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Front. Microbiol. | doi: 10.3389/fmicb.2017.02324

A polyphasic approach for phenotypic and genetic characterisation of the fastidious aquatic pathogen Francisella noatunensis subsp. orientalis

 Jose Gustavo Ramirez-Paredes1*,  Kimberly D. Thompson2,  Matthijs Metselaar3,  Khalid Shahin1,  Esteban Soto4, Randolph H. Richards5, David J. Penman5, Duncan J. Colquhoun6 and  Alexandra Adams1
  • 1Institute of Aquaculture, University of Stirling, United Kingdom
  • 2Aquaculture Research Group, Moredun Research Institute, United Kingdom
  • 3The Fish Vet Group UK, United Kingdom
  • 4School of Veterinary Medicine, University of California Davis, United States
  • 5Institute of Aquaculture, University of Stirling, United Kingdom
  • 6Section for Bacteriology, Norwegian Veterinary Institute, Norway

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 fulfilment 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 8 housekeeping genes and the 16S rRNA-ITS-23S rRNA sequence. The phenotypic profiling indicated a high degree of similarity between the Fno taxon as all were able to metabolise 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 analysed. In all the phylogenetic trees the Fno strains clustered together in independent branches confirming a high degree of homogeneity. Interestingly in five of the individual trees i.e mutS, putA, rpoB, the concatenated sequence and 16S rRNA-ITS-23S rRNA genes the two Francisella noatunensis ssp. diverged more from each other than from the closely related human pathogen Francisella philomiragia (Fp). The phenotypic and genetic characterisation 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 characterise strains of Fnn and Fp and verify their current taxonomic rank of Fno.

Keywords: Francisellosis in tilapia, Francisella characterisation, tilapia diseases, Francisella noatunensis subsp. orientalis, Fno antimicrobial resitance, Francisellosis diagnosis

Received: 16 Mar 2017; Accepted: 10 Nov 2017.

Edited by:

Hongyue Dang, Xiamen University, China

Reviewed by:

Martin W. Hahn, University of Innsbruck, Austria
Henning Sørum, Norwegian School of Veterinary Science, Norway
Sintia Almeida, University of São Paulo, Brazil  

Copyright: © 2017 Ramirez-Paredes, Thompson, Metselaar, Shahin, Soto, Richards, Penman, Colquhoun and Adams. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: DVM, PhD. Jose Gustavo Ramirez-Paredes, University of Stirling, Institute of Aquaculture, Institute of Aquaculture, Pathfoot Building, University of Stirling, Stirling, FK9 4LA, Scotland, United Kingdom,