Computational protocol: Genomic and Phenotypic Characterization of Vibrio cholerae Non-O1 Isolates from a US Gulf Coast Cholera Outbreak

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Protocol publication

[…] Clinical V. cholerae isolates that were epidemiologically linked to consumption of oysters harvested from the Apalachicola Bay, FL were obtained from the Florida Department of Health Bureau of Public Health Laboratories in Jacksonville, FL. The genomes described in this study were either obtained from the NCBI Genbank database or, in the case of strains CP1110, 1111, 1112, 1113, 1114, 1115, 1116 and 1117, were sequenced using the Genome Analyzer IIx system (Illumina, Inc., San Diego, CA) according to the manufacturer's methods. Raw reads of these genomes were assembled with CLC Genomics Workbench. Genome-to-genome comparisons, identification and characterization of molecular genetic elements (MGEs), as well as core genome phylogenetics were performed by using methods described previously . Genomes of V. cholerae strains CP1110 to CP1117 were annotated using Rapid Annotation using Subsystem Technology . For in silico genomic island BLASTN and phylogenetic analyses the RAST-annotated ORFs of V. cholerae CP1110 were used as a reference. PCR analyses of virulence factors not resolved by genome sequencing (rstR alleles, nanH, and ctxB biotype) were done using the methods of Choi et al. , Vora et al. , and Nusrin et al. . Phenotypic assays (proteolysis, hemolysis, biofilm formation, and motility) were conducted following methods standardized for V. cholerae . Hemolysis, biofilm formation, motility, and proteolysis assays were done in nine replicates. BiOLOG phenotypic microarrays (PM1, PM2A, PM9, and PM10) were conducted in duplicate following the manufacturer's instructions (BiOLOG, Hayward, CA). Substrate metabolism was scored by dividing the area under the curve by the background values. Scores >2 were considered positive for metabolism of that substrate.For the Caenorhabditis elegans model, SS104 glp-4 (bn2) temperature sensitive sterile strain was acquired from the Caenorhabditis Genetics Center (CGC). SS104 worms were maintained at 16°C, and experiments were performed at 25°C. Worms were cultured in C. elegans habitation media (CeHM) in tissue culture flasks on a platform shaker . Adult nematodes were bleached (0.5 M NaOH, 1% Hypochlorite) to collect eggs, which were incubated in M9 media for 24 hours to bring them to synchronized L1 stage, and then transferred to CeHM. L4 stage worms were transferred to assay plates for survival experiments. Pathogen lawns for survival assays along with control bacteria E. coli OP50 were prepared by inoculating Nematode Growth Medium (NGM), in 6-cm Petri dishes, with 50 µl of an overnight V. cholerae culture. Plates were incubated overnight at room temperature before worms were added. Temperature sensitive sterile worms (SS104 glp-4(bn2)) strain, obtained from Caenorhabditis Genetics Center were transferred to NGM plates containing V. cholerae wild type strains E7946, CP1112, CP1114, CP1115 or E. coli OP 50 bacterial lawns and incubated at 25°C with ∼20–30 L4 stage worms added to each plate. Animals were scored every 24 h for survival. Animals were considered dead when they no longer responded to a gentle prod with a platinum wire. C. elegans survival was plotted using Kaplan-Meier survival curves and analyzed by log rank test using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Survival curves resulting in p values of <0.05 relative to control were considered significantly different . Strains and genomes used in this study are listed in . […]

Pipeline specifications

Software tools CLC Genomics Workbench, RAST, BLASTN
Application Phylogenetics
Organisms Vibrio cholerae, Caenorhabditis elegans
Diseases Cholera, Infection