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1 Department of Soil Environmental Science, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, Taiwan
2 Department of Seafood Science, National Kaohsiung Marine University, 142 Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan
Correspondence
Wen-Ming Chen
p62365{at}ms28.hinet.net
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ABSTRACT |
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Electrophoretic protein patterns of the novel strains and related bacteria and tables showing phenotypic characteristics and antibiograms of members of the genus Trabulsiella are available as supplementary material in IJSEM Online.
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belongs to the family Enterobacteriaceae, a member of the Gammaproteobacteria. At the time of writing, the genus Trabulsiella encompasses a single species, Trabulsiella guamensis. Five similar strains, including the type strain of T. guamensis, were isolated from dust collected inside a vacuum cleaner, whereas other strains similar to T. guamensis have been isolated from soil samples (one strain) and human faeces (two strains) (McWhorter et al., 1991). Strains of T. guamensis were previously classified as Centers for Disease Control and Prevention (CDC) Enteric Group 90 (based on their biochemical similarity to strains of Salmonella). However, strains of T. guamensis did not agglutinate any of the Salmonella typing antisera O or H. Based on DNA relatedness results, McWhorter et al. (1991) clearly separated T. guamensis from 62 tested strains in the family Enterobacteriaceae. In the addendum section, these workers described two different biochemical patterns in the T. guamensis strains, but they did not name them as biogroups. The eight strains from the USA and Guam were negative for indole production, gelatin hydrolysis and aesculin hydrolysis, whereas the four strains from Germany and Malaysia were positive for these reactions, and whole genome DNA–DNA hybridization between these four strains and the type strain was 98 %. Hence, based on these data, two different biogroups of T. guamensis were identified (Lindquist & Farmer, 2005). Recently, J. J. Farmer (personal communication) found a second group of three strains in the Enterics Reference Laboratory at CDC that were H2S-negative and may represent a second species in this genus (Lindquist & Farmer, 2005). The aim of the present study was to determine the taxonomic position of two novel H2S-negative strains, Eant 3-9T and Eant 3-3, isolated from the termite gut. Eant 3-9T and Eant 3-3 were isolated from the gut of the termite Odontotermes formosanus Shiraki, collected from a decayed bamboo tree located in Pingtung County in southern Taiwan. For this purpose, the termites were surface-sterilized with 75 % ethanol for 10 s and 0.1 % (w/v) mercuric chloride for 10 min, rinsed several times in sterile distilled water, crushed and streaked on desoxycholate agar (BD Difco) plates and incubated at 25 °C. The isolates thus obtained were further maintained and subcultivated on nutrient agar (BD Difco).
Bacterial cultures were observed in the lag, exponential and stationary phases of growth under a phase-contrast microscope to ascertain the shape and motility (hanging drop method) of the cells. Gram staining was tested using a Gram Stain Set S kit (BD). The optimum pH range for growth was examined by measuring the OD595 of the culture grown in tryptic soy broth (BD Difco) and nutrient broth adjusted to various pH values (pH 4–10 at intervals of 0.5 pH units) using the appropriate biological buffers (Chung et al., 1995). Tolerance of strains to various NaCl levels was tested in nutrient broth adjusted to different NaCl concentrations (0, 0.5 % and 1.0–5.0 %, w/v, at intervals of 1.0 %). The optimum temperature for growth of the strains was examined in tryptic soy broth and nutrient broth adjusted to pH 7. Growth was examined by measuring the turbidity of cultures grown at various temperatures (4–45 °C). Growth under anaerobic conditions was determined after incubating the strains in the Oxoid AnaeroGen system. Growth was monitored by measuring the optical density of the cultures with respect to time.
Phenotypically and morphologically, strains Eant 3-9T and Eant 3-3 showed similar characteristics. Good growth was observed aerobically and anaerobically in complex media including nutrient medium, Luria–Bertani medium and tryptic soy medium, indicating that strains Eant 3-9T and Eant 3-3 were facultatively anaerobic. Cells of Eant 3-9T and Eant 3-3 were Gram-negative, motile, non-spore-forming and rod-shaped (0.5–0.7 µm in diameter and 1.0–1.5 µm in length). Eant 3-9T and Eant 3-3 formed visible circular, convex, semi-transparent colonies with entire edges. The colony size of Eant 3-9T and Eant 3-3 was 0.5–2.0 mm in diameter on nutrient agar after 48 h incubation at 28 °C. Good growth was observed at 15–42 °C, 0–5 % NaCl and pH 5–10. Optimum growth was observed at 28–35 °C, 0–1 % NaCl and pH 7.0.
Extraction of genomic DNA, PCR amplification and 16S rRNA gene sequencing was carried out as described previously (Chen et al., 2001). Sequence analysis was achieved using a DNA sequencer (ABI PRISM 310; Applied Biosystems) and sequence assembly was carried out by using the Fragment Assembly System program from Wisconsin Package 9.1 supplied by the National Health Research Institute of Taiwan. The resultant sequences were compared with available 16S rRNA gene sequences in the EMBL, GenBank and RDP II databases. Multiple sequence alignment of strains Eant 3-9T and Eant 3-3 with their closest relatives was performed using BIOEDIT software (Hall, 1999). Phylogenetic trees were inferred using the maximum-parsimony and neighbour-joining (Saitou & Nei, 1987) tree-making algorithms. An evolutionary distance matrix was generated for the neighbour-joining algorithm using the Jukes & Cantor (1969) distance model and bootstrap analysis (1000 resamplings).
Nearly complete 16S rRNA gene sequences of 1450 and 1449 nt, respectively, were obtained for strains Eant 3-9T and Eant 3-3. The 16S rRNA gene sequence similarity between Eant 3-9T and Eant 3-3 was 99.6 %. A comparison of the sequences with those of representatives of genera classified in the family Enterobacteriaceae of the Gammaproteobacteria showed that the organisms fell within the evolutionary radiation occupied by the genus Trabulsiella (Fig. 1). The overall topologies of phylogenetic trees obtained by the neighbour-joining and maximum-parsimony (data not shown) methods were similar. According to sequence similarity calculations, strain Eant 3-9T was phylogenetically most closely related to T. guamensis DSM 16940T (98.1 % similarity), Citrobacter koseri CDC 3613-63T (97.6 % similarity), Salmonella enterica subsp. enterica ATCC 9150 (97.6 % similarity), Salmonella typhimurium ATCC 13311T (97.6 % similarity), Raoultella planticola ATCC 33558T (97.0 % similarity), Enterobacter cloacae subsp. cloacae ATCC 13047T (96.7 % similarity), Klebsiella pneumoniae subsp. pneumoniae ATCC 13883T (96.6 % similarity) and Serratia liquefaciens JCM 1245T (96.6 % similarity). The high bootstrap value (100 %) provides strong support for the inclusion of strain Eant 3-9T in the genus Trabulsiella.
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between strains Eant 3-9T, Eant 3-3, T. guamensis DSM 16940T and Salmonella typhimurium ATCC 13311T. The degree of hybridization was calculated by means of triplicate experiments. The DNA–DNA relatedness value between Eant 3-9T and Eant 3-3 was 96±4 %. The high degree of DNA–DNA relatedness and the high levels of 16S rRNA gene similarity between the two isolates indicate that the two strains represent a single species. The separate species status of strain Eant 3-9T was demonstrated by the DNA–DNA hybridization values obtained with T. guamensis DSM 16940T and Salmonella typhimurium ATCC 13311T (48±5 and 14±3 %, respectively). The thermal stability of DNA was determined further by the methods outlined by Fernandez et al. (1989). A lower melting temperature (
Tm value, less than 1.0 °C) was observed between Eant 3-9T and Eant 3-3, whereas a higher melting temperature (Tm value, 6.5±1.0 °C) was observed between T. guamensis DSM 16940T and Eant 3-9T. The DNA–DNA relatedness between strain Eant 3-9T and its closest phylogenetic neighbour, T. guamensis DSM 16940T, is well below the 70 % (Tm value, 5 °C) cut-off point recommended for assignment of strains to the same genomic species (Wayne et al., 1987). Based on the above DNA–DNA relatedness and DNA thermal stability data, isolate Eant 3-9T warrants separate species status in the genus Trabulsiella.
For G+C content calculations, a DNA sample was prepared and degraded enzymically into nucleosides as described by Mesbah et al. (1989). The nucleoside mixture obtained was then separated by HPLC. The G+C contents of strains Eant 3-9T and T. guamensis DSM 16940T were 55.7±0.2 and 54.9±0.3 mol%, respectively.
Further differentiation of strains Eant 3-9T and Eant 3-3 from their closest phylogenetic neighbours was examined by protein electrophoretic patterns and fatty acid profiling. Preparation of whole-cell proteins and SDS-PAGE were carried out as described by Pot et al. (1994). Whole-cell protein profiles of strains Eant 3-9T and Eant 3-3 were highly similar, but differed from those of T. guamensis DSM 16940T and Salmonella typhimurium ATCC 13311T (Supplementary Fig. S1, available in IJSEM Online). Biomass for the preparation of fatty acid methyl esters was grown in shake flasks of nutrient broth for 24 h at 28 °C, checked for purity, harvested by centrifugation, washed twice with distilled water and freeze-dried. Fatty acid methyl esters were then prepared, separated and identified according to the instructions of the Microbial Identification System (MIDI; Microbial ID). The fatty acid compositions of strains Eant 3-9T and T. guamensis DSM 16940T are shown in Table 1. The fatty acid profile of Eant 3-9T was similar to that of T. guamensis DSM 16940T, but the fatty acid proportions differed (Table 1).
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) (Supplementary Table S1). T. guamensis DSM 16940T, Eant 3-9T and Eant 3-3 were susceptible to ampicillin (10 µg), chloramphenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), novobiocin (30 µg), rifampicin (5 µg), streptomycin (10 µg), sulfamethoxazole (23.75 µg) plus trimethoprim (1.25 µg) and tetracycline (30 µg), but were resistant to penicillin G (10 U).
Strains Eant 3-9T and Eant 3-3 and T. guamensis DSM 16940T were examined for a broad range of phenotypic properties, notably for the features known to be of value in Enterobacteriaceae systematics (Farmer et al., 1985) using conventional tests. Additional biochemical tests were performed by Biolog-GN2, API ZYM (bioMérieux) and API 20E (bioMérieux) microtest systems according to the methods outlined by the manufacturers. The results of physiological and biochemical characteristics of the strains are listed in Supplementary Table S2 and in the species description. It is apparent from the results (Table 2) that strains Eant 3-9T and Eant 3-3 can be distinguished clearly from their closest phylogenetic relative T. guamensis DSM 16940T by nitrate reduction, an H2S-negative reaction, acetoin production (Voges–Proskauer reaction) and weak tyrosine clearing activity (Table 2). Despite physiological and biochemical attributes, results of DNA–DNA hybridization, fatty acid composition and whole-cell protein profiles clearly distinguished strains Eant 3-9T and Eant 3-3 from T. guamensis DSM 16940T.
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Description of Trabulsiella odontotermitis sp. nov.
Trabulsiella odontotermitis (o.don.to.ter'mi.tis. N.L. n. Odontotermes the scientific name of a genus of termite; N.L. gen. n. odontotermitis of a termite of the genus Odontotermes).
Facultatively anaerobic, Gram-negative, motile, non-spore-forming and rod-shaped. After 24 h growth on nutrient agar at 25 °C, the mean cell size is about 0.5–0.7x1.0–1.5 µm. Growth is observed at 28, 30, 37 and 42 °C. Positive (API 20E) for catalase, ONPG test, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, acetoin production and acid production from glucose, mannitol, sorbitol, rhamnose, amygdalin and arabinose. Negative for oxidase, nitrate reduction, H2S production, urease, tryptophan deaminase, indole production, gelatinase, inositol, sucrose and melibiose. Positive (API ZYM) for alkaline phosphatase, C4 esterase, leucine arylamidase, trypsin, 
-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, 
-galactosidase, -glucosidase, -glucosidase and -fucosidase, but negative for C8 lipase, C14 lipase, valine arylamidase, -galactosidase and -glucuronidase. The following compounds (Biolog GN2) are oxidized: dextrin, glycogen, Tween 40, Tween 80, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, L-arabinose, D-cellobiose, D-fructose, D-galactose, gentiobiose, -D-glucose, maltose, D-mannitol, D-mannose, D-psicose, L-rhamnose, D-sorbitol, D-trehalose, methyl pyruvate, monomethyl succinate, acetic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, p-hydroxyphenylacetic acid, DL-lactate, D-saccharic acid, succinic acid, bromosuccinic acid, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, L-ornithine, L-phenylalanine, L-proline, L-serine, urocanic acid, inosine, uridine, thymidine, putrescine, glycerol, DL--glycerol phosphate, glucose 1-phosphate and glucose 6-phosphate. The following compounds are not oxidized: -cyclodextrin, adonitol, D-arabitol, i-erythritol, myo-inositol, -D-lactose, lactulose, D-melibiose, methyl -D-glucoside, D-raffinose, sucrose, turanose, xylitol, cis-aconitic acid, -hydroxybutyric acid, -hydroxybutyric acid, 
-hydroxybutyric acid, itaconic acid, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, malonic acid, propionic acid, sebacic acid, succinamic acid, glucuronamide, L-alaninamide, hydroxy-L-proline, L-leucine, L-pyroglutamic acid, D-serine, L-threonine, DL-carnitine, -aminobutyric acid, phenylethylamine, 2-aminoethanol and 2,3-butanediol. Utilization of L-fucose, formic acid and quinic acid is strain-specific and differs between the two strains isolated. The major fatty acids are 16 : 0, 17 : 0 cyclo, 18 : 1
7c, summed feature 2 (14 : 0 3-OH and/or 16 : 1 iso I) and summed feature 3 (16 : 17c and/or 15 iso 2-OH).
The type strain is Eant 3-9T (=BCRC 17577T=LMG 23580T). Strain Eant 3-9T and reference strain Eant 3-3 were isolated from the gut of the termite Odontotermes formosanus Shiraki from southern Taiwan. The DNA G+C contents of strains Eant 3-3 and Eant 3-9T are 55.5 and 55.7 mol%, respectively.
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REFERENCES |
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Chen, W. M., Laevens, S., Lee, T. M., Coenye, T., de Vos, P., Mergeay, M. & Vandamme, P. (2001). Ralstonia taiwanensis sp. nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient. Int J Syst Evol Microbiol 51, 1729–1735.[Abstract]
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Lindquist, J. A. & Farmer, J. J., III (2005). Genus XXXVIII. Trabulsiella. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 2, part B, pp. 827–828. Edited by D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer.
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Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
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