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Complete genome sequences of Francisella noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190: a fish pathogen with genomic clonal behavior.

ABSTRACT: The genus Francisella is composed of Gram-negative, pleomorphic, strictly aerobic and non-motile bacteria, which are capable of infecting a variety of terrestrial and aquatic animals, among which Francisella noatunensis subsp. orientalis stands out as the causative agent of pyogranulomatous and granulomatous infections in fish. Accordingly, F. noatunensis subsp. orientalis is responsible for high mortality rates in freshwater fish, especially Nile Tilapia. In the current study, we present the genome sequences of F. noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190. The genomes include one circular chromosome of 1,859,720 bp, consisting of 32 % GC content, 1538 coded proteins and 363 pseudogenes for FNO12; one circular chromosome of 1,862,322 bp, consisting of 32 % GC content, 1537 coded proteins and 365 pseudogenes for FNO24; and one circular chromosome of 1,859,595 bp, consisting of 32 % GC content, 1539 coded proteins and 362 pseudogenes for FNO190. All genomes have similar genetic content, implicating a clonal-like behavior for this species.

SUBMITTER: Goncalves LA

PROVIDER: S-EPMC4828924 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Complete genome sequences of Francisella noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190: a fish pathogen with genomic clonal behavior.

Gonçalves Lucas Amorim LA de Castro Soares Siomar S Pereira Felipe Luiz FL Dorella Fernanda Alves FA de Carvalho Alex Fiorini AF de Freitas Almeida Gabriel Magno GM Leal Carlos Augusto Gomes CA Azevedo Vasco V Figueiredo Henrique César Pereira HC

Standards in genomic sciences 20160412

The genus Francisella is composed of Gram-negative, pleomorphic, strictly aerobic and non-motile bacteria, which are capable of infecting a variety of terrestrial and aquatic animals, among which Francisella noatunensis subsp. orientalis stands out as the causative agent of pyogranulomatous and granulomatous infections in fish. Accordingly, F. noatunensis subsp. orientalis is responsible for high mortality rates in freshwater fish, especially Nile Tilapia. In the current study, we present the ge ...[more]

PMID: 27073591

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Project description:Francisella noatunensis subsp. orientalis (Fno) is the causative agent of piscine francisellosis, an emerging infectious disease in Asia and Latin America. In this study two outbreaks of francisellosis were diagnosed in the UK on the basis of histopathology, electron microscopy, PCR, bacterial isolation and fulfillment of Koch's postulates. Furthermore, a phenotypic fingerprint based on biochemical analyses, metabolic activity, chemotaxonomic composition, and antimicrobial assays was generated for the novel isolates, the Fno type strain Ehime-1 from Asia and other Fno from Latin America. The genetic relatedness between the novel Fno and other Francisellaceae species was investigated by sequencing and comparing the 16SrRNA gene, 8 housekeeping genes (individually and concatenated) and the 16SrRNA-ITS-23SrRNA sequence. The phenotypic profiling indicated a high degree of similarity among the Fno strains as all were able to metabolize dextrin, N-acetyl-D glucosamine, D-fructose, ?-D-glucose, D-mannose, methyl pyruvate, acetic acid, ?-keto butyric acid, L-alaninamide, L-alanine, L-alanylglycine, L-asparagine, L-glutamic acid, L-proline, L-serine, L-threonine, inosine, uridine, glycerol, D L-?-glycerol phosphate, glucose-1-phosphate, and glucose-6-phosphate. The chemotaxonomic analyses indicated that 24:1 (20.3%), 18:1n-9 (16.9%), 24:0 (13.1%) 14:0 (10.9%), 22:0 (7.8%), 16:0 (7.6%), and 18:0 (5.5%) were the predominant structural fatty acids in Fno. The antimicrobial assays showed little variation between the isolates and high susceptibility to enrofloxacin, gentamicin, neomycin, streptomycin, amikacin, ciprofloxacin, gatifloxacin, nitrofurantoin, tobramycin, kanamycin, tetracycline, oxytetracycline, florfenicol, oxolinic acid, and streptomycin in all the Fno analyzed. In all the phylogenetic trees the Fno strains clustered together in independent branches confirming a high degree of homogeneity. Interestingly in five of the 11 trees i.e., mutS, putA, rpoB, 16SrRNA-ITS-23SrRNA, and concatenated sequence the two Francisella noatunensis ssp. diverged more from each other than from the closely related Francisella philomiragia (Fp). The phenotypic and genetic characterization confirmed the Fno isolates represent a solid phylo-phenetic taxon that in the current context of the genus seems to be misplaced within the species Fn. We propose the use of the present polyphasic approach in future studies to characterize strains of Fnn and Fp and verify their current taxonomic rank of Fno and other aquatic Francisella spp.

Project description:Francisella noatunensis subsp. orientalis (Fno) is the causative agent of piscine francisellosis in warm water fish including tilapia. The disease induces chronic granulomatous inflammation with high morbidity and can result in high mortality. Early and accurate detection of Fno is crucial to set appropriate outbreak control measures in tilapia farms. Laboratory detection of Fno mainly depends on bacterial culture and molecular techniques. Recombinase polymerase amplification (RPA) is a novel isothermal technology that has been widely used for the molecular diagnosis of various infectious diseases. In this study, a recombinase polymerase amplification (RPA) assay for rapid detection of Fno was developed and validated. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in tilapia farms, but also by screening 78 Nile tilapia and 5 water samples. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other tested bacteria. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The RPA gave quicker results in approximately 6 min in contrast to the qPCR that needed about 90 min to reach the same detection limit, taking only 2.7-3 min to determine Fno in clinical samples. Moreover, RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to controlling the infection through prompt on-site detection of Fno.

Project description:BackgroundSince Francisella noatunensis was first isolated from cultured Atlantic cod in 2004, it has emerged as a global fish pathogen causing disease in both warm and cold water species. Outbreaks of francisellosis occur in several important cultured fish species making a correct management of this disease a matter of major importance. Currently there are no vaccines or treatments available. A strain typing system for use in studies of F. noatunensis epizootics would be an important tool for disease management. However, the high genetic similarity within the Francisella spp. makes strain typing difficult, but such typing of the related human pathogen Francisella tullarensis has been performed successfully by targeting loci with higher genetic variation than the traditional signature sequences. These loci are known as Variable Numbers of Tandem Repeat (VNTR). The aim of this study is to identify possible useful VNTRs in the genome of F. noatunensis.ResultsSeven polymorphic VNTR loci were identified in the preliminary genome sequence of F. noatunensis ssp. noatunensis GM2212 isolate. These VNTR-loci were sequenced in F. noatunensis isolates collected from Atlantic cod (Gadus morhua) from Norway (n = 21), Three-line grunt (Parapristipoma trilineatum) from Japan (n = 1), Tilapia (Oreochromis spp.) from Indonesia (n = 3) and Atlantic salmon (Salmo salar) from Chile (n = 1). The Norwegian isolates presented in this study show both nine allelic profiles and clades, and that the majority of the farmed isolates belong in two clades only, while the allelic profiles from wild cod are unique.ConclusionsVNTRs can be used to separate isolates belonging to both subspecies of F. noatunensis. Low allelic diversity in F. noatunensis isolates from outbreaks in cod culture compared to isolates wild cod, indicate that transmission of these isolates may be a result of human activity. The sequence based MLVA system presented in this study should provide a good starting point for further development of a genotyping system that can be used in studies of epizootics and disease management of francisellosis.

Dataset Information

Complete genome sequences of Francisella noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190: a fish pathogen with genomic clonal behavior.

Publications

Complete genome sequences of Francisella noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190: a fish pathogen with genomic clonal behavior.

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