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Letter
Cholera in Mozambique, Variant
of Vibrio cholerae
M. Ansaruzzaman,* N.A. Bhuiyan,* G. Balakrish Nair,* David A. Sack,*
Marcelino Lucas,† Jacqueline L. Deen,‡ Julia Ampuero,§¶ Claire-Lise Chaignat,#
and The Mozambique Cholera Vaccine Demonstration Project Coordination
Group1
*Centre for Health and Population Research, Dhaka, Bangladesh;
†Ministério da Saude, Maputo, Mozambique; ‡International Vaccine Institute,
Seoul, Korea; §Médecins Sans Frontières, Geneva, Switzerland; ¶Epicentre,
Paris, France; and #World Health Organization, Geneva, Switzerland
Suggested
citation for this article
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 CTXΦ filamentous phage (1),
in which the cholera toxin genes are contained, differs between the classical
and El Tor biotypes. CTXclassΦ is found in classical strains,
CTXETΦ is present in El Tor and O139 strains, and CTXcalcΦ
is found in resurgent O139 strains. The diversity of CTXΦ 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.
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Click to view
enlarged image
Figure. Amino acid sequence alignment of
CT-B subunit of Vibrio cholerae O1 classical, El Tor, and
Mozambique (B33 and B65) strains...
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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 (rstRclass), 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 CT-B 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.
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 CTXΦ, 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 Tor 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
- Waldor MK, Mekalanos JJ. Lysogenic
conversion by a filamentous phage encoding cholera toxin. Science.
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in Bangladesh. J Clin Microbiol. 2002;40:3296–9.
1The group includes participants from the:
Ministério 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); Médecins Sans Frontières, Geneva, Switzerland (Claude Mahoudeau,
Bruno Lab, Gérard 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
Touré and Pierre Kahozi).
Suggested citation
for this article:
Ansaruzzaman M, Bhuiyan
NA, Nair GB, Sack DA, Lucas M, Deen JL, et al. Cholera in Mozambique,
variant of Vibrio cholerae [letter]. Emerg Infect Dis [serial on
the Internet]. 2004 Nov [date cited]. Available from http://www.cdc.gov/ncidod/EID/vol10no11/04-0682.htm
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