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

Abstract

<i>Francisella noatunensis</i> subsp. <i>orientalis</i> (<i>Fno</i>) 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 <i>Fno</i> type strain Ehime-1 from Asia and other <i>Fno</i> from Latin America. The genetic relatedness between the novel <i>Fno</i> and other <i>Francisellaceae</i> 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 <i>Fno</i> 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 <i>Fno</i>. 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 <i>Fno</i> analyzed. In all the phylogenetic trees the <i>Fno</i> strains clustered together in independent branches confirming a high degree of homogeneity. Interestingly in five of the 11 trees i.e., <i>mutS, putA, rpoB</i>, 16SrRNA-ITS-23SrRNA, and concatenated sequence the two <i>Francisella noatunensis</i> ssp. diverged more from each other than from the closely related <i>Francisella philomiragia</i> (<i>Fp</i>). The phenotypic and genetic characterization confirmed the <i>Fno</i> isolates represent a solid phylo-phenetic taxon that in the current context of the genus seems to be misplaced within the species <i>Fn</i>. We propose the use of the present polyphasic approach in future studies to characterize strains of <i>Fnn</i> and <i>Fp</i> and verify their current taxonomic rank of <i>Fno</i> and other aquatic <i>Francisella</i> spp.