Population and Genetic Study of <i>Vibrio cholerae</i> from the Amazon Environment Confirms that the <i>WASA-1</i> Prophage Is the Main Marker of the Epidemic Strain that Circulated in the Region

<div><p><i>Vibrio cholerae</i> is a natural inhabitant of many aquatic environments in the world. Biotypes harboring similar virulence-related gene clusters are the causative agents of epidemic cholera, but the majority of strains are harmless to humans. Since 1971, environmental surveillance for potentially pathogenic <i>V. cholerae</i> has resulted in the isolation of many strains from the Brazilian Amazon aquatic ecosystem. Most of these strains are from the non-O1/non-O139 serogroups (NAGs), but toxigenic O1 strains were isolated during the Latin America cholera epidemic in the region (1991-1996). A collection of environmental <i>V. cholerae</i> strains from the Brazilian Amazon belonging to pre-epidemic (1977-1990), epidemic (1991-1996), and post-epidemic (1996-2007) periods in the region, was analyzed. The presence of genes related to virulence within the species and the genetic relationship among the strains were studied. These variables and the information available concerning the strains were used to build a Bayesian multivariate dependency model to distinguish the importance of each variable in determining the others. Some genes related to the epidemic strains were found in environmental NAGs during and after the epidemic. Significant diversity among the virulence-related gene content was observed among O1 strains isolated from the environment during the epidemic period, but not from clinical isolates, which were analyzed as controls. Despite this diversity, these strains exhibited similar PFGE profiles. PFGE profiles were significant while separating potentially epidemic clones from indigenous strains. No significant correlation with isolation source, place or period was observed. The presence of the <i>WASA-1</i> prophage significantly correlated with serogroups, PFGE profiles, and the presence of virulence-related genes. This study provides a broad characterization of the environmental <i>V. cholerae</i> population from the Amazon, and also highlights the importance of identifying precisely defined genetic markers such as the <i>WASA-1</i> prophage for the surveillance of cholera.</p> </div

Geographical distribution of <i>V. cholerae</i> isolates.

<p>The geographical location of rivers, streams, and wastewater plants from where the strains that were used in this study were isolated are indicated in the map. The sizes of markers indicate the number of strains in each location, markers are centered in the cities where the strains were isolated (see Table S1). Belem (yellow), Barcarena (light green), Maruda (pink), Macapá (dark green), Oiapoque (light blue), Manaus (red), Tabatinga (light blue), Rio Branco (purple), and Santa Rosa (orange). Quantities of strains isolated in each period are indicated in the bar graphs. </p

Distribution of genotypes among NAG strains.

<p>The presence or absence of virulence-related genes are represented, respectively, by blue and white squares. The histogram below each figure correspond to the frequency of each gene. The colors highlighting the strains’ keys correspond to the isolation sources. Strains highlighted pink were isolated from wastewater, blue from superficial water, green from superficial stream water, yellow from fish, and brown from copepods.</p

Dependency model of multivariate data from strains.

<p>Bayesian network representing conditional probabilities of variables that were available for the strains. Arcs are colored according to the impact in the posterior probability of the model when the arc is removed. The network represents the end result of the evaluation of 4.5 * 10⁷ different topologies, in which the last 1.4 * 10⁷ evaluations did not yield a better model. The network was constructed using the online B-Course software [42]. </p

O1 genotypes.

<p>The presence and absence of virulence-related genes are represented, respectively, by blue and white squares. The strains are grouped in colored bars according to their PFGE cluster (Fig. 4): from top to bottom are groups 1 (purple), 2 (red), 3 (blue), 9 (yellow), 6 (orange), and 8 (green). The colors highlighting the strain keys correspond to the isolation sources. Strains highlighted pink were isolated from wastewater, blue from superficial water, green from superficial stream water, and black from clinical sources.</p