Bull. Eur. Ass. Fish Pathol., 33(2) 2013, 67 NOTE Psychrobacter sp. isolated from the kidney of salmonids at a number of aquaculture sites in Scotland Ú. McCarthy*, H. Stagg, K. Donald, A. Garden and S. J. Weir ȱȱǰȱȱ¢ǰȱǰȱŗŗȱşȱǰȱ Abstract In October 2011, a Psychrobacter sp. was isolated as the predominant bacterial type from the kidney ȱȱęǰȱȱȱȱȱȱǻSalmo salar) site and one rainbow trout (Onco- rhynchus mykiss Walbaum) site, at two geographically and commercially separate seawater loca- tions in Scotland. The organism was subsequently isolated from kidney, and skin lesions or gills of moribund salmon at three further farm sites. While psychrobacter species have frequently been ęȱȱęȱȱȱȱĚǰȱȱ¢ȱȱȱȱȱȱȱȱ from the kidney of compromised individuals suggests it has the capacity to establish opportunistic infections in salmonids. growth was examined at regular intervals during In the late summer/autumn of 2011, marine colony development to determine the predomi- salmon farms in Scotland experienced high nant colony types based on colony morphology levels of mortality caused by amoebic gill (colour, size, shape, consistency, margins) and disease (AGD). As part of routine surveillance bacterial cell morphology (eg. straight or curved ȱȱȱȱęȱȱȱ ǰȱǰȱęȱǼǯȱȱ ǰȱȱȱȱǻǼȱěȱ of predominant colonies were subcultured to ȱȱȱȱęȱȱ- ȱȱȱȱęȱǯȱ- fected farms for testing to rule out the involve- terial isolates were then subjected to frontline ment of other factors in the mortalities. ęȱȱǯǯȱȱȱ ȱȱ 15°C or 22°C, for media containing 1.5 % or 2 On site, samples of kidney material were taken % NaCl, Gram appearance, motility, presence with sterile disposable loops and inoculated asep- of cytochrome oxidase and catalase enzymes, tically onto tryptone soya agar (TSA), TSA plus oxidative or fermentative metabolism on modi- 1.5% NaCl (TSAS) and Flexibacter Maritimus ęȱȂȱȱǻȱȱǯǰȱŗşŞśǼȱȱ Medium (FMM) for bacterial recovery. On receipt sensitivity to vibriostat O/129. Based on results at the Marine Laboratory, agar plates were incu- ȱȱǰȱȱęȱ ȱȱ bated at 15°C for 10 days. The resultant bacterial out using a selection of standard biochemical * Corresponding author’s email: [email protected] 68, Bull. Eur. Ass. Fish Pathol., 33(2) 2013 tests, including including miniaturised tests from ęȱȱȱ ȱȱ ȱ¡- bioMérieux (API 20E, API 20NE or API ZYM, ȱ ȱǻ ǰȱŘŞŜŖŜǼǯȱȱȱ bioMérieux UK), supplementing media to 2 % were performed using the GenomeLab DTCS ęȱȱȱ ȱ ǯȱȱ ȱȱ ȱǻȱǼȱȱȱ activity of isolates was tested on TSAS contain- 8800 Sequencher (Beckman Coulter), according ing 1 % Tween 80 and haemolysin production to the manufacturer’s instructions, using the was tested on sheep blood agar (blood agar base ȱȱȱȱȱȱęǯȱȱ ǻ¡ȱŖŞŚśǼǰȱ ȱśȱƖȱęȱȂȱ sequences were manually checked and edited ȱȱŘȱƖȱęȱȱǼǯȱȱ using BioEdit software (Hall, 1999). The con- an isolate was of particular interest based on sensus sequence was matched with sequences ȱȱȱęȱȱ available in NCBI using BLAST (Altschul et al., the foregoing tests, genomic DNA was extracted, 1997). For phylogenetic comparisons, 1361 bases to allow sequencing of the 16S rRNA gene, using from 16S rRNA gene sequences of a range of ȱ¢ȱȱȱȱ ȱǻ ǰȱŜşśŖŜǼȱ Psychrobacter species were aligned using Clustal and following the manufacturer’s instructions for W2 (Larkin et al., 2007). Distance matrices were Gram-positive bacteria. generated by DNADIST using the assumptions of Kimura (1980) and dendrograms constructed Sections of DNA within the 16S rRNA gene using the neighbour-joining method (Saitou and ȱęȱ¢ȱȱȱȱȱȱ ǰȱŗşŞŝǼȱȱ ȱȱřǯŜşȱǻĴDZȦȦ primer pairs to achieve sequence coverage of evolution.gs.washington.edu/phylip/getme. the gene between positions 24 and 1540 (Yumoto html accessed 15/10/2012). Bootstrap values et al., 1999, see Table 1 for the list of primers). were obtained from 1000 trees generated with The production of a single band of the expected ȱȱǯȱ ȱȱ £ȱ¢ȱȱȱȱ ȱęȱ¢ȱ 1.6.6 (Page 1996) was used to visualise trees. electrophoresis on a 1.5 % agarose gel. The ęȱȱ ȱ¡ȱȱȱȱȱ In October 2011, a previously unknown cocco- Table 1. Positions and sequences of primers used in this study; F – forward, R – reverse. Primer name Sequence (5’ – 3’) 24Fa GCA GGC YTA ACA CAT GCA AGT CGA 357F CTC CTA CGG GAG GCA GCA 806R GGA CTA CCA GGG TAT CTA AT 1080R GAG CTG ACG ACA RCC ATG CA 1240R TAC CGD CCA TTG TAG CAC GTG TGT A 1540R AAG GAG GTG ATC CAG CCG CA a based on numbering of E. coli 16S rRNA gene Bull. Eur. Ass. Fish Pathol., 33(2) 2013, 69 bacillus was the most prevalent isolate, cultured 1496 (numbering corresponds to the Escherichia on TSAS from the kidney of 4 out of 5 sampled coli 16S rRNA gene). The results of phylogenetic ęȱȱ ȱ¢ȱȱ¢ȱ analysis are shown in Figure 1. Sequences used separate sea sites, one Atlantic salmon (Salmo in the analysis included a range of isolates from salar) and one rainbow trout (Oncorhynchus marine organisms as well as two sequences mykiss Walbaum). Subsequently, the organism with similarities to isolates obtained by Steinum was isolated on TSAS from skin lesions of 3 et al. (2009) in their study of Atlantic salmon ȱȱȱ¢ȱȱŗȱȱȱęȱȱȱśȱ gill-associated bacteria, namely Accession Nos. ęȱȱȱ¢ȱŘŖŗŘDzȱȱȱȱȱ ŚřřřřŘȱȱŜŝŝŞŜŞǯȱ a single salmon sampled in March; from the kidney of 1 of 2 salmon sampled in July. In all The genus Psychrobacter incorporates a group of 5 cases, the isolates exhibited the same pheno- aerobic, non-motile, oxidase and catalase posi- typical and biochemical characteristics . In cases tive, non-pigmented, mainly psychrotrophic 1, 2, 4 and 5 the isolate was sequenced and the Gram-negative rods or coccobacilli (Juni and 16S rRNA sequence was identical. Heym, 1986). Members of the genus have been isolated from a variety of low-temperature envi- The organism forms creamy-white, opaque, ronments including polar seas, sea ice and soils low convex 1 mm diameter colonies at 48 h on ȱȱȱȱȱȱȱęȱȱ TSAS and prefers growth at 22°C to 15°C. It cold storage (Bowman et al., 1996; González et is a Gram-negative, non-motile coccobacillus al., 2000; Bozal et al., 2003; Romanenko et al., occurring in pairs, oxidase and catalase posi- 2004). Psychrobacters have also been isolated tive and resistant to vibriostat O/129. In oxida- from the faeces of seals (Yassin and Busse, 2009) tion-fermentation tests the organism is inert and of pigeons (Kämpfer et al., 2002), and a and it is negative in sulphide-indole-motility strain designated as P. pulmonis was isolated (SIM) and gas from glucose (GfG) tests (both from the lungs of infected lambs (Vela et al., of these media supplemented with NaCl to a 2003). Human isolates, other than P. phenylpyru- ęȱȱȱŘƖǼǯȱȱ ȱŘŖȱęȱ vicus, mostly belong to the species ǯȱ and is 0201004, i.e. positive for citrate utilisation, P. pulmonis (Deschaght et al., 2012). Voges-Proskauer reaction and oxidase. The ȱŘŖȱęȱȱŖŖŖŖŖŖŚǰȱǯȱȱȱ Psychrobacters are found as a normal compo- all tests except for oxidase. In API ZYM, the ȱȱȱĚȱȱęȱȱȱȱǻ ȱ isolate is positive for alkaline phosphatase, C4 and Heym, 1986; Scholes and Shewan 1964; esterase, C8 esterase-lipase, leucine and valine ĴDZȦȦǯ ǯȦȦśŘŜȱ peptidases and phosphoamidase. Tween 80 is accessed 8/09/2012). They have been reported hydrolysed but the organism is non-haemolytic from the gut of Atlantic salmon, Arctic charr on sheep blood agar. Blast analysis of the partial (Salvelinus alpinus L.), Atlantic cod (Gadus morhua ŗŜȱȱȱǻȱśŖȬŗśŖŖǼȱęȱ L.) (Kristiansen et al., 2011; Ringø et al., 2006a; the organism as Psychrobacter sp. with 100 % Ringø et al., 2006b) and from fast growing ju- similarity to Psychrobacter sp. 146Z4-2 (GenBank venile grouper (Epinephilus coioides) (Sun, 2009). řŗŖŘŜŚǼǰȱȱȱȱȱȱśŖȱȮȱ ęȱȱǯȱǻŘŖŖşǼȱȱ¢ȱ 70, Bull. Eur. Ass. Fish Pathol., 33(2) 2013 EU433332 EU433332 Psychrobacter sp. (Arctic marine sediments) AF468405 AF468405 Arctic sea ice bacterium HE800832 HE800832 P. okhotskensis (Arctic Ocean) PSYSAL Psychrobacter salmon isolate Scotland JX310264 JX310264 Psychrobacter sp. (Southern Ocean) GU574735 GU574735 Psychrobacter sp. (Bering Sea) FJ785515 FJ785515 Psychrobacter sp. (Pacific Ocean) 675 HM584277 HM584277 P. glacincola (red tanner crab) 989 NR024806 NR_024806 P. okhotskensis (north-eastern Pacific Ocean) 902 JQ691541 JQ691541 Psychrobacter sp. (fish gut microbiome) DQ677868 DQ677868 Psychrobacter sp. (glacier, Antarctica) 938 JQ691535 JQ691535 Psychrobacter sp. (fish gut microbiome) EU000245 511 EU000245 P. maritimus (Arctic marine bacterium) 701 977 AB302185 AB302185 (diseased Antarctic krill) AB302184 AB302184 (diseased Antarctic krill) NR042221 NR_042221 P. urativorans type strain DSM 14009 662 HM212666 HM212666 P. sanguinis (human) 1000 HM212668 HM212668 P. sanguinis (human) 940 FJ463826 FJ463826 Psychrobacter sp. (chicken) 919 PRwf 1 NC_009524 Psychrobacter sp. PRwf-1 (silk snapper skin and gills) 1000 GQ370385 GQ370385 Psychrobacter sp. (herring gut) 559 NR041688 NR_041688 P. fulvigenes (marine crustacean) HQ698584 HQ698584 P. pulmonis (human) HQ698570 HQ698570 P. faecalis (human) Figure 1. Phylogenetic relationship of the Psychrobacter sp. isolated in this study with species from marine ȱȱǰȱȱȱȱȱȱęȱȱȱȱǯȱȬ joining tree based on the alignment of 1361 nucleotides of the 16S rRNA gene, using the assumptions of Kimura. Numbers at nodes are the bootstrap values from 1000 replications (only values greater than 500 are Ǽǯȱȱȱęȱ¢ȱȱ ȱȱǰȱȱȱǯ species from the intestine of rainbow trout fed a ming motility, oxidase and catalase positive, diet rich in soybean meal, but not from trout fed fermentative, sensitive to O/129 and exhibiting ęǯȱ ȱȱ¢ȱȱȱȱ ȱ ȱęȱȱȱ ȱŘŖȱǯȱȱ proliferative gill disease of salmon in Norway Moritella viscosa and Allivibrio spp. have been (Steinum et al., 2009), psychrobacter was one of ęȱȱȱȱȱ¡ȱǯȱǰȱ the predominant species isolated from the gills the coincidental initial isolation of Psychrobacter ȱȱěȱȱȬěȱęǯȱ as the predominant species in 4 of 5 sampled ęȱȱ ¢ȱȱȱȱ With this widespread and common association ȱȱ¢ȱȱȱęȱȱȱȱ ȱȱȱ ȱęǰȱȱȱȱȱȱ chance occurrence. Furthermore, isolation of ęȱȱȱĴȱǯȱ ǰȱȱȱ the bacterium from kidney implies that it can hands, bacteria typically recovered from the access and establish a presence in the internal kidney of salmonids in seawater are mixed iso- ȱȱȱęǯȱ lates of the Vibrionaceae family ie.