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Brackiella Oedipodis Gen. Nov., Sp. Nov., Gram- Negative, Oxidase-Positive Rods That Cause Endocarditis of Cotton-Topped Tamarin (Saguinus Oedipus)

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Brackiella Oedipodis Gen. Nov., Sp. Nov., Gram- Negative, Oxidase-Positive Rods That Cause Endocarditis of Cotton-Topped Tamarin (Saguinus Oedipus) International Journal of Systematic and Evolutionary Microbiology (2002), 52, 179–186 Printed in Great Britain Brackiella oedipodis gen. nov., sp. nov., Gram- negative, oxidase-positive rods that cause endocarditis of cotton-topped tamarin (Saguinus oedipus) 1 Laboratorium voor Anne Willems,1 Helga Gilhaus,2 W. Beer,3 Henriette Mietke,4 Microbiologie, Faculteit 5 3 3 3 Wetenschappen, H. R. Gelderblom, Ba$ rbel Burghardt, W. Voigt and R. Reissbrodt Universiteit of Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium Author for correspondence: R. Reissbrodt. Tel: j49 3943 679 258. Fax: j49 3943 679 207. e-mail: reissbrodtr!rki.de 2 Deutsches Primatenzentrum GmbH, Kellnerweg 4, D-37077 Go$ ttingen, Germany A Gram-negative, oxidase-positive, rod-shaped bacterium isolated from the heart of a cotton-topped tamarin was characterized by 16S rDNA sequence 3 Robert Koch-Institut, Wernigerode, Burgstr. 37, analysis, SDS-PAGE of whole-cell proteins, fatty acid analysis and biochemical D-38855 Wernigerode, tests. Outer-membrane proteins, iron-regulated outer-membrane proteins, Germany lipopolysaccharides and siderophore production were studied. On the basis of 4 Sa$ chsische Landesanstalt the results, the organism belongs to the β-Proteobacteria where it forms a fu$ r Landwirtschaft, Gustav- separate line of descent, for which a novel genus and species are proposed, Ku$ hn-Str. 8, D-04159 T T T Leipzig, Germany Brackiella oedipodis (LMG 19451 l DSM 13743 l NCIMB 13739 ). Nearest phylogenetic neighbours of the new genus are Taylorella, Pelistega, Bordetella, 5 Robert Koch-Institut Berlin, Nordufer 20, D-13353 Alcaligenes and Achromobacter. Berlin, Germany Keywords: Brackiella oedipodis gen. nov., sp. nov., new genus, endocarditis INTRODUCTION ences; even minor environmental changes may cause transient diarrhoea. The most dramatic diseases of The German Primate Center Go$ ttingen (GPC) was captive cotton-topped tamarins are mucoid colonic established in 1978 as a biomedical institute for cancers and chronic colitis, both of unknown cause. research on non-human primates. The centre is also Like all callitrichids, they are prone to mesangioproli- engaged in the breeding and preservation of certain ferative nephropathies. Remarkable bacterial infec- highly endangered primate species that are not in- tions of the GPC cotton-topped tamarin colony in the tended for experimental use. One of these endangered past have been repeated clusters of Bordetella bron- species is the cotton-topped tamarin (Saguinus oedi- chiseptica bronchopneumonias, occasional Erysipelo- pus), a small neotropical primate of approximately thrix rhusiopathiae septicaemias or Yersinia pseudotu- 300–400 g body mass. The natural habitat of this berculosis intestinal infections. primate is in the secondary forests of north-western Colombia. Habitat destruction by extensive defor- During a routine post-mortem necropsy investigation estation has resulted in the dramatic decline of the wild of the heart of a cotton-topped tamarin, which had S. oedipodis populations. suddenly died during a tooth extraction, an unknown Gram-negative, oxidase-positive, rod-shaped bac- Since 1978, the GPC has housed a colony of approxi- terium was isolated. The captive-bred male tamarin mately 60 cotton-topped tamarins with satisfactory had not previously displayed any obvious signs of breeding success. The animals are kept strictly indoors disease when it died aged 10 years, 4 months and 12 d. in small family groups for breeding purposes. Cotton- It had lived behind a barrier system at 25–27 mC and topped tamarins are very sensitive to outside influ- 70% humidity and was fed on a diet of commercial marmoset pellets, enriched by cream cheese and a ................................................................................................................................................. variety of fruits depending on the season. During its Abbreviations: FT-IR spectra, Fourier-transform infrared spectra; IROMP, iron-regulated outer-membrane protein; LPS, lipopolysaccharide; OMP, life, it suffered from several bouts of diarrhoea, outer-membrane protein. common with all tamarins. When such infections were The EMBL accession number for the 16S rDNA sequence of Brackiella shown to be associated with Giardia lamblia or oedipodis LMG 19451T is AJ277742. Campylobacter, they were successfully treated with 01670 # 2002 IUMS 179 A. Willems and others antibiotics or probiotics (Symbioflor 1; Symbio- suspended in distilled water. Negative staining of the pharm). The monkey sired several offspring, the last bacteria followed routine techniques using 400 mesh copper ones being 40 d before its sudden death. grids, covered with Pioloform, reinforced with a 15 nm layer of carbon and made hydrophilic before use by a glow The bacterium isolated from the heart tissue could not discharge (Gelderblom et al., 1985). Cells were adsorbed be assigned to any of the known bacterial species or from a 50 µl droplet of the bacterial suspension on the grids genera. In this paper, isolation and characterization and negatively contrasted using 1% uranyl acetate and of this organism by 16S rDNA analysis, analysis evaluated at 80 kV under a Zeiss EM 10 A transmission of whole-cell proteins by SDS-PAGE, fatty acid ana- electron microscope. Measurements were made on the lysis, characterization of the outer-membrane pro- electron microscopy negatives using a 10-fold magnifying lens. teins (OMPs), iron-regulated outer-membrane proteins (IROMPs) and siderophore production as well as Phenotypic characterization. Physiological and biochemical lipopolysaccharides (LPSs) and Fourier-transform IR characteristics were investigated by classical methods (FT-IR) spectra are reported. Based on these findings, (Murray et al., 1999; Vandamme et al., 1998) by the Biolog creation of a novel genus and species for this organism, identification system (Micro-Station system release 3.50; GN MicroPlate) and by the 47 biochemical reactions of the Brackiella oedipodis gen. nov., sp. nov., in the β- Bactid-Identification system for Enterobacteriaceae kindly Proteobacteria is proposed. provided by J. J. Farmer, III, Centers for Diseases Control and Prevention, Atlanta, GA, USA. For all test systems, METHODS bacterial strains that are known to give either positive or negative reactions were included to verify test conditions. Isolation of the organism. Dissection of the dead cotton- The tests were repeated at least twice. topped tamarin was performed immediately after its sudden Analysis of proteins by SDS-PAGE. Bacteria were grown on death according to the standardized GPC protocol. In this buffered nutrient agar at 37 mC for 48 h. Cellular protein case, bacteriological examination was limited to the heart extracts were prepared as described previously (Pot et al., only, because the abdominal cavity was opened without 1994). SDS-PAGE, digitization, normalization and numeri- sterile precautions assuming an intestinal neoplastic con- cal analyses of the protein patterns were performed with dition from the colony and individual anamnesis. That 4.0 software (Applied Maths, Kortrijk, Belgium) assumption, however, was not confirmed by the necropsy as described by Pot et al. (1994). results. Instead, at thoracotomy, cardiomegaly and chronic epicarditis were evident with at least the latter requiring Sequencing and phylogenetic analysis of the 16S rDNA. A bacteriological examination. The cardiac surface was heat- large part (corresponding to positions 39–1521 of the E. coli sterilized immediately following thoracotomy, the left ven- rDNA) of the 16S rRNA gene sequences was amplified by tricle was opened using a sterile scalpel and a loop of cardiac PCR using conserved primers 5h-CTGGCTCAGGAYGA- blood was streaked onto the surface of a Columbia agar ACGCTG (19–38) and 5h-AAGGAGGTGATCCAGCCG- plate for initial cultivation and purification. Cultivation CA (1522–1541). The PCR product was purified using a under aerobic conditions was at 37 mC for 24 h. Qiaquick PCR purification kit (Qiagen) and sequenced using conserved primers and a BigDye Dideoxy Terminator Cycle Bacterial strains. Achromobacter xylosoxidans subsp. xylo- Sequencing kit (PE Biosystems) and an ABI 310 Genetic soxidans DSM 2402T, Alcaligenes defragrans DSM 12141T, T Analyzer (PE Biosystems) according to the manufacturer’s Bordetella avium DSM 11332 , Bordetella parapertussis instructions. The consensus sequence was constructed using DSM 13415T, Alcaligenes faecalis subsp. faecalis DSM T T the software (PE Biosystems). A 30030 and Taylorella equigenitalis DSM 10668 were search was performed to identify the sequences most similar purchased from the Deutsche Sammlung fu$ r Mikro- to the new data. Sequences of related taxa were retrieved organismen und Zellkulturen (DSMZ), Braunschweig, Ger- from the EMBL database for comparison. Sequences were many. The siderophore indicator strains Escherichia coli AB aligned using the program of the GCG software 2847, Salmonella typhimurium enb-7, E. coli LG 1522, (Devereux et al., 1984) and phylogenetic analyses were Pseudomonas aeruginosa PAO 6609 and Microbacterium performed using the package (Van de Peer & De (formerly Aureobacterium) flavescens JG-9 were taken from Wachter, 1994). Distances were calculated using the Kimura the stock collection of the Robert Koch-Institut Werni- correction and clustering was performed with the neighbour- gerode. joining algorithm. Bootstrap analysis was performed using Bacterial growth and growth media. To examine bacterial 500 replications.
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