A Polyphasic Approach for Phenotypic and Genetic Characterization of the Fastidious Aquatic Pathogen Francisella noatunensis subsp. orientalis

Abstract

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, α-D-glucose, D-mannose, methyl pyruvate, acetic acid, α-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-α-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.

Molecular characterization and pathogenicity of Francisella noatunensis subsp. orientalis isolated from cultured tilapia (Oreochromis sp.) in Taiwan

“…Four housekeeping genes, designated, malate dehydrogenase ( mdh ), DNA‐directed RNA polymerase alpha subunit ( rpo A),triosephosphate‐isomerase alpha subunit ( tpi A) and peptide chain release factor 2 beta subunit ( prf B) were selected for amplification and sequencing. Primers used to amplify the mdh , rpo A, tpi A and prf B genes were identical to those from the previous study (Ramírez‐Paredes et al, ) (Table ). PCR was carried out as followed: initial denaturation at 94°C for 10 min followed by 35 cycles at 95°C for 45 s, 55°C for 45 s, 72°C for 45 s; and a final extension at 72°C for 10 min.…”

“…Tilapia francisellosis is a systemic visceral granulomatous disease that causes high mortality and huge economic losses in tilapia farming worldwide, including in Taiwan (Hsieh, Tung, Tu, Chang, & Tsai, ), USA (Birkbeck, Feist, & Verner‐Jeffreys, ; Colquhoun & Duodu, ; Mauel et al, ; Soto, Baumgartner, Wiles, & Hawke, ), Brazil (Leal, Tavares, & Figueiredo, ), Columbia (Iregui, Vasquez, Rey, & Verjan, ), Costa Rica (Soto, Hawke, Fernandez, & Morales, ), UK (Jeffery, Stone, Feist, & Verner‐Jeffreys, ; Ramírez‐Paredes et al, ; Shahin et al, ), Mexico (Ortega et al, ), China (Lin et al, ) and Thailand (Jantrakajorn & Wongtavatchai, ; Nguyen, Dong, Senapin, Pirarat, & Rodkhum, ). The aetiologic agent of tilapia francisellosis is a Gram‐negative facultative intracellular bacterium belonging to F. noatunensis subsp.…”

Whole cell inactivated autogenous vaccine effectively protects red Nile tilapia (Oreochromis niloticus) against francisellosis via intraperitoneal injection

“…In farmed Nile tilapia ( Oreochromis niloticus L.), outbreaks caused by Francisella noatunensis subsp. orientalis usually occur in intensive culture systems with high stocking densities when water temperature drops below 25°C (Ramírez‐Paredes, Thompson, et al, ; Soto, Abrams, & Revan, ; Soto, Fernandez, & Hawke, ; Soto et al, ). Acute episodes of francisellosis in tilapia are commonly triggered by husbandry handling procedures and result in high levels of morbidity (80%–90%) and mortality (50%–90%) that can affect fish at all production stages (Birkbeck, Feist, & Verner‐Jeffreys, ; Colquhoun & Duodu, ).…”

“…Francisella noatunensis subsp. orientalis is one of the most pathogenic bacteria for tilapia (Klinger‐Bowen et al, ; Pulpipat, Lin, Chen, Wang, & Chen, ; Ramírez‐Paredes, Thompson, et al, ; Soto et al, ) and one of the main challenges that this industry currently faces globally. Nevertheless, despite the relevance of this pathogen, there are still no commercial vaccines available for its prevention and control.…”

Draft Genome Sequence of Francisella noatunensis subsp. orientalis STIR-GUS-F2f7, a Highly Virulent Strain Recovered from Diseased Red Nile Tilapia Farmed in Europe

“…In November 2012, a highly virulent strain of F. noatunensis subsp. orientalis , STIR-GUS-F2f7, was recovered from diseased red Nile tilapia (~10 g/5.5 cm) farmed in a recirculating system in Europe, where mortality rates were around 60% (2). …”