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Vibrio cholerae O1 Amazonia is a pathogen that was isolated from cholera-like diarrhea cases in at least two countries, Brazil and Ghana. Based on multilocus sequence analysis, this lineage belongs to a distinct profile compared to strains from El Tor and classical biotypes. The genomic analysis revealed that it contains Vibrio pathogenicity island 2 and a set of genes related to pathogenesis and fitness, such as the type VI secretion system, present in choleragenic V. cholerae strains.
Vibrio cholerae O1 Amazonia was identified as the cause of cholera cases in villages in the Brazilian Amazon region in 1991, during the seventh cholera pandemic that reached South America and Brazil. Unlike the major human pathogen V. cholerae O1 El Tor biotype, these strains were characterized as not carrying the cholera toxin gene (ctxAB) or the tcpA gene, encoding the structural protein of the TCP pilus, the major virulence determinants of the epidemic strains, and they presented a distinct genomic profile (1). Multilocus sequence analysis (MLSA) revealed that the V. cholerae Amazonia strains belong to a profile distinct from the epidemic/pandemic strains, clustering with some isolates from cholera cases in Ghana in Africa (9). In order to reveal the genomic elements present in this lineage that could explain its pathogenesis and fitness, we sequenced the complete genome of a V. cholerae Amazonia strain.
The chromosomes of V. cholerae O1 Amazonia R-18332 were sequenced by using the Roche 454 pyrosequencing system according to the methods described Margulies et al. (4). Genomic DNA was extracted using a previously described method (8). The coverage was 20-fold, and raw reads were assembled using the Newbler software, generating 63 contigs. The genome was annotated and analyzed using the RAST annotation server (6). The estimated size of the V. cholerae O1 Amazonia R-18332 genome, comprising the two chromosomes, is 4,005,357 bp with an average G+C content of 47%. The number of coding sequences (CDS) is 3,622.
None of the genes of the two major virulence determinants, CTXPhi, the lysogenic filamentous bacteriophage that carries the cholera toxin genes, and Vibrio pathogenicity island 1 (VPI-1), which harbors the tcpA gene, were found in the V. cholerae O1 Amazonia R-18332 genome. However, a sequence was identified that showed 91% and 96% identity at the nucleotide and amino acid levels, respectively, with the VPI-1 integrase gene from V. cholerae O1 pandemic lineages. Vibrio pathogenicity island 2 (VPI-2), carried by cholera-producing strains (7), was also present in this Amazonia strain. Most of the 57-kb region corresponding to VPI-2 in Amazonia presented 98% nucleotide identity with the pandemic lineages, but some genes, like nanH (neuraminidase) and hsdM (putative DNA methylase), presented distinct alleles. The genome sequence analysis also revealed 61 genes known to be related to virulence, disease, and defense. Some of these genes are related to invasion and intracellular resistance (e.g., metalloprotease, lipase precursor, cytolysin, hemolysin [hlyA], pore-forming toxin, methyl-accepting chemotaxis protein, hemolysin secretion protein [hlyB], lipase activator protein, and lipase-specific foldase). There is experimental evidence for both cytotoxic and enterotoxic activities of the hemolysin (2). The type VI secretion system (T6SS), recently identified in V. cholerae and associated with its survival in aquatic environments (3), was identified in V. cholerae O1 Amazonia R-18332. Another feature found in this strain was the intI4 gene, the genomic signature of the chromosomal V. cholerae superintegron (5).
V. cholerae O1 Amazonia R-18332 has been deposited in the BCCM/LMG Bacteria Collection at Ghent University and in the LGMM Vibrio Collection at Oswaldo Cruz Institute Collection (Rio de Janeiro, Brazil). The results of the whole-genome shotgun project have been deposited with DDBJ/EMBL/GenBank under accession number AFSV00000000. The version described in this paper is the first version, AFSV01000000.
This work was sponsored by CAPES, CNPq, and FAPERJ. R.A.E. acknowledges support of NSF grant DBI 0850356 from the Division of Biological Infrastructure. B.E.D. is supported by a Dutch Science Foundation (NWO) Veni grant (016.111.075).