Draft Genome Sequences of Citrobacter freundii Strains CF04 and A41 Isolated from Moribund, Septicemic Giant Gourami (Osphronemus goramy) in Sri Lanka

Citrobacter freundii is a Gram-negative opportunistic pathogen associated with many infectious conditions including septicemia in humans and animals. Here, we announce the draft genome sequences of two multidrug-resistant C. freundii strains (CF04 and A41) isolated from septicemic giant gourami (Osphronemus goramy) collected from aquaria in Sri Lanka.

systemic infections in farmed food fish and aquarium fish (1,2). During an investigation to identify septicemia causing bacterial pathogens of fresh water ornamental fish in Sri Lanka, the occurrence of Citrobacter spp. was only second to Aeromonas spp. (3). C. freundii has received increasing attention as an opportunistic pathogen that causes diarrhea, urinary tract infections, and septicemia in humans (4). However, to our knowledge the information on genome sequences of C. freundii isolated from diseased fish was not available. Further, C. freundii has shown a strong ability to developing resistance to antibiotics used in human therapy and aquaculture (5,6). It is therefore imperative to advance our understanding of pathogenic, multidrug-resistant C. freundii strains associated with fish in order to identify antimicrobial resistance and virulence determinants.
Two C. freundii strains, CF04 and A41, were isolated from the kidneys of two moribund giant gourami (Osphronemus goramy) fish showing signs of generalized septicemia collected from aquaria in Kandy, Sri Lanka.
Genomic DNA was extracted with the DNeasy Blood and Tissue kit (Qiagen). Genomic DNA libraries were constructed using the Ion Xpress Plus fragment library kit and sequenced on an Ion Torrent PGM (Life Technologies) platform using the ion 318 chip and the 400-bp kit following standard protocols. A total of 1,328,195 reads equivalent to 340,760,015 bp of data and 1,170,722 reads equivalent to 330,995,850 bp of data were, respectively, generated from C. freundii CF04 and A41 DNA samples. The reads acquired were assembled separately using MIRA version 4.0.2 (7) and improved with CLC Genomics Workbench version 8 by alignment against a reference genome (accession no. CP007557). The qualities of the assemblies were evaluated using QUAST version 3.0 (8).
The assembly of the C. freundii CF04 strain resulted in 58 con-tigs over 1,000 bp (largest contig 1,274,500 bp; N 50 ϭ 679,933 bp) with a total length of 5,138,911 bp and 51.49% GϩC content. The reads of C. freundii A41 were assembled to 153 contigs larger than 1,000 bp (largest contig 548,564 bp; N 50 ϭ 366,314 bp) with a total length of 5,301,254 bp and 51.5% GϩC content. The total size of the draft genomes and GϩC contents are in agreement with published C. freundii genomes (4.9 to 5.0 Mb and 51.6% GϩC) (9-11). Functional annotation using RAST (12) revealed 5,777 and 5,253 coding sequences (CDS) in the draft genomes of CF04 and A41, respectively. The presence of genes involved with virulence, disease and defense, quorum sensing, and biofilm formation as well as genes encoding resistance to different antibiotics confirms the clinical significance of these isolates. Moreover, RAST indicated that both organisms carry genes encoding resistance to antibiotics that include fluoroquinolon, macrolides, beta-lactams, rifampin, chloramphenicol, tetracycline, aminocoumarin, aminoglycoside, polymyxin, and vancomycin. PHAST, a phage search tool (13), predicted six intact phages in CF04 and seven intact phages in A41.
Accession number(s). The draft genome sequences of C. freundii A41 and CF04 have been deposited in DDBJ/EMBL/ GenBank under the accession numbers BDFK01000001 to BDFK01000153 and BDFL01000001 to BDFL01000058, respectively.

FUNDING INFORMATION
This work, including the efforts of Shuichi Asakawa, was funded by Japan Society for the Promotion of Science (JSPS) (24248034). This work, including the efforts of Shuichi Asakawa, was funded by JST | Core Research for Evolutional Science and Technology (CREST).