastInaba predominance in Calcutta was observed in 1989 (17).The isolation of V. cholerae O1 belonging to the Inaba serotypebecame rare; the only isolation reported was from a choleraoutbreak in Warangal, which was due to nontoxigenic V. chol-erae O1 Inaba El Tor biotype strains (20). We began receivingsome representative strains belonging to the Inaba serotypefrom Delhi in December 1998 and from Sewagram in Novem-ber 1999. This study is an extensive molecular characterizationof the recently isolated Inaba strains to determine their clonal-

ity and to evaluate their similarity to Inaba strains that wereisolated in Calcutta in 1989.

The present study is part of the continuing nationwide sur-veillance program on cholera of the National Institute of Chol-era and Enteric Diseases (NICED), Calcutta, India. In Decem-ber 1998, we received a representative set of strains fromDelhi, two of which were identified as V. cholerae O1 Inaba. InNovember 1999 we received a set of seven strains from Sewa-gram, six of which were identified as V. cholerae O1 Inaba andone of which was V. cholerae O1 Ogawa. The purity and iden-tity of the strains were then confirmed by previously publishedprocedures (15).

The strains were examined for resistance to ampicillin (10mg), chloramphenicol (30 mg), cotrimoxazole (25 mg), cipro-floxacin (5 mg), furazolidone (100 mg), gentamicin (10 mg),neomycin (30 mg), nalidixic acid (30 mg), norfloxacin (10 mg),streptomycin (10 mg), and tetracycline (30 mg) with commer-cial disks (HiMedia, Mumbai, India). Antimicrobial suscepti-bility analysis was carried out as described previously (15).

The 7.5-kb BamHI fragment of plasmid pKK 3535 contain-ing the 16S and 23S rRNA genes of Escherichia coli was usedas the rRNA probe (4). The ctxA probe consisted of a 540-bpXbaI-ClaI fragment of ctxA cloned in pKTN901 using EcoRIlinkers (11). The DNA probe for the RS element was a 2.7-kbNotI fragment from plasmid pCT5A11 (10). Genomic DNAfor ribotyping and for studying the structure and organizationof the CTX prophage was extracted as described previously(1). The transfer of digested DNA from a gel to a Hybond N1

membrane (Amersham Pharmacia Biotech, Little Chalfont,Buckinghamshire, England) and hybridizations with the rRNAprobe for ribotyping and with the ctxA and RS1 probes forCTX genotyping were performed as described previously (1)using the ECL Nucleic Acid Detection System (AmershamPharmacia Biotech). The membranes were then washed, ex-posed to Kodak Biomax film (Eastman Kodak Co., Rochester,N.Y.), and developed according to the manufacturer’s instruc-tions. Autoradiograms were digitally processed for documen-tation using the Gel Doc 2000 gel documentation system (Bio-Rad, Richmond, Calif.).

Pulsed-field gel electrophoresis (PFGE) was performed withthe genomic DNA of the V. cholerae strains by preparing aga-

* Corresponding author. Mailing address: National Institute ofCholera and Enteric Diseases, P-33, CIT Rd., Scheme XM, Beliaghata,Calcutta 700 010, India. Phone: 91-33-3505533. Fax: 91-33-3505066.E-mail: gbnair@vsnl.com.

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rose plugs as described previously (12, 28). PFGE of the NotI(Takara, Shuzo Co., Ltd., Shiga, Japan)-digested inserts wasperformed by using the contour-clamped homogenous electricfield (CHEF) method on the CHEF Mapper system (Bio-Rad)with 1% PFGE grade agarose in 0.53 TBE (44.5 mM Tris-HCl, 44.5 mM boric acid, 1.0 mM EDTA [pH 8.0]) for 40 h, 24min. Run conditions were generated by the autoalgorithmmode of the CHEF Mapper PFGE system using a size range of20 to 300 kb. The gels were stained for 30 min in Elix MilliQwater (Millipore Corporation, Bedford, Mass.) containing 1.0mg of ethidium bromide per ml, then destained in Elix waterfor 15 min and photographed under UV light using the GelDoc 2000 gel documentation system (Bio-Rad).

For molecular characterization we included two Inabastrains, V2 and V13, isolated in 1989 in Calcutta, two Inabastrains from Delhi isolated in 1998, six Inaba strains and oneOgawa strain isolated in 1999 in Sewagram, and four Ogawastrains from Calcutta isolated in 1998. Our rationale for exam-ining these strains was to determine whether the Inaba strainsthat reappeared recently in Delhi and Sewagram bore anyresemblance to the Inaba strains last isolated in Calcutta in1989 or whether they belonged to a new clone.

The antibiotic susceptibility patterns of these strains re-vealed that V2 and V13, representing the Inaba strains isolatedin 1989, were sensitive to nalidixic acid and streptomycin (16,17), while the Inaba strains recently isolated in Sewagram andDelhi were resistant to these antibiotics (Table 1). Overall, theantibiograms of the recent Inaba strains matched those of theprevailing O1, Ogawa strains (9, 22). Resistance to cotrimox-azole, furazolidone, and streptomycin suggests the possibilityof the presence of the SXT element in recently isolated Inabastrains. The SXT element is an approximately 62.0-kb self-transmissible, chromosomally integrating genetic elementwhich carries resistances to the antibiotics sulfamethoxazole,trimethoprim, streptomycin, and furazolidone (26). The SXTelement integrates into the chromosome by a site-specificmechanism independent of recA. The properties of the SXTelement are very similar to those of the conjugative trans-posons. The widespread use of cotrimoxazole, a very popularand useful antimicrobial drug combination, may have providedan additional selective pressure for the sporadic emergence ofthe V. cholerae O1 Inaba strains in Delhi and Sewagram.

BglI ribotyping showed that the Inaba strains recently iso-

lated in Delhi and Sewagram had a ribotype different fromthose of the Inaba strains isolated in 1989. The ribotype pat-terns of the Inaba strains isolated in 1989, V2 and V13 (Fig. 1,lanes 4 and 5), were different from the ribotype patterns shownby the V. cholerae reference strains SG24 (O139) (Fig. 1, lane1), 569B (O1 classical Inaba) (Fig. 1, lane 2), and CO840 (O1Ogawa El Tor) (Fig. 1, lane 3). On the other hand, the recentInaba strains DO182 and DO183 isolated in Delhi in 1998 (Fig.1, lanes 6 and 7) and the Inaba strains isolated in Sewagram in1999, SO86 and SO90 (Fig. 1, lanes 8 and 9), showed ribotypessimilar to that of the prevailing V. cholerae O1 Ogawa strainCO840 (the slight difference in the mobility of the bands is dueto a gel anomaly), which is the RIII type, first described bySharma et al. (22). The ribotypes of the recent V. choleraeInaba strains indicate that these strains have molecular traitsidentical to those of the prevailing V. cholerae O1 Ogawa

TABLE 1. Antibiograms, ribotype patterns, and sizes of restriction enzyme-digested DNAs of V. cholera O1 strains isolated in India

Strain no.(serotype) Isolation yr, place Antibiograma RPb

Fragment size(s) (kb) with the following probe and restriction enzyme:

ctxA RS1

BglI HindIII PstI BglII PstI

V2, (Inaba) 1989, Calcutta A Co Fz N S PD 18.5 20.0 10.0 20.0, 7.0, 3.6 30.0, 10.0V13 (Inaba) 1989, Calcutta A Co Fz N PD 19.0 23.0 10.0 16.0, 7.0, 3.6, 2.7 30.0, 10.0DO182 (Inaba) 1998, Delhi A Co Fz Na S RIII 14.0 21.0 6.2 16.0, 8.4, 2.7 30.0DO183 (Inaba) 1998, Delhi A Co Fz N Na S RIII 23.0 23.0 16.0 16.0, 7.0, 3.6, 2.7 30.0, 16.0SO85 (Inaba) 1999, Sewagram A Co Fz N Na S RIII 12.0 19.5 6.2 16.0, 8.4, 2.7 30.0SO86 (Inaba) 1999, Sewagram A Co Fz N Na S T RIII 13.0 19.5 6.3 16.0, 8.4, 2.7 30.0SO87 (Inaba) 1999, Sewagram A Cf Co Fz N Na S RIII 13.0 20.0 6.3 16.0, 8.4, 2.7 30.0SO88 (Ogawa) 1999, Sewagram A Co Fz N Na S RIII 14.0 20.5 6.2 16.0, 8.4, 2.7 30.0SO89 (Inaba) 1999, Sewagram A Co Fz N S T RIII 13.0 13.0 6.2 16.0, 8.4, 2.7 30.0SO90 (Inaba) 1999, Sewagram A Cf Co Fz Na S RIII 14.0 21.0 6.3 16.0, 8.4, 2.7 30.0SO84 (Inaba) 1999, Sewagram A Co Fz N Na S T ND ND ND ND ND NDPG81 (Ogawa) 1998, Calcutta A Co Fz N Na RIII ND 26.0 5.6 18.5, 8.6, 3.1 31.0PG117 (Ogawa) 1998, Calcutta A C Co Fz Na S RIII ND 26.0 5.6 17.0, 8.6, 3.1 22.0CO840 (Ogawa) 1995, Calcutta A Co Fz N Na S RIII ND 23.0 6.0 ND 23.0

a A, ampicillin; Cf, ciprofloxacin; Co, cotrimoxazole; Fz, furazolidone; N, neomycin; Na, nalidixic acid; S, streptomycin; T, tetracycline.b RP, ribotype pattern; PD, previously described by Dalsgaard et al. (6); ND, not done.

FIG. 1. Ribotypes of representative V. cholerae strains. Lanes: 1, SG24(O139); 2, 569B (O1, classical Inaba); 3, CO840 (O1, El Tor Ogawa); 4, V2 (O1,Inaba); 5, V13 (O1, Inaba); 6, DO182 (O1, Inaba); 7, DO183 (O1, Inaba); 8,SO86 (O1, Inaba); 9, SO90 (O1, Inaba); 10, SO88 (O1, Ogawa). The positions ofl HindIII molecular size markers are indicated on the left.

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strains. This result also points out that the recent Inaba strainsare quite different from the Inaba strains isolated in 1989,when Inaba was the dominant serotype (16, 17).

Southern hybridization with the ctxA probe usingHindIII-digested genomic DNA detected only one band, but ofvarying size, in the Inaba strains. This result indicates that theCTX prophage is located at a single site in the chromosome, asHindIII does not have any recognition site within the CTXprophage (14). The arrangement and number of copies of theCTX prophage were determined by analysis of the Southernhybridization pattern generated separately with ctxA and RS1probes using other restriction enzymes which do cut within theCTX prophage but not in ctxA. ctxA RFLP patterns generatedwith BglI- and PstI-digested genomic DNA showed a singleband in all the strains (Table 1), suggesting the presence of asingle copy of the CTX prophage. The CTX prophage genomehas two regions: a 4.6-kb “core region” that includes ctxAB anda 2.4-kb region termed RS2. The integrated CTX prophage isfrequently flanked by an element known as RS1, which isrelated to RS2 (25). These related elements contain threenearly identical open reading frames (ORFs), while RS1 con-tains an additional ORF (25). Southern hybridization with theRS1 probe was carried out to determine the organization ofthe RS sequences upstream and downstream of the core re-gion. The restriction endonuclease PstI, which cuts within thecore region (14), was used to digest genomic DNA, which wasthen hybridized with the RS1 probe. RS1 RFLP patterns gen-erated with PstI-digested genomic DNA exhibited the presenceof two bands of 30.0 and 10.0 kb in strains V2 and V13 and of30.0 and 16.0 kb in strain DO183, while a single band of 30.0kb appeared in the remaining strains (Table 1). Therefore,strains V2, V13, and DO183 each contain at least one copy of

RS1 at both sides of the core region. When the results ofhybridization of PstI-digested genomic DNA with the ctxA andRS1 probes were compared, a band common to both wasobserved for strains V2, V13, and DO183. The size of thecommon band was 10.0 kb in strains V2 and V13 and 16.0 kbin strain DO183 (Table 1). This result confirms the presence ofan RS element downstream of the CTX prophage. The BglIIrestriction enzyme has a site in the RS region (25). As ex-pected, when BglII-digested genomic DNA was hybridizedwith the RS1 probe, three bands were observed for V2 andfour bands were observed for V13 and DO183; one of these, a7.0-kb band, is actually the size of the CTX prophage (25). Thepresence of tandemly arranged RS sequences is not uncom-mon in toxigenic V. cholerae, and the size of RS1 is reported tobe 2.7 kb (25). Since hybridization with the RS1 probe nevershowed the presence of a 2.7-kb band in V2, the possibility oftandemly arranged RS1 on either side of the core region can beexcluded. The presence of a 2.7-kb band in V13 and DO183indicates the possibility of tandemly arranged RS1 on eitherside of the core region. When the Inaba strains isolated inSewagram and strain DO182 were digested with PstI, theRFLP data showed a single band upon hybridization with boththe ctxA and RS1 probes (Table 1), indicating the presence ofa single copy of the CTX prophage, while BglII digestion andhybridization with the RS1 probe showed a band of 2.7 kb,indicating the presence of two tandemly arranged copies of theRS1 element in these strains. Thus, strain DO183 and theSewagram strains have a single copy of CTX prophage withone RS1 element upstream of the prophage. This organizationis very similar to that of the prevailing Ogawa strains (22).Figure 2 shows schematic representations, based on RFLPanalysis, of the CTX prophage of the Inaba strain V2, isolated

FIG. 2. Schematic representations of the organization of the CTX prophage (not to scale) as deduced by Southern hybridization data for V. cholerae O1 Inabastrains represented by V2 (a) and V. cholerae O1 Inaba strains represented by SO85 (b). Arrows and boxes represent the RS elements and the core region of the CTXprophage, respectively, with the hatched portion of the box representing the ctxAB gene. Restriction site abbreviations: BI, BglI; BII, BglII; P, PstI; H, HindIII.

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in 1989 (Fig. 2a), and of strain SO85, which is typical of theInaba strains recently isolated in Delhi and Sewagram (Fig.2b).

Analysis of PFGE patterns showed that the Inaba strainsisolated recently (Fig. 3, lanes 3 to 7) had a profile differentfrom those of strains V2 (Fig. 3, lane 1) and V13 (Fig. 3, lane2), representing the Inaba strains isolated in 1989. The recentInaba strains (Fig. 3, lanes 3 to 7) differed from strain CO840(Fig. 3, lane 8) by the presence of more than one band in the145.5-kb region. Interestingly, O1 Ogawa strains isolated dur-ing 1998 (Fig. 3, lanes 9 to 12) had a PFGE profile identical tothat of the recently isolated Inaba strains, indicating that theInaba strains that have emerged recently are similar to theprevailing O1 Ogawa strains.

V. cholerae O1 strains are known to interconvert between theOgawa and Inaba forms (5, 8, 24). The frequency of conversionof Ogawa to Inaba is approximately 1025 (3), whereas conver-sion from Inaba to Ogawa is rare and may be strain dependent(13). In vivo seroconversion correlates well with the host im-mune response, and this is supported by observations withgerm-free mice (19) and a clinical study carried out by Sheehyet al. (23). The wbeT gene of the highly conserved wb region isresponsible for the serotype conversion, as there is a single-nucleotide change within the gene, resulting in a TGA stopcodon and a truncated 32-kDa wbeT protein (24). The productof the wbeT gene of V. cholerae O1 is not essential for Oantigen biosynthesis but is required for determining the Ogawaserotype specificity (24). A variety of changes in wbeT couldproduce an Inaba strain by leading to truncated wbeT proteinsof various sizes due to reading frameshifts. Thus, Inaba strainsare effectively wbeT mutants and presumably have arisen as aresult of selection due to the immune response during cholerainfection (24). This study demonstrates that V. cholerae O1Inaba, after predominating until 1989 (17), suddenly disap-

peared and that the Inaba strains that have emerged in differ-ent parts of the country in 1998 to 1999 could have evolvedfrom the prevailing V. cholerae O1 Ogawa El Tor biotype.

This work was supported, in part, by the Japan International Coop-eration Agency (JICA/NICED project O54-1061-E-O).

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