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

Genome of the Latin American epidemic <i>Vibrio cholerae</i> marker phage.

<p>Graphical representation of the genes that correspond to the genome of a bacteriophage that is present in all the Latin American epidemic strains tested in this study. The CDS are the blue arrows that are pointed towards the direction they are coded in the genome. The putative protein functions are listed below, with a corresponding number to its localization in the image. (Images generated in the Geneious software – reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037283#pone.0037283-Drummond1" target="_blank">[24]</a>)</p

General genome features of the <i>V. cholerae</i> strain IEC224.

<p>General genome features of the <i>V. cholerae</i> strain IEC224.</p

Classification of the <i>V. cholerae</i> Latin American epidemic phage in the dsDNA Phage Proteomic Tree.

<p>Neighbor Joining comparison of all the proteins of 733 dsDNA phages grouped the Latin American epidemic phage with the genus of LUZ24-Like viruses. These are phages from the Podoviridae family that conform a separate cluster, which is different from all other phages classified in the tree. The group containing these phages is highlighted pink. On the right is a table with the color keys of the phages that are colored in the tree according to their ICTV classification <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037283#pone.0037283-King1" target="_blank">[36]</a>.</p

The altered sucrose phenotype of the strain IEC224 is due to a frame-shift mutation.

<p>The characteristic phenotype of <i>Vibrio cholerae</i> is to produce shiny gold colonies after 48 h incubation on TCBS agar (as pictured on the right side). The strain IEC224 fails to ferment sucrose and colonies remain green (pictured on the left). The only genomic difference between a functioning sucrose fermenter and the IEC224 strain is an insertion in the gene coding for the sucrose-specific IIB domain of the PTS system, which is shown above in the aligned fragments of the mutated IEC224 gene and the functioning N16961 gene. A diagram with the metabolic role of this protein is illustrated in the center, showing that it is a carrier that selectively transports sucrose into the cell and phosphorylates it to signal downstream reactions. A model of the functioning protein structure is shown on the top left, as well as a model for the altered structure can be seen in the top right. (Model generated by PHYRE², following the pipeline of reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037283#pone.0037283-Kelley1" target="_blank">[25]</a>)</p

Variants of the Latin American epidemic phage in sequenced V. cholerae genomes.

<p>The assembly of sequencing reads from 16 other El Tor <i>V. cholerae</i> genomes from Latin America, and their putative ancestor strain from Angola, revealed the presence of the Latin American epidemic phage in all strains. The genomic variations were in 8 sites numbered from bases 0 through 49,291 (right). Collectively these strains formed 10 variants of the phage, with the variant A being the most abundant. This variant is shared with the putative ancestor strain. All strains accumulated at least one SNP after 1992.</p