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Taxonomic Note |
1 National Collection of Type Cultures, Health Protection Agency Centre for Infections, Colindale, London, UK
2 Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ghent, Belgium
3 BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, Ghent, Belgium
Correspondence
Peter Vandamme
Peter.Vandamme{at}UGent.be
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ABSTRACT |
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A comparison of the fatty acid profiles of seven strains assigned to P. phragmitetus is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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discerned six phena that clustered separately from the reference strains of named species. They designated these strains as Achromobacter groups A–F. These six taxa were included in the probabilistic identification matrix of Holmes et al. (1986). Subsequent studies (Holmes et al., 1988) established groups A, C and D as biovars of a single novel species, for which the name Ochrobactrum anthropi was proposed. Some genotypically (but not phenotypically) distinct strains in O. anthropi were subsequently proposed as a separate species Ochrobactrum intermedium by Velasco et al. (1998).
Thirty-two strains of Achromobacter groups B, E and F were studied by numerical analysis of their electrophoretic protein patterns (Holmes et al., 1990a). Two major clusters were observed, one comprising 30 strains of groups B and E and the other containing the two strains of group F. A similar observation was made by a study of representative strains of groups B, E and F by comparison of their DNA restriction endonuclease digest and rRNA gene probe patterns (Holmes et al., 1990b). Thus, Achromobacter groups B and E appeared to be biovars of a single unnamed taxon, which was confirmed by subsequent characterization of representative strains of the two biovars by cellular fatty acid analysis (Holmes et al., 1993).
In the present study, we describe how a comparison of 16S rRNA gene sequences indicated that the Achromobacter group B and E taxon corresponded to the organism described by Borsodi et al. (2003, 2005) as Pannonibacter phragmitetus, an alkalitolerant bacterium isolated from decomposing reed rhizomes in a Hungarian soda lake. We present the characteristics of this organism so that its role in human clinical infections may be properly determined.
The strains examined in the present study include P. phragmitetus LMG 22736T (=NCTC 13350T), Achromobacter group B strains LMG 5410 (=CL264/81), LMG 5411 (=CL299/79), LMG 5412 (=CL43/77) and LMG 5421 (=CL622/77) and Achromobacter group E strains LMG 5430 (=CL731/80) and LMG 5431 (=CL616/80). All Achromobacter group B and E strains were isolated from blood cultures in the UK. Further details of the origin of these isolates were provided by Holmes et al. (1990a). All strains were grown aerobically on ordinary nutrient agar at 37 °C.
DNA for 16S rRNA gene sequencing was prepared by heating one or two colonies at 95 °C for 15 min in 20 µl lysis buffer containing 0.25 % (w/v) SDS and 0.05 M NaOH. Following lysis, 180 µl distilled water was added to the lysate. The 16S rRNA gene was amplified using oligonucleotide primers complementary to highly conserved regions of bacterial 16S rRNA genes. The forward primer was 5'-AGAGTTTGATCCTGGCTCAG-3' (hybridizing at positions 8–27, according to the Escherichia coli numbering system) and the reverse primer was 5'-AAGGAGGTGATCCAGCCGCA-3' (hybridizing at positions 1541–1522). PCR products were purified by using a NucleoFast 96 PCR Clean-up kit (Macherey-Nagel). Sequencing reactions were performed by using a BigDye Terminator cycle sequencing kit (Applied Biosystems) and purified by using a Montage SEQ96 sequencing reaction cleanup kit (Millipore). Sequencing was performed by using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). The eight sequencing primers used are listed in Coenye et al. (1999). Sequence assembly was performed using the program AutoAssembler (Applied Biosystems). Sequences were compared and aligned with sequences retrieved from GenBank using CLUSTAL_X (Thompson et al., 1997). Phylogenetic analyses were subsequently performed using the BioNumerics 4.5 software package (Applied Maths).
Nearly complete 16S rRNA gene sequences were determined for Achromobacter group B strains LMG 5410, LMG 5411, LMG 5412 and LMG 5421 and Achromobacter group E strains LMG 5430 and LMG 5431. The 16S rRNA gene sequences of all Achromobacter group B and E strains were >99.8 % similar and about 99.5 % similar to that of P. phragmitetus LMG 22736T (GenBank accession no. AJ400704), suggesting that they represent the same species.
The G+C contents of representatives of Achromobacter groups B and E and of P. phragmitetus were determined as described by Mesbah et al. (1989). DNA was degraded enzymically into nucleosides. The nucleoside mixture obtained was then separated by HPLC using a Waters Symmetry Shield C8 column thermostatted at 37 °C. The solvent was 0.02 M NH4H2PO4 (pH 4.0) with 1.5 % acetonitrile. Non-methylated lambda phage (Sigma) was used as the calibration reference. The DNA G+C contents of strains LMG 5412 (63.6 mol%), LMG 5421 (63.2 mol%), LMG 5431 (63.0 mol%) and LMG 22736T (63.2 mol%) proved very similar and agree well with the value of 64.6 mol% determined for the type strain of P. phragmitetus by Borsodi et al. (2003).
In order to confirm the tentative identification of Achromobacter groups B and E as P. phragmitetus, we performed DNA–DNA hybridization experiments among strains LMG 5412 and LMG 5421 (Achromobacter group B), LMG 5430 and LMG 5431 (Achromobacter group E) and P. phragmitetus LMG 22736T. High-molecular-mass DNA was prepared as described by Pitcher et al. (1989) and DNA–DNA hybridizations were performed with photobiotin-labelled probes in microplate wells as described by Ezaki et al. (1989) using an HTS7000 Bio Assay Reader (Perkin-Elmer) for the fluorescence measurements. The hybridization temperature was 45 °C. Reciprocal experiments were performed for every pair of strains. All hybridization values obtained were in the range of 73 to 96 % (data not shown).
Cellular fatty acid analysis was carried out with a loopful of well-grown cells after an incubation period of 48 h. Fatty acid methyl esters were prepared, separated and identified using the Microbial Identification System (Microbial ID, Inc.) as described by Vandamme et al. (1992). All seven strains tested proved highly similar in their cellular fatty acid profiles, with dominant features 14 : 0 3-OH, 16 : 0, 17 : 0, 18 : 0, 18 : 1
7c, 18 : 0 3-OH (see Supplementary Table S1 in IJSEM Online). These results are comparable with those of Holmes et al. (1993). The observed quantitative differences may be explained by different cultivation and incubation conditions.
All strains were characterized biochemically in most of a range of 68 conventional biochemical tests by methods described previously by Holmes et al. (1975). The features presented below are based on data obtained for a total of 33 strains of Achromobacter groups B and E studied by Holmes et al. (1990a, b, 1993), including the isolates of the present study and the P. phragmitetus type strain. Colonies are circular, low-convex, entire, opaque or translucent, shiny and smooth. All strains tested were positive for acid production (in ammonium salt sugar medium) from glucose, adonitol, arabinose, cellobiose, fructose, inositol, maltose, rhamnose and xylose, catalase production, cytochrome oxidase production, 
-galactosidase production (ONPG test), growth at 37 °C, growth at room temperature (18–22 °C), growth on -hydroxybutyrate, growth on MacConkey agar, motility at room temperature (hanging drop preparation at 18–22 °C), urease production and utilization of citrate (Christensen's medium). Most strains tested (exceptions given in parentheses) were positive for acid from ethanol (LMG 5430, LMG 5431, CL515/79, CL158/81), glycerol (CL109/78), mannitol (LMG 22736T, LMG 5430, LMG 5431, CL515/79, CL221/85, CL139/87), salicin (LMG 22736T, LMG 5431), sorbitol (LMG 22736T, LMG 5430, LMG 5431, CL515/79), sucrose (LMG 22736T) and trehalose (CL221/85), aesculin hydrolysis (CL90/88), growth at 42 °C (LMG 5411, CL775/77, CL547/78, CL515/79), hydrolysis of Tween 20 (LMG 22736T, LMG 5410, LMG 5412, LMG 5430, CL 299/79, CL515/79, CL734/79, CL752/80), hydrolysis of tyrosine (LMG 5410, LMG 5411, CL373/78, CL515/79, CL734/79, CL752/80, CL26/84, CL27/84, CL28/84, CL43/84, CL137/87, CL139/87, CL221/85, CL90/88, CL329/88), lipid inclusions after growth on -hydroxybutyrate (LMG 5411, LMG 5430, LMG 5431, CL556/75, CL166/76, CL109/78, CL373/78, CL752/80, CL329/88), motility at 37 °C (hanging drop preparation) (CL166/76, CL176/87), nitrate reduction (CL90/88), nitrite reduction (LMG 5430, LMG 5431, CL90/88), oxidative metabolism in the Hugh and Leifson O-F test (LMG 5431, CL775/77, CL43/84, CL98/88) and utilization of citrate (Simmons' medium; LMG 22736T, LMG 5431, CL556/75, CL98/76, CL514/77, CL775/77, CL815/77, CL373/78, CL547/78, CL515/79, CL100/80, CL752/80). All strains tested were negative for acid production from dulcitol and raffinose, acid from 10 % (w/v) lactose, both acid and gas production from glucose in peptone water medium, casein digestion, fluorescence on King's B medium, gelatinase production (plate and stab methods), gluconate oxidation, growth at 5 °C, H2S production (by both lead acetate paper and triple-sugar iron agar methods), hydrolysis of starch and of Tween 80, lysine decarboxylase production, malonate utilization, ornithine decarboxylase production, phenylalanine deamination, pigment production, production of extracellular DNase, production of 3-ketolactose and reduction of 0.4 % (w/v) selenite. Most strains tested (exceptions in parentheses) were negative for acid from lactose (CL98/76, CL166/76, CL514/77) and from 10 % (w/v) glucose (LMG 5410, LMG 5431, CL317/77, CL514/77, CL815/77, CL734/79, CL158/81, CL221/85, CL176/87, CL98/88), arginine dihydrolase production (by Moeller's method) (LMG 5430, LMG 5431, CL556/75, CL98/76, CL515/79, CL100/80, CL137/87, CL139/87, CL221/85, CL90/88), arginine dihydrolase production (by Thornley's method) (LMG 5430, LMG 5431, CL98/76), growth on cetrimide agar (LMG 22736T, CL556/75, CL514/77, CL815/77, CL109/78, CL515/79, CL734/79, CL27/84, CL221/85, CL176/87, CL90/88, CL98/88), KCN tolerance (CL28/84, CL90/88), lecithinase production (CL373/78) and production of a brown melanin-like pigment on tyrosine agar (LMG 5411, CL556/75, CL109/78, CL547/78). These results are comparable with those obtained by Borsodi et al. (2003) except that we found that most strains hydrolysed aesculin. The type strain is somewhat atypical in that it was the only one of the 33 to fail to produce acid from sucrose and was one of only two that failed to produce acid from salicin.
In summary, the 16S rRNA gene sequence data, DNA–DNA hybridization results, G+C content determinations, cellular fatty acid analysis and biochemical characterizations presented above all confirm that Achromobacter group B, Achromobacter group E and P. phragmitetus are the same taxon. P. phragmitetus is thus an organism that has the potential to cause serious bloodstream infections in humans. A case of replacement valve endocarditis (McKinley et al., 1990) and two cases of septicaemia (Holmes et al., 1992) have been reported. P. phragmitetus belongs to the Alphaproteobacteria and is closely related to taxa such as Agrobacterium and Ochrobactrum that occur in human clinical specimens. Differential characteristics between P. phragmitetus and phenotypically similar organisms from human clinical material (Agrobacterium tumefaciens and O. anthropi) are shown in Table 1.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Borsodi, A. K., Micsinai, A., Rusznyák, A., Vladár, P., Kovács, G., Tóth, E. M. & Márialigeti, K. (2005). Diversity of alkaliphilic and alkalitolerant bacteria cultivated from decomposing reed rhizomes in a Hungarian soda lake. Microb Ecol 50, 9–18.[CrossRef][Medline]
Coenye, T., Falsen, E., Vancanneyt, M., Hoste, B., Govan, J. R. W., Kersters, K. & Vandamme, P. (1999). Classification of some Alcaligenes faecalis-like isolates from the environment and human clinical samples as Ralstonia gilardii sp. nov. Int J Syst Bacteriol 49, 405–413.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Holmes, B. & Dawson, C. A. (1983). Numerical taxonomic studies on Achromobacter isolates from clinical material. In Gram Negative Bacteria of Medical and Public Health Importance: Taxonomy–Identification–Applications, pp. 331–341. Proceedings of symposium, Lille, 25–27 May 1983. Colloque INSERM, vol. 114. Edited by H. Leclerc. Paris: INSERM.
Holmes, B., Lapage, S. P. & Malnick, H. (1975). Strains of Pseudomonas putrefaciens from clinical material. J Clin Pathol 28, 149–155.
Holmes, B., Pinning, C. A. & Dawson, C. A. (1986). A probability matrix for the identification of Gram-negative, aerobic, non-fermentative bacteria that grow on nutrient agar. J Gen Microbiol 132, 1827–1842.
Holmes, B., Popoff, M., Kiredjian, M. & Kersters, K. (1988). Ochrobactrum anthropi gen. nov., sp. nov. from human clinical specimens and previously known as Group Vd. Int J Syst Bacteriol 38, 406–416.
Holmes, B., Costas, M., Wood, A. C. & Kersters, K. (1990a). Numerical analysis of electrophoretic protein patterns of "Achromobacter" group B, E and F strains from human blood. J Appl Bacteriol 68, 495–504.[Medline]
Holmes, B., Costas, M., Wood, A. C., Owen, R. J. & Morgan, D. D. (1990b). Differentiation of Achromobacter-like strains from human blood by DNA restriction endonuclease digest and ribosomal RNA gene probe patterns. Epidemiol Infect 105, 541–551.[Medline]
Holmes, B., Lewis, R. & Trevett, A. (1992). Septicaemia due to Achromobacter group B: a report of two cases. Med Microbiol Lett 1, 177–184.
Holmes, B., Moss, C. W. & Daneshvar, M. I. (1993). Cellular fatty acid compositions of "Achromobacter groups B and E". J Clin Microbiol 31, 1007–1008.
McKinley, K. P., Laundy, T. J. & Masterton, R. G. (1990). Achromobacter Group B replacement valve endocarditis. J Infect 20, 262–263.[CrossRef][Medline]
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 109–114.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Vandamme, P., Vancanneyt, M., Pot, B. & 10 other authors (1992). Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens. Int J Syst Bacteriol 42, 344–356.
Velasco, J., Romero, C., López-Goñi, I., Leiva, J., Díaz, R. & Moriyón, I. (1998). Evaluation of the relatedness of Brucella spp. and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp. nov., a new species with a closer relationship to Brucella spp. Int J Syst Bacteriol 48, 759–768.
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