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Characteristics of epidemic and sporadic Flavobacterium psychrophilum sequence types
Characteristics of epidemic and sporadic Flavobacterium psychrophilum sequence types Krister Sundell, Tom Wiklund PII: DOI: Reference:
S0044-8486(15)00081-2 doi: 10.1016/j.aquaculture.2015.02.010 AQUA 631548
To appear in:
Aquaculture
Received date: Revised date: Accepted date:
26 January 2015 5 February 2015 7 February 2015
Please cite this article as: Sundell, Krister, Wiklund, Tom, Characteristics of epidemic and sporadic Flavobacterium psychrophilum sequence types, Aquaculture (2015), doi: 10.1016/j.aquaculture.2015.02.010
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ACCEPTED MANUSCRIPT Characteristics of epidemic and sporadic Flavobacterium
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psychrophilum sequence types
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Krister Sundell* and Tom Wiklund
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Laboratory of Aquatic Pathobiology, Environmental and Marine Biology, Department of
*
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Correspondence: Krister Sundell
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Biosciences, Åbo Akademi University, Tykistökatu 6, 20520 Turku, Finland
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Email: [email protected] Telephone: +358-2-2154634
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Fax: +358-2-2154748
Keywords: Flavobacterium psychrophilum, multilocus sequence typing, sequence type, epidemic, sporadic
Submitted to: Aquaculture
ACCEPTED MANUSCRIPT Abstract Population genetic analysis of Flavobacterium psychrophilum using multilocus sequence typing
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(MLST) data has identified a genetic lineage consisting of sequence types (STs) associated with
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epidemic spread and high mortality in farmed rainbow trout (Oncorhynchus mykiss). To identify
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phenotypic characteristics that discriminate epidemic F. psychrophilum STs from sporadically occurring STs, a set of traits of isolates/STs (n=32) with distinct genetic background and clinical
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impact was studied in vitro. The results showed that epidemic and sporadic STs could not be discriminated by their resistance to povidone-iodine or iron starvation or by their ability to hydrolyze
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elastin and gelatin. Antimicrobial resistance against flumequine, oxolinic acid and oxytetracycline was significantly more prevalent (p<0.05), although not a prerequisite, in epidemic STs. The two
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groups showed equal biofilm forming abilities, while the epidemic STs were associated with higher
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adherence (p<0.05) indicating that adhesion and antimicrobial resistance could be factors contributing to the epidemic potential of F. psychrophilum. All isolates/STs of the largest clonal
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complex CC-ST2 were distinguished from others by the bacterial insertion sequence IS256 in the collagenase encoding gene. IS256 might thus constitute a novel molecular marker for rapid
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differentiation of epidemiologically important CC-ST2 genotypes.
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ACCEPTED MANUSCRIPT 1. Introduction Since a standardized multilocus sequence typing (MLST) scheme based on the sequences of seven
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loci was released for the fish pathogen Flavobacterium psychrophilum (Nicolas et al., 2008), several
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studies have implemented the method for genetic typing of isolates from diverse origins (Siekoula-
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Nguedia et al., 2012; Apablaza et al., 2013; Fujiwara-Nagata et al., 2013; Strepparava et al., 2013; Avendaño-Herrera et al., 2014; Nilsen et al., 2014). A web-accessible MLST database (http://pubmlst.org/fpsychrophilum/) has also been established which allows comparable typing
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between laboratories. Population genetic studies of F. psychrophilum using MLST data have
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unambiguously supported the hypothesis of an epidemic population structure where successful clonal complexes (CCs) have expanded within a generally recombinant bacterial population (Nicolas et al.,
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2008; Siekoula-Nguedia et al., 2012; Nilsen et al., 2014).
Common to previously published MLST studies of F. psychrophilum is the identification of a large
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globally spread CC, hereafter referred to as CC-ST2 according to its currently predicted ancestral sequence type (ST), with an apparent affinity to the rainbow trout (Oncorhynchus mykiss) host. STs
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within CC-ST2 have been responsible for severe bacterial cold water disease (BCWD) outbreaks in many European countries for several consecutive years (Siekoula-Nguedia et al., 2012; Strepparava et al., 2013; Nilsen et al., 2014). CCs genetically distinct from CC-ST2 have been associated with repeated disease outbreaks in Atlantic salmon (Salmo salar) in Norway (Nilsen et al., 2014) and in ayu (Plecoglossus altivelis altivelis) in Japan (Fujiwara-Nagata et al., 2013), suggesting either geographical restriction or host adaptation within certain clonal groups. More sporadically occurring F. psychrophilum STs appearing as singletons or minor CCs are usually isolated from a broader host range and are considered to be of a lesser clinical impact (Siekoula-Nguedia et al., 2012; Nilsen et al., 2014). The reason for the epidemiological success of the putatively virulent CC-ST2 is not known, but its worldwide distribution has been associated with the international trade of fish and
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ACCEPTED MANUSCRIPT associated products (Nicolas et al., 2008; Fujiwara-Nagata et al., 2013; Avendaño-Herrera et al., 2014; Nilsen et al., 2014). Evidence has linked the rapid increase in F. psychrophilum infections in
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Chile and Norway to a recent introduction of epidemic CC-ST2 genotypes, particularly to ST2
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important genotypes can become a long term endemic problem.
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(Avendaño-Herrera et al., 2014; Nilsen et al., 2014). Once established, these epidemiologically
The global distribution and dominance of CC-ST2 might be due to changes in properties determining
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the survival of F. psychrophilum in the aquatic environment or in the rainbow trout host. In
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aquaculture environments, where antimicrobials are commonly used, inherent resistance or an enhanced ability to form biofilms (Álvarez et al., 2006; Sundell and Wiklund, 2011) could provide a
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selective advantage over susceptible F. psychrophilum populations. In host-pathogen interactions,
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the ability to obtain iron (Álvarez et al., 2008) and to adhere to (Nematollahi et al., 2003) and degrade host tissue (Madsen and Dalsgaard, 1998; Duchaud et al., 2007) could be crucial factors for
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the outcome of BCWD.
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The main objective of this study was to identify discriminative phenotypic characteristics for F. psychrophilum STs of the geographically most widely spread genetic lineage. For this purpose, we compared the ability of F. psychrophilum STs with distinct genetic background and clinical impact to resist antimicrobial agents and iron starvation, to adhere and to form biofilms and to hydrolyze elastin and gelatin. In addition, we studied the potential of the bacterial insertion sequence IS256 as a molecular marker for rapid differentiation of epidemiologically important F. psychrophilum genotypes.
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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. eBURST analysis of the global F. psychrophilum MLST database
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The complete allelic profile list from the F. psychrophilum MLST database
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(http://pubmlst.org/fpsychrophilum/) was downloaded (accessed on 28 March 2014) and a
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population snapshot of the 995 isolates and 166 STs was created using the eBURSTv3 algorithm (Fig. 1). CCs were identified based on sharing 5 (relaxed) or 6 (default) out of 7 alleles using
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standard eBURST methodology (http://eburst.mlst.net/).
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2.2. Selection of bacterial isolates and culture conditions
Thirty-two F. psychrophilum isolates representing 16 epidemic and 16 sporadic STs (Table 1) were
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selected from the MLST database based on eBURST grouping (Fig. 1). Epidemic STs were
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genetically associated with the globally spread CC-ST2 (Fig. 1) and were considered part of the same lineage whereas sporadic STs consisted of singletons and minor CCs genetically distinct from CC-
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ST2. The colony morphology of F. psychrophilum is either rough or smooth (Högfors-Rönnholm and Wiklund, 2010) when grown on tryptone yeast extract salts (TYES) (Holt et al., 1993) agar. The
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rough and smooth F. psychrophilum morphotypes have been shown to express different phenotypic characteristics in vitro (Högfors-Rönnholm 2014). Hence, only the smooth morphotype was selected because of its higher prevalence within BCWD outbreaks (Sundell et al., 2013). Although none of several screened ST2 isolates showed typical smooth morphological characteristics, this genotype was included in the study because of its significance and predominance in the global MLST database. All isolates were grown on TYES agar for 5 days at 15°C prior to testing. An approximate 109 CFU mL-1 stock culture of each F. psychrophilum isolate was prepared by inoculating 5-day-old colonies from TYES agar into TYES broth to an optical density of 1.0 at 520 nm (Unicam, Heλios β) and maintained at -70°C. Dilutions from the stock cultures were used in all analyses unless otherwise stated.
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ACCEPTED MANUSCRIPT 2.3. Minimum inhibitory concentration (MIC) testing Antimicrobial resistance of the isolates was tested by measuring the MIC of the aquaculture drugs
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flumequine (FLU), oxolinic acid (OXA) and oxytetracycline (OTC) by broth microdilution in round
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bottomed 96-well microtiter plates (Nunc). FLU and OTC were obtained from Sigma-Aldrich and
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OXA from Santa Cruz Biotechnology Inc. In addition we tested the MIC of Betadine® (Leiras Takeda), a povidone-iodine based chemical used for fish egg surface disinfection. The MIC of the iron chelator 2,2′-Dipyridyl (DPD), obtained from Sigma-Aldrich, was used for studying the
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capability of epidemic and sporadic F. psychrophilum STs to resist iron starvation.
Antimicrobial stock solutions (1000 µg mL-1) of FLU, OXA and OTC were prepared by dissolving in 0.1 M NaOH, 1 M NaOH and methanol respectively and making up to volume with TYES broth.
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Approximately 105 CFU of each isolate were exposed to triplicate two-fold serial dilutions of FLU, OTC and OXA ranging from 64 to 0.125 µg mL-1. The concentrations of Betadine® and DPD diluted
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in TYES broth were 1000, 800, 600, 400, 200, 100 µg mL-1 and 31.2, 27.3, 23.4, 19.5, 15.6, 11.7, 7.8, 3.9 µg mL-1, respectively. TYES broth was used as negative control for each compound. After
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static incubation for 96 h at 15°C, the MIC of the antimicrobial was interpreted as the lowest concentration that prevented visible growth. Because of the slow bacterial growth in iron restricted media the MIC of DPD was determined after 10 days of incubation. To separate fully susceptible wild-type isolates from those with a decreased susceptibility, microbiological criteria were used to define resistance (EUCAST, 2000; Kahlmeter et al., 2003). A tentative wild-type cut-off value (COWT) was visually assessed from the MIC distribution as the modal MIC ± 1 twofold dilution (the inherent variability of the test). An isolate was regarded as being resistant to a specific antimicrobial when the MIC was above the wild-type distribution.
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ACCEPTED MANUSCRIPT 2.4. In vitro biofilm formation assay The ability to form biofilms was evaluated by the micro titer dish biofilm formation assay described
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by O´Toole (2011), modified for the growth requirements of F. psychrophilum. Briefly, 100 µl
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TYES broth containing 105 CFU of F. psychrophilum was added in triplicate into wells of untreated
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flat-bottomed 96-well microtiter plates (Nunc) and incubated statically for 7 days at 15°C to allow biofilm formation. Following incubation, the plates were rinsed by submersion in a container of tap water to remove non-adherent cells. A 125 μL volume of a 0.1% crystal violet (CV) solution was
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then added to each well and incubated at room temperature for 45 min. After discarding the contents,
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the plates were rinsed three times as previously with water and air dried. Then, 150 μL of 96% ethanol was added to each well and incubated at room temperature for 15 min. A 100 μL volume of
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the solubilized CV was transferred to a flat bottomed microtiter plate and the absorbance was
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quantified in a microplate reader (Victor2, Wallac) at 595 nm using 96% ethanol as the blank. The test was repeated four times and the mean absorbance of the replicates was used to compare the
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biofilm forming ability between the epidemic and the sporadic group.
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2.5. Adhesion to mucus coated and uncoated polystyrene Adhesion to polystyrene, both uncoated and coated with rainbow trout mucus, was examined in flatbottomed 96-well microtiter plates (Nunclon ∆ Surface, Nunc) according to the method described by Högfors-Rönnholm et al. (2014). Briefly, mucus coated wells were prepared by adding 50 μL of a sterile filtered (0.22 μm) rainbow trout mucus suspension with a protein concentration of 0.5 mg mL−1 (BCA Protein Assay Kit, Pierce). After incubation over night the wells were washed twice with 0.5% NaCl and air dried. F. psychrophilum cells grown on TYES agar were suspended in sterile lake water and 100 μL of this suspension containing approximately 108 CFU was added to both uncoated and mucus coated wells while sterile lake water was used as a negative control. After static incubation at 15 °C for 1 h and washing of the wells, the adhesion ability of the bacterial cells
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ACCEPTED MANUSCRIPT was quantified following the staining and measuring procedures described above. Each isolate was
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examined in triplicate and the experiment was repeated three times.
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2.6. Elastin and gelatin degradation
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The ability to hydrolyze elastin and gelatin was studied by streaking each isolate on TYES agar supplemented with 0.1 % and 3 % (w/v) of elastin and gelatin (Sigma) respectively. After incubation at 15°C for 5 days, a positive reaction was indicated by a clear zone in the surrounding turbid agar
2.7. Bacterial insertion sequence IS256
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medium around the inoculum.
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The presence of the bacterial insertion sequence IS256 in each F. psychrophilum isolate and the
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integrity of the collagenase-encoding gene (Duchaud et al., 2007) were studied by PCR in a thermal cycler (ArktikTM, Finnzymes). The DNA template was prepared by suspending a F. psychrophilum
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colony from a pure culture in a 50 µl volume of double-distilled water (ddH2O). Each reaction contained 1×Phire® reaction buffer (Thermo Scientific), 0.5 μM of the primers coll_fw
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AAATTGCTGCCGATGCTTGG and coll_rev CATATCCTGTTTTACCATCCCA (Duchaud et al., 2007), 200 μM of each dNTP, 0.4 μl Phire® HotStart II DNA polymerase (Thermo Scientific), 1 μl DNA template and ddH2O to a total volume of 20 μl. Samples were initially denatured for 30 s at 98°C, followed by 30 amplification cycles including denaturation for 5 s at 98°C, annealing of primers for 5 s at 60°C, and extension for 30 s at 72°C. After the last cycle, the PCR mixture was incubated at 72°C for 60 s. The amplicons were electrophoresed (4 V cm-1, 30 min) on a 1% agaroseTris-borate-EDTA gel stained with ethidium bromide and detected with ultraviolet transillumination. The presence or absence of IS256 was indicated by a 2418 or 1046 bp amplicon respectively (Duchaud et al., 2007).
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ACCEPTED MANUSCRIPT 2.8. Statistical analyses Statistical analyses were performed using the GraphPad Prism5 software. An unpaired t-test with
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Welch’s correction for groups with unequal variances was used to determine significant (p<0.05)
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differences between the epidemic and the sporadic groups. When appropriate, values from the
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Fisher’s exact test were used to compare the prevalence of characteristics between the two groups.
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3. Results 3.1. eBURST
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The global distribution of F. psychrophilum STs (Fig. 1) corresponds to a dominant epidemic lineage including the clonal complexes CC-ST2, CC-ST21, CC-ST124 and CC-ST125 together with four
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singleton STs against a background of smaller CCs and singletons.
3.2. MICs
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The MIC of Betadine® was 400 µg mL-1 for all isolates. Of the other aquaculture compounds, FLU had a broader MIC range (0.25–64 µg mL-1) compared to OTC and OXA (1–32 µg mL-1). The
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estimated COWT was ≤ 2 µg mL-1 for OTC and OXA, ≤ 1 µg mL-1 and ≤ 15.6 µg mL-1 for FLU and DPD respectively. Based on the COWT half of the epidemic, but only one (6%) of the sporadic isolates was resistant to the two quinolones (Table 1). All except one epidemic isolate (94%), but only two of the sporadic ones (13%) showed a reduced susceptibility to OTC. Only one isolate had a MIC value above the COWT for DPD. Acquired resistance indicated by the decreased susceptibility against FLU, OTC and OXA (Table 2) was more prevalent in the epidemic group compared to the sporadic group (Fisher’s test, p<0.05).
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ACCEPTED MANUSCRIPT 3.3. In vitro adhesion and biofilm formation assays The epidemic and sporadic F. psychrophilum isolates showed equal biofilm forming abilities in vitro
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(p=0.745) (Table 2). Although all isolates were able to adhere, the adhesion capacity was
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significantly higher in the epidemic group both on an uncoated (p=0.039) and on a rainbow trout
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mucus coated polystyrene surface (p=0.027).
3.4. Presence of IS256
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IS256 was present in half of the epidemic, but in none of the sporadic isolates. Interestingly, all
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isolates that contained IS256 belonged to CC-ST2, shared 4 or more MLST alleles and had a decreased susceptibility against oxytetracycline (Table 1). The Fisher’s exact test showed that IS256
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was significantly more prevalent in the epidemic group compared to the sporadic (p=0.002) (Table
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2). When the test was repeated by treating CC-ST2 (n=8) and the other isolates (n=24) as two
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separate groups the p-value dropped to <0.0001.
3.5. In vitro elastin and gelatin hydrolysis
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Half of both the epidemic and the sporadic isolates were able to hydrolyze elastin, while all isolates were able to hydrolyze gelatin (Table 1 and 2). Six out of the eight isolates belonging to CC-ST2 were elastinase producing. All isolates from CC-ST125 were unable to degrade elastin.
4. Discussion The dedicated F. psychrophilum MLST database (http://pubmlst.org/fpsychrophilum/) allows for global epidemiological surveillance and identification of STs associated with disease outbreaks in particular fish species. Still, MLST is laborious and expensive and requires multiple PCRs and sequence analyses before generating a typing result useful for disease outbreak investigation and control. Before the F. psychrophilum MLST database was established, the work of Duchaud et al.
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ACCEPTED MANUSCRIPT (2007) showed the presence of IS256 in a disrupted collagenase encoding gene of F. psychrophilum in several putatively virulent rainbow trout isolates originating from different parts of the world.
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According to the published MLST data available today the isolates harboring IS256 belonged to CC-
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ST2, corroborating their hypothesis of a clonal dissemination of virulent F. psychrophilum strains
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containing the IS256 disrupted gene. Our study showed that the IS256 disrupted collagenase encoding gene was limited to CC-ST2 indicating that the STs in the genetically related CC-ST21, CC-ST124 and CC-ST125 are not clonal descendants of ST2. Nevertheless, our study implies that
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IS256 could serve as a molecular marker for rapid differentiation of epidemiologically important
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CC-ST2 genotypes. While it has been suggested that IS256 can contribute to virulence by influencing phenotypic variation, antimicrobial resistance and niche adaptation of bacterial
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pathogens (Conlon et al., 2004; Kozitskaya et al., 2004; Gu et al., 2005), the consequences of its
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integration in the collagenase encoding gene and/or other parts of the genome of F. psychrophilum is not yet understood. Hence a more detailed comparative genomic analysis of distinct F.
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psychrophilum STs is necessary.
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Because bacterial attachment is a prerequisite for colonization of surfaces or host cells, the adhesion of epidemic and sporadic isolates was compared. The higher adherence of epidemic F. psychrophilum STs to inert and mucus coated surfaces might allow for a more efficient colonization of fish, tank and equipment surfaces in aquaculture environments. The higher ability to adhere to rainbow trout mucus could also be indicative of specific host-adaptation mechanisms in epidemic STs. Although our findings do not allow us to make any associations between adherence and virulence, a positive correlation between F. psychrophilum adhesion to gill arches of rainbow trout and virulence has previously been reported (Nematollahi et al., 2003). It is also known that the genome of the virulent F. psychrophilum isolate JIP02/86 contains several (n=27) genes encoding proteins related to adhesion (Duchaud et al., 2007). Thus, our findings provide a compelling
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ACCEPTED MANUSCRIPT argument for continuing research into genetic factors underlying specific mechanisms of adhesion, since the higher adherence of specific F. psychrophilum lineages could be a factor contributing to the
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potential to cause epidemic BCWD outbreaks.
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Biofilm formation is known to improve F. psychrophilum survival during antimicrobial exposure (Sundell and Wiklund, 2011), but the association between biofilm formation and genetic background has not been studied before. Our analysis did not detect any differences in the biofilm forming ability
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between the epidemic and the sporadic group, suggesting that biofilm formation as a trait is not any
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more important in epidemic F. psychrophilum lineages. However, as the in vitro culture environment is artificial it does not necessarily represent in vivo conditions; thus biofilm formation cannot be
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excluded as an important virulence determinant of F. psychrophilum. Another example of this relates
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to iron availability, which is essential for pathogen survival and growth in the low-iron environment of the fish host where iron is limited due to host production of iron-binding proteins (Otto et al.,
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1992). Even though our results did not show any differences in resistance to iron starvation between the two groups, specific iron uptake mechanisms could still be important virulence factors in F.
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psychrophilum.
The proteolytic activity of F. psychrophilum has been studied as a potential discriminatory marker for pathogenicity with inconclusive results (Bertolini et al., 1994; Faruk 2000; Madetoja et al., 2002). Previous studies with experimentally injected rainbow trout have suggested that the production of elastinase and gelatinase (Madsen and Dalsgaard 1998; Ostland et al., 2000) may be important for the pathogenicity of F. psychrophilum. Our results showed that epidemic and sporadic STs could not be distinguished from each other by their ability to hydrolyze gelatin or elastin. All isolates were gelatin-producing and some of the elastinase-negative F. psychrophilum isolates represented STs (CC-ST125 and ST92) previously associated with high virulence in rainbow trout (Nilsen et al.,
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ACCEPTED MANUSCRIPT 2014). Our results indicated that the ability of F. psychrophilum to hydrolyze elastin or gelatin in
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vitro does not correlate with propensity to cause BCWD.
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Resistance to aquaculture drugs particularly to quinolones has been repeatedly reported from F.
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psychrophilum outbreaks in different parts of the world (Izumi and Aranishi 2004; Henríquez-Nuñez et al., 2012; Shah et al., 2012; Sundell et al., 2013). Quinolone resistant F. psychrophilum genotypes associated with disease in juvenile rainbow trout have also been isolated from eggs (Sundell et al.,
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2013). In routine hatchery practices, povidone-iodine is used at a concentration of 100 µg mL-1
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which has proven ineffective for complete disinfection of eggs (Wagner et al., 2012). In this study, growth for each F. psychrophilum isolate was observed in nutrient broth containing povidone-iodine
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at twice the concentration used in hatcheries, indicating that currently used egg disinfection strategies
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are both inefficient for elimination of F. psychrophilum and that resistant genotypes could efficiently
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be spread through trade of contaminated eggs.
At present, there are no internationally accepted criteria to be applied for establishing COWT values
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(Silley, 2012), but evaluation of the MIC distributions (Kahlmeter et al., 2003) is widely adopted for detection of bacterial isolates with reduced susceptibility. In our study, a division in isolate susceptibility to FLU, OTC and OXA was characterized by the bimodal distribution of MIC values, with a minimum gap of one two-fold dilution between susceptible and resistant STs. We found a significantly higher prevalence of resistance against the quinolones FLU and OXA and the tetracycline OTC among epidemic STs (Table 1 and 2). However, susceptible wild-type isolates were also present in the epidemic group and vice versa (Table 1 and 2), indicating that antimicrobial resistance is not a prerequisite for epidemic STs. Still, our results indicate that antimicrobial resistance is strongly associated with epidemic F. psychrophilum STs and may therefore constitute one of the main explanations for their epidemiological success and dominance in aquaculture
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ACCEPTED MANUSCRIPT environments. Whether this association could be a result of co-selection of resistance and virulence, a high transmissibility of epidemic STs with intrinsic resistance or of a more efficient acquisition and
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expression of resistance determinants remains to be elucidated.
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In conclusion, our study showed that STs of an epidemic lineage were discriminated from sporadically occurring STs by a higher ability to adhere and to resist antimicrobial agents commonly used in aquaculture. The results suggest that specific adhesion mechanisms and antimicrobial
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resistance may be key factors contributing to the success of epidemic F. psychrophilum clones in fish
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farm environments. To identify and limit the spread of epidemiologically important F. psychrophilum genotypes, IS256 could serve as a rapid molecular marker for differentiation of CC-
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ST2 genotypes. The knowledge gained from this work provides further research direction for
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discovering the underlying genetic mechanisms contributing to the emergence and characteristics of
Acknowledgements
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epidemic F. psychrophilum lineages.
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This publication made use of the F. psychrophilum MLST database developed by Keith Jolley at the University of Oxford. Funding was provided by the Finnish Ministry of Agriculture and Forestry as part of the EU EMIDA ERA-NET project “Control Flavobacteriaceae infections in European fish farms” (2114/311/210). The authors wish to thank all partners of the consortium. We are grateful to J-F. Bernardet, I. Dalsgaard, E. Duchaud and H. Nilsen for kindly providing isolates.
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Kahlmeter, G., Brown, D.F., Goldstein, F.W., MacGowan, A.P., Mouton, J.W., Osterlund, A., Rodloff, A., Steinbakk, M., Urbaskova, P., Vatopoulos, A., 2003. European harmonization of MIC breakpoints for antimicrobial susceptibility testing of bacteria. J. Antim. Chem. 52, 145–148.
Kozitskaya, S., Cho, S.H., Dietrich, K., Marre, R., Naber, K., Ziebuhr, W., 2004. The bacterial insertion sequence element IS256 occurs preferentially in nosocomial Staphylococcus epidermidis isolates: association with biofilm formation and resistance to aminoglycosides. Inf. Imm. 72, 1210– 1215.
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ACCEPTED MANUSCRIPT Madetoja, J., Dalsgaard, I., Wiklund, T., 2002. Occurrence of Flavobacterium psychrophilum in fish-
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farming environments. Dis. Aquat. Org. 52, 109−118.
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Madsen, L., Dalsgaard, I., 1998. Characterization of Flavobacterium psychrophilum; comparison of
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proteolytic activity and virulence of strains isolated from rainbow trout (Oncorhynchus mykiss). In A. C. Barnes, G. A. Davidson, M. P. Hiney, & D. McIntosh (Eds.), Methodology in Fish Diseases
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Research. (pp. 45–52). Aberdeen: Fisheries Research Services.
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Nematollahi, A., Decostere, A., Pasmans, F., Ducatelle, R., Haesebrouck, F., 2003. Adhesion of high and low virulence Flavobacterium psychrophilum strains to isolated gill arches of rainbow
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trout Oncorhynchus mykiss. Dis. Aquat. Org. 55, 101–107.
Nicolas, P., Mondot, S., Achaz, G., Bouchenot, C., Bernardet, J.F., Duchaud, E., 2008. Population
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74, 3702–3709.
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structure of the fish-pathogenic bacterium Flavobacterium psychrophilum. Appl. Environ. Microbiol.
Nilsen, H., Sundell, K., Duchaud, E., Nicolas, P., Dalsgaard, I., Madsen, L., Aspán, A., Jansson, E., Colquhoun, D.J., Wiklund, T., 2014. Multilocus sequence typing identifies epidemic clones of Flavobacterium psychrophilum in Nordic countries. Appl. Environ. Microbiol. 80, 2728–2736.
Ostland, V.E., Byrne, P.J., Hoover, G., Ferguson, H.W., 2000. Necrotic myositis of rainbow trout, Oncorhynchus mykiss (Walbaum): proteolytic characteristics of a crude extracellular preparation from Flavobacterium psychrophilum. J. Fish Dis. 23, 329–336.
O'Toole, G.A., 2011. Microtiter dish biofilm formation assay. J. Vis. Exp. 47, 2437.
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ACCEPTED MANUSCRIPT Otto, B.R., Verweij-van Vught, A.M., MacLaren, D.M., 1992. Transferrins and heme-compounds as
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iron sources for pathogenic bacteria. Crit. Rev. Microbiol. 18, 217–233.
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Shah, S.Q.A., Nilsen, H., Bottolfsen, K., Colquhoun, D.J., Sørum, H., 2012. DNA gyrase and
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topoisomerase IV mutations in quinolone-resistant Flavobacterium psychrophilum isolated from diseased salmonids in Norway. Microb. Drug. Res. 18, 207–214.
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Siekoula-Nguedia, C., Blanc, G., Duchaud, E., Calvez, S., 2012. Genetic diversity of Flavobacterium
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psychrophilum isolated from rainbow trout in France: predominance of a clonal complex. Vet.
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Microbiol. 161, 169–178.
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Silley, P., 2012. Susceptibility testing methods, resistance and breakpoints: what do these terms
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really mean? Rev. Sci. Tech. 31, 33–41.
Strepparava, N., Nicolas, P., Wahli, T., Segner, H., Petrini, O., 2013. Molecular epidemiology of
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Flavobacterium psychrophilum from Swiss fish farms. Dis. Aquat. Org. 105, 203–210.
Sundell, K., Heinikainen, S., Wiklund, T., 2013. Structure of Flavobacterium psychrophilum populations infecting farmed rainbow trout (Oncorhynchus mykiss). Dis. Aquat. Org. 103, 111–119.
Sundell, K., Wiklund, T., 2011. Effect of biofilm formation on antimicrobial tolerance of Flavobacterium psychrophilum. J. Fish Dis. 34, 373–383. Wagner, E., Oplinger, R., Bartley, M., 2012. Penicillin-G: Efficacy against Flavobacterium psychrophilum and evaluation of lethal dose limits for rainbow trout. OJAS 2, 150–158.
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Table and figure legends
Table 1. Origin, source and in vitro properties of epidemic and sporadic F. psychrophilum isolates/STs selected for the study.
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Table 2. In vitro activities of epidemic versus sporadic F. psychrophilum isolates/STs.
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Figure 1. Population snapshot of the F. psychrophilum MLST database with default eBURSTv3
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settings (accessed on 28 March 2014). Each dot represents an individual ST and dot size reflects its abundance. Linked clusters represent CCs, singleton STs are unlinked. Clonal founders are shown in blue, subgroup founders in yellow. CCs and STs within the shaded area are linked with the use of a
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relaxed group definition. Epidemic STs selected for this study are indicated with red numbers,
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sporadic with green respectively.
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Figure 1
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ACCEPTED MANUSCRIPT Table 1
Sporadic
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Source ____________________ Host Country Year RT DEN 1994 RT FIN 1999 RT FIN 1996 RT FRA 1986 AS SCO 2007 RT FIN 1996 RT FIN 2000 RT FIN 2009 RT FIN 2010 RT FIN 2011 RT FIN 2007 RT FIN 2011 RT FIN 2003 RT FIN 2008 RT FIN 2000 RT FIN 2000 T FRA 1992 W FIN 2000 P FIN 2006 RT FIN 1994 RT FIN 1983 P FIN 2006 W FIN 2000 AS FIN 1999 AS NOR 2009 AS NOR 2012 RT DEN 2006 Flo DEN 2000
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MLST data ____________________________________ ST CC Allelic profile ST2 CC-ST2 2, 2, 2, 2, 2, 2, 2 ST10 CC-ST2 2, 2, 2, 8, 2, 2, 2 ST12 CC-ST2 2, 2, 2, 8, 2, 2, 7 ST20 CC-ST2 2, 2, 2, 8, 2, 8, 2 ST21 CC-ST21 10, 2, 2, 2, 2, 2, 7 ST73 CC-ST124 1, 15, 14, 13, 1, 8, 7 ST79 CC-ST2 2, 8, 2, 8, 2, 2, 2 ST91 CC-ST2 2, 2, 2, 2, 2, 2, 41 ST92 CC-ST2 2, 2, 2, 2, 2, 3, 41 ST124 CC-ST124 1, 15, 14, 13, 1, 2, 7 ST125 CC-ST125 2, 15, 2, 13, 2, 1, 7 ST126 CC-ST124 1, 15, 16, 13, 1, 2, 7 ST128 CC-ST2 2, 2, 2, 8, 2, 2, 48 ST129 CC-ST124 1, 2, 14, 13, 1, 2, 7 ST131 CC-ST125 2, 15, 2, 1, 2, 1, 7 ST135 CC-ST125 2, 15, 2, 13, 2, 1, 3 ST18 CC-ST8 6, 9, 3, 6, 5, 4, 6 ST23 Singleton 12, 11, 7, 13, 9, 9, 13 ST34 CC-ST34 18, 15, 3, 20, 16, 1, 6 ST36 Singleton 20, 8, 10, 22, 6, 13, 20 ST122 Singleton 14, 12, 10, 48, 10, 10, 47 ST127 Singleton 18, 8, 2, 37, 36, 8, 47 ST132 Singleton 3, 2, 11, 49, 3, 11, 3 ST141 Singleton 42, 8, 7, 23, 38, 21, 51 ST169 Singleton 1, 15, 1, 3, 1, 8, 1 ST171 Singleton 43, 8, 17, 51, 33, 7, 17 ST180 Singleton 44, 8, 7, 21, 28, 35, 19 ST195 Singleton 3, 22, 3, 54, 40, 18, 3
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Isolate ____ Code DK008 FI152 FI055 JIP02/86 FI166 FI155 FI140 FI091 FI001 FI024 FI049 FI058 FI095 FI104 FI132 FI161 LVDJXP189 FI147 FI071 FI181 FI016 FI074 FI146 FI198 NO061 NO111 DK061 DK098
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Epidemic
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Origin, source and properties of the 16 epidemic and sporadic F. psychrophilum isolates/STs selected for the study.
MIC [µg mL-1] _________________________ BET DPD FLU OTC OXA 400 7.8 2r 16r 4r r r 400 7.8 2 4 4r 400 7.8 0.25 4r 1r r r 400 7.8 8 8 32r r r 400 7.8 32 16 32r r r 400 11.7 2 4 8r r 400 7.8 0.25 4 1 400 7.8 64r 16r 32r 400 7.8 64r 16r 32r r 400 7.8 0.25 32 1 400 7.8 0.25 4r 1 400 11.7 0.25 8r 1 r r 400 7.8 2 16 4r r 400 11.7 0.25 32 1 400 7.8 0.25 4r 1 400 7.8 0.25 1 1 400 11.7 0.25 1 1 400 11.7 0.25 1 1 400 11.7 0.25 1 1 400 11.7 0.25 1 1 400 11.7 0.25 1 1 r 400 19.5 0.25 1 1 400 7.8 0.25 4r 1 400 7.8 0.25 1 1 400 7.8 0.25 1 1 400 7.8 0.25 1 1 400 7.8 0.25 1 1 400 11.7 8r 1 32r
Hydrolysis __________ ELA GEL + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
IS256 ____ Present Yes Yes Yes Yes No No Yes Yes Yes No No No Yes No No No No No No No No No No No No No No No
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CC-ST35 Singleton Singleton Singleton
19, 8, 7, 21, 17, 12, 7 42, 8, 10, 28, 17, 2, 19 53, 28, 2, 21, 18, 38, 20 54, 15, 2, 62, 25, 28, 1
RT AS RT AS
FIN FIN DEN NOR
2008 1999 1995 2012
400 400 400 400
7.8 7.8 7.8 7.8
0.25 0.25 0.25 0.25
32r 1 1 1
1 1 1 1
+ + -
+ + + +
No No No No
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FI102 FI197 DK152 NO114
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ST, sequence type; CC, clonal complex; MLST, multilocus sequence typing; Allelic profile (trpB, gyrB, dnaK, fumC, murG, tuf, atpA); RT, rainbow trout; AS, Atlantic salmon; T, tench; W, water; P, perch; Flo, flounder; DEN, Denmark; FIN, Finland; FRA, France; SCO, Scotland; NOR, Norway; MIC, minimum inhibitory concentration; IS, insertion sequence; BET, Betadine®; DPD, 2,2’-Dipyridyl; FLU, Flumequine; OTC, oxytetracycline; OXA, oxolinic acid; superscript r, resistant; ELA, elastin; GEL, gelatin; +, positive reaction; -, negative reaction
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ACCEPTED MANUSCRIPT Table 2
Absorbance at 595 nm 1.256±0.052
Adhesion to rainbow trout mucusa
1.458±0.056
Biofilm formationb
0.112±0.008
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Adhesion to polystyrenea
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No. of isolates (%) with resistance to Betadine®
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Flumequine Oxolinic acid
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Oxytetracycline
1.099±0.051
0.039*
1.267±0.060
0.027*
0.108±0.009
0.745
p-valued 0 (0%)
N/A
0 (0%)
1 (6%)
1.000
8 (50%)
1 (6%)
0.016*
8 (50%)
1 (6%)
0.016*
15 (94%)
2 (13%)
<0.0001*
p-valued
No. of isolates (%) positive for
Gelatin hydrolysis IS256
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Elastin hydrolysis
8 (50%)
8 (50%)
1.000
16 (100%)
16 (100%)
N/A
8 (50%)
0 (0%)
0.002*
a
values are means ± SEM of 3 replicates from 3 independent experiments
b
values are means ± SEM of 3 replicates from 4 independent experiments
c
p-value from the unpaired t-test with Welch’s correction
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p-value from Fisher’s exact test
* indicates significance between groups at a 95% confidence interval N/A not applicable
p-valuec
0 (0%)
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Iron starvation
Sporadic STs (n=16)
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Epidemic STs (n=16)
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In vitro properties of epidemic versus sporadic F. psychrophilum isolates/sequence types (STs).
ACCEPTED MANUSCRIPT Highlights
MLST data reveals an epidemic F. psychrophilum lineage with a worldwide distribution.
Characteristics of epidemic and sporadic F. psychrophilum STs are compared.
Epidemic STs show a higher ability to adhere and to resist antimicrobial agents.
IS256 is a potential molecular marker for STs of the largest clonal complex CC-ST2.
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S0044-8486(15)00081-2 doi: 10.1016/j.aquaculture.2015.02.010 AQUA 631548
To appear in:
Aquaculture
Received date: Revised date: Accepted date:
26 January 2015 5 February 2015 7 February 2015
Please cite this article as: Sundell, Krister, Wiklund, Tom, Characteristics of epidemic and sporadic Flavobacterium psychrophilum sequence types, Aquaculture (2015), doi: 10.1016/j.aquaculture.2015.02.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Characteristics of epidemic and sporadic Flavobacterium
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psychrophilum sequence types
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Krister Sundell* and Tom Wiklund
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Laboratory of Aquatic Pathobiology, Environmental and Marine Biology, Department of
*
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Correspondence: Krister Sundell
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Biosciences, Åbo Akademi University, Tykistökatu 6, 20520 Turku, Finland
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Email: [email protected] Telephone: +358-2-2154634
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Fax: +358-2-2154748
Keywords: Flavobacterium psychrophilum, multilocus sequence typing, sequence type, epidemic, sporadic
Submitted to: Aquaculture
ACCEPTED MANUSCRIPT Abstract Population genetic analysis of Flavobacterium psychrophilum using multilocus sequence typing
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(MLST) data has identified a genetic lineage consisting of sequence types (STs) associated with
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epidemic spread and high mortality in farmed rainbow trout (Oncorhynchus mykiss). To identify
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phenotypic characteristics that discriminate epidemic F. psychrophilum STs from sporadically occurring STs, a set of traits of isolates/STs (n=32) with distinct genetic background and clinical
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impact was studied in vitro. The results showed that epidemic and sporadic STs could not be discriminated by their resistance to povidone-iodine or iron starvation or by their ability to hydrolyze
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elastin and gelatin. Antimicrobial resistance against flumequine, oxolinic acid and oxytetracycline was significantly more prevalent (p<0.05), although not a prerequisite, in epidemic STs. The two
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groups showed equal biofilm forming abilities, while the epidemic STs were associated with higher
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adherence (p<0.05) indicating that adhesion and antimicrobial resistance could be factors contributing to the epidemic potential of F. psychrophilum. All isolates/STs of the largest clonal
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complex CC-ST2 were distinguished from others by the bacterial insertion sequence IS256 in the collagenase encoding gene. IS256 might thus constitute a novel molecular marker for rapid
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differentiation of epidemiologically important CC-ST2 genotypes.
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ACCEPTED MANUSCRIPT 1. Introduction Since a standardized multilocus sequence typing (MLST) scheme based on the sequences of seven
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loci was released for the fish pathogen Flavobacterium psychrophilum (Nicolas et al., 2008), several
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studies have implemented the method for genetic typing of isolates from diverse origins (Siekoula-
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Nguedia et al., 2012; Apablaza et al., 2013; Fujiwara-Nagata et al., 2013; Strepparava et al., 2013; Avendaño-Herrera et al., 2014; Nilsen et al., 2014). A web-accessible MLST database (http://pubmlst.org/fpsychrophilum/) has also been established which allows comparable typing
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between laboratories. Population genetic studies of F. psychrophilum using MLST data have
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unambiguously supported the hypothesis of an epidemic population structure where successful clonal complexes (CCs) have expanded within a generally recombinant bacterial population (Nicolas et al.,
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2008; Siekoula-Nguedia et al., 2012; Nilsen et al., 2014).
Common to previously published MLST studies of F. psychrophilum is the identification of a large
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globally spread CC, hereafter referred to as CC-ST2 according to its currently predicted ancestral sequence type (ST), with an apparent affinity to the rainbow trout (Oncorhynchus mykiss) host. STs
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within CC-ST2 have been responsible for severe bacterial cold water disease (BCWD) outbreaks in many European countries for several consecutive years (Siekoula-Nguedia et al., 2012; Strepparava et al., 2013; Nilsen et al., 2014). CCs genetically distinct from CC-ST2 have been associated with repeated disease outbreaks in Atlantic salmon (Salmo salar) in Norway (Nilsen et al., 2014) and in ayu (Plecoglossus altivelis altivelis) in Japan (Fujiwara-Nagata et al., 2013), suggesting either geographical restriction or host adaptation within certain clonal groups. More sporadically occurring F. psychrophilum STs appearing as singletons or minor CCs are usually isolated from a broader host range and are considered to be of a lesser clinical impact (Siekoula-Nguedia et al., 2012; Nilsen et al., 2014). The reason for the epidemiological success of the putatively virulent CC-ST2 is not known, but its worldwide distribution has been associated with the international trade of fish and
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ACCEPTED MANUSCRIPT associated products (Nicolas et al., 2008; Fujiwara-Nagata et al., 2013; Avendaño-Herrera et al., 2014; Nilsen et al., 2014). Evidence has linked the rapid increase in F. psychrophilum infections in
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Chile and Norway to a recent introduction of epidemic CC-ST2 genotypes, particularly to ST2
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important genotypes can become a long term endemic problem.
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(Avendaño-Herrera et al., 2014; Nilsen et al., 2014). Once established, these epidemiologically
The global distribution and dominance of CC-ST2 might be due to changes in properties determining
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the survival of F. psychrophilum in the aquatic environment or in the rainbow trout host. In
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aquaculture environments, where antimicrobials are commonly used, inherent resistance or an enhanced ability to form biofilms (Álvarez et al., 2006; Sundell and Wiklund, 2011) could provide a
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selective advantage over susceptible F. psychrophilum populations. In host-pathogen interactions,
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the ability to obtain iron (Álvarez et al., 2008) and to adhere to (Nematollahi et al., 2003) and degrade host tissue (Madsen and Dalsgaard, 1998; Duchaud et al., 2007) could be crucial factors for
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the outcome of BCWD.
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The main objective of this study was to identify discriminative phenotypic characteristics for F. psychrophilum STs of the geographically most widely spread genetic lineage. For this purpose, we compared the ability of F. psychrophilum STs with distinct genetic background and clinical impact to resist antimicrobial agents and iron starvation, to adhere and to form biofilms and to hydrolyze elastin and gelatin. In addition, we studied the potential of the bacterial insertion sequence IS256 as a molecular marker for rapid differentiation of epidemiologically important F. psychrophilum genotypes.
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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. eBURST analysis of the global F. psychrophilum MLST database
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The complete allelic profile list from the F. psychrophilum MLST database
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(http://pubmlst.org/fpsychrophilum/) was downloaded (accessed on 28 March 2014) and a
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population snapshot of the 995 isolates and 166 STs was created using the eBURSTv3 algorithm (Fig. 1). CCs were identified based on sharing 5 (relaxed) or 6 (default) out of 7 alleles using
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standard eBURST methodology (http://eburst.mlst.net/).
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2.2. Selection of bacterial isolates and culture conditions
Thirty-two F. psychrophilum isolates representing 16 epidemic and 16 sporadic STs (Table 1) were
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selected from the MLST database based on eBURST grouping (Fig. 1). Epidemic STs were
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genetically associated with the globally spread CC-ST2 (Fig. 1) and were considered part of the same lineage whereas sporadic STs consisted of singletons and minor CCs genetically distinct from CC-
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ST2. The colony morphology of F. psychrophilum is either rough or smooth (Högfors-Rönnholm and Wiklund, 2010) when grown on tryptone yeast extract salts (TYES) (Holt et al., 1993) agar. The
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rough and smooth F. psychrophilum morphotypes have been shown to express different phenotypic characteristics in vitro (Högfors-Rönnholm 2014). Hence, only the smooth morphotype was selected because of its higher prevalence within BCWD outbreaks (Sundell et al., 2013). Although none of several screened ST2 isolates showed typical smooth morphological characteristics, this genotype was included in the study because of its significance and predominance in the global MLST database. All isolates were grown on TYES agar for 5 days at 15°C prior to testing. An approximate 109 CFU mL-1 stock culture of each F. psychrophilum isolate was prepared by inoculating 5-day-old colonies from TYES agar into TYES broth to an optical density of 1.0 at 520 nm (Unicam, Heλios β) and maintained at -70°C. Dilutions from the stock cultures were used in all analyses unless otherwise stated.
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ACCEPTED MANUSCRIPT 2.3. Minimum inhibitory concentration (MIC) testing Antimicrobial resistance of the isolates was tested by measuring the MIC of the aquaculture drugs
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flumequine (FLU), oxolinic acid (OXA) and oxytetracycline (OTC) by broth microdilution in round
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bottomed 96-well microtiter plates (Nunc). FLU and OTC were obtained from Sigma-Aldrich and
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OXA from Santa Cruz Biotechnology Inc. In addition we tested the MIC of Betadine® (Leiras Takeda), a povidone-iodine based chemical used for fish egg surface disinfection. The MIC of the iron chelator 2,2′-Dipyridyl (DPD), obtained from Sigma-Aldrich, was used for studying the
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capability of epidemic and sporadic F. psychrophilum STs to resist iron starvation.
Antimicrobial stock solutions (1000 µg mL-1) of FLU, OXA and OTC were prepared by dissolving in 0.1 M NaOH, 1 M NaOH and methanol respectively and making up to volume with TYES broth.
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Approximately 105 CFU of each isolate were exposed to triplicate two-fold serial dilutions of FLU, OTC and OXA ranging from 64 to 0.125 µg mL-1. The concentrations of Betadine® and DPD diluted
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in TYES broth were 1000, 800, 600, 400, 200, 100 µg mL-1 and 31.2, 27.3, 23.4, 19.5, 15.6, 11.7, 7.8, 3.9 µg mL-1, respectively. TYES broth was used as negative control for each compound. After
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static incubation for 96 h at 15°C, the MIC of the antimicrobial was interpreted as the lowest concentration that prevented visible growth. Because of the slow bacterial growth in iron restricted media the MIC of DPD was determined after 10 days of incubation. To separate fully susceptible wild-type isolates from those with a decreased susceptibility, microbiological criteria were used to define resistance (EUCAST, 2000; Kahlmeter et al., 2003). A tentative wild-type cut-off value (COWT) was visually assessed from the MIC distribution as the modal MIC ± 1 twofold dilution (the inherent variability of the test). An isolate was regarded as being resistant to a specific antimicrobial when the MIC was above the wild-type distribution.
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ACCEPTED MANUSCRIPT 2.4. In vitro biofilm formation assay The ability to form biofilms was evaluated by the micro titer dish biofilm formation assay described
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by O´Toole (2011), modified for the growth requirements of F. psychrophilum. Briefly, 100 µl
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TYES broth containing 105 CFU of F. psychrophilum was added in triplicate into wells of untreated
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flat-bottomed 96-well microtiter plates (Nunc) and incubated statically for 7 days at 15°C to allow biofilm formation. Following incubation, the plates were rinsed by submersion in a container of tap water to remove non-adherent cells. A 125 μL volume of a 0.1% crystal violet (CV) solution was
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then added to each well and incubated at room temperature for 45 min. After discarding the contents,
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the plates were rinsed three times as previously with water and air dried. Then, 150 μL of 96% ethanol was added to each well and incubated at room temperature for 15 min. A 100 μL volume of
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the solubilized CV was transferred to a flat bottomed microtiter plate and the absorbance was
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quantified in a microplate reader (Victor2, Wallac) at 595 nm using 96% ethanol as the blank. The test was repeated four times and the mean absorbance of the replicates was used to compare the
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biofilm forming ability between the epidemic and the sporadic group.
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2.5. Adhesion to mucus coated and uncoated polystyrene Adhesion to polystyrene, both uncoated and coated with rainbow trout mucus, was examined in flatbottomed 96-well microtiter plates (Nunclon ∆ Surface, Nunc) according to the method described by Högfors-Rönnholm et al. (2014). Briefly, mucus coated wells were prepared by adding 50 μL of a sterile filtered (0.22 μm) rainbow trout mucus suspension with a protein concentration of 0.5 mg mL−1 (BCA Protein Assay Kit, Pierce). After incubation over night the wells were washed twice with 0.5% NaCl and air dried. F. psychrophilum cells grown on TYES agar were suspended in sterile lake water and 100 μL of this suspension containing approximately 108 CFU was added to both uncoated and mucus coated wells while sterile lake water was used as a negative control. After static incubation at 15 °C for 1 h and washing of the wells, the adhesion ability of the bacterial cells
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ACCEPTED MANUSCRIPT was quantified following the staining and measuring procedures described above. Each isolate was
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examined in triplicate and the experiment was repeated three times.
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2.6. Elastin and gelatin degradation
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The ability to hydrolyze elastin and gelatin was studied by streaking each isolate on TYES agar supplemented with 0.1 % and 3 % (w/v) of elastin and gelatin (Sigma) respectively. After incubation at 15°C for 5 days, a positive reaction was indicated by a clear zone in the surrounding turbid agar
2.7. Bacterial insertion sequence IS256
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medium around the inoculum.
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The presence of the bacterial insertion sequence IS256 in each F. psychrophilum isolate and the
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integrity of the collagenase-encoding gene (Duchaud et al., 2007) were studied by PCR in a thermal cycler (ArktikTM, Finnzymes). The DNA template was prepared by suspending a F. psychrophilum
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colony from a pure culture in a 50 µl volume of double-distilled water (ddH2O). Each reaction contained 1×Phire® reaction buffer (Thermo Scientific), 0.5 μM of the primers coll_fw
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AAATTGCTGCCGATGCTTGG and coll_rev CATATCCTGTTTTACCATCCCA (Duchaud et al., 2007), 200 μM of each dNTP, 0.4 μl Phire® HotStart II DNA polymerase (Thermo Scientific), 1 μl DNA template and ddH2O to a total volume of 20 μl. Samples were initially denatured for 30 s at 98°C, followed by 30 amplification cycles including denaturation for 5 s at 98°C, annealing of primers for 5 s at 60°C, and extension for 30 s at 72°C. After the last cycle, the PCR mixture was incubated at 72°C for 60 s. The amplicons were electrophoresed (4 V cm-1, 30 min) on a 1% agaroseTris-borate-EDTA gel stained with ethidium bromide and detected with ultraviolet transillumination. The presence or absence of IS256 was indicated by a 2418 or 1046 bp amplicon respectively (Duchaud et al., 2007).
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ACCEPTED MANUSCRIPT 2.8. Statistical analyses Statistical analyses were performed using the GraphPad Prism5 software. An unpaired t-test with
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Welch’s correction for groups with unequal variances was used to determine significant (p<0.05)
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differences between the epidemic and the sporadic groups. When appropriate, values from the
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Fisher’s exact test were used to compare the prevalence of characteristics between the two groups.
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3. Results 3.1. eBURST
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The global distribution of F. psychrophilum STs (Fig. 1) corresponds to a dominant epidemic lineage including the clonal complexes CC-ST2, CC-ST21, CC-ST124 and CC-ST125 together with four
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singleton STs against a background of smaller CCs and singletons.
3.2. MICs
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The MIC of Betadine® was 400 µg mL-1 for all isolates. Of the other aquaculture compounds, FLU had a broader MIC range (0.25–64 µg mL-1) compared to OTC and OXA (1–32 µg mL-1). The
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estimated COWT was ≤ 2 µg mL-1 for OTC and OXA, ≤ 1 µg mL-1 and ≤ 15.6 µg mL-1 for FLU and DPD respectively. Based on the COWT half of the epidemic, but only one (6%) of the sporadic isolates was resistant to the two quinolones (Table 1). All except one epidemic isolate (94%), but only two of the sporadic ones (13%) showed a reduced susceptibility to OTC. Only one isolate had a MIC value above the COWT for DPD. Acquired resistance indicated by the decreased susceptibility against FLU, OTC and OXA (Table 2) was more prevalent in the epidemic group compared to the sporadic group (Fisher’s test, p<0.05).
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ACCEPTED MANUSCRIPT 3.3. In vitro adhesion and biofilm formation assays The epidemic and sporadic F. psychrophilum isolates showed equal biofilm forming abilities in vitro
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(p=0.745) (Table 2). Although all isolates were able to adhere, the adhesion capacity was
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significantly higher in the epidemic group both on an uncoated (p=0.039) and on a rainbow trout
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mucus coated polystyrene surface (p=0.027).
3.4. Presence of IS256
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IS256 was present in half of the epidemic, but in none of the sporadic isolates. Interestingly, all
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isolates that contained IS256 belonged to CC-ST2, shared 4 or more MLST alleles and had a decreased susceptibility against oxytetracycline (Table 1). The Fisher’s exact test showed that IS256
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was significantly more prevalent in the epidemic group compared to the sporadic (p=0.002) (Table
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2). When the test was repeated by treating CC-ST2 (n=8) and the other isolates (n=24) as two
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separate groups the p-value dropped to <0.0001.
3.5. In vitro elastin and gelatin hydrolysis
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Half of both the epidemic and the sporadic isolates were able to hydrolyze elastin, while all isolates were able to hydrolyze gelatin (Table 1 and 2). Six out of the eight isolates belonging to CC-ST2 were elastinase producing. All isolates from CC-ST125 were unable to degrade elastin.
4. Discussion The dedicated F. psychrophilum MLST database (http://pubmlst.org/fpsychrophilum/) allows for global epidemiological surveillance and identification of STs associated with disease outbreaks in particular fish species. Still, MLST is laborious and expensive and requires multiple PCRs and sequence analyses before generating a typing result useful for disease outbreak investigation and control. Before the F. psychrophilum MLST database was established, the work of Duchaud et al.
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ACCEPTED MANUSCRIPT (2007) showed the presence of IS256 in a disrupted collagenase encoding gene of F. psychrophilum in several putatively virulent rainbow trout isolates originating from different parts of the world.
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According to the published MLST data available today the isolates harboring IS256 belonged to CC-
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ST2, corroborating their hypothesis of a clonal dissemination of virulent F. psychrophilum strains
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containing the IS256 disrupted gene. Our study showed that the IS256 disrupted collagenase encoding gene was limited to CC-ST2 indicating that the STs in the genetically related CC-ST21, CC-ST124 and CC-ST125 are not clonal descendants of ST2. Nevertheless, our study implies that
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IS256 could serve as a molecular marker for rapid differentiation of epidemiologically important
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CC-ST2 genotypes. While it has been suggested that IS256 can contribute to virulence by influencing phenotypic variation, antimicrobial resistance and niche adaptation of bacterial
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pathogens (Conlon et al., 2004; Kozitskaya et al., 2004; Gu et al., 2005), the consequences of its
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integration in the collagenase encoding gene and/or other parts of the genome of F. psychrophilum is not yet understood. Hence a more detailed comparative genomic analysis of distinct F.
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psychrophilum STs is necessary.
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Because bacterial attachment is a prerequisite for colonization of surfaces or host cells, the adhesion of epidemic and sporadic isolates was compared. The higher adherence of epidemic F. psychrophilum STs to inert and mucus coated surfaces might allow for a more efficient colonization of fish, tank and equipment surfaces in aquaculture environments. The higher ability to adhere to rainbow trout mucus could also be indicative of specific host-adaptation mechanisms in epidemic STs. Although our findings do not allow us to make any associations between adherence and virulence, a positive correlation between F. psychrophilum adhesion to gill arches of rainbow trout and virulence has previously been reported (Nematollahi et al., 2003). It is also known that the genome of the virulent F. psychrophilum isolate JIP02/86 contains several (n=27) genes encoding proteins related to adhesion (Duchaud et al., 2007). Thus, our findings provide a compelling
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ACCEPTED MANUSCRIPT argument for continuing research into genetic factors underlying specific mechanisms of adhesion, since the higher adherence of specific F. psychrophilum lineages could be a factor contributing to the
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potential to cause epidemic BCWD outbreaks.
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Biofilm formation is known to improve F. psychrophilum survival during antimicrobial exposure (Sundell and Wiklund, 2011), but the association between biofilm formation and genetic background has not been studied before. Our analysis did not detect any differences in the biofilm forming ability
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between the epidemic and the sporadic group, suggesting that biofilm formation as a trait is not any
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more important in epidemic F. psychrophilum lineages. However, as the in vitro culture environment is artificial it does not necessarily represent in vivo conditions; thus biofilm formation cannot be
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excluded as an important virulence determinant of F. psychrophilum. Another example of this relates
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to iron availability, which is essential for pathogen survival and growth in the low-iron environment of the fish host where iron is limited due to host production of iron-binding proteins (Otto et al.,
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1992). Even though our results did not show any differences in resistance to iron starvation between the two groups, specific iron uptake mechanisms could still be important virulence factors in F.
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psychrophilum.
The proteolytic activity of F. psychrophilum has been studied as a potential discriminatory marker for pathogenicity with inconclusive results (Bertolini et al., 1994; Faruk 2000; Madetoja et al., 2002). Previous studies with experimentally injected rainbow trout have suggested that the production of elastinase and gelatinase (Madsen and Dalsgaard 1998; Ostland et al., 2000) may be important for the pathogenicity of F. psychrophilum. Our results showed that epidemic and sporadic STs could not be distinguished from each other by their ability to hydrolyze gelatin or elastin. All isolates were gelatin-producing and some of the elastinase-negative F. psychrophilum isolates represented STs (CC-ST125 and ST92) previously associated with high virulence in rainbow trout (Nilsen et al.,
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ACCEPTED MANUSCRIPT 2014). Our results indicated that the ability of F. psychrophilum to hydrolyze elastin or gelatin in
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vitro does not correlate with propensity to cause BCWD.
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Resistance to aquaculture drugs particularly to quinolones has been repeatedly reported from F.
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psychrophilum outbreaks in different parts of the world (Izumi and Aranishi 2004; Henríquez-Nuñez et al., 2012; Shah et al., 2012; Sundell et al., 2013). Quinolone resistant F. psychrophilum genotypes associated with disease in juvenile rainbow trout have also been isolated from eggs (Sundell et al.,
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2013). In routine hatchery practices, povidone-iodine is used at a concentration of 100 µg mL-1
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which has proven ineffective for complete disinfection of eggs (Wagner et al., 2012). In this study, growth for each F. psychrophilum isolate was observed in nutrient broth containing povidone-iodine
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at twice the concentration used in hatcheries, indicating that currently used egg disinfection strategies
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are both inefficient for elimination of F. psychrophilum and that resistant genotypes could efficiently
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be spread through trade of contaminated eggs.
At present, there are no internationally accepted criteria to be applied for establishing COWT values
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(Silley, 2012), but evaluation of the MIC distributions (Kahlmeter et al., 2003) is widely adopted for detection of bacterial isolates with reduced susceptibility. In our study, a division in isolate susceptibility to FLU, OTC and OXA was characterized by the bimodal distribution of MIC values, with a minimum gap of one two-fold dilution between susceptible and resistant STs. We found a significantly higher prevalence of resistance against the quinolones FLU and OXA and the tetracycline OTC among epidemic STs (Table 1 and 2). However, susceptible wild-type isolates were also present in the epidemic group and vice versa (Table 1 and 2), indicating that antimicrobial resistance is not a prerequisite for epidemic STs. Still, our results indicate that antimicrobial resistance is strongly associated with epidemic F. psychrophilum STs and may therefore constitute one of the main explanations for their epidemiological success and dominance in aquaculture
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ACCEPTED MANUSCRIPT environments. Whether this association could be a result of co-selection of resistance and virulence, a high transmissibility of epidemic STs with intrinsic resistance or of a more efficient acquisition and
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expression of resistance determinants remains to be elucidated.
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In conclusion, our study showed that STs of an epidemic lineage were discriminated from sporadically occurring STs by a higher ability to adhere and to resist antimicrobial agents commonly used in aquaculture. The results suggest that specific adhesion mechanisms and antimicrobial
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resistance may be key factors contributing to the success of epidemic F. psychrophilum clones in fish
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farm environments. To identify and limit the spread of epidemiologically important F. psychrophilum genotypes, IS256 could serve as a rapid molecular marker for differentiation of CC-
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ST2 genotypes. The knowledge gained from this work provides further research direction for
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discovering the underlying genetic mechanisms contributing to the emergence and characteristics of
Acknowledgements
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epidemic F. psychrophilum lineages.
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This publication made use of the F. psychrophilum MLST database developed by Keith Jolley at the University of Oxford. Funding was provided by the Finnish Ministry of Agriculture and Forestry as part of the EU EMIDA ERA-NET project “Control Flavobacteriaceae infections in European fish farms” (2114/311/210). The authors wish to thank all partners of the consortium. We are grateful to J-F. Bernardet, I. Dalsgaard, E. Duchaud and H. Nilsen for kindly providing isolates.
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Sundell, K., Wiklund, T., 2011. Effect of biofilm formation on antimicrobial tolerance of Flavobacterium psychrophilum. J. Fish Dis. 34, 373–383. Wagner, E., Oplinger, R., Bartley, M., 2012. Penicillin-G: Efficacy against Flavobacterium psychrophilum and evaluation of lethal dose limits for rainbow trout. OJAS 2, 150–158.
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Table and figure legends
Table 1. Origin, source and in vitro properties of epidemic and sporadic F. psychrophilum isolates/STs selected for the study.
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Table 2. In vitro activities of epidemic versus sporadic F. psychrophilum isolates/STs.
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Figure 1. Population snapshot of the F. psychrophilum MLST database with default eBURSTv3
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settings (accessed on 28 March 2014). Each dot represents an individual ST and dot size reflects its abundance. Linked clusters represent CCs, singleton STs are unlinked. Clonal founders are shown in blue, subgroup founders in yellow. CCs and STs within the shaded area are linked with the use of a
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relaxed group definition. Epidemic STs selected for this study are indicated with red numbers,
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sporadic with green respectively.
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Figure 1
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ACCEPTED MANUSCRIPT Table 1
Sporadic
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Source ____________________ Host Country Year RT DEN 1994 RT FIN 1999 RT FIN 1996 RT FRA 1986 AS SCO 2007 RT FIN 1996 RT FIN 2000 RT FIN 2009 RT FIN 2010 RT FIN 2011 RT FIN 2007 RT FIN 2011 RT FIN 2003 RT FIN 2008 RT FIN 2000 RT FIN 2000 T FRA 1992 W FIN 2000 P FIN 2006 RT FIN 1994 RT FIN 1983 P FIN 2006 W FIN 2000 AS FIN 1999 AS NOR 2009 AS NOR 2012 RT DEN 2006 Flo DEN 2000
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MLST data ____________________________________ ST CC Allelic profile ST2 CC-ST2 2, 2, 2, 2, 2, 2, 2 ST10 CC-ST2 2, 2, 2, 8, 2, 2, 2 ST12 CC-ST2 2, 2, 2, 8, 2, 2, 7 ST20 CC-ST2 2, 2, 2, 8, 2, 8, 2 ST21 CC-ST21 10, 2, 2, 2, 2, 2, 7 ST73 CC-ST124 1, 15, 14, 13, 1, 8, 7 ST79 CC-ST2 2, 8, 2, 8, 2, 2, 2 ST91 CC-ST2 2, 2, 2, 2, 2, 2, 41 ST92 CC-ST2 2, 2, 2, 2, 2, 3, 41 ST124 CC-ST124 1, 15, 14, 13, 1, 2, 7 ST125 CC-ST125 2, 15, 2, 13, 2, 1, 7 ST126 CC-ST124 1, 15, 16, 13, 1, 2, 7 ST128 CC-ST2 2, 2, 2, 8, 2, 2, 48 ST129 CC-ST124 1, 2, 14, 13, 1, 2, 7 ST131 CC-ST125 2, 15, 2, 1, 2, 1, 7 ST135 CC-ST125 2, 15, 2, 13, 2, 1, 3 ST18 CC-ST8 6, 9, 3, 6, 5, 4, 6 ST23 Singleton 12, 11, 7, 13, 9, 9, 13 ST34 CC-ST34 18, 15, 3, 20, 16, 1, 6 ST36 Singleton 20, 8, 10, 22, 6, 13, 20 ST122 Singleton 14, 12, 10, 48, 10, 10, 47 ST127 Singleton 18, 8, 2, 37, 36, 8, 47 ST132 Singleton 3, 2, 11, 49, 3, 11, 3 ST141 Singleton 42, 8, 7, 23, 38, 21, 51 ST169 Singleton 1, 15, 1, 3, 1, 8, 1 ST171 Singleton 43, 8, 17, 51, 33, 7, 17 ST180 Singleton 44, 8, 7, 21, 28, 35, 19 ST195 Singleton 3, 22, 3, 54, 40, 18, 3
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Isolate ____ Code DK008 FI152 FI055 JIP02/86 FI166 FI155 FI140 FI091 FI001 FI024 FI049 FI058 FI095 FI104 FI132 FI161 LVDJXP189 FI147 FI071 FI181 FI016 FI074 FI146 FI198 NO061 NO111 DK061 DK098
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Origin, source and properties of the 16 epidemic and sporadic F. psychrophilum isolates/STs selected for the study.
MIC [µg mL-1] _________________________ BET DPD FLU OTC OXA 400 7.8 2r 16r 4r r r 400 7.8 2 4 4r 400 7.8 0.25 4r 1r r r 400 7.8 8 8 32r r r 400 7.8 32 16 32r r r 400 11.7 2 4 8r r 400 7.8 0.25 4 1 400 7.8 64r 16r 32r 400 7.8 64r 16r 32r r 400 7.8 0.25 32 1 400 7.8 0.25 4r 1 400 11.7 0.25 8r 1 r r 400 7.8 2 16 4r r 400 11.7 0.25 32 1 400 7.8 0.25 4r 1 400 7.8 0.25 1 1 400 11.7 0.25 1 1 400 11.7 0.25 1 1 400 11.7 0.25 1 1 400 11.7 0.25 1 1 400 11.7 0.25 1 1 r 400 19.5 0.25 1 1 400 7.8 0.25 4r 1 400 7.8 0.25 1 1 400 7.8 0.25 1 1 400 7.8 0.25 1 1 400 7.8 0.25 1 1 400 11.7 8r 1 32r
Hydrolysis __________ ELA GEL + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
IS256 ____ Present Yes Yes Yes Yes No No Yes Yes Yes No No No Yes No No No No No No No No No No No No No No No
ACCEPTED MANUSCRIPT ST197 ST198 ST231 ST232
CC-ST35 Singleton Singleton Singleton
19, 8, 7, 21, 17, 12, 7 42, 8, 10, 28, 17, 2, 19 53, 28, 2, 21, 18, 38, 20 54, 15, 2, 62, 25, 28, 1
RT AS RT AS
FIN FIN DEN NOR
2008 1999 1995 2012
400 400 400 400
7.8 7.8 7.8 7.8
0.25 0.25 0.25 0.25
32r 1 1 1
1 1 1 1
+ + -
+ + + +
No No No No
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FI102 FI197 DK152 NO114
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ST, sequence type; CC, clonal complex; MLST, multilocus sequence typing; Allelic profile (trpB, gyrB, dnaK, fumC, murG, tuf, atpA); RT, rainbow trout; AS, Atlantic salmon; T, tench; W, water; P, perch; Flo, flounder; DEN, Denmark; FIN, Finland; FRA, France; SCO, Scotland; NOR, Norway; MIC, minimum inhibitory concentration; IS, insertion sequence; BET, Betadine®; DPD, 2,2’-Dipyridyl; FLU, Flumequine; OTC, oxytetracycline; OXA, oxolinic acid; superscript r, resistant; ELA, elastin; GEL, gelatin; +, positive reaction; -, negative reaction
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ACCEPTED MANUSCRIPT Table 2
Absorbance at 595 nm 1.256±0.052
Adhesion to rainbow trout mucusa
1.458±0.056
Biofilm formationb
0.112±0.008
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Adhesion to polystyrenea
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No. of isolates (%) with resistance to Betadine®
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Flumequine Oxolinic acid
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Oxytetracycline
1.099±0.051
0.039*
1.267±0.060
0.027*
0.108±0.009
0.745
p-valued 0 (0%)
N/A
0 (0%)
1 (6%)
1.000
8 (50%)
1 (6%)
0.016*
8 (50%)
1 (6%)
0.016*
15 (94%)
2 (13%)
<0.0001*
p-valued
No. of isolates (%) positive for
Gelatin hydrolysis IS256
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Elastin hydrolysis
8 (50%)
8 (50%)
1.000
16 (100%)
16 (100%)
N/A
8 (50%)
0 (0%)
0.002*
a
values are means ± SEM of 3 replicates from 3 independent experiments
b
values are means ± SEM of 3 replicates from 4 independent experiments
c
p-value from the unpaired t-test with Welch’s correction
d
p-value from Fisher’s exact test
* indicates significance between groups at a 95% confidence interval N/A not applicable
p-valuec
0 (0%)
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Iron starvation
Sporadic STs (n=16)
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Epidemic STs (n=16)
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In vitro properties of epidemic versus sporadic F. psychrophilum isolates/sequence types (STs).
ACCEPTED MANUSCRIPT Highlights
MLST data reveals an epidemic F. psychrophilum lineage with a worldwide distribution.
Characteristics of epidemic and sporadic F. psychrophilum STs are compared.
Epidemic STs show a higher ability to adhere and to resist antimicrobial agents.
IS256 is a potential molecular marker for STs of the largest clonal complex CC-ST2.
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