Clonal analysis of non-toxigenic Vibrio cholerae 01 associated with the outbreak of cholera

Clonal analysis of non-toxigenic Vibrio cholerae 01 associated with the outbreak of cholera

Pal, Amit

Accepted May 20,1999

We examined the clonal relationships among eight clinical isolates of non-toxigenic (NT) V cholerae Ol associated with a cluster of cases of cholera in Warangal, Andhra Pradesh in south India and compared their relatedness to toxigenic O1 strains of classical and ElTor biotypes and with 0139 Bengal strains of If cholerae by pulsed-field gel electrophoresis (PFGE). Phylogentic analysis of the NotI restriction fragment length polymorphism showed that all the NT. lf cholerae O1 strains formed a tight cluster with more than 80 per cent similarity. Interestingly, the NT V cholerae O1 cluster was more closely related to V cholerae 0139 than to classical and ElTor biotypes of V cholerae O1 indicating closer genetic relationships between NT V cholerae 01 and 0139 Bengal strains that were isolated during the same time-frame.

Key words Non-toxigenic (NT) Vibrio cholerae 01 – phylogenetic analysis – pulsed-field gel electrophoresis (PFGE)

Epidemics of cholera caused by toxigenic Vibrio cholerae Ol and 0139 Bengal represent a major public health problem in many developing countries1-3. Nontoxigenic (NT) V cholerae, defined as strains of V. cholerae OI which do not produce cholera toxin (CT) and which do not have the CTX genetic element, have been isolated from sporadic cases of cholera-like disease and also from non-human sources4. Molecular epidemiological studies have shown the NT V. cholerae strains to be genetically diverse5,6 and at times indistinguishable from their toxigenic counterparts5. Incidence of genetically identical toxigenic and NT V cholerae Ol strains have recently been reported7. The pathogenesis of NT V cholerae is not clear although its role is indisputable4. Molecular epidemiological surveillance of cholera and comparative analysis of clonal relationships among toxigenic and NT V cholerae strains collected during epidemic and inter-epidemic periods are essential for identifying potential epidemic strains and for studying the evolution and spread of epidemic strains. While the 0139 serogroup was spreading through cholera endemic areas in India during 1993, we received a set of NT V cholerae Ol strains from Warangal, Andhra Pradesh, south India, which was associated with a cluster of cholera cases8. In this study, we have attempted to determine the lineage of the unusual NT V cholerae strains using pulsed field-gel electrophoresis (PFGE).

Material & Methods

During June 1993, an outbreak of cholera was recorded in the city of Warangal, in the state of Andhra Pradesh. The causative organism was identified as V cholerae Ol belonging to the El Tor biotype, Inaba8, and tHe virulence properties of these strains were retrospectively analysed using standard techniques1,9. A highly sensitive CT bead-ELISA for the detection of in vitro production of cholera toxin (CT) and a battery of DNA probes specific for several virulence genes including ctxA, zot, ace and stn and PCR specific for EAST gene failed to identify any of the established virulence markers8. These strains were labelled as NT because they did not possess the genetic potential to produce CT. However, the NT strains showed impressive Isc activity as detected in the Ussing chambers and therefore induced secretory diarrhoea by a hitherto unknown mechanism8.

The objective of the present study was to ascertain whether restriction fragments produced by cleavage of the genomes of NT V cholerae could classify these strains as clonal and to determine the extent of relatedness with standard toxigenic strains of 01 and 0139 by PFGE. Eight strains of NT V cholerae O1 (WO-3, WO-5, WO-6, WO-7, WO-8 WO-10, WO-11 and WO-21), along with standard reference strains of V cholerae belonging to classical (569B) and ElTor (V-5) biotypes and V cholerae 0139 Bengal (SG-26 and VO-2) were examined by PFGE.

To perform PFGE, the genomic DNA of V cholerae strains was prepared in agarose plugs as described previously10. Digestion of genomic DNA was performed with 50U NotI (Takara, Shuzo Co, Ltd., Japan) in accordance with the manufacturer’s instructions. PFGE of inserts was performed in a CHEF DR-ITM apparatus (Bio-Rad, Calif., USA) with 1per cent PFGE grade agarose in 0.5 X TBE (44.5 mM Tris HCI, 44.5 mM boric acid, 1.0 mM EDTA, pH 8.0). Agarose gels were electrophoresed at 14 deg C using a constant voltage of 200 V at 120 deg with two different switch ramptimes. During the first run of 22 h, the timings were 5 to 50 sec to separate larger DNA fragments; the second run with 1.0 to 10 sec to separate smaller DNA fragments for 12h. A DNA size standard (k ladder, Bio-Rad, Calif., USA) was used as the molecular weight marker. After the completion of PFGE, the gels were stained in distilled water containing 1.0 pg ethidium bromide per ml for 30 min, rinsed several times and photographed under UV light.

The PFGE profiles were compared directly after scanning the photograph in a 420oe optically enhanced densitometer/scanner interfaced with a computer in which the program Diversity one(TM) (Version 1.6) was installed (pdi Inc., Huntington Station, NY, USA). Comparisons of different NoI digested DNA patterns were made to ascertain the phylogenetic relationships between the strains, using a software that runs on SunTM work station (pdi Inc., Huntington Station, NY, USA). The unrooted dendrogram was constructed on the basis of neighbour-joining method11,12 wiLi- percentage of matched bands ranging between 97 and 436 kbp. Plasmid extraction was done by alkaline lysis method’3.

Results & Discussion

In the present study, we analyzed genomic DNA restriction patterns of the V cholerae strains after NotI restriction digestion. This approach allowed us to determine and compare the extent of clonal relationship among the NT V cholerae Ol strains and between the NT O1 strains and standard V cholerae Ol strains belonging to the classical and ElTor biotypes and V cholerae 0139 strains. Since none of the NT V cholerae Ol strains harboured any plasmids, the DNA bands obtained after NotI restriction digestion are of chromosomal origin. Under the set conditions, the PFGE of different strains of V cholerae gave 10 to 13 DNA bands (Fig. 1). Three strains of NT V cholerae Ol in lanes 11 to 13 (strains WO-8, WO-21, and WO-5 respectively) showed slight polymorphism when compared to the remaining NT V cholerae O1 strains by 3 to 4 DNA bands (Fig. 1). However, these strains formed a part of the outbreak and were categorised as `possibly related14. The genomic DNA restriction patterns of individual strains were compared quantitatively, and the per cent similarity in restriction patterns between the strains was estimated and is represented as a dendrogram (Fig. 2). In the cluster analysis we found that the strains of NT V cholerae O1 had one polymorphic lineage with six pulso types, which were related to each other at levels between 80 and 100 per cent (Fig. 2). The most distant relation as revealed by the analysis separated both classical and ElTor biotypes of V cholerae O1 strains. Since, the DNA banding pattern of the two V cholerae 0139 strains SG26 and VO-2 were identical, the latter was excluded from the phylogenetic analysis. There is ample evidence that the 0139 Bengal strain has evolved from a V cholerae O1 ElTor strains by horizontal transfer of genes coding for synthesis of cell surface polysaccharides15,l6. In the present study, we observed that the NT V cholerae Ol biotype El Tor strain cluster was more closely related (55% similarity) to the toxigenic V cholerae 0139 strain (SG-26) tl,ar. to the Ol classical (569B) or ElTor (V-5) biotypes (Fig. 2). This genetic similarity as detected by PFGE was further supported by epidemiological data since at the time when the V cholerae 0139 serogroup was in the process of rapidly spreading through vast areas of India2, the cluster of cholera cases caused by NT V cholerae Ol serotype Inaba, biotype El Tor occurred.

While studying the population structure and molecular evolution of V cholerae as a species by examining the asd (chromosomal housekeeping gene), locus, Karaolis et al17 inferred the horizontal transfer of O antigen genes, since isolates with near identical asd sequences had different O antigens, and isolates with the same O antigen did not group together but were found in different lineages. According to Bik et al18, closely related strains may contain gene cluster coding for different serotypes and that horizontal transfer of O-antigen determining genes occurs among V cholerae strains. The origin of NT V cholerae strains that caused the cholera outbreak in Warangal remains cryptic. Viewed in retrospect and by the chronology of events, we assume that (i) the V cholerae 0139 Bengal strains could have been the precursor of NT V cholerae Ol in which the CTX genetic element was deleted and the db genes (determinants of somatic antigens) were altered or (ii) it is also possible that the two lineages have diverged from a common ancestor. However, with the existing immune pressure in the population against the Ol antigen of an endemic area, the NT V cholerae 01 serotype Inaba may have,been suppressed and did not spread further. Either hypothesis would be difficult to test. There appears to be some correlation between pathogenecity of NT V cholerae Ol and AC (Inaba) antigen. In parallel to the observation of Rodrigues et al4, in the present study, we observed that all the NT V cholerae Ol strains were classified as serotype Inaba. Further extensive surveillance is needed to substantiate this hypothesis and to generate additional information in the role of NT V cholerae O1 or 0139 in the evolution of emerging clones of V cholerae.


This work was supported, in part, by the Japan International Cooperation Agency, Tokyo, Japan.


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Amit Pal, P.K. Saha, G.B. Nair, Shinji Yamasaki”, Tae Takeda*, Yoshifumi Takeda**, S. K. Bhattacharya & T. Ramamurthy

National Institute of Cholera & Enteric Diseases, Calcutta, *Department of Infectious Diseases Research, National Children’s Medical Research Center, Tokyo & **Research Institute, International Medical Center of Japan, Tokyo, Japan

Reprint requests:

Dr T. Ramamurthy, Assistant Director, National Institute of Cholera and Enteric Diseases P-33, C.I.T. Road, Scheme XM, Beliaghata, Calcutta 700010

Copyright Indian Council of Medical Research Jun 1999

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