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Assessing clonality of Vibrio cholerae Inaba isolates by characterization of nonsense mutations in wbeT

¹Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar EN6 3QG, UK ²National Institute of Cholera and Enteric Diseases, Calcutta, India ³Laboratory Sciences Division, ICDDR, B: Centre for Health and Population Research, Mohakhali, Dhaka 1212, Bangladesh

Correspondence Sjoerd G. Rijpkema srijpkema{at}nibsc.ac.uk

Received May 17, 2004
Accepted July 29, 2004

The transferase gene wbeT of six clinical isolates of Vibrio cholerae O1 biotype El Tor was analysed. Two unique mutations were identified in the wbeT gene of three Inaba isolates. Due to their random nature, mutations in wbeT can be used to determine the clonal origin of clinical Inaba isolates.

	Introduction

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Vibrio cholerae serotypes O1 Inaba, O1 Ogawa and the recently described O139 Bengal serogroup have been associated with widespread cholera outbreaks which occur intermittently over decades and have established cholera as a global disease (Sack et al., 2004). The occurrence of a novel serotype of V. cholerae, designated O139, in the Bay of Bengal in 1992 showed that this strain was able to spread rapidly through areas where cholera is endemic (Ramamurthy et al., 1993). The current hypothesis is that the O139 serogroup must have first evolved from the O22 serotype and, subsequently, the gene responsible for O-antigen biosynthesis in O139 was transferred into an O1 El Tor epidemic strain of V. cholerae, resulting in the emergence of an epidemic strain of O139 (reviewed by Faruque et al., 2003). Serotype switching also occurs within V. cholerae O1. In mice vaccinated with Ogawa cells, infection with V. cholerae serotype Ogawa results in a rapid conversion of the bacterial population from Ogawa to Inaba, followed by the appearance of rough strains (Sack & Miller, 1969). The switch in O-antigen from Ogawa to Inaba to rough LPS is caused by selective pressure of anti-Ogawa antibodies and the emergence of anti-Inaba antibodies; Gangarosa et al. (1967) described a similar phenomenon occurring in the human host. Stroeher et al. (1992) identified the product of rfbT [now designated wbeT (Reeves et al., 1996)] as a transferase that is responsible for the expression of the B determinant, which is Ogawa-specific. Nucleotide sequence analysis of the wbeT gene of eight Inaba strains revealed that unique mutations had occurred at different locations; a total of three deletions, two insertions and two substitutions resulted in truncated and inactive transferases, and the strains expressed the C determinant specific for Inaba (Stroeher et al., 1992; Ito et al., 1993). We investigated if the inactivation of wbeT by apparent random mutations can be used to establish the clonal origin of three clinical isolates of V. cholerae Inaba. The wbeT sequences were compared to published sequences from V. cholerae strains Ogawa NIH41 and Inaba 35A3 (Ito et al., 1993).

	Methods

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DNA was isolated from V. cholerae as described previously by Garg et al. (2000), alternatively DNA was liberated by boiling whole cells for 5 min. The wbeT gene was amplified by PCR in a 50 µl mixture consisting of 5 µl (10–20 ng) DNA, 200 µM dNTPs, 1 mM MgCl₂, 1.25 U Taq (Sigma) and 1 µM each of reverse and forward primers. Three combinations were used: VFBT-F and VFBT-R, VFBT-F1 and VFBT-R2, and VFBT-F3 and VFBT-R4 (Table 1). Samples were subjected to 25 cycles of 94 °C for 30 s, 40 °C for 1 min and 72 °C for 2 min, with a final extension step at 72 °C for 3 min. The size of the PCR product was analysed by electrophoresis on a 1 % agarose gel and the DNA was purified using the QIAquick PCR purification kit (Qiagen). The nucleotide sequences of the PCR products were determined at least once on each DNA strand by cycle sequencing with BigDye Terminator Cycle Sequencing reagent, version 3.1 (Applied Biosystems), used in accordance with the manufacturer's instructions. Extension reactions were performed with the amplification primers (Table 1). Unincorporated dye terminators were removed by precipitation of the termination products with 95 % (v/v) ethanol and the labelled extension products were separated and detected with an ABI 3100 Genetic Analyser (Applied Biosystems). The STADEN suite of computer programs version 1999.0 by Staden (1996) was used to assemble sequences from the resultant chromatograms. Sequence analysis was carried out with the GCG program, version 10.

	Results and Discussion

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The nucleotide sequence of wbeT from six clinical isolates of V. cholerae El Tor, three Inaba and three Ogawa strains, was determined. The sequences were deposited in GenBank under the accession numbers AY458621–AY458626 (Table 2). We observed unique mutations for Inaba strains 35A3, SO84 and V2, which all resulted in a truncated and inactivated gene product. Identical mutations were found for strains SO84 and SO90. By contrast, Ogawa isolates only had a silent substitution (Table 2). In an attempt to understand the sudden appearance of the Inaba serotype in India after a lapse of several years, Garg et al. (2000) used molecular typing methods to investigate the relationship of local V. cholerae El Tor isolates. They showed that Inaba isolate SO90 and Ogawa isolates PG117 and PG81 had the same pulsed-field gel electrophoresis (PFGE) pattern. These isolates were thought to originate from the same clone that also generated Inaba isolate SO84. By contrast, isolates CO840 (Ogawa) and V2 (Inaba) had different PFGE patterns. This allowed Garg et al. (2000) to conclude that Inaba isolates, represented by SO90, originated from the prevailing Ogawa isolates and were not a recrudescence of Inaba isolates, such as V2, that were prevalent several years ago. The occurrence of random mutations in wbeT supports the association of SO84 and SO90 with one clone that was responsible for an epidemic that covered at least 2 years and two locations (Garg et al., 2000). We conclude that nucleotide sequence determination of wbeT can be used for serotyping of V. cholerae O1. However, a larger panel of both related and unrelated Inaba isolates will have to be analysed to determine if mutations in wbeT can be used as a marker of clonality for V. cholerae Inaba strains.

	ACKNOWLEDGEMENTS

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We are grateful to Ms Sarah Mitchell for technical assistance.

	References

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