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Int J Syst Evol Microbiol 57 (2007), 755-760; DOI  10.1099/ijs.0.64714-0
© 2007 International Union of Microbiological Societies

Description of Pseudochrobactrum kiredjianiae sp. nov.

Peter Kämpfer1, Holger Scholz2, Birgit Huber3, Kathrin Thummes1, Hans-Jürgen Busse3, Elizabeth W. Maas4 and Enevold Falsen5

1 Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany
2 Bundeswehr Institute of Microbiology, D-80937 Munich, Germany
3 Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität, A-1210 Wien, Austria
4 National Institute of Water & Atmospheric Research Ltd, Taihoro Nukurangi Greta Point, PO Box 14-901, Kilbirnie, Wellington, New Zealand
5 Culture Collection University Göteborg, Department of Clinical Bacteriology, S-41346 Göteborg, Sweden

Correspondence
Peter Kämpfer
peter.kaempfer{at}agrar.uni-giessen.de


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A Gram-negative, rod-shaped, oxidase-positive, non-spore-forming, non-motile bacterium (strain CCUG 49584T), isolated from a seafood processing plant sample in New Zealand, was subjected to a polyphasic taxonomic study. On the basis of 16S rRNA and recA gene sequence similarities, the isolate was allocated to the genus Pseudochrobactrum. This was confirmed by fatty acid data (major fatty acids: C18 : 1{omega}7c and C19 : 0 cyclo {omega}8c), a polar lipid profile exhibiting major characteristics of Pseudochrobactrum (phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine), quinone system Q-10 and a polyamine pattern with the predominant compounds spermidine and putrescine. DNA–DNA hybridization with the type strains of the two established species of Pseudochrobactrum and physiological and biochemical data clearly differentiated the isolate from established Pseudochrobactrum species. As a consequence, this organism represents a novel species, for which the name Pseudochrobactrum kiredjianiae sp. nov. is proposed, with the type strain CCUG 49584T (=CIP 109227T).

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and recA gene sequences of strain CCUG 49584T are AM263420 and AM263419, respectively.


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The genus Pseudochrobactrum was proposed recently by Kämpfer et al. (2006)Go, comprising the species Pseudochrobactrum saccharolyticum and Pseudochrobactrum asaccharolyticum. This genus could be clearly differentiated from Ochrobactrum and from Brucella species based on its phylogenetic position and the presence of a combination of certain phenotypic traits (Kämpfer et al., 2006Go).

Strain CCUG 49584T was isolated in 1995 from stainless-steel vent covers from a seafood processing plant in Nelson, New Zealand, on standard plate count agar (Oxoid) at 30 °C. Subcultivation was done on tryptone soy agar (TSA) at 28 °C for 48 h. On this agar, the organism also grew at 15–45 °C, but not at 10 or 50 °C. Growth at 30 °C was also observed on MacConkey agar and R2A agar (all from Oxoid).

Gram-staining was performed as described by Gerhardt et al. (1994)Go. Cell morphology was observed under a Zeiss light microscope at x1000, with cells grown for 3 days at 30 °C on nutrient agar. The 16S rRNA gene was analysed as described by Kämpfer et al. (2003)Go. Phylogenetic analysis was performed using the software package MEGA (Molecular Evolutionary Genetics Analysis) version 2.1 (Kumar et al., 2001Go) after multiple alignment of data using CLUSTAL_X (Thompson et al., 1997Go). Distances (distance options according to the Kimura two-parameter model on the basis of 1360 bp) and clustering with the neighbour-joining method were performed by using bootstrap values based on 1000 replications (Fig. 1Go). The sequenced length of the 16S rRNA gene of strain CCUG 49584T was 1387 bp (GenBank accession no. AM263420). The sequence similarities with the two Pseudochrobactrum species were 99.4 % to P. saccharolyticum CCUG 33852T and 99.5 % to P. asaccharolyticum CCUG 46016T. Nucleotide sequence similarities to all established species of the genus Bartonella ranged from 94.2 to 94.3 %. Sequence similarities to established species of Brucella and Ochrobactrum were 94.6–94.8 and 93.8–94.6 %, respectively. The 16S rRNA gene-based phylogenetic tree shown in Fig. 1Go resulted from a neighbour-joining reconstruction using the Kimura two-parameter correction and 1000 resamplings for bootstrap analysis.


Figure 1
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  		Fig. 1. Phylogenetic analysis based on 16S rRNA gene sequences available from GenBank/EMBL/DDBJ (accession numbers are given in parentheses) constructed after multiple alignment of data using CLUSTAL_X (Thompson et al., 1997Go). Distances (distance options according to the Kimura two-parameter model) and clustering with the neighbour-joining method were performed by using MEGA version 2.1 (Kumar et al., 2001Go). Bootstrap percentages based on 1000 replications are given at branch points. Bar, 0.005 substitutions per nucleotide position.

 
A partial recA sequence of strain CCUG 49584T was analysed according to Scholz et al. (2006)Go. PCR was performed with primers RecA-PsOchro-f (5'-AAGGCTCTGGACGCGGCACT-3') and RecA-PsOchro-r (5'-CGCAAGGTCAGTTCAATCTCAT-3'). The similarities of recA between strain CCUG 49584T and the two other Pseudochrobactrum species were 90.5 % for P. saccharolyticum CCUG 33852T and 91.3 % for P. asaccharolyticum CCUG 46016T within 897 nt. Lower similarity values were observed with Brucella species (76.7 %), Ochrobactrum species (77.3–81.4 %), Bartonella (71.0 %) and Rickettsia felis (62.3 %) (Fig. 2Go). Strain CCUG 49584T showed several motifs within recA that are absent from Bartonella, Brucella and Ochrobactrum species, but are present in Pseudochrobactrum species (Kämpfer et al., 2006Go).


Figure 2
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  		Fig. 2. Phylogenetic analysis based on recA gene sequences (909 nt) available from GenBank/EMBL/DDBJ (accession numbers are given in parentheses) constructed after multiple alignment of data using CLUSTAL_X (Thompson et al., 1997Go). Distances (distance options according to the Kimura two-parameter model) and clustering with the neighbour-joining method were performed by using MEGA version 2.1 (Kumar et al., 2001Go). Bootstrap percentages based on 1000 replications are given at branch points. Bar, 0.1 substitutions per nucleotide position.

 
Strain CCUG 49584T formed a separate branch in the phylogenetic tree of recA sequences (897 nt) (Fig. 2Go). The tree was constructed as described above for the 16S rRNA gene.

Results of the fatty acid analysis are shown in Table 1Go. Fatty acids were analysed according to Kämpfer & Kroppenstedt (1996)Go. The fatty acid profile of strain CCUG 49584T mainly comprised C19 : 0 cyclo {omega}8c (53.0 %), C18 : 1{omega}7c (33.4 %), C18 : 0 (7.7 %) and C16 : 0 (2.8 %). A clear differentiation from the other Pseudochrobactrum species was possible based on more than 2-fold greater amounts of C18 : 1{omega}7c in the Pseudochrobactrum reference strains and more than 3.5-fold greater amounts of C19 : 0 cyclo {omega}8c in strain CCUG 49584T. A quinone system with ubiquinone Q-10 predominant, which was detected after extraction and analysis as reported by Tindall (1990)Go and Altenburger et al. (1996)Go, but using an HPLC-apparatus consisting of a JASCO PU 2080 Plus Pump and a JASCO UV-2075 Plus UV/VIS-Detector, was in agreement with the characteristics of the two established Pseudochrobactrum species. Polyamines were extracted and analysed by HPLC according to Busse & Auling (1988)Go, employing JASCO PU 2080 Plus Pump and a JASCO model 821-FP spectrofluorometric detector. Similar to P. asaccharolyticum CCUG 46016T, the polyamine pattern of strain CCUG 49584T consisted predominantly of spermidine and putrescine. Polar lipids were analysed according to Ventosa et al. (1993)Go from biomass that was grown overnight in PYE medium (0.3 % peptone from casein, 0.3 % yeast extract, pH 7.2). The polar lipid profile of strain CCUG 49584T shared the major characteristics reported recently for the two Pseudochrobactrum species (Kämpfer et al., 2006Go). It consisted of major components phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, moderate amounts of phosphatidylmonomethylethanolamine and an unknown aminolipid AL1, and small to trace amounts of three unknown polar lipids [L3, L4 and L5, which have been reported to be present in minor amounts in the two established Pseudochrobactrum species; see Supplementary Fig. S2 of Kämpfer et al. (2006)Go in IJSEM Online]. In addition, trace amounts of an unknown aminolipid were detected, which was not reported to be present in the established Pseudochrobactrum species (Kämpfer et al., 2006Go), and phosphatidyldimethylethanolamine, an unknown phospholipid and two unknown polar lipids, present in the reference species, were not detected. In contrast to Kämpfer et al. (2006)Go, reanalysis of the polar lipid profile of P. asaccharolyticum CCUG 46016T from newly grown biomass, as for strain CCUG 49584T, also revealed the absence of phosphatidyldimethylethanolamine, the unknown phospholipid, the two unknown polar lipids and the presence of trace amounts of the unknown aminolipid. These observations indicate that the presence/absence of phosphatidyldimethylethanolamine, the unknown phospholipid, the unknown aminolipid and the two unknown polar lipids may be related to the physiological conditions of the cells when harvested for polar lipid extraction. Based on these results, strain CCUG 49584T shares the polar lipid characteristics that differentiate Pseudochrobactrum species from Ochrobactrum and Brucella species (Kämpfer et al., 2006Go).


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  		Table 1. Major fatty acid compositions (%) of strain CCUG 49584T and type strains of Pseudochrobactrum and Ochrobactrum species

Strains: 1, CCUG 49584T (Pseudochrobactrum kiredjianiae sp. nov.); 2, P. asaccharolyticum CCUG 46016T; 3, P. saccharolyticum CCUG 33852T; 4, O. gallinifaecis Iso 196T; 5, O. intermedium LMG 3301T; 6, O. anthropi CIP 14970T; 7, O. grignonense DSM 13338T; 8, O. lupini LUP21T; 9, O. tritici LMG 18957T. All strains were grown on trypticase soy broth agar at 28 °C for 48 h prior to fatty acid analysis. For unsaturated fatty acids, the position of the double bond is located by counting from the methyl ({omega}) end of the carbon chain; cis and trans isomers are indicated by the suffixes c and t, respectively. –, Not detected.

 
Results of the physiological characterization are given in the species description and in Table 2Go. The methods used were the same as those described previously (Kämpfer et al., 1991Go). The organism could be clearly differentiated from other Pseudochrobactrum and Ochrobactrum species on the basis of several tests.


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  		Table 2. Physiological characteristics of strain CCUG 49584T and type strains of Pseudochrobactrum and Ochrobactrum species

Strains: 1, CCUG 49584T (Pseudochrobactrum kiredjianiae sp. nov.), 2, P. asaccharolyticum CCUG 46016T; 3, P. saccharolyticum CCUG 33852T; 4, O. gallinifaecis Iso 196T; 5, O. intermedium LMG 3301T; 6, O. anthropi CIP 14970T; 7, O. grignonense DSM 13338T; 8, O. lupini LUP21T; 9, O. tritici LMG 18957T. +, Positive; –, negative; (+), weakly positive; ND, not determined; pNA, p-nitroanilide; pNP, p-nitrophenyl. All strains (except O. lupini LUP21T, for which only the data given in the table are available) are positive for hydrolysis of L-alanine pNA and weakly positive for hydrolysis of bis-pNP phosphate. All strains (except O. lupini LUP21T) are negative for hydrolysis of aesculin, pNP beta-D-galactopyranoside, pNP beta-D-glucuronide, pNP {alpha}-D-glucopyranoside, pNP beta-D-glucopyranoside, pNP phosphoryl choline and 2-deoxythymidine-5'-pNP phosphate. All strains (except O. lupini LUP21T) are positive for assimilation of acetate, oxoglutarate, L-alanine, L-proline and L-serine. All strains are negative for assimilation of p-arbutin, salicin, putrescine, L-phenylalanine, L-tryptophan, 3-hydroxybenzoate, adipate, itaconate, mesaconate, phenylacetate, {alpha}-D-melibiose and azelate. Hydrolysis of aesculin and pNP beta-D-galactopyranoside and assimilation of oxoglutarate, L-serine, phenylacetate and {alpha}-D-melibiose were also tested using a different method by Holmes et al. (1998)Go with O. anthropi; results were in agreement with those of this study. Assimilation of adipate was also tested using a different method by Velasco et al. (1998)Go with O. intermedium; results were in agreement with those of this study.

 
DNA–DNA hybridization experiments were performed with strain CCUG 49584T and the type strains of the two established Pseudochrobactrum species using the method described by Ziemke et al. (1998)Go, except that, for nick translation, 2 µg DNA was labelled during a 3 h incubation at 15 °C. Strain CCUG 49584T showed relatively low DNA–DNA relatedness to P. asaccharolyticum CCUG 46016T (37.8 %) and P. saccharolyticum CCUG 33852T (22.8 %).

From the results of 16S rRNA gene and recA sequencing, DNA–DNA hybridization data, fatty acid analyses and from the physiological characteristics it is evident that strain CCUG 49584T is different from the two established species of Pseudochrobactrum. Hence, strain CCUG 49584T represents a novel species of the genus Pseudochrobactrum, for which we propose the name Pseudochrobactrum kiredjianiae.

Description of Pseudochrobactrum kiredjianiae sp. nov.
Pseudochrobactrum kiredjianiae (ki.red.ji.a'ni.ae. N.L. fem. gen. n. kiredjianiae of Kiredjian, named after Martine Kiredjian, a contemporary French microbiologist, for her numerous contributions to the taxonomy of Ochrobactrum and related organisms).

Shares all characteristics listed in the genus description. Good growth occurs on R2A agar, TSA, nutrient agar and MacConkey agar at 25–30 °C. Beige, translucent and shiny colonies with entire edges form within 24 h, with a diameter of approximately 2 mm. Quinone system is ubiquinone Q-10 (99 %) and Q-9 (1 %). The polar lipid profile consists of the major components phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylcholine, moderate amounts of phosphatidylmonomethylethanolamine and unknown aminolipid AL1 and trace amounts of three unknown lipids. Polyamine profile consists of spermidine [60.3 µmol (g dry weight)–1], putrescine [42.7 µmol (g dry weight)–1], 1,3-diaminopropane [2.0 µmol (g dry weight)–1] and spermine [1.5 µmol (g dry weight)–1]. Carbon source utilization and hydrolysis of chromogenic substrates (including characteristics that differentiate Ochrobactrum species) are given in Table 2Go.

The type strain is CCUG 49584T (=CIP 109227T), which was isolated in 1995 from a stainless-steel vent cover, from a seafood processing plant in Nelson, New Zealand.


   ACKNOWLEDGEMENTS
 
We thank E. R. B. Moore for helpful discussions and Gundula Will and Maria Sowinsky for excellent technical assistance. The work of B. H. and H.-J. B. was supported by a Contract-Research-Project for the Bundeswehr Medical Service.


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