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Department of Molecular and Cell Biology, University of Cape Town, Private Bag 1, Rondebosch, 7701, Cape Town, South Africa
Correspondence
Paul R. Meyers
pmeyers{at}science.uct.ac.za
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Published online ahead of print on 8 October 2004 as DOI 10.1099/ijs.0.63359-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Actinomadura napierensis B60T is AY568292.
A more complete neighbour-joining tree for strain B60T and closely related Actinomadura species is available as a supplementary figure in IJSEM Online.
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). The problem is exacerbated by the fact that the country has a low rate of treatment success (65 %), which has resulted in a 14 % increase in multidrug-resistant TB (MDR-TB) between 2001 and 2002 (WHO, 2004). The TB crisis is further compounded by human immunodeficiency virus (HIV) co-infection, which is the greatest risk factor for the progression from latent infection with Mycobacterium tuberculosis to the active disease. TB is the leading cause of death among people who are HIV positive in South Africa (WHO, 2000). New drugs are needed to combat the threat to public health that MDR-TB strains pose.
The genus Actinomadura is one of four genera belonging to the family Thermomonosporaceae (Stackebrandt et al., 1997; Zhang et al., 1998). Although the genus is not well recognized as a producer of antibiotics, several antibiotics (of diverse structural classes) have in fact been described from Actinomadura species (http://www.nih.go.jp/
jun/NADB/search.html).
An actinomycete, designated strain B60T, was isolated from cultivated soil collected in the town of Napier, Western Cape province, South Africa, in 2002 (34° 27·736' S 19° 54·003' E). One millilitre of sterile distilled water was added to 0·1 g soil, vortexed vigorously for 1 min and allowed to stand for 5 min. Serial 10-fold dilutions of this soil suspension were prepared in sterile distilled water and 0·1 ml volumes were plated on Middlebrook 7H9 agar (Difco Laboratories), Czapek solution agar (CZ) (Atlas, 1993) and yeast extract–malt extract agar (ISP 2) plates containing cycloheximide (100 µg ml–1). Plates were incubated at 28 °C for 14 days. Tests for morphological and physiological characterization of strain B60T were carried out using CZ agar and using various media and culture methods described by Shirling & Gottlieb (1966). All characterization test cultures were incubated for 14 days at 28 °C. The diaminopimelic (DAP) acid isomer and whole-cell sugar pattern were analysed using the methods described by Hasegawa et al. (1983). Analysis of phospholipids was performed according to the method of Komagata & Suzuki (1987). Fatty acid methyl esters were analysed according to the method described by Chou et al. (1998). The DNA G+C content was determined using the method of Mandel & Marmur (1968). Determination of mycolic acids was carried out using the method described by Minnikin et al. (1975).
Antibiotic activity was initially determined by agar overlay tests. Strain B60T was stab inoculated into ISP 2, Middlebrook 7H9 agar and CZ agar and incubated for 14 days at 28 °C. Antibiotic activity was tested against Escherichia coli ATCC 25922, Enterococcus faecium (a VanA vancomycin-resistant clinical isolate) and Mycobacterium aurum A+, overlaid in 6 ml Luria sloppy agar (Sambrook et al., 1989). Antibiotic extraction and purification was performed on a 14-day broth culture of strain B60T grown at 30 °C in 500 ml Hacène–Lefebvre medium [comprising (l–1) 5·0 g glucose, 10·0 g malt extract, 2·0 g yeast extract, 1·0 g NaCl (pH 7·0); Hacène & Lefebvre, 1995] with agitation. The culture was filtered through a coffee filter and the culture filtrate was extracted with 200 ml ethyl acetate at room temperature with continuous stirring for 24 h. The ethyl acetate fraction was concentrated to 10 ml and applied to a 10 ml glass column containing silica gel 60 (Merck). The sample was eluted with benzene/ethyl acetate (70 : 30, v/v). Fractions (2 ml) were collected, concentrated 10-fold and tested by bioautography on silica gel 60 TLC plates (Merck) for antibiotic activity against the test bacteria (Betina, 1973). Fractions that exhibited activity were pooled and concentrated a further 10-fold. Purity of the antibiotic was assessed by TLC in benzene/ethyl acetate (70 : 30, v/v) followed by cerium ammonium sulphate oxidation to detect organic molecules. Cerium ammonium sulphate (63 g cerium ammonium sulphate l–1, 30 ml H2SO4 l–1) was lightly applied to the TLC plate, after which the plate was heated at 110 °C for 10 min. The partially purified, concentrated antibiotic was spot-tested on TLC plates against M. tuberculosis H37Rv (ATCC 27294) over the range 0·3–20·0 µg per spot. The spot tests against M. tuberculosis H37Rv were performed in a Biosafety Level 3 laboratory.
Strain B60T was isolated on Middlebrook 7H9 agar. It produced grey aerial and brown–grey substrate mycelia on this medium. Strain B60T was rapidly identified as belonging to one of four genera Actinomadura, Actinocorallia, Saccharothrix or Spirillospora by the method of Cook & Meyers (2003). Based on an examination of colony morphology using a light microscope, the non-fragmenting, asporangiate strain B60T could not belong to the genus Saccharothrix, which exhibits aerial and substrate mycelium fragmentation (Labeda et al., 1984), or the sporangiate genus Spirillospora (Goodfellow, 1989).
The 16S rRNA gene was amplified by PCR using primers F1 and R5 (Cook & Meyers, 2003). These primers were used to amplify a nearly full-length 16S rRNA gene sequence. Using BLAST analysis of the 1346 bp 16S rRNA gene sequence of strain B60T revealed high sequence similarity to species belonging to the genus Actinomadura. The highest similarity (all 97 %) was found to Actinomadura latina DSM 43382T, Actinomadura madurae DSM 43067T, Actinomadura meyerae A288T and Actinomadura formosensis DSM 43997T. A phylogenetic tree, based on neighbour-joining analysis (Saitou & Nei, 1987) of Actinomadura 16S rRNA gene sequences, is shown in Fig. 1, and an extended tree is available as supplementary material in IJSEM Online. Given the low similarity values, DNA–DNA hybridization was not considered necessary to confirm that strain B60T represents a novel species of Actinomadura (Stackebrandt & Goebel, 1994; Keswani & Whitman, 2001).
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shows a comparison of the phenotypic characteristics of strain B60T and the three most closely related species based on the neighbour-joining analysis.
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) and included diphosphatidylglycerol, phosphatidylinositol and phosphatidylglycerol. Hexadecanoic acid (28 % of total fatty acids) and 10-methyloctadecanoic acid (tuberculostearic acid, 36 %) were the major fatty acids. The G+C content of the DNA of strain B60T was 70·0 mol%. Mycolic acids were not detected. Strain B60T exhibited strong antibacterial activity against M. aurum A+ on Middlebrook 7H9 agar. No activity was detected against Escherichia coli ATCC 25922 or Enterococcus faecium (vancomycin-resistant) on any of the three agar media tested. A purity assessment of the antibiotic by cerium ammonium sulphate treatment showed one spot with the same Rf value as the antibacterial activity spot. This partially purified antibiotic exhibited activity against all three reference bacteria by the more sensitive bioautographic analysis. The minimal inhibitory concentrations determined were: Escherichia coli 0·4 µg, Enterococcus faecium 0·3 µg and M. aurum 0·3 µg. Strain B60T showed no activity against M. tuberculosis H37Rv up to 20 µg per spot.
Based on the rapid identification method of Cook & Meyers (2003), the presence of genus-specific chemotaxonomic markers and its phylogenetic position, strain B60T is a member of the genus Actinomadura. Although phylogenetically related to Actinomadura latina DSM 43382T, Actinomadura madurae DSM 43067T and Actinomadura meyerae A288T, B60T is distinguished from these species based on phenotypic differences. The name Actinomadura napierensis sp. nov. is proposed.
Description of Actinomadura napierensis sp. nov.
Actinomadura napierensis (na.pier.en'sis. N.L. fem. adj. napierensis of Napier in South Africa, where the type strain was isolated).
Gram-positive. Grey aerial and browny-grey substrate mycelia develop on ISP 2, Middlebrook 7H9 agar and Czapek solution agar. White aerial and substrate mycelia develop on ISP 4. After 14 days of incubation at 28 °C, spiral spore chains composed of smooth spores are observed on the aerial mycelium. The diagnostic amino acid of the peptidoglycan is meso-DAP. Cell hydrolysates contain galactose, glucose, madurose, mannose and ribose. Arabinose and xylose are absent. Phospholipids include diphosphatidylglycerol, phosphatidylinositol and phosphatidylglycerol. The fatty acid methyl esters mainly comprise C16 : 0 and 10-methyl C18 : 0 (tuberculostearic acid). Mycolic acids are not present. A faint blue pigment is produced on ISP 7 after 21 days. No melanin is produced on peptone–yeast extract–iron agar (ISP 6) and ISP 7. Positive for pectin hydrolysis and negative for hippurate hydrolysis. It degrades aesculin, arbutin, casein, cellulose, gelatin, hypoxanthine, starch, L-tyrosine, Tween 80 and urea, but does not degrade allantoin, guanine, xanthine or xylan. In addition to the results shown in Table 1
, (+)-L-arabinose, dextrin, (–)-D-fructose, (+)-D-galactose, glycerol, maltose, sucrose and (+)-D-trehalose are utilized as sole carbon sources, but (+)-D-melibiose is not. Able to grow in the presence of 2 % NaCl (but not 5 %). It exhibits weak H2S production, does not reduce nitrate and does not exhibit lecithinase activity. Exhibits protease and lipase activity on egg yolk medium. The DNA G+C content is 70·0 mol%.
The type strain, B60T (=DSM 44846T=NRRL B-24319T), was isolated from soil in Napier, Western Cape province, South Africa.
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ACKNOWLEDGEMENTS |
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