Pseudomonas Lundensis, a New Bacterial Species Isolated from Meat G

INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1986, p. 339-342 Vol. 36, No. 2 0020-7713/86/020339-04$02.00/0 Copyright 0 1986, International Union of Microbiological Societies

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Pseudomonas lundensis, a New Bacterial Species Isolated from Meat G. MOLIN,' A. TERNSTROM,l* AND J. URSING2 Swedish Meat Research Institute, S-244 00 Kavlinge, and Department of Medical Microbiology, University of Lund, Malmii General Hospital, S-214 01 Malmo,2 Sweden

We propose the name Pseudomonas lundensis for a new species of gram-negative, polarly flagellated, chemoorganotrophic, rod-shaped bacteria that were isolated from refrigerated meat. Strains of P. lundensis are capable of respiratory but not fermentative metabolism; they grow at O'C, produce fluorescent pigments, catalase, and cytochrome c oxidase, and possess an arginine dihydrolase system. The mean guanine-plus- cytosine content of the deoxyribonucleic acids (DNAs) of 12 strains was 59.1 mol% (standard deviation, 0.7 mol%). Numerical phenotypic studies performed with 60 strains and DNA-DNA hybridization experiments with 12 strains (data separately published) revealed a tight phenotypic and genotypic cluster. P. lundensis is related to Pseudomonas fragi and the Pseudomonasfluorescens DNA-DNA homology group, and characteristics which differentiate these organisms and Pseudomonas aeruginosa are given. The type strain is strain 138 of Molin and Ternstrom (= 573 = CCM 3503); it was isolated from beef and has a guanine-plus-cytosinecontent of 60 mol%.

In a numerical study of psychrotrophic Pseudomonas more than 80% intragroup relatedness at 70°C, and the strains isolated from refrigerated meat, Molin and Ternstrom members of this group were 50% or less related to type (3) described a homogeneous cluster of organisms constitut- strains of other Pseudomonas species. One strain (strain ing a major phenon, which was designated cluster 2. It was 132) was related to the reference strain at a level of 70%, and suspected that these strains might represent a new Pseu- the remaining two strains (strains 102 and 99) were related to domonas species. Similar strains were also isolated from the reference strain at levels of about 50%. The strains tested refrigerated, spoiled meat in Great Britain (6) and from and the levels of DNA relatedness with P. lundensis CCM spoiled fish (G. Molin and I.-M. Stenstrom, unpublished 3503T (T = type strain) are shown in Table 2. data). The guanine-plus-cytosine content of strain CCM 3503T In a second numerical study, Molin and Ternstrom (4) was 60 mol%, and the mean value for 10 strains was 59 mol% found that the organism in question constituted the prime (range, 58 to 60 mol% [Ursing, in press]). spoilage organism for 20% of cold stored beef and pork in Description of Pseudomonas lundensis. Pseudomonas Sweden. A total of 40 strains, including 2 cluster 2 strains of lundensis sp. nov. (1und.en'sis. M.L. adj. lundensis referring Molin and Ternstrom (3), formed a cluster at a similarity to the city of Lund, Sweden). An extensive phenotypic level of 78% when the Jaccard similarity coefficient and characterization of the species and lists of strains have been group average clustering were used. This cluster merged published by Molin and Ternstrom (3, 4). A detailed geno- with a Pseudomonas fragi complex at the 65% level. A typic characterization has been published by Ursing (in centroid analysis confirmed that the nearest cluster was the press). Two peripheral strains of cluster 2 of Molin and P. fragi complex, followed by clusters corresponding to the Ternstrom (3), strains 99 and 102, are not considered to various biovars of Pseudomonasjluorescens and Pseudomo- belong to P. lundensis. These strains did not cluster with nas aureofaciens-Pseudomonas chlororaphis. other strains when the Jaccard coefficient and the clustering Thus, a new group of Pseudomonas strains can be differ- level definition of Molin and Ternstrom were used (4); they entiated from previously recognized species of the genus are less vigorous, exhibit several atypical reactions, and, both phenotypically and genotypically (see below). We furthermore, were not included in P. lundensis when genetic propose the new species Pseudomonas lundensis to accom- criteria were used (Ursing, in press). The description below modate these strains. is based on 60 strains. Phenotypic characteristics that are useful for differenti- Cells grown in batch cultures are typically 0.7 to 1.0 by 1.5 ating the new species from P. fiuorescens, P. fragi, Pseu- to 3.0 pm, but cells in their natural habitat on meat and cells domonas putida, and Pseudomonas aeruginosa are given in grown in continuous cultures are frequently quite small (0.5 Table 1. to 0.7 by 1.0 to 1.5 km). The cells are asporogenous, gram Genotypic classification. Twelve strains of cluster 2 of negative, rod shaped, and motile at 25°C by means of a polar Molin and Ternstrom (3) were included in a deoxyribonu- flagellum. Colonies on nutrient agar (Oxoid Ltd.) are 1 to 5 cleic acid (DNA)-DNA hybridization study (Ursing, Curr. mm in diameter after 3 days of incubation at 25"C, circular, Microbiol., in press). Nine of the isolates formed a group and smooth. Pigments are usually not formed on nutrient with agar, but a greenish tinge due to the formation of fluorescent pigments may be observed. Fluorescent pigments are pro- * Corresponding author. duced by all strains on King B agar, although detection of

339 340 NOTES INT. J. SYST. BACTERIOL.

TABLE 1. Differentiation of P. lundensis from the P.fragi complex, the P.Juorescens-P. putida complex, and P. aeruginosa P. fragi P. fragi P. lundensis SenSu stricto subclusters B1 Characteristic P. jluorescens P. putidad P. aeruginosad (n = 60>" through B3 and allied taxad.e (n = 183)b (n = 33)c Growth at 0°C + (100) + (+) Fluorescent pigments Visual scoring D (15) (+) + Fluorimetric assay D (15) + + Gelatin liquefaction D (39) + - Acid from maltose + (100) -E -R Assimilation of 2-Aminodeox yglucose D (79) + + - - D- Arabinose + (94) - Arabitol + (97) + DL-Carnitine + (92) + + Creatine + (94) - V Deox ycholate + (97) - - D-Galactonate + (100) + D D-Glucuronate + (100) D + 4-H ydrox ybenzoate D (79) + + H ydrox y-L-proline ( + 1 (87) (+I + Inosine + (97) + D meso-Inositol + (100) + - Malonate - (0) + + D-Mannitol + (95) + V Mucate + (97) + + D-Quinate + (100) + + D-Saccharate + (100) D + D-Xylose + (100) D V

~~~~~ ~~ ~ Data from references (3 and 4) and from Molin and Stenstrom (unpublished data). Data from references 3 and 4. Data from reference 4. Data from references 3-5. Includes P. aureofaciens and P. chlororaphis. f +, 90 to 100% of the strains are positive; (+), 75 to 89% of the strains are positive; v, 26 to 74% of the strains are positive; (-), 11 to 25% of the strains are positive; -, 0 to 10% of the strains are positive; D, the percentage of positive strains differs between biovars. The numbers in parentheses are the percentages of isolates showing positive reactions. p In contrast with our results, Hugh and Gilardi (2) state that 20 to 40% of the P.jluorescens and P. putida strains are positive for this characteristic.

pigments may require a fluorimetric assay. Metabolism is (strain 478), D-fructose, fumarate, D-gluconate (strains 321, respiratory and never fermentative. Oxygen is the terminal 322, 323, and 325), D-glucose, L-glutamate, L-glutamine, electron acceptor; nitrate does not serve as an alternate glycerate (strain 573T), glycerol (strain 478), heptanoate, electron acceptor. The temperature range for growth is L-histidine, meso-inositol (strains 137, 358, 424, 425, 433, usually 0 to 33"C, but 30% of the strains are capable of slow and 436), L-lactate (strains 313 and 326), L-leucine (strain growth at 37°C. The optimal growth temperature is 25°C. 359), L-malate (strains 8 and 178), L-ornithine, 2- There is no growth at pH 4.5. No growth factors are okoglutarate, pelargonate, L-phenylalanine, L-proline, required. No flocks with dendritic outgrowths are formed. propionate, putrescine, pyruvate, succinate, DL-tyrosine, The organism is strictly chemoorganotrophic. Poly-P-hy- and m-valine (strain 359). The assimilation of the following droxybutyrate is not accumulated as a carbon reserve mate- substances is variable (the percentages of positive strains are rial. Arginine dihydrolase is present. The cells are catalase indicated in parentheses): D-arabinose (77%), benzoate and cytochrome oxidase positive. All strains except strains (70%), carnosine (32%), creatine (78%), deoxycholate (85%), 134, 313,436, and 500 liquefy gelatin. No strain except strain D-galactose (13%), DL-hydroxybutyrate (16%), L-isoleucine 469 produces lipase against Tween 80. All strains produce (89%), D-malate (21%), D-ribose (43%), sarcosine (89%), and acid but no gas from L-arabinose, D-galactose, D-glucose, taurocholate (88%). Assimilation of butyrate, caproate, L- D-mannose, D-ribose, cellobiose, maltose, melibiose, and lysine, D-mannose, and trehalose is slow, and the results D-xylose; the last four sugars do not serve as single-carbon depend on the incubation time. sources in assimilation tests. The characteristics and substances assimilated by less Production of acid from glycerol and L-rhamnose varies ihari 10% of the strains are given below in the type strain between strains, as does the production of urease and description. proteolytic attack of egg yolk and casein. The mean guanine-plus-cytosine content of the DNAs of The following substances are assimilated by at least 90% 10 strains is 59 mol% (range, 58 to 60 mol%). of the strains within 7 days of incubation at 25°C (negative Strains 338 and 385 of Molin and Ternstrom (4) have been strains are indicated in parentheses): acetate, L-a-alanine, deposited in the Czechoslovak Collection of Microorga- p-alanine, 4-aminobutyrate, 2-aminoethanol (strain 178), 5- nisms. aminopentanoate, L-arabinose (strain 43, L-arginine, L- Description of the type strain. Type strain 138 (= 573 = asparagine, L-aspartate, betaine (strains 65 and 137), citrate CCM 3503) was isolated from beef. Most of the tests (see VOL. 36, 1986 NOTES 341 below) were performed on three different occasions in two TABLE 2. Levels of DNA relatedness of P. lundensis CCM laboratories. 3503T to Pseudornonas strains isolated from meat by Molin and Cell characteristics. The cells are gram-negative, motile by Ternstrom and to different type strains of Pseudomonas spp." means of one polarly inserted flagellum (although no statis- Level of relative tical evaluation has been performed), 0.7 to 0.8 by 1.5 to 3.0 binding (%) with pm, asporogenous, and rod shaped. Source of unlabeled DNA labeled DNA from Colonial characteristics. Colonies on nutrient agar are strain CCM 3503T at: smooth, circular, and nonpigmented. They are nonhemolytic 70°C 85°C on ox blood agar and do not glide on cytophaga agar. Biochemical and physiological characteristics. P. lundensis CCM 3503T 100 100 Accumula- P. lundensis 128 92 a3 tion of poly-P-hydroxybutyrate as intracellular inclusions P. lundensis 131 90 78 does not occur. The temperature range for growth is 0 to P. lundensis 178 89 80 33"C, with optimal growth at 25°C. Growth occurs in the P. lundensis 8 88 91 absence of NaCl, but at least 6% NaCl is tolerated when cells P. lundensis 136 88 90 are grown on plates and approximately 5% NaCl (but not 6% P. lundensis 139 87 74 NaC1) is tolerated when cells are grown in broth. P. lundensis 45 86 72 Arginine dihydrolase, catalase, and cytochrome c oxidase P. lundensis 129 84 93 P. lundensis 135 84 88 are produced. A fluorescent pigment (pyoverdine) is pro- P. lundensis 65 84 82 duced on King B medium. Phenazine pigments are not P. lundensis 132 70 45 produced on King A medium. Growth occurs on Simmons Pseudomonas sp. strain 102 52 17 citrate agar, hippurate is hydrolyzed, and tyrosine is decom- Pseudomonas sp. strain 99 46 10 posed. Gelatin is liquefied, although this capability seems to P. chlororaphis DSM 50083T 51 11 be unstable after subculturing. Small amounts of proteases P.fragi ATCC 4973T 48 14 against casein are also produced, but the standard method P. aureofaciens ATCC 13985T 42 NDb (1) frequently fails to detect them. Action on egg yolk is P. putida DSM 502Q2T 39 ND characterized by a weak egg yolk reaction and a definite P.fluorescens ATCC 13525T 35 ND P. aeruginosa CCM 19WT 25 ND clearing zone. There is no action against DNA or ribonucleic Pseudomonas delafieldii DSM 50403T 8 ND acid. No lipases against Tween 20, Tween 40, Tween 60, Tween 80, Tween 85, or tributyrin are produced. a Data from Ursing (in press). Also see reference 3. There is no growth on alginate and pectate agar; phenyl- ND, Not determined. alanine is not deaminated; lysine and ornithine are not decarboxylated; esculin and starch are not hydrolyzed; aminopentane, DL-2-aminopentanoate (norvaline), 2- hydrogen sulfide (triple sugar iron), indole, phosphatase, and aminotoluene, 3-amino-~-tyrosine,amygdalin, anthranilate, urease are not produced; nitrate is not reduced to nitrogen D-arabitol, arbutin, ascorbate, azelate, benzylformate, 2,3- gas; trimethylamineoxide is not reduced; extracellular butanediol, 2-butanol, DL-carnitine, cellobiose, citraconate, polysaccharides are not synthesized; and methyl red and L-citrulline, creatinine, crotonate, L-cysteine, L-cystine, de- Voges-Proskauer reactions are negative. oxycholate, dextrin, i-erythritol, esculin, 1,2-ethanediol, Acid without gas is produced aerobically but never ethanol, D-fucose, galactitol, D-galactonate, D-galactose, anaerobically from L-arabinose, cellobiose, D-galactose, D- geraniol, glucitol, D-glucose 6-phosphate, D-glucuronate, glucose, maltose, D-mannose, melibiose, D-ribose, and D- glycine, glycolate, l-hexanol, hippurate, histamine xylose on modified Hugh-Leifson medium (4). Acid is not diphosphate, 2-hydroxybenzoate, 3-hydroxybenzoate, 4- produced from amygdalin, dextrin, D-fructose, galactitol, hydroxybenzoate, 3-hydroxybutanone (acetoin), 3-hydroxy- glucitol, glycogen (oyster), rneso-inositol, inulin, lactose, 3-met h y lglu t arat e , 1- h y d rox y naph t h ale ne , hydroxy-L - D-mannitol, melezitose, raffinose, ribitol, salicin, L-sorbose, proline, inosine, inulin, itaconate, kynurenate, lactose, starch, sucrose, trehalose, or xylitol. No significant amount lactulose, laevulinate, D-lyxose, maleate, malonate, of acid (no zone) is produced from glycerol, although the maltose, mandelate, D-mannitol, melezitose, melibiose, bacterial growth may turn yellow. The data for acid produc- menthol, methanol, L-methionine, 3-methylbutanoate (iso- tion from L-rhamnose are conflicting. valerate), 3-methyl-l-butanol, cx-methyl-D-glucoside, a- Assimilation of carbon compounds. The following carbon methyl-D-mannoside, 2-methyl-2,4-pentanediol, 2- sources are assimilated: acetate, D-alanine, L-alanine, methylpropan-1-01, mucate, nicotinate, oxalate, palmitate, p-alanine, 4-aminobutyrate (GABA), 2-aminoethanol, 5- pectate, l-pentanol, phenol, phenylacetate, phthalate, aminopentanoate, D-arabinose, L-arabinose, L-arginine, L- pimelate, propane-l,2-diol, l-propanol, 2-propanol, 2,6- asparagine, L-aspartate, benzoate, betaine, butyrate, capr- pyridine decarboxylate, pyridoxal, quinate, raffinose, L- ate, caproate, caprylate, carnosine, citrate, creatine, D- rhamnose, ribitol, riboflavine, D-saccharate, salicin, sebac- fructose, fumarate, D-gluconate, D-glucose, L-glutamate, ate, L-serine, L-sorbose, starch, suberate, sucrose, D- L-glutamine, glutarate, glycerol, heptanoate, L-histidine, tartrate, L-tartrate, meso-tartrate, taurine, testosterone, ~~-3-hydroxybutyrate,rneso-inositol, isoleucine, L-lactate, trigonelline, tryptamine, D-tryptophan, L-tryptophan, L-leucine, L-lysine, L-malate, D-mannose, L-ornithine, 2- turanose, valerate, xylitol, D-xylose, and L-xylose. oxoglutarate, pelargonate, L-phenylalanine, propionate, L- The data for the assimilation of l-butanol, D-malate, and proline, putrescine, pyruvate, D-ribose, sarcosine, trehalose are in conflict. spermine, succinate, taurocholate, DL-tyrosine, and DL- The guanine-plus-cytosine content of the DNA of the type valine. strain is 59.7 mol% (standard deviation, 0.3 mol%). The following carbon sources are not assimilated: acetamide, cis-aconitate, adipate, alginate, l-aminobutane, We thank Birger Bergh, Department of Classical Studies, Univer- ~~-2-aminobutyrate, ~~-3-aminobutyrate, 2-amino- sity of Lund, Sweden, for his kind help with the latinization of the deoxyglucose, DL-2-aminohexanoate (norleucine), 1- epithet. 342 NOTES INT. J. SYST.BACTERIOL.

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