Whole-Genome Sequencing and Annotation of Aeromonas veronii Isolates from Channel Catfish

ABSTRACT

The genomes of seven Aeromonas veronii strains isolated from tissues of healthy or diseased channel catfish obtained from Alabama, USA, fish farms were sequenced and annotated. These genome sequences will enable comparative analyses to determine the roles these bacteria play in catfish aquaculture and the development of new preventative or management strategies.

ANNOUNCEMENT

Aeromonas veronii, a Gram-negative bacterium, is ubiquitous in various aquatic environments, including fishponds. The bacterium is frequently isolated from both apparently healthy fish and diseased fish in which A. veronii was recognized as either a primary or secondary pathogen. Multiple types of virulence factors have been identified in A. veronii isolates (1–5). During a study that surveyed farmed catfish with disease symptoms indicative of motile Aeromonas septicemia (MAS) caused by hypervirulent Aeromonas hydrophila (vAh) in Alabama, USA, many isolates of A. veronii were cultured from internal tissues of either diseased or apparently healthy fish (Table 1). All bacteria were isolated at the Alabama Fish Farming Center (32.69084, −87.59172) between 2009 and 2014 from fish provided by farms in the local area. Fish were aseptically dissected, a sterile loop was inserted into a tissue, and the sample was spread onto tryptic soy agar supplemented with sheep blood (Remel, Lenexa, KS, USA). Plates were then incubated at 28°C for ~24 h. Isolates were confirmed by blastn analyses of PCR-generated 16S rRNA and gyrB gene sequences, using primer pairs 16S_f and 16S_r (5′-AGAGTTTGATCMTGGCTCAG-3′ and 5′-GGTTACCTTGTTACGACTT-3′, respectively) and gyrB_f and gyrB_r (5′-CCCAGACCAAGGACAAGCTGGT-3′ and 5′-CCAGCCCTTTATAGCGCTGGAT-3′, respectively) and the PCR protocol described previously (6).

TABLE 1.

Details from the sequencing and assembly effort for seven strains of Aeromonas veronii^a

Isolate	Read count	Coverage (×)	No. of scaffolds (>500 bp)	GC content (%)	Total length (bp)	N 50	No. of predicted genes	Tissue	16S top hit by BLAST
ALD17-04	9,512,505	314	70	58.62	4,588,282	168,651	4,254	Gut (H)	NR_119045.1
ALG10-089	7,834,582	259	50	58.54	4,609,237	204,327	4,172	Kidney (D)	NR_119045.1
ALH17-19	5,201,984	172	85	58.38	4,828,371	126,561	4,479	Gut (D)	NR_044845.1
ALH17-23	6,810,867	225	73	58.47	4,693,559	145,811	4,418	Kidney (D)	NR_044845.1
ALP17-17	9,736,740	322	251	58.56	4,789,795	163,362	4,539	Kidney (D)	NR_044845.1
ALP17-18	7,967,005	263	78	58.82	4,605,149	134,287	4,206	Kidney (D)	NR_044845.1
ML10-202	7,651,987	253	88	58.64	4,633,676	122,253	4,324	Kidney (D)	NR_104824.1

^aRead counts are total numbers of paired-end reads from Illumina sequencing. The number of scaffolds, GC content, total length, and N₅₀ are the results from SPAdes analyses. The total numbers of genes were predicted from PGAP analyses. “Tissue” indicates the tissue from which bacteria was isolated and whether the fish was asymptomatic (healthy [H]) or symptomatic for MAS (diseased [D]). The 16S top hit by BLAST indicates the accession number from top-hit blastn results using the identified 16S rRNA gene sequence from each strain as a query.

For sequencing, individual bacterial strains were cultured in tryptic soy broth at 29°C while shaking at 200 rpm for 16 h. Bacterial cells were harvested following centrifugation at 10,000 × g and were subjected to genomic DNA isolation using a DNeasy Blood & Tissue kit (Qiagen, Germantown, MD, USA). Quality control (QC) analysis of DNA was performed using the Qubit DNA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and gel electrophoresis. Sequencing libraries were prepared for all samples using the Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA) per the manufacturer’s instructions. QC of sequencing libraries was performed by Bioanalyzer high-sensitivity DNA analysis (Agilent, Santa Clara, CA, USA). Paired-end sequencing (2 × 75 bp) on an Illumina NextSeq 1000 was performed via a service provider (Applied Biological Materials, Vancouver, Canada). The sequencing output is shown in Table 1. For computational analyses, unless otherwise noted, software was executed within the OmicsBox platform (7) with default parameters selected. Preprocessing and QC of raw reads prior to genome assembly were performed using Trimmomatic v.0.38 (8). SPAdes v.3.15.5 (9) was used for de novo assembly of QC reads (pipeline option: --careful). The resultant scaffolds for each isolate were annotated via the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v.6.3 with the annotation method best-placed reference protein set (GeneMarkS-2+) (10).

Assuming an A. veronii genome size of ~4.54 Mb, the final genome sequencing coverage ranged from 172× to 322× (Table 1). For all seven strains, GC content was an average of 58%. Information on the number of predicted genes is found in Table 1. BUSCO analysis (11–13) of coding sequences against the lineage data set gammaproteobacterial_odb10 indicated substantial completeness of these genomes, with estimates ranging from 82.79% to 83.06%. Characterization of the functional genomic diversity of these strains will assist in our understanding of the roles A. veronii plays in aquaculture and particularly in pathogenesis of MAS, which will aid in the development of new disease management strategies.

Data availability.

This Whole Genome Shotgun project has been deposited in DDBJ/ENA/GenBank under accession numbers JAOTOB000000000, JAOTPP000000000, JAOTPQ000000000, JAOTPR000000000, JAOTPS000000000, JAOTPT000000000, and JAOTPU000000000. The versions described in this paper are versions JAOTOB010000000, JAOTPP010000000, JAOTPQ010000000, JAOTPR010000000, JAOTPS010000000, JAOTPT010000000, and JAOTPU010000000, respectively. Raw reads can be found at the NCBI SRA under accession numbers SRR21799103, SRR21802829, SRR21802948, SRR21803002, SRR21803011, SRR21803683, and SRR21803762.