Molecular Analyses of Vibrio cholerae O1 Clinical Strains, Including New Nontoxigenic Variants Isolated in Mexico during the Cholera Epidemic Years between 1991 and 2000
Résumé
We studied the evolution of Vibrio cholerae O1 during the 1991 to 2000 cholera epidemic in Mexico by biochemical, serological, and molecular characterization of strains collected during this period. Strains were divided into toxigenic and nontoxigenic groups according to the presence or absence of genes encoding cholera toxin. As previously reported, we characterized two populations among toxigenic strains, which were present from the first year of the epidemic. BglI rRNA analysis revealed that these strains had ribotype profiles, denoted M5 and M6 in our study, that were identical to those previously designated Koblavi B5 or Popovic 5 and Popovic 6a or Tamayo B21a, respectively. Ribotype M5 was isolated between 1991 and 1993. This ribotype had a low level of genetic variation as detected by pulsed-field gel electrophoresis (PFGE). Ribotype M6 persisted from 1991 to 2000. However, PFGE profiles suggested that two epidemiologically unrelated strains coexisted within this single ribotype from 1995 until the end of the epidemic. We identified three new BglI ribotypes, Mx1, Mx2, and Mx3, from nontoxigenic V. cholerae O1 strains isolated between 1998 and 2000; one of them grouped strains positive for the toxin-coregulated pilus island. They differed from nontoxigenic clones isolated in Latin America and on the U.S. Gulf Coast and are probably autochthonous Mexican V. cholerae O1 variants. Most of these new variants were isolated from states surrounding the Gulf of Mexico, where the highest incidence of cholera in the country was recorded. Thus, the Mexican Gulf Coast, like the U.S. Gulf Coast, may act as an environmental reservoir of V. cholerae O1. The seventh cholera pandemic, characterized by Vibrio chol-erae O1 biotype El Tor, is still present around the world. Moreover, the number of reported cholera cases has continuously increased since 2004. The World Health Organization (WHO) (http://www.who.int/wer) reported a 30% increase in cases of cholera worldwide between 2004 (101,383 cases) and 2005 (131,943 cases) and a further 79% increase between 2005 and 2006 (236,860 cases), whereas the number of countries reporting cases has remained constant. At the same time, the global case-fatality rate rose from 1.72% in 2005 to 2.66% in 2006. However, the actual numbers of cholera cases globally are estimated to be much higher than officially reported, due to underreporting and other limitations of surveillance systems. In 2006, the total number of cases reported in Africa accounted for 99% of the global total; Africa has been the continent with the highest number of officially reported cholera cases since 1996. However, in America the number of cases has greatly decreased since 1999, with only 10 cases reported from Canada and the United States (http://www.who.int/wer/2007 /wer8231/en/index.html), 4 of which were indigenous to the United States. Two basic facts distinguish the seventh cholera pandemic from the other six, proposed by Pollitzer (26), occurring before 1961: (i) the epidemic clone shifted from the classic to the El Tor biotype, with an origin in Indonesia rather than the Indian subcontinent, and (ii) the seventh pandemic evolved in two waves, with the first one spreading throughout Asia between 1961 and 1966 and the second spreading to Asia, Africa, and Latin America around 1970 (20). Before the 1991 epidemic, cholera was mostly absent for a century in South, Central, and North America; a few sporadic cases were reported between 1973 and 1992 on the U.S. Gulf Coast (2), and one case was reported in 1983 in Cancun, Mexico, in a tourist from the United States (1). The epidemic began in 1991 in the coastal regions of Peru and spread rapidly to the eastern, northern, and southern countries of the continent , forming a branch of the seventh pandemic. The appearance of the disease in coastal areas of Peru remains a mystery; some studies suggest maritime transport as the source, whereas others suggest a local source (36). Following the first report in Peru in January 1991, cholera was reported in Mexico in June 1991. The number of reported cases in Mexico increased from 1991 until 1993, decreased substantially in 1994, but rose to its highest level ever recorded in 1995 (15,526 cases). Thereafter, cholera cases consistently decreased, with no cases of cholera reported by the Mexican Instituto Nacional de Diagnóstico y Referencia Epidemio-lógica (INDRE) (National Diagnostics and Epidemiological Reference Institute) since 2002 (32). Therefore, two epidemic cycles have been identified: between 1991 and 1994 and between 1995 and 2001. Strains responsible for the cholera epidemic in Latin Amer-ica have been extensively characterized using various molecular methods such as restriction endonuclease digestion of plas-mids or chromosomal DNA, ribotyping, multilocus enzyme
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