Cholera in Mozambique, variant of Vibrio cholerae.

To the Editor: Cholera outbreaks caused by toxigenic Vibrio cholerae serogroup O1 frequently occur in many sub-Saharan African countries. The serogroup O1 is classified into two biotypes, classical and El Tor. The seventh and current pandemic of cholera is caused by the El Tor biotype; the classical biotype is believed to be extinct. The classical and El Tor biotypes of V. cholerae O1 are closely related in their O-antigen biosynthetic genes but differ in other regions of the genome. The genomic structure of the CTX [PHI] filamentous phage (1), in which the cholera toxin genes are contained, differs between the classical and El Tor biotypes. CT[X.sup.class][PHI] is found in classical strains, CT[X.sup.ET][PHI] is present in El Tor and O139 strains, and CT[X.sup.calc][PHI] is found in resurgent O139 strains. The diversity of CTX [PHI] among biotypes is mainly due to the variations in the repeat sequence elements, particularly in the rstR gene region (2).

While conducting surveillance in the cholera treatment center in Beira, the second largest city in Mozambique, we examined 175 rectal swabs or stool samples from January 7 to March 8, 2004, using standard published procedures. During this period, we isolated 58 strains of V. cholerae O1. The isolates were transported to the Enteric Microbiology Unit of the International Center for Diarrhoeal Disease Research in Dhaka, Bangladesh (ICDDRD,B), for further phenotypic and genotypic characterization to determine serotype, biotype, and presence of important virulence genes. All 58 strains were identified as V. cholerae O1 of the Ogawa serotype. Forty strains selected for detailed characterization were resistant to polymyxin B, agglutinated chicken cells, yielded a positive Voges-Proskauer reaction, were positive for the El Tor hemolysin by the tube agglutination method, and were sensitive to group IV El Tor phage but resistant to the classical group V phage and were therefore classified as the El Tor biotype. The antimicrobial susceptibility of 15 of the 40 isolates examined showed that the strains were sensitive to tetracycline, ampicillin, furazolidone, erythromycin, and ciprofloxacin but resistant to trimethoprim-sulfamethoxazole, and also to the vibriostatic compound 0/129.

By using polymerase chain reaction (PCR), we established that all 40 strains carried the ctxA gene (constituent gene of the CTX prophage) and the tcpA gene (the El Tor type), a constituent gene of the vibrio pathogenicity island. We then focused on the rstR gene because of its diversity between the two biotypes. All of the 40 El Tor strains produced a 500-bp PCR product of the rstR gene of the classical type (rst[R.sup.class]), despite belonging to the El Tor biotype. Nucleotide sequence analysis of the rstR gene of two representative Mozambique strains showed 100% homology to the classical rstR gene of classical reference strain O395. The amino acid sequence of the B-subunit of classical and El Tor biotypes have distinct signature sequences (3). We amplified the ctxB gene using specific primers and found that the deduced amino acid sequence of the CT-B subunit of the Mozambique strains varied from the El Tor CT-B subunit at positions 39 (histidine replaces tyrosine in El Tor) and 68 (threonine replaces isoleucine in El Tor), and the amino acid residues at these positions are identical to those of the classical CTB subunit (Figure). The nucleotide sequences obtained for ctxB of the two Mozambique strains B33 and B65 were deposited in GenBank under accession no. AY648939 and AY6448940, respectively. Therefore, the Mozambique strains of V. cholerae O1 displayed typical traits of the El Tor biotype overall but carried the classical CTX prophage.

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Our findings that El Tor strains of V. cholerae O1 from Mozambique are carrying the classical prophage shows the presence of genetic materials associated with the classical biotype in Mozambique. Further, these findings provide the first circumstantial evidence of transmission of the classical CTX prophage. The CTX prophages in El Tor strains give rise to infectious phage particles (1), but neither of the two CTX prophages integrated at two different sites of the classical genome give rise to phage particles (4). Subsequent studies have shown that, although the genes of the classical prophages encode functional forms of all of the proteins needed for production of CTX[PHI], the CTX prophage does not yield virions because of the atypical arrangement of its prophage arrays (4).

Genetic hybrids between El Tor and classical biotypes of O1 V. cholerae were reported among sporadic isolates earlier in Bangladesh (5) and were named the Matlab variants after the place where they were first isolated. The Mozambique strains of V. cholerae likely evolved from an El Tor strain, which shed its CTX phage and acquired the classical prophage. Alternatively, strains like the Matlab variant may have spread to the African subcontinent. Whether introducing the CTX prophage in the El Tot genome background will increase pathogenicity, affect genomic stability, or enhance the epidemic-causing potential is uncertain. This subtle genetic change might also alter the effectiveness of current cholera vaccines which stimulate antitoxic as well as antibacterial immunity.

Acknowledgments

We thank J.J. Mekalanos for useful comments on this manuscript and Motiur Rahman for help with the nucleotide sequencing. We also thank the International Vaccine Institute funded by the Bill and Melinda Gates Foundation (Diseases of the Most Impoverished Program).

The ICDDR,B is supported by the aid agencies of the governments of Australia, Bangladesh, Belgium, Canada, Japan, Kingdom of Saudi Arabia, the Netherlands, Sweden, Sri Lanka, Switzerland, and the United States.

References

(1.) Waldor MK, Mekalanos JJ. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science. 1996;272:1910-4.

(2.) Kimsey HH, Nair GB, Ghosh A, Waldor MK. Diverse CTXOs and evolution of new pathogenic Vibrio cholerae. Lancet. 1998;352:457-8.

(3.) Popovic T, Fields PI, Olsvik O. Detection of cholera toxin genes. In: Wachsmuth IK, Blake PA, Olsvik O, editors. Vibrio cholerae and cholera: molecular to global perspectives. Washington: American Society for Microbiology; 1994. p. 41-52.

(4.) Davis BM, Waldor MK. Filamentous phages linked to virulence of Vibrio cholerae. Curr Opin Microbiol. 2003;6:3542.

(5.) Nair GB, Faruque SM, Bhuiyan NA, Kamruzzaman M, Siddique AK, Sack DA. New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh. J Clin Microbiol. 2002;40:3296-9.

Address for correspondence: David Sack, Executive Director, ICDDR,B, Centre for Health and Population Research, P.O. Box 128, Mohakhali, Dhaka 1000, Bangladesh; fax: [phone]; email: [e-mail]

M. Ansaruzzaman, * N.A. Bhuiyan, * G Balakrish Nair, * David A. Sack, * Marcelino Lucas, ([dagger]) Jacqueline L. Deen, ([double dagger]) Julia Ampuero, ([subsections]) ([paragraph]) Claire-Lise Chaignat, (#) and The Mozambique Cholera Vaccine Demonstration Project Coordination Group (1)

* Centre for Health and Population Research, Dhaka, Bangladesh; ([dagger]) Ministerio da Saude, Maputo, Mozambique; ([double dagger]) International Vaccine Institute, Seoul, Korea; ([subsections]) Medecins Sans Frontieres, Geneva, Switzerland; ([paragraph]) Epicentre, Paris, France; and (#) World Health Organization, Geneva, Switzerland

(1) The group includes participants from the: Ministerio da Saude, Maputo, Mozambique (Avertino Barreto, Juvenaldo Amos, Catarina Mondlane, and Raul Vaz); International Vaccine Institute (John D. Clemens, Lorenz von Seidlein, Xuan-Yi Wang, Mohammad Ali, and Mahesh K Puri); Medecins Sans Frontieres, Geneva, Switzerland (Claude Mahoudeau, Bruno Lab, Gerard Bedock, Valerie Perroud, and Margaret McChesney); Epicentre, Paris, France (Philippe J. Guerin, Dominique Legros, and Philippe Cavailler); World Health Organization, Geneva, Switzerland (Marie-Paule Kieny and Duncan Steele); and World Health Organization, Maputo, Mozambique (Bocar Toure and Pierre Kahozi).