Pseudomonas chlororaphis subsp. piscium subsp. nov., isolated from freshwater fish

The past two decades have seen a rapid increase in the aquaculture of freshwater finfish. With this has come increasing interest in the use of probiotic, or beneficial bacteria, as a means of controlling bacterial disease in farmed fish. The genus Pseudomonas comprises a heterologous group of organisms commonly isolated from water and soil environments. During two recent studies, Pseudomonas sp. strain JF3835^T, isolated from the intestine of a European perch (Perca fluviatilis L.), was suggested as a potential probiotic to control bacterial disease in freshwater fish (Goldschmidt-Clermont et al., 2008; Gobeli et al., 2009). Phylogenetic analysis based on 16S rRNA gene sequences revealed 100 % sequence similarity between strain JF3835^T and Pseudomonas sp. strain JF4413, isolated from the intestinal lumen of a rainbow trout (Oncorhynchus mykiss, Walbaum). During the present study, strains JF3835^T and JF4413 were investigated to clarify their taxonomic position. While DNA–DNA hybridization data suggested that both strains belonged to the species Pseudomonas chlororaphis, multilocus sequence analysis indicated that the strains formed a distinct cluster within this species.

Strain JF3835^T was isolated in 2005 from the distal intestine of a European perch, which was caught in the Lake of Neuchâtel, Switzerland. The strain was recovered following 7 days incubation on tryptic soy agar (TSA) containing 5 % sheep blood at 18 °C. Strain JF4413 was isolated in 2008 from the intestinal lumen of a rainbow trout, which had been caught in the same lake. Both strains were routinely cultivated on Luria–Bertani (LB) agar or in LB broth at 25 °C and were stored at −80 °C in modified Hogness freezing medium [final concentration: 36 mM K₂HPO₄, 13.2 mM KH₂PO₄, 0.4 mM MgSO₄, 1.7 mM Na₃-citrate, 6.8 mM (NH₄)₂SO₄, 4.4% (v/v) glycerol]. Cell morphology and Gram-staining properties were observed using light microscopy following 2 days growth on LB agar. Motility was observed by the hanging drop method (Koneman et al., 1997). Oxidase and catalase tests were performed using commercial reagents (Becton Dickinson and Company). Pyocyanin and pyoverdine production were examined on Pseudomonas Agar P and Pseudomonas Agar F (Difco), respectively. Resistance to irgasan was determined on Pseudomonas Isolation Agar (Difco). Carbohydrate assimilation, enzyme production and additional phenotypic characteristics were assessed using ID32 GN and API 20 NE strips (bioMérieux) and read after 48 h incubation at 25 °C. Phenotypic characteristics were compared with those of P. chlororaphis subsp. chlororaphis DSM 50083^T, P. chlororaphis subsp. aureofaciens DSM 6698^T, P. chlororaphis subsp. aurantiaca NCIMB 10068^T, P. lundensis DSM 6252^T, P. fragi DSM 3456^T, P. putida DSM 291^T, P. cichorii DSM 50259^T, P. brassicacearum DSM 13227^T, P. mediterranea CFBP 5447^T, P. congelans P 538/23^T, P. syringae DSM 10604^T, P. frederiksbergensis JAJ28^T, P. lini CFBP 5737^T, P. thivervalensis DSM 13194^T, P. corrugata DSM 7228^T, P. fluorescens DSM 50090^T, P. grimontii DSM 17515^T and P. marginalis DSM 13124^T analysed concurrently.

Cells of strain JF3835^T and JF4413 were Gram-negative, rod-shaped and motile. Oxidase and catalase tests were positive. Both strains grew aerobically on LB agar at 18, 25 and 30 °C. Growth at 37 °C was weak. No growth was seen after 5 days incubation under anaerobic conditions. Both isolates produced yellow–green, diffusible, fluorescent pigment on Pseudomonas Agar F. No pyocyanin production was seen when the strains were grown on Pseudomonas Agar P. Strains JF3835^T and JF4413 both grew on Pseudomonas Isolation Agar indicating resistance to irgasan. When grown on LB agar, both strains produced a non-diffusible pigment that was visible after 4–5 days incubation at 25 and 30 °C (Fig. 1⇓). Both strains could be distinguished from related species of the genus Pseudomonas by their ability to assimilate 3-hydroxybenzoate and their inability to assimilate arabinose (Table 1⇓). Positive tests for 5-ketogluconate and phenylacetate assimilation and a negative test for arginine dihydrolase production were also characteristic. Other phenotypic properties are given in Table 1⇓.

Determination of the DNA G+C content of strain JF3835^T by HPLC was performed by the DSMZ according to the method of Mesbah et al. (1989). The DNA G+C content of strain JF3835^T was 62.9 mol%, which was in agreement with the range reported previously for species of the genus Pseudomonas (Palleroni, 2005).

GC of cellular fatty acid methyl esters (FAME) and their identification using the Sherlock Microbial Identification System was carried out by the DSMZ. For this purpose, strains JF3835^T, P. lundensis DSM 6252^T, P. taetrolens DSM 3456^T, P. fragi DSM 3456^T, P. putida DSM 291^T, P. cichorii DSM 50259^T, P. brassicacearum DSM 13227^T, P. mediterranea CFBP 5447^T, P. congelans P 538/23^T, P. syringae DSM 10604^T, P. frederiksbergensis JAJ28^T, P. lini CFBP 5737^T, P. thivervalensis DSM 13194^T, P. corrugata DSM 7228^T, P. fluorescens DSM 50090^T, P. grimontii DSM 17515^T and P. marginalis DSM 13124^T were cultivated on trypticase soy broth agar at 28 °C for 24 h. The predominant fatty acids of all strains analysed were summed feature 3 (comprising C_16 : 1ω7c and/or C_15 : 0 iso 2-OH), C_16 : 0 and C_18 : 1ω7c (see Supplementary Table S1 in IJSEM Online). However, the proportion of the total fatty acids represented by these three fatty acids was lowest in strain JF3835^T, where they accounted for 68 % of the total fatty acids as compared with 84 % in P. lundensis DSM 6252^T, P. putida DSM 291^T, P. cichorii DSM 50259^T and P. fluorescens DSM 50090^T. Strain JF3835^T could be further distinguished from all other species of the genus Pseudomonas tested by the presence of trace amounts of C_13 : 1 (Supplementary Table S1). The presence of fatty acids C_10 : 0 3-OH, C_12 : 0 3-OH and C_12 : 0 2-OH supported the classification of strain JF3835^T as a member of the genus Pseudomonas (Oyaizu & Komagata, 1983).

Total DNA was prepared according to the procedure of Wilson (1987). DNA–DNA hybridization was performed by the LMG using the method of Ezaki et al. (1989) and a hybridization temperature of 47 °C. Values reported are the means of a minimum of four hybridizations (see Supplementary Table S2). The degree of DNA–DNA relatedness between strains JF3835^T and JF4413 was 97 %. In comparison with named species, strain JF3835^T shared the highest degree of DNA–DNA relatedness with members of the species Pseudomonas chlororaphis: P. chlororaphis subsp. aurantiaca NCIMB 10068^T, 81 %; P. chlororaphis subsp. chlororaphis DSM 50083^T, 80 % and P. chlororaphis subsp. aureofaciens DSM 6698^T, 74 %. As the recommended threshold value for the definition of a bacterial species is 70 % DNA–DNA relatedness (Wayne et al., 1987), strains JF3835^T and JF4413 are regarded as belonging to the species P. chlororaphis.

The 16S rRNA gene sequence of strains JF3835^T, JF4413 and P. chlororaphis subsp. chlororaphis DSM 50083^T were determined according to the method of Kuhnert et al. (2002). Amplification and sequencing of the atpD, carA and recA genes from strains JF3835^T and JF4413 was performed as described by Hilario et al. (2004). Phylogenetic analyses were performed in Bionumerics v. 5.1 (Applied Maths). For comparisons with related species, previously published reference sequences were used (Hilario et al., 2004; Peix et al., 2007).

Sequence analysis of the 16S rRNA gene of strains JF3835^T and JF4413 revealed 100 % sequence similarity. The gene sequence similarity values of strain JF3835^T with P. chlororaphis subsp. aurantiaca NCIMB 10068^T, P. chlororaphis subsp. chlororaphis DSM 50083^T and P. chlororaphis subsp. aureofaciens DSM 6698^T were 99.75, 99.84 and 99.4 % respectively. Comparative phylogenetic analysis with selected members of the genus Pseudomonas placed strains JF3835^T and JF4413 in the P. chlororaphis cluster (Fig. 2⇓). There was a discrepancy observed between the 16S rRNA gene sequence of P. chlororaphis subsp. chlororaphis strain DSM 50083^T (Peix et al., 2007) and strain ICMP 13613^T (Hilario et al., 2004), both based on the type strain, due to 10 base pair mismatches. We therefore resequenced the 16S rRNA gene of strain DSM 50083^T and found one base pair mismatch to GenBank entry Z76673 (DSM 50083^T) and 11 base pair mismatches to entry AJ308301 (ICMP 13613^T). The 16S rRNA gene sequence of P. chlororaphis subsp. chlororaphis strain DSM 50083^T generated in the present study was deposited at GenBank under accession number FJ544575 and was used to construct the neighbour-joining phylogenenetic tree shown in Fig. 2⇓. Additional phylogenetic trees constructed with the 16S rRNA gene sequences but using the maximum-parsimony and maximum-likelihood algorithms are available as Supplementary Figs 1 and 2 in IJSEM Online.

Additional housekeeping genes atpD, carA and recA, previously suggested to improve the resolution among species of the genus Pseudomonas, were sequenced (Hilario et al., 2004). Sequence analysis of the atpD, carA and recA genes indicated 100 % sequence similarity between strains JF3835^T and JF4413. Neighbour-joining trees constructed for each of the three genes also placed strains JF3835^T and JF4413 within the P. chlororaphis cluster (see Supplementary Figs S3–S5). A combined phylogenetic tree of the three genes clearly placed strains JF3835^T and JF4413 on a distinct branch within the P. chlororaphis cluster (Fig. 3⇓).

The results of phenotypic characterization, DNA–DNA relatedness, fatty acid analysis and 16S rRNA gene sequencing supported the classification of strains JF3835^T and JF4413 in the species P. chlororaphis. However, multilocus sequencing of selected housekeeping genes placed the strains on a distinct branch within this species. It is therefore proposed that strains JF3835^T and JF4413 represent a novel subspecies of P. chlororaphis. The name Pseudomonas chlororaphis subsp. piscium subsp. nov. is proposed.