Supplementary Materials:
Materials and Methods
Genome sequencing, assembly, and annotation. Genomic DNA of Chinese Carp isolates ZC1 and Mississippi catfish isolate S04-690 were extracted according to the methods described previously (1). A barcoded Illumina library for strain ZC1 was constructed using a Nextera DNA Sample Prep Kit (Epicentre, Madison, WI), and sequenced using an Illumina MiSeq (Illumina, Inc., San Diego, CA). A barcoded Illumina library for strain S04-690 was constructed using a NxSeq® DNA Sample Prep Kit (Lucigen, Middleton, WI) and sequenced using an Illumina HiSeq 2000. The Illumina sequence reads were trimmed for quality and de novo assembled using the CLC Genomics Workbench (CLCBio, Cambridge, MA). Gene prediction and annotation were carried out using GeneMark (2) and the RAST annotation server (3), respectively. The raw sequence reads for strains ZC1 and S04-690 were deposited within the NCBI’s Short Read Archive under the accession numbers SRR1031999 and SRR1032027, respectively.
PCR amplification and sequencing of house-keeping genes. PCR primers for PCR amplification and sequencing of the six different house-keeping genes gyrA, gyrB, recA, dnaJ, dnaX and rpoD from Aeromonas isolates and the PCR conditions used were described previously (4).
gyrB phylogeny. For single gene analyses, concatenated sequences were aligned and analyzed using MEGA5 (5). Genetic distances were computed by the Kimura two-parameter model, and phylogenetic trees were constructed using the neighbor-joining method, with 1,000 bootstrap replications. The gyrB phylogeny was constructed using gyrB sequences from 264 different Aeromonas strains: 55 isolates (Table S1) and 210 Aeromonas strains downloaded from the Aeromonas MLST database (www.pubmlst.org/aeromonas) (6). The gyrB sequences derived from Chinese carp isolates and US catfish isolates (7) were obtained from the GenBank nr/nt database. In addition, a further gyrB phylogenetic analysis was constructed from a collection of 190 Aeromonas isolates: 151 isolates and 39 Aeromonas reference strains (www.pubmlst.org/aeromonas). The gyrB sequences that we determined in this study were deposited in GenBank under the accession numbers KF913677-KF913679.
Multilocus sequence based phylogeny. The MLST scheme proposed by Martinez-Murcia and colleagues (4) was applied to 33 Aeromonas isolates used in this study (Table S2). Each of the six house-keeping loci (gyrA, gyrB, recA, dnaJ, dnaX and rpoD) were aligned and trimmed at the same nucleotide positions using the CLC Genomics Workbench (version 4.9). Gene sequences for each isolates were concatenated using CLC Genomics Workbench and aligned using the ClustalW alignment algorithm in MEGA5 (5). The evolutionary history was inferred by constructing a phylogenetic tree using the Neighbor-Joining method in MEGA5 (5) with 1,000 bootstrap replications including a total of 146 Aeromonas strains: 33 isolates and 113 Aeromonas strains from the dataset published by Martinez-Murcia and colleagues (4). The complete deletion inference method was used. The six house-keeping gene sequences from each isolate used in this analysis were deposited within the GenBank nr/nt database under the accession numbers KF873622 to KF873761.
Phylogenomics of A. hydrophila epidemic-specific regions. A total of 303,863 bp of epidemic-specific sequences present in recent VAh isolates (7), Chinese carp isolate ZC1 and Mississippi isolate S04-690 were used for a phylogenomic analysis. Paired-end Illumina reads from A. hydrophila isolates ML09-121, ML09-122, PB10-118, AL09-79, AL10-121, ZC1 and S04-690 were separately reference mapped against the complete genome sequence of A. hydrophila epidemic isolate ML09-119 (8) using default parameters in CLC Genomics Workbench. Homologous sequences specific to the epidemic-specific genomic regions were collected from the consensus sequences of each isolates and were concatenated using CLC Genomics Workbench. Concatenated sequences from all isolates were aligned with the ClustalW alignment algorithm in MEGA5 (5). The evolutionary history was inferred with the Neighbor-joining method using MEGA5 (5).
BLASTp matrix. Proteome homology searching of ZC1 and S04-690 isolates with ATCC 7966 (9) and previously sequenced 11 A. hydrophila isolates (7) was conducted using an all-against-all BLASTp algorithm according to previously described methods (10). This BLASTp matrix analysis determines the percent similarities between two isolates by measuring the ratio of the number of conserved protein families shared between isolates to the total number of protein families in the isolates. The pairwise distribution of the conserved protein families within the genome of 14 different A. hydrophila isolates was presented in a triangle-shaped matrix.
Determination of the virulence of A. hydrophila strains in channel catfish and grass carp. The virulence of ML09-119, ZC1 and AL06-06 in channel catfish (Ictalurus punctatus) (11.8 g ± 1.2 g) and Grass carp (Ctenopharyngodon idella) (11.6 g ± 1.4 g) were determined according to the methods described previously (7). All fish with known disease histories were obtained from the E. W. Shell Fisheries Research Station, Auburn, AL, USA. Ten channel catfish and 10 grass carp were stocked and cohabitated in each aquarium, with five aquaria randomly picked for each bacterial strain treatment. During the disease challenge, fish were maintained in flow-through aquaria (0.2 L/min) systems at 30oC using underground water sources with constant aeration. Fish were acclimated for three days and fed 4% body weight daily with commercial feed before challenge. A cryostock of strain ML09-119, ZC1 and AL06-06 was incubated separately in 5 ml of Tryptic Soy Broth (TSB) overnight at 30oC with shaking at 150 rpm, and then sub-cultured into 100 ml TSB until the optical density (OD) of the culture at 600 nm (OD600) reached approximately 1.0. The bacterial suspension was subjected to centrifugation at 10,000 x g for 8 min, and then the supernatant was discarded and the bacterial pellet was re-suspended in fresh TSB media to an OD600 = 1.0 using a spectrophotometer (Thermo Spectronic, Rochester, NY, USA). A 1:100 (v/v) dilution was performed using fresh TSB, and the bacterial cultures were placed on ice and used for challenge within 3 hours. To inoculate each fish, 100 ml of the cell culture was injected into the intraperitoneal (I.P.) cavity of each fish. Fish in the control group were injected with TSB medium alone. Plate counts of serially diluted bacterial cultures were performed to determine the actual dose injected into fish. The dosage used in this study was 2 x 106 CFU/fish. Mortalities were recorded daily for 14 days post challenge, and any moribund or dead fish were removed from the system daily and suitable fish necropsied for bacteriological identification. Statistical significance was determined by a 3 (bacteria) x 2 (fish species) factorial analysis of variance (ANOVA) and Tukey’s multiple comparisons to compare individual treatment groups.
Prediction of Genomic Islands. Genomic islands of A. hydrophila isolates ZC1 and S04-690 were predicted using IslandViewer (11). The concatenated contigs of ZC1 and S04-690 that comprised approximately 5.0 Mbp each were converted to GenBank format using the Sequin program (version 11.9). The GenBank formatted sequence files were scanned using IslandViewer to determine the presence of predicted genomic islands. To identify ZC1 and S04-690-specific GIs that share homology with that of the VAh isolates, the nucleotide sequences of all GIs predicted within the genome of ML09-119 isolates were forced joined and used as a reference sequence to conduct a reference mapping against trimmed pair-end Illumina reads of strains ZC1 or S04-690.
Myo-inositol utilization assay. The myo-inositol utilization capacity of A. hydrophila ZC1 and S04-690 was tested according to methods described previously (7). A. hydrophila isolates ML09-119 and AL06-06 were used as positive and negative control, respectively, for this assay.
Figure Legends:
Supplementary Figure 1. gyrB phylogeny using sequences from 169 A. hydrophila isolates, including strains of Asian and US origin. Colored ranges refer to the geographical origin of the isolates. The red segments indicate the epidemic strains. Tree figure was generated using the interactive Tree of Life web application (itol.embl.de) (12).
Supplementary Figure 2. Phylogenetic analysis based on the virulent A. hydrophila (VAh)-specific sequences. The evolutionary history was inferred using the Neighbor-joining method. The evolutionary distances are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 303,863 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 with 1000 repetitions to generate bootstrap support (5).
Supplementary Figure 3. BLASTp MATRIX of 14 different A. hydrophila isolates. The proteomes of each of the A. hydrophila isolates were compared pair-wise using all-against-all BLASTp as described previously (10). This matrix illustrates the output from the pairwise comparison of conserved protein families of each of the isolates to each other. The green color represents the relative % homology between proteomes and the red color represents relative % homology within the isolate’s own proteome. The dense green pyramid on the right indicates greater homology (>97%) between the proteomes of VAh isolates from US catfish epidemics, Chinese carp isolate ZC1 and Mississippi catfish isolate S04-690.
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