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1 National Veterinary Institute, Oslo, Norway
2 National Veterinary Institute, Bergen, Norway
Correspondence
J. Mikalsen
jarle.mikalsen{at}vetinst.no
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ABSTRACT |
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Supplementary tables detailing the primers used in this study, the gene sequence similarities and Kimura two-parameter genetic distances between the isolates from Atlantic cod and other members of the genus Francisella and the GenBank accession numbers for the 16S rRNA, groEL, shdA, rpoB, rpoA, pgm and atpA gene sequences reported in this study are available with the online version of this paper.
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Hollis et al., 1989; Sjöstedt, 2005), Francisella novicida, Francisella tularensis and Francisella philomiragia, although evidence for other species exists (Barns et al., 2005; Hsieh et al., 2006; Kay et al., 2006; Nylund et al., 2006). It has been suggested that F. novicida represents a subspecies of F. tularensis (Sjöstedt, 2005), but this reclassification has not been formally proposed and the name ‘Francisella tularensis subsp. novicida’ has not been validly published. In 2005, the National Veterinary Institute (NVI) in Norway isolated a Gram-negative, facultatively intracellular bacterium, genetically and phenotypically consistent with the genus Francisella, from farmed Atlantic cod (Gadus morhua L.) displaying granulomatous disease (Olsen et al., 2006). The isolation procedures employed and the characteristics of the disease have been described previously (Olsen et al., 2006) and strain 2005/50/F292-6CT was deposited in the NCIMB and LMG culture collections (=NCIMB 14265T=LMG 23800T). In this study, seven isolates of an unidentified Francisella species isolated from different disease outbreaks in Atlantic cod along the Norwegian coast were characterized. In addition F. philomiragia strains CCUG 12603, CCUG 13404 and CCUG 19701 from the Culture Collection of the University of Göteborg, Sweden (CCUG) and F. philomiragia ATCC 25015T (=DSM 735T) were investigated.
Phenotypic characterizations were performed following standard protocols combined with enzymic reactions using the commercial kits API ZYM, Rapid ID 32A and Rapid ID 32E (bioMérieux) unless otherwise noted. These tests were performed at both 37 °C and 22 °C, reflecting the optimal growth temperatures of the isolates studied. Tests were read after 4 h and 24 h incubation, respectively.
Primers targeting the groEL, atpA, rpoA and pgm genes were constructed from the draft whole genome sequence of F. philomiragia ATCC 25017 (https://maple.lsd.ornl.gov/microbial) and supplemented by primers previously described for the detection of the shdA gene (Barns et al., 2005) and the variable region of the rpoB gene from Legionella pneumophila (Ko et al., 2002). The 16S rRNA sequences were generated as reported by Olsen et al. (2006), following standard protocols. Primers and PCR programs developed during the present study are presented in Supplementary Table S1 (available with the online version of this paper). Gene fragments were amplified and sequenced in both directions on separate days. DNA–DNA hybridization of Atlantic cod strain 2005/50/F292-6CT with F. philomiragia ATCC 25015T was performed in 2x SSC at 62 °C at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Germany) in duplicate experiments as described by Lopez-Cortes et al. (2006).
Sequences were aligned and compiled using BioEdit software (Hall, 1999). Two matrices were constructed for phylogenetic analyses. The first was concatenated, encompassing the groEL, atpA, pgm, rpoB, shdA and rpoA gene sequences from seven Atlantic cod isolates, five F. philomiragia isolates and five isolates of F. tularensis, including two recognized subspecies and ‘F. tularensis subsp. novicida’. The other matrix covered the majority of the small-subunit rRNA gene (16S rRNA) from a larger number of strains. Both matrices were subjected to maximum-likelihood analysis in PAUP version 4.0b10 (Swofford, 2002) using GTR+I+G and HKY+G models for concatenated and 16S rRNA gene sequence matrices, respectively, as selected by MODELTEST 3.7 software (Posada & Crandall, 1998). Trees were constructed using 10x random addition of the sequences in heuristic searches with tree bisection-reconnection branch swap. To assess the stability of tree branching patterns, bootstrap analyses (100 pseudoreplicates) were performed using evolutionary models and tree building strategy as described above.
Results and Discussion
Phenotypical characterization of established Francisella species is somewhat limited due to their fastidious nature. No reaction was registered on inoculation of the strains isolated from Atlantic cod into phenol red broth base (Difco) supplemented with 1 % (w/v) glucose, ribose, maltose, mannitol, sorbitol, trehalose, sucrose or arabinose, respectively, after incubation for 14 days at 22 °C. Production of 
-lactamase was detected by the Gots test (Gots, 1945), a feature shared by nearly all isolates of the genus Francisella (Sjöstedt, 2005). Using Rapid ID 32A and Rapid ID 32E test kits, all strains isolated from Atlantic cod were positive for the degradation of glucose, mannose, alanine and proline, features shared with the F. philomiragia strains tested (Table 1). However, in contrast to F. philomiragia, maltose, sucrose, leucine, glycine and trehalose were not degraded. The incubation temperature did not affect the outcome of these tests. In common with F. tularensis, and in contrast to F. philomiragia, the novel isolates tested cytochrome oxidase-negative. The novel isolates, in common with F. philomiragia, did not show cross-reactivity toward F. tularensis antiserum (Clarridge et al., 1996; Olsen et al., 2006).
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), no visible growth of the novel strains from Atlantic cod was detected on media without cysteine supplement after 14 days incubation. The isolates from Atlantic cod grew relatively poorly on cysteine heart agar (CHA, Difco); after 6 days incubation at 22 °C, no single colonies could be detected. On addition of 5 % sheep blood (CHAB), single colonies of approximately 1 mm in diameter were observed after 4 days of incubation at 22 °C. To determine the optimal growth temperature for the novel isolates, six different temperatures (4, 10, 15, 22, 30 and 37 °C) were tested. Optimal growth was observed at 22 °C, while only slight growth was detected at 30 °C on CHAB plates. All isolates of the genus Francisella previously recovered from various fish species appear to have upper cardinal temperatures in this range (Ottem et al., 2007; Hsieh et al., 2006). The F. philomiragia isolates grew well at 37 °C on both blood agar and CHAB in clear contrast to the isolates from Atlantic cod. Although Hollis reported three F. philomiragia-like bacteria with optimum growth temperatures of 25 °C (Hollis et al., 1989), these strains were able to grow at 35 °C in contrast to the isolates from Atlantic cod.
BLAST searches (Altschul et al., 1997) of the 16S rRNA gene sequence (1416 bp) of strain 2005/50/F292-6CT from Atlantic cod (GenBank accession number DQ295795) revealed similarities of 99.3 % and 99.2 % with F. philomiragia ATCC 25015T (AY928394) and F. philomiragia ATCC 25017 (AY928395), respectively. The remaining F. philomiragia strains studied had 16S rRNA gene sequences identical to F. philomiragia ATCC 25017. The isolates from Atlantic cod showed highest similarity to ‘F. tularensis subsp. novicida’ (AY968237) with 97.9 % gene sequence similarity. Further, 16S rRNA gene sequence similarities of 100 % (DQ309246, 1416/1416), 99.9 % (AM403242, 1414/1416) and 99.2 % (AF385857, 1404/1416) were identified with Francisella spp. reported from Atlantic cod from Norway, Atlantic salmon (Salmo salar) from Chile and tilapia (Oreochromis sp.) from Asia, respectively (Hsieh et al., 2006; Nylund et al., 2006). A 2 bp insertion was observed in the strains isolated from Atlantic cod compared with those of the F. philomiragia strains and this indel was shared with all Francisella tularensis subspecies and the three aforementioned unidentified Francisella species from fish.
All housekeeping loci were analysed individually and as concatenated sequences. Identical sequences were identified for all seven isolates from Atlantic cod. Sequencing and alignment of genes generated in-frame fragments of 1461 bp, 775 bp, 575 bp, 261 bp, 289 bp and 502 bp from groEL, atpA, pgm, shdA, rpoB and rpoA genes, respectively. Sequences were concatenated in the aforementioned order (total length: 3863 bp). The housekeeping gene sequence alignment was expanded to include homologous gene sequences from ‘F. tularensis subsp. novicida’ U112 (CP000439), F. tularensis subsp. holarctica OSU18 (CP000437), F. tularensis subsp. holarctica LVS (AM233362), F. tularensis subsp. tularensis Schu 4 (AJ49949), F. tularensis subsp. tularensis FCS 198 (AM286280) and F. philomiragia ATCC 25017. The ratio of mean nonsynonymous (dN) to synonymous substitutions (dS) per site (dN : dS ratio) within the selected genes was calculated using START software (Jolley et al., 2001). Housekeeping genes are believed to be either selectively neutral or subject to purifying selection. Thus, the rate of synonymous substitutions (dS) should be equal to or slightly greater than the rate of nonsynonymous substitutions (dN), giving a dN : dS ratio of <1 (Viscidi & Demma, 2003). The dN : dS ratio varied from 0.0071 (rpoB) to 0.0706 (groEL), indicating purifying selection in all loci investigated and demonstrating their suitability for use in phylogenetic analysis. No gaps were observed in the alignments of the housekeeping genes. Sequence similarity values were calculated for individual gene fragments and for a concatenated sequence (see Supplementary Table 2 in IJSEM Online). The groEL gene sequence obtained for the isolates from Atlantic cod shared between 96.6 and 97.3 % similarity with the F. philomiragia strains examined, whereas the atpA gene sequences shared between 96.8 and 97.2 % similarity, pgm gene sequences between 95.0 and 96.3 % similarity, shdA gene sequences between 97.7 and 98.5 % similarity, rpoB gene sequences shared between 97.9 and 99.0 % similarity and the rpoA gene sequences shared between 92.2 and 92.8 %. These levels of average nucleotide identity (ANI), with the exception of rpoA, lie just above the 95 % level suggested by Konstantinidis et al. (2006) to represent separate species and can be compared with similarity values of 98.7–98.9 % for groEL, 98.5–99.2 % for atpA, 95.1–98.4 % for pgm, 97.7–100 % for shdA, 97.7–99.3 % for rpoB and 99–99.8 % for rpoA observed among established F. philomiragia strains. The concatenated sequence obtained for the strains from Atlantic cod shared between 96.1 and 96.7 % similarity with those of F. philomiragia, whereas the concatenated sequences obtained from different F. philomiragia strains shared between 98.3 and 99.2 % similarity with one another.
Phylogenetic analysis confirmed the close relationship between the isolates from Atlantic cod and F. philomiragia (Fig. 1). In the 16S rRNA gene sequence analysis, there was 92 % bootstrap support for the separation of the Francisella species isolated from fish, including the isolates from Atlantic cod, from established F. philomiragia isolates (Fig. 1a). Maximum-likelihood analysis of concatenated housekeeping gene fragments (not including other fish isolates as this information is not available) verified the separation of the isolates from Atlantic cod from F. philomiragia (Fig. 1b), with 100 % bootstrap support. Analyses of individual genes did not identify congruous relationships in every case (data not shown). Nevertheless, concatenation of housekeeping genes has been shown to produce more reliable phylogenetic trees than single gene approaches (Gadagkar et al., 2005). Further, a Kimura two-parameter distance matrix (see Supplementary Table S3 in IJSEM Online) based upon the concatenated sequence was generated in PAUP version 4.0b10 (Swofford, 2002). The mean genetic distance between the isolates from Atlantic cod and F. philomiragia isolates was 0.03766 and was 0.01275 between the F. philomiragia isolates. In comparison, the greatest genetic distance between subspecies of F. tularensis was 0.0136, detected between ‘F. tularensis subsp. novicida’ and F. tularensis subsp. holarctica strain LVS. DNA–DNA reassociation values between strain 2005/50/F292-6CT and F. philomiragia ATCC 25015T gave a mean value of 68 % (duplicate experiments, 65.2+70.8 %), values close to those found by Hollis et al. (1989) when comparing different F. philomiragia isolates (72–83 % at 65 °C). Thus, this novel strain lies in a ‘grey area’ regarding the recommendations of Stackebrandt et al. (2002) in relation to recognition as an independent species.
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). Strains of F. philomiragia have less fastidious metabolic requirements than the isolates from Atlantic cod, suggesting a different ecological niche for the pathogenic fish isolates. All F. philomiragia isolates are able to grow at 35 °C, whereas the isolates from Atlantic cod did not thrive at 30 °C or above. Thus, the novel strains are not expected to cause disease in warm-blooded animals and are therefore not considered to be a zoonotic threat. All isolates belonging to the same 16S rRNA gene clade as the isolates from Atlantic cod are directly linked to diseases in fish (Ottem et al., 2007; Hsieh et al., 2006) and it is likely that this phylogenetic clade represents an ecological separation from previously recognized F. philomiragia strains.
Considering differences in growth requirements, DNA–DNA reassociation values, phylogenetic analysis and the genetic distance of evolutionarily conserved genes, we consider that the isolates from Atlantic cod merit subspecies status within F. philomiragia. The name Francisella philomiragia subsp. noatunensis subsp. nov. is proposed for the isolates from Atlantic cod. According to Rule 40b of the Bacteriological Code, the creation of this subspecies automatically creates the subspecies Francisella philomiragia subsp. philomiragia subsp. nov. Tests for the differentiation of related species are listed in Table 1
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Description of Francisella philomiragia subsp. noatunensis subsp. nov.
Francisella philomiragia subsp. noatunensis [no.at.un.en.sis. N.L. n. noatun (enclosure of ships) was the coastal abode of the Norse god of fisheries and seamanship; L. fem. suffix -ensis suffix meaning ‘belonging to’; N.L. fem. adj. noatunensis belonging to the coast/sea].
Cells are weakly Gram-negative, facultatively intracellular, weakly catalase positive and strictly aerobic coccobacilli. Cells do not grow on blood agar and colonies on CHAB are low convex, whitish, slightly translucent and mucoid in appearance. Single colonies of approximately 1 mm in diameter are observed after 4 days incubation at 22 °C. Growth is observed in the temperature range 10–30 °C, with limited growth in the upper limits of the range and no growth is observed at 37 °C. Optimal growth takes place at 22 °C on CHAB agar. Using the Rapid ID 32A and Rapid ID 32E tests, degradation of glucose and mannose and hydrolysis of proline and alanine is detected. API ZYM tests indicate the presence of alkaline phosphatase, esterase C4, esterase lipase C8, acid phosphatase and naphthol-AS-BI-phosphohydrolase.
The type strain, strain 2005/50/F292-6CT (=NCIMB 14265T=LMG 23800T), was isolated from Atlantic cod.
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REFERENCES |
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-galactosidase, 