Molecular Analyses of Vibrio cholerae O1 Clinical Strains, Including New Nontoxigenic Variants Isolated in Mexico during the Cholera Epidemic Years between 1991 and 2000

International audienceWe 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 enzym

Whole-genome comparison between reference sequences and oyster Vibrio vulnificus C-genotype strains.

Whole-genome sequences of Vibrio vulnificus clinical genotype (C-genotype) from the CICESE Culture Collection, isolated from oysters, were compared with reference sequences of CMCP6 and YJ016 V. vulnificus C-genotype strains of clinical origin. The RAST web server estimated the whole genome to be ~4.8 Mb in CICESE strain 316 and ~4.7 Mb in CICESE strain 325. No plasmids were detected in the CICESE strains. Based on a phylogenetic tree that was constructed with the whole-genome results, we observed high similarity between the reference sequences and oyster C-genotype isolates and a sharp contrast with environmental genotype (E-genotype) reference sequences, indicating that the differences between the C- and E-genotypes do not necessarily correspond to their isolation origin. The CICESE strains share 3488 genes (63.2%) with the YJ016 strain and 3500 genes (63.9%) with the CMCP6 strain. A total of 237 pathogenicity associated genes were selected from reference clinical strains, where-92 genes were from CMCP6, 126 genes from YJ016, and 19 from MO6-24/O; the presence or absence of these genes was recorded for the CICESE strains. Of the 92 genes that were selected for CMCP6, 67 were present in both CICESE strains, as were as 86 of the 126 YJ016 genes and 13 of the 19 MO6-24/O genes. The detection of elements that are related to virulence in CICESE strains-such as the RTX gene cluster, vvhA and vvpE, the type IV pili cluster, the XII genomic island, and the viuB genes, suggests that environmental isolates with the C-genotype, have significant potential for infection

Crecimiento y sobrevivencia de Vibrio parahaemolyticus en ostión americano (Crassostrea virginica) almacenada en refrigeración

Objetivo. Cuantificar las densidades de Vibrio parahaemolyticus en ostión americano (Crassostrea virginica) almacenado en refrigeración. Material y métodos. Se almacenaron 320 ostiones a 7 °C durante nueve días y se determinaron las densidades totales y patogénicas mediante la técnica NMP-PCR. Resultados. Se observaron densidades de V. parahaemolyticus tlh+ en los días 0, 3 y 6 de almacenamiento con 1.134, 2.764 y 0.785 log10NMP/g, respectivamente, y en los días 0 y 3 la densidad patogénica trh+ con 0.477 y 0.519 log10NMP/g, respectivamente; las densidades patogénicas tdh+ (0.519 log10NMP/g), tdh+/trh+ (0.519 log10NMP/g) y tdh+/orf8+ (-0.444 log10NMP/g) se detectaron al tercer día de almacenamiento. Conclusión. Los resultados sugieren que el crecimiento de V. parahaemolyticus y la ocurrencia de genes patogénicos a 7 °C involucran cambios en la expresión génica como una respuesta al estrés por frío. Esto contribuye a la sobrevivencia y virulencia de V. parahaemolyticus, lo cual representa un riesgo a la salud pública

Genetic characterization of <i>Vibrio vulnificus</i> strains isolated from oyster samples in Mexico

<div><p><i>Vibrio vulnificus</i> strains were isolated from oysters that were collected at the main seafood market in Mexico City. Strains were characterized with regard to <i>vvhA</i>, <i>vcg</i> genotype, PFGE, multilocus sequence typing (MLST), and <i>rtxA1</i>. Analyses included a comparison with <i>rtxA1</i> reference sequences. Environmental (<i>vcg</i>E) and clinical (<i>vcg</i>C) genotypes were isolated at nearly equal percentages. PFGE had high heterogeneity, but the strains clustered by <i>vcg</i>E or <i>vcg</i>C genotype. Select housekeeping genes for MLST and primers that were designed for <i>rtxA1</i> domains divided the strains into two clusters according to the E or C genotype. Reference <i>rtxA1</i> sequences and those from this study were also clustered according to genotype. These results confirm that this genetic dimorphism is not limited to <i>vcg</i> genotyping, as other studies have reported. Some environmental C genotype strains had high similarity to reference strains, which have been reported to be virulent, indicating a potential risk for oyster consumers in Mexico City.</p></div

Genetic Analysis of <i>Vibrio parahaemolyticus</i> O3:K6 Strains That Have Been Isolated in Mexico Since 1998

<div><p><i>Vibrio parahaemolyticus</i> is an important human pathogen that has been isolated worldwide from clinical cases, most of which have been associated with seafood consumption. Environmental and clinical toxigenic strains of <i>V</i>. <i>parahaemolyticus</i> that were isolated in Mexico from 1998 to 2012, including those from the only outbreak that has been reported in this country, were characterized genetically to assess the presence of the O3:K6 pandemic clone, and their genetic relationship to strains that are related to the pandemic clonal complex (CC3). Pathogenic <i>tdh</i>⁺ and <i>tdh</i>⁺/<i>trh</i>⁺ strains were analyzed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Also, the entire genome of a Mexican O3:K6 strain was sequenced. Most of the strains were <i>tdh</i>/ORF8-positive and corresponded to the O3:K6 serotype. By PFGE and MLST, there was very close genetic relationship between ORF8/O3:K6 strains, and very high genetic diversities from non-pandemic strains. The genetic relationship is very close among O3:K6 strains that were isolated in Mexico and sequences that were available for strains in the CC3, based on the PubMLST database. The whole-genome sequence of CICESE-170 strain had high similarity with that of the reference RIMD 2210633 strain, and harbored 7 pathogenicity islands, including the 4 that denote O3:K6 pandemic strains. These results indicate that pandemic strains that have been isolated in Mexico show very close genetic relationship among them and with those isolated worldwide.</p></div

Comparative results between CICESE-170 and RIMD 2210633.

<p>Comparative results between CICESE-170 and RIMD 2210633.</p

Primers used for the molecular characterization of <i>V</i>. <i>parahaemolyticus</i>.

<p>Primers used for the molecular characterization of <i>V</i>. <i>parahaemolyticus</i>.</p

<i>Vibrio parahaemolyticus</i> strains from CICESE and CAIM culture collections.

<p><i>Vibrio parahaemolyticus</i> strains from CICESE and CAIM culture collections.</p

Genetic Analysis of <i>Vibrio parahaemolyticus</i> O3:K6 Strains That Have Been Isolated in Mexico Since 1998 - Fig 1

<p>Dendrograms of CICESE and CAIM <i>V</i>. <i>parahaemolyticus</i> O3:K6 strains for: a) the neighbor-joining tree (UPGMA) by PFGE analysis after digestion with NotI and b) the neighbor-joining tree (Kimura’s 2 parameters) by MLST of the concatenated sequences of 7 loci.</p