International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1629–1634
DOI 10.1099/ijs.0.051334-0
Nocardia sungurluensis sp. nov., isolated from soil Mustafa Camas, Aysel Veyisoglu and Nevzat Sahin Correspondence Nevzat Sahin
Department of Biology, Faculty of Art and Science, Ondokuz Mayis University, 55139 Kurupelit-Samsun, Turkey
[email protected]
A novel Gram-reaction-positive, rod-shaped, non-motile and mycolic acid-containing strain, CR3272T, isolated from soil, was studied using a polyphasic approach. The organism showed a combination of chemotaxonomic and morphological properties typical of the genus Nocardia. The cell wall contained meso-diaminopimelic acid (type IV) and whole-cell sugars were galactose, glucose, arabinose and xylose. The predominant menaquinone was MK-8(H4cyc). The major phospholipids were diphosphatidylglycerol and phosphatidylinositol mannosides. Major fatty acids were C16 : 0, C18 : 1cis9, C18 : 0 10-methyl (TBSA) and C16 : 1cis9. The novel strain formed distinct phyletic line in the Nocardia 16S rRNA gene tree and was closely associated with Nocardia goodfellowii A2012T (98.6 % 16S rRNA gene sequence similarity), Nocardia alba YIM 30243T (98.5 %) and Nocardia caishijiensis F829T (97.9 %). However, DNA–DNA relatedness values and phenotypic data demonstrated that strain CR3272T was clearly distinguished from all closely related species of the genus Nocardia. It is concluded that the organism be classified as representing a novel species of the genus Nocardia, for which the name Nocardia sungurluensis is proposed. The type strain is CR3272T (5DSM 45714T5KCTC 29094T).
The genus Nocardia belongs to the family Nocardiaceae and was proposed by Trevisan (1889). Members of the genus are aerobic, Gram-reaction-positive, mycolic acid-containing actinomycetes that form extensively branched mycelia and substrate hyphae that fragment into rod-shaped, nonmotile elements (Goodfellow & Lechevalier, 1989). Characteristics of the genus Nocardia comprising chemotaxonomic, molecular genetic and numerical phenetic methods are well-defined (Goodfellow et al., 1999). At the time of writing, the genus encompasses more than 90 species with validly published names (http://www.bacterio.net/n/ nocardia.html) including the recently described Nocardia amikacinitolerans (Ezeoke et al., 2013), Nocardia goodfellowii and Nocardia thraciensis (Sazak et al., 2012), Nocardia grenadensis (Ka¨mpfer et al., 2012), Nocardia rhamnosiphila (Everest et al., 2011). Members of the genus Nocardia are known to be human and animal pathogens (Brown-Elliott et al., 2006; Ezeoke et al., 2013), although it is also known that they play functional roles in natural habitats, notably soil (Maldonado et al., 2000), and some species produce secondary metabolites such as nocardicin, Abbreviations: FAME, fatty acid methyl esters; ISP, International Streptomyces Project. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CR3272T is JN989289. The GenBank/EMBL/ DDBJ accession numbers for the gyrB sequences of strain CR3272T and Nocardia goodfellowii A2012T are KF818381 and KF818382, respectively. Five supplementary figures and a supplementary table are available with the online version of this paper.
051334 G 2014 IUMS
Printed in Great Britain
tubelactomicin A and brasilicardin A (Aoki et al., 1976; Komaki et al., 1999; Igarashi et al., 2000). In the present study, the taxonomic position of the soil isolate CR3272T was clarified by using a polyphasic approach. Strain CR3272T was isolated from soil samples collected from Sungurlu-Corum, Turkey using Stevenson’s Medium No. 3 (Tan et al., 2006) supplemented with cycloheximide (50 mg ml21), nystatin (50 mg ml21), nalidixic acid (10 mg ml21) and novobiocin (10 mg ml21), and incubated for 21 days at 28 uC. The strain was maintained on yeast extract-malt extract agar slants [International Streptomyces Project (ISP) Medium No. 2; (Shirling & Gottlieb, 1966)] and stored in glycerol suspensions (20 %, v/v) at 220 uC. Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and purification of the PCR product was carried out following Chun & Goodfellow (1995). The almost-complete 16S rRNA gene sequence (1462 bp) of strain CR3272T was determined using an ABI PRISM 3730 XL automatic sequencer (Applied Biosystems). The identification of phylogenetic neighbours and the calculation of pairwise 16S rRNA gene sequence identities were achieved using the EzTaxon-e server (http://eztaxon-e. ezbiocloud.net/; Kim et al., 2012). CLUSTAL W software, version 1.8 (Thompson et al., 1994) was used to align the sequences of strain CR3272T and related taxa retrieved from public databases. Phylogenetic trees were inferred using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) tree-making algorithms from the MEGA version 5.0 program (Tamura et al., 2011), and the maximum-likelihood method (Felsenstein, 1629
M. Camas, A. Veyisoglu and N. Sahin
1981) from the PHYLIP suite of programs (Felsenstein, 1993). Evolutionary distances were calculated using model of Jukes & Cantor (1969). The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1000 replicates. The methods for genomic DNA extraction and PCRmediated amplification of the 1265 bp gyrB gene fragment, primers and the sequencing protocol were described previously by Takeda et al. (2010). Phylogenetic analysis was conducted using MEGA5 software with bootstrap percentages based on 1000 replicates as previously described (Tamura et al., 2011). DNA–DNA relatedness values between strain CR3272T and the related type strains Nocardia goodfellowii DSM 45516T, Nocardia caishijiensis JCM 11508T and Nocardia alba DSM 44684T were determined by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zelkulturen (DSMZ), Braunschweig, Germany. DNA was isolated using a French pressure cell (Thermo Spectronic) and was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA–DNA hybridization was carried out as described by De Ley et al. (1970) and incorporating the modifications described by Huss et al. (1983), using a model Cary 100 Bio UV/VISspectrophotometer equipped with a Peltier-thermostatted 666 multicell changer and a temperature controller with in situ temperature probe (Varian). Biomass for chemotaxonomic studies was prepared by growing strain CR3272T in ISP 2 broth at 160 r.p.m. for 10 days at 28 uC; cells were harvested by centrifugation, washed twice in distilled water, recentrifuged and freezedried. Whole-cell amino acids and sugars were prepared according to Lechevalier & Lechevalier (1970) and analysed by thin layer chromatography (Staneck & Roberts, 1974). The N-acyl type of muramic acid in the cell-wall peptidoglycan of strain CR3272T was determined with the glycolate test described by Uchida & Seino (1997). Mycolic acids were detected with TLC as described by Minnikin et al. (1980). Polar lipid and respiratory quinone analyses were carried out by the Identification Service of the DSMZ. Respiratory quinones were extracted from 100 mg of freeze-dried cells based on the two-stage method described by Tindall (1990a, 1990b). Respiratory quinones were separated into their different classes by thin layer chromatography on silica gel (Macherey-Nagel Art. no. 805 023), using hexane/tert-butylmethylether (9 : 1, v/v) as the solvent. UV-absorbing bands corresponding to menaquinones were removed from the plate and further analysed by HPLC. This step was carried out on a LDC Analytical HPLC (Thermo Separation Products) fitted with a reversephase column (Macherey-Nagel, 2 mm6125 mm, 3 mm, RP18) using methanol as the eluant; respiratory quinones were detected at 269 nm. Polar lipids were extracted and analysed by the method of Minnikin et al. (1984) as modified by Kroppenstedt & Goodfellow (2006). For the extraction of whole-cell fatty acids, cells were grown in 1630
20 ml Trypticase Soy Broth (TSB) at 28 uC with shaking at 150 r.p.m. After 5 days incubation, 5 ml seed culture was inoculated into 50 ml TSB. The inoculated flask was incubated as before for 5 days. After harvesting by cellulose membrane filtration (0.45 mm), 200 mg wet cells were placed in an extraction tube. Cellular fatty acids were extracted, derivatized to their fatty acid methyl esters (FAME) and separated by the Microbial Identification System (MIDI; Microbial ID), utilizing an Agilent Technologies 6890N gas chromatograph with a G2614A autosampler and a 6783 injector (Sasser, 1990; Ka¨mpfer & Kroppenstedt, 1996). FAME peaks were analysed using the TSBA 5.0 database. The DNA G+C content of strain CR3272T was determined following the procedure developed by Gonzalez & Saiz-Jimenez (2005). The morphological and physiological features of strain CR3272T were determined in comparison with the phylogenetically closely related type strains, N. goodfellowii DSM 45516T, N. caishijiensis JCM 11508T and N. alba DSM 44684T Cultural characteristics of strain CR3272T were determined after incubation at 28 uC for 14 days on various media as described by Shirling & Gottlieb (1966): yeast extract-malt extract agar (ISP 2), oatmeal agar (ISP 3), inorganic salt-starch agar (ISP 4), glycerol-asparagine agar (ISP 5), peptone-yeast extract-iron agar (ISP 6), tyrosine agar (ISP 7), modified Bennett’s agar (MBA; Jones, 1949) and Czapek’s Agar (Waksman, 1967). National Bureau of Standards (NBS) Colour Name Charts (Kelly, 1964) were used for determining colour designation and names. Growth tolerance was determined for temperatures (10–45 uC), pH (5.0–10.0) and NaCl concentrations (0–5 %; w/v) using ISP 2 media. Decomposition of various compounds was examined using glucose-yeast extract agar (GYEA) recommended by Athalye et al. (1985). In addition, degradation of Tweens 20, 40 and 80 (1.0 %, w/v) was examined using Sierra medium (Sierra, 1957). Utilization of sole carbon sources was determined as described by Goodfellow (1971) using Stevenson’s basal medium (Stevenson, 1967) supplemented with a final concentration of 1.0 % (w/v) of the tested carbon sources and 0.1 % (w/v) propionic acids/succinic acids. Nitrogen source utilization was examined using the basal medium recommended by Williams et al. (1983) supplemented with a final concentration of 0.1 % (w/v) of the tested nitrogen sources. Antimicrobial susceptibility testing was performed using antimicrobial susceptibility test discs (Bioanalyse) according to the manufacturer’s instructions. Colony morphology and micromorphological properties of isolate CR3272T were determined by examining gold-coated dehydrated specimens of 14-day cultures from ISP 2 medium using a JSM 6060 (JEOL) scanning electron microscope. The almost-complete 16S rRNA gene sequence (1462 nt) of strain CR3272T determined in this study was compared with the corresponding sequences of members of the genus Nocardia. The phylogenetic tree based on the neighbourjoining algorithm showed that strain CR3272T formed a distinct branch from other species of the genus Nocardia, International Journal of Systematic and Evolutionary Microbiology 64
Nocardia sungurluensis sp. nov.,
DSM 44484T (98.2 %; 27 nt differences at 1457 locations), Nocardia exalbida IFM 0803T (98.1 %; 27 nt differences at 1413 locations), Nocardia shimofusensis IFM 10311T (98.1 %; 28 nt differences at 1462 locations), Nocardia thailandica IFM 10145T (98.0 %; 28 nt differences at 1430 locations), Nocardia anaemiae IFM 0323T (97.95 %; 30 nt differences at 1462 locations), Nocardia neocaledoniensis SBHR OA6T (97.91 %; 30 nt differences at 1437 locations) and N. caishijiensis F829T (97.90 %; 30 nt differences at 1426 locations). Lower sequence similarities (,97.90 %) were found with all other species of the genus Nocardia with validly published names.
notably from its nearest neighbours of the genus, N. goodfellowii A2012T, N. caishijiensis F829T and N. alba YIM 30243T (Fig. 1). The relationship between strain CR3272T and its closest neighbours, N. goodfellowii A2012T, N. caishijiensis F829T and N. alba YIM 30243T, was supported by the maximum-likelihood algorithm but not a high bootstrap value, however, this relationship was not supported by maximum-parsimony or by high bootstrap values (Supplementary Figs S1 and S2). Sequence similarity of identity calculations indicated that the closest relatives of strain CR3272T were N. goodfellowii A2012T (98.6 %; 20 nt differences at 1462 locations), N. alba YIM 30243T (98.5 %; 21 nt differences at 1442 locations), Nocardia asteroides ATCC 19247T (98.3 %; 25 nt differences at 1455 locations), Nocardia abscessus IMMIB D-1592T (98.2 %; 26 nt differences at 1447 locations), Nocardia lijiangensis YIM 33378T (98.2 %; 27 nt differences at 1462 locations), Nocardia cyriacigeorgica
The similarity of the gyrB gene (1265 nt) between strain CR3272T and N. goodfellowii A2012T, N. asteroides ATCC 19247T, N. neocaledoniensis DSM 44717T, N. thailandica JCM 12356T and N. niwae W9241T was 95.07 %, 90.86 %, 90.62 %, 90.62 % and 90.22 %, respectively. The novel strain showed gyrB sequence similarity values of ,90.0 %
N. goodfellowii A2012T (HQ157183)
93
N. caishijiensis F829T (AF459443)
0.01
N. alba YIM 30243T (AY222321) Nocardia sungurluensis CR3272T (JN989289) 59
N. nova JCM 6044T (Z36930) N. anaemiae IFM 0323T (AB162801) 69
N. vinacea MK703-102F1T (AB024312)
N. paucivorans IMMIB D-1632T (AF179865) N. niwae W9241T (FJ765056) N. exalbida IFM 0803T (AB187522) 99 N. gamkensis CZH20T (DQ235272) N. takedensis MS1-3T (AB158277) N. lijiangensis YIM 33378T (AY779043) N. xishanensis AS 4.1860T (AY333115) N. shimofusensis IFM 10311T (AB108775)
86 59
N. farcinica ATCC 3318T (Z36936) N. thailandica IFM 10145T (AB126874) N. asteroides ATCC 19247T (Z36934)
76
N. neocaledoniensis SBHR OA6T (AY282603) N. abscessus IMMIB D-1592T (AF218292) N. cyriacigeorgica DSM 44484T (AF430027) N. grenadensis GW5-5797T (FR729900) 94 99
N. carnea DSM 43397T (Z36929) N. testacea JCM 12235T (AB192415) N. flavorosea JCM 3332T (Z46754) N. rhamnosiphila 202GMOT (EF418604)
N. transvalensis DSM 43405T (Z36926) N. altamirensis OFN S17T (EU006090) 79 54
N. brasiliensis ATCC 19296T (Z36935) N. iowensis UI 122540T (DQ925490) Streptomyces somaliensis DSM 40738T (AJ007403)
Fig. 1. Neighbour-joining phylogenetic tree based on a comparative analysis of 16S rRNA gene sequences, showing the relationships between strain CR3272T and closely related species of the genus Nocardia. Numbers at nodes indicate the levels of bootstrap support (% of 1000 replications); only values ¢50 % are shown. GenBank accession numbers are given in parentheses. Streptomyces somaliensis DSM 40738T was used as an outgroup. Bar, 0.01 substitutions per site. http://ijs.sgmjournals.org
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M. Camas, A. Veyisoglu and N. Sahin
with the type strains of other species of the genus Nocardia (Fig. S3). DNA–DNA relatedness values between strain CR3272T and its closest phylogenetic neighbours were 37.8±1.06 % with N. goodfellowii DSM 45516T, 24.4±4.8 % with N. alba DSM 44684T and 23.7±4.12 % with N. caishijiensis JCM 11508T (values are mean of duplicate determinations). These values are clearly well below the 70 % cut-off point recommended for the assignment of strains to the same genomic species (Wayne et al., 1987). Strain CR3272T grew well on all of the media tested. Whitish/yellowish aerial mycelium was formed on ISP 2, ISP 3, ISP 4, ISP 5, ISP 7, Czapek’s, modified Bennett’s and nutrient agars. The colour of the substrate mycelium was orange–yellow. Diffusible pigments were not produced. Melanoid pigments were not produced on ISP 6 or ISP 7 medium. Strain CR3272T produced extensively branched, irregular rod-shaped elements (Goodfellow & Lechevalier, 1989) (Fig. 2). The physiological properties that distinguish strain CR3272T from closely related species of the genus Nocardia are listed in Table 1. Strain CR3272T also indicated typical markers of members of the genus Nocardia. It contained meso-diaminopimelic acid as the cell-wall diamino acid and whole-cell sugars included arabinose, galactose, glucose and xylose (Type IV; Lechevalier & Lechevalier, 1970). The acyl type of strain CR3272T was N-glycolated. The polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unknown glycolipid (Fig. S4). Onedimensional TLC of whole-cell acid methanolysates of strain CR3272T revealed the presence of mycolic acids as identified by its RF value (0.41; Fig. S5). The predominant menaquinone of strain CR3272T was MK-8(H4cyc) (85 %); MK-8 (H4) (2.0 %) and two minor unidentified components were also detected. The major cellular fatty acids were C16 : 0 (34.2 %), C18 : 1cis9 (19.7 %), C18 : 0 10-methyl (TBSA)
Table 1. Phenotypic properties of strain CR3272T and type strains of the most closely related species of the genus Nocardia Strains: 1, CR3272T; 2, N. goodfellowii DSM 45516T; 3, N. alba DSM 44684T; 4, N. caishijiensis JCM 11508T. All strains were positive for nitrate reduction; growth with D-fructose (1.0 %), maltose (1.0 %) and propionic acid (0.1 %) as sole carbon sources; utilization of Lproline, L-serine and L-valine (all 0.1 %) as sole nitrogen sources; and growth at pH 10 and with 1–3 % NaCl. All strains were negative for degradation of adenine (0.5 %), casein (1.0 %), chitin (0.5 %), elastin (0.3 %), guanine (0.05 %), RNA (0.5 %), Tween 80 (1.0 %) and xylan (0.4 %); utilization of D-arabinose, cellobiose, melezitose, melibiose, dextran, inulin, L-sorbose, L-rhamnose, lactose, Lglutamate, myo-inositol, raffinose, starch, sucrose and xylitol (all 1.0 %) as sole carbon sources; utilization of DL-phenylalanine, Larginine, L-methionine and L-threonine (all 0.1 %) as sole nitrogen sources; and for growth at 45 uC. All data were obtained in this study. Characteristic Hydrolysis of: Aesculin Arbutin Allantoin Urea Degradation of: DNA (0.5 %) Gelatin (0.5 %) Hypoxanthine (0.4 %) Tween 20 (1.0 %) Growth on sole carbon sources (1.0 %, w/v) Methyl a-D-glycoside Adonitol D-Salicin D-Sorbitol D-Galactose D-Mannose D-Mannitol Dextrin L-Arabinose Succinic acid (0.1 %) Xylose Growth on sole nitrogen sources (0.1 %, w/v) a-Isoleucine L-Alanine L-Cysteine L-Histidine L-Hydroxyproline Growth at/with: pH 5 10 uC 4 % (w/v) NaCl 5 % (w/v) NaCl
1
2
3 4
+ + + +
+ + 2 +
+ + 2 2
2 2 2 +
+ 2 + +
2 + + 2
2 + 2 2
2 + 2 2
+ 2 + 2 + + 2 2 + + +
2 + 2 + + + + 2 2 + 2
2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 + + + 2 + 2
+ + + + +
+ + + + 2
2 + 2 2 2
2 2 + 2 2
2 + + 2
+ + + +
2 2 2 2
+ + + +
Fig. 2. Scanning electron micrograph of strain CR3272T grown on ISP 2 agar at 28 6C for 14 days. Bar, 1 mm.
(19.3 %), C16 : 1cis9 (13.4 %); minor amounts of C18 : 0 (7.5 %), C14 : 0 (1.8 %) and C15 : 0 (1.4 %) were also present (Table S1). The G+C content of the DNA was 69.8 mol%.
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International Journal of Systematic and Evolutionary Microbiology 64
Nocardia sungurluensis sp. nov.,
It is evident from the genotypic and phenotypic data that isolate CR3272T represents a novel species within the genus Nocardia, for which the name Nocardia sungurluensis sp. nov. is proposed.
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Nocardia sungurluensis (sun. gur. lu.en9sis. N.L. fem. adj. sungurluensis of or belonging to Sungurlu, Corum, Turkey, source of the organism).
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Aerobic, Gram-reaction-positive, non-acid-fast, non-motile actinomycete which forms an extensively branched, orange– yellow substrate mycelia that bear white or pale yellow aerial mycelia on several tested media. Aerial mycelia fragment into irregular rod-shaped elements. Diffusible pigments are not produced. Melanoid pigments are not produced on ISP 6 or ISP 7 agars. Growth occurs at pH 6.0–10.0 and 10– 37 uC, but not pH 5.0 or 45 uC. Optimal growth occurs at 28 uC and pH 7.0. Growth is observed in the presence of 0–4 % (w/v) NaCl. Aesculin, allantoin, arbutin and urea hydrolysis and nitrate reduction are positive. DNA, hypoxanthine and Tween 20 are degraded, but adenine, casein, elastin, chitin, gelatin, guanine, RNA, Tween 80 and xylan are not. Methyl a-D-glycoside, L-arabinose, D-fructose, D-galactose, maltose, D-mannose, D-salicin, xylose, propionic acid and succinic acid are utilized as sole carbon and energy sources, but not adonitol, cellobiose, D-sorbitol, starch, melezitose, melibiose, D-mannitol, dextrin, dextran, inulin, L-sorbose, L-rhamnose, lactose, myo-inositol, sucrose, L-glutamate or xylitol. Utilizes a-isoleucine, L-alanine, L-cysteine, L-histidine, L-hydroxyproline, L-proline, L-serine, L-tyrosine and L-valine as sole nitrogen sources, but not D-phenylalanine, L-phenylalanine, L-arginine, L-methionine or L-threonine. Growth does not occur in the presence of filter paper discs soaked in amikacin (30 mg), amoxicilin/ clavulanate (30 mg), ceftriaxone (30 mg), ciprofloxacin (5 mg), imipenem (10 mg), linezolid (30 mg), tigecycline (15 mg), trimethoprim/sulfamethoxazole (25 mg), gentamicin (10 mg), streptomycin (10 mg) and penicillin (10 U). MK-8(H4cyc) is the major menaquinone. The phospholipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and an unknown glycolipid. Major fatty acids are C16 : 0, C18 : 1cis9, C18 : 0 10-methyl (TBSA) and C16 : 1cis9.
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The type strain, CR3272 (5DSM 45714 5KCTC 29094 ) was isolated from soil in Sungurlu, Corum, Turkey. The G+C content of the genomic DNA of the type strain is 69.8 mol%.
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Acknowledgements
membered lactone antibiotic, from Nocardia sp. I. Taxonomy, production, isolation and biological properties. J Antibiot (Tokyo) 53, 1096–1101.
T
T
T
This research was supported by Ondokuz Mayis University (OMU), project no. PYO. FEN. 1901.09.003.
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Brasilicardin A, a new terpenoid antibiotic from pathogenic Nocardia brasiliensis: fermentation, isolation and biological activity. J Antibiot (Tokyo) 52, 13–19. Kroppenstedt, R. M. & Goodfellow, M. (2006). The family Thermomonosporaceae: Actinocorallia, Actinomadura, Spirillispora and Thermomonospora. In The Prokaryotes: a Handbook on the Biology of Bacteria, 3rd edn, vol. 3, pp. 682–724. Edited by M. Dworkin, S. Falkow, E. Rosenberg, K. H. Schleifer & E. Stackebrandt. New York: Springer. Lechevalier, M. P. & Lechevalier, H. A. (1970). The chemotaxonomy
of actinomycetes. In Actinomycete Taxonomy, (Special Publication no. 6), pp. 227–291. Edited by A. Dietz & J. Thayer. Arlington, VA: Society for Industrial Microbiology. Maldonado, L., Hookey, J. V., Ward, A. C. & Goodfellow, M. (2000).
Arthrobacter spp. Can J Microbiol 13, 205–211. Takeda, K., Kang, Y., Yazawa, K., Gonoi, T. & Mikami, Y. (2010).
Phylogenetic studies of Nocardia species based on gyrB gene analyses. J Med Microbiol 59, 165–171. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739. Tan, G. Y., Ward, A. C. & Goodfellow, M. (2006). Exploration of
Amycolatopsis diversity in soil using genus-specific primers and novel selective media. Syst Appl Microbiol 29, 557–569. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
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The Nocardia salmonicida clade, including descriptions of Nocardia cummidelens sp. nov., Nocardia fluminea sp. nov. and Nocardia soli sp. nov. Antonie van Leeuwenhoek 78, 367–377.
Tindall, B. J. (1990b). Lipid composition of Halobacterium lacu-
Minnikin, D. E., Hutchinson, I. G., Caldicott, A. B. & Goodfellow, M. (1980). Thin-layer chromatography of methanolysates of mycolic
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various actinomycete strains based on the acyl types of the muramyl residue in cell wall peptidoglycans examined in a glycolate test. Int J Syst Bacteriol 47, 182–190.
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method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406– 425. Sasser, M. (1990). Identification of bacteria by gas chromatography of
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International Journal of Systematic and Evolutionary Microbiology 64
DOI 10.1099/ijs.0.051334-0
Nocardia sungurluensis sp. nov., isolated from soil Mustafa Camas, Aysel Veyisoglu and Nevzat Sahin Correspondence Nevzat Sahin
Department of Biology, Faculty of Art and Science, Ondokuz Mayis University, 55139 Kurupelit-Samsun, Turkey
[email protected]
A novel Gram-reaction-positive, rod-shaped, non-motile and mycolic acid-containing strain, CR3272T, isolated from soil, was studied using a polyphasic approach. The organism showed a combination of chemotaxonomic and morphological properties typical of the genus Nocardia. The cell wall contained meso-diaminopimelic acid (type IV) and whole-cell sugars were galactose, glucose, arabinose and xylose. The predominant menaquinone was MK-8(H4cyc). The major phospholipids were diphosphatidylglycerol and phosphatidylinositol mannosides. Major fatty acids were C16 : 0, C18 : 1cis9, C18 : 0 10-methyl (TBSA) and C16 : 1cis9. The novel strain formed distinct phyletic line in the Nocardia 16S rRNA gene tree and was closely associated with Nocardia goodfellowii A2012T (98.6 % 16S rRNA gene sequence similarity), Nocardia alba YIM 30243T (98.5 %) and Nocardia caishijiensis F829T (97.9 %). However, DNA–DNA relatedness values and phenotypic data demonstrated that strain CR3272T was clearly distinguished from all closely related species of the genus Nocardia. It is concluded that the organism be classified as representing a novel species of the genus Nocardia, for which the name Nocardia sungurluensis is proposed. The type strain is CR3272T (5DSM 45714T5KCTC 29094T).
The genus Nocardia belongs to the family Nocardiaceae and was proposed by Trevisan (1889). Members of the genus are aerobic, Gram-reaction-positive, mycolic acid-containing actinomycetes that form extensively branched mycelia and substrate hyphae that fragment into rod-shaped, nonmotile elements (Goodfellow & Lechevalier, 1989). Characteristics of the genus Nocardia comprising chemotaxonomic, molecular genetic and numerical phenetic methods are well-defined (Goodfellow et al., 1999). At the time of writing, the genus encompasses more than 90 species with validly published names (http://www.bacterio.net/n/ nocardia.html) including the recently described Nocardia amikacinitolerans (Ezeoke et al., 2013), Nocardia goodfellowii and Nocardia thraciensis (Sazak et al., 2012), Nocardia grenadensis (Ka¨mpfer et al., 2012), Nocardia rhamnosiphila (Everest et al., 2011). Members of the genus Nocardia are known to be human and animal pathogens (Brown-Elliott et al., 2006; Ezeoke et al., 2013), although it is also known that they play functional roles in natural habitats, notably soil (Maldonado et al., 2000), and some species produce secondary metabolites such as nocardicin, Abbreviations: FAME, fatty acid methyl esters; ISP, International Streptomyces Project. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CR3272T is JN989289. The GenBank/EMBL/ DDBJ accession numbers for the gyrB sequences of strain CR3272T and Nocardia goodfellowii A2012T are KF818381 and KF818382, respectively. Five supplementary figures and a supplementary table are available with the online version of this paper.
051334 G 2014 IUMS
Printed in Great Britain
tubelactomicin A and brasilicardin A (Aoki et al., 1976; Komaki et al., 1999; Igarashi et al., 2000). In the present study, the taxonomic position of the soil isolate CR3272T was clarified by using a polyphasic approach. Strain CR3272T was isolated from soil samples collected from Sungurlu-Corum, Turkey using Stevenson’s Medium No. 3 (Tan et al., 2006) supplemented with cycloheximide (50 mg ml21), nystatin (50 mg ml21), nalidixic acid (10 mg ml21) and novobiocin (10 mg ml21), and incubated for 21 days at 28 uC. The strain was maintained on yeast extract-malt extract agar slants [International Streptomyces Project (ISP) Medium No. 2; (Shirling & Gottlieb, 1966)] and stored in glycerol suspensions (20 %, v/v) at 220 uC. Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and purification of the PCR product was carried out following Chun & Goodfellow (1995). The almost-complete 16S rRNA gene sequence (1462 bp) of strain CR3272T was determined using an ABI PRISM 3730 XL automatic sequencer (Applied Biosystems). The identification of phylogenetic neighbours and the calculation of pairwise 16S rRNA gene sequence identities were achieved using the EzTaxon-e server (http://eztaxon-e. ezbiocloud.net/; Kim et al., 2012). CLUSTAL W software, version 1.8 (Thompson et al., 1994) was used to align the sequences of strain CR3272T and related taxa retrieved from public databases. Phylogenetic trees were inferred using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) tree-making algorithms from the MEGA version 5.0 program (Tamura et al., 2011), and the maximum-likelihood method (Felsenstein, 1629
M. Camas, A. Veyisoglu and N. Sahin
1981) from the PHYLIP suite of programs (Felsenstein, 1993). Evolutionary distances were calculated using model of Jukes & Cantor (1969). The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1000 replicates. The methods for genomic DNA extraction and PCRmediated amplification of the 1265 bp gyrB gene fragment, primers and the sequencing protocol were described previously by Takeda et al. (2010). Phylogenetic analysis was conducted using MEGA5 software with bootstrap percentages based on 1000 replicates as previously described (Tamura et al., 2011). DNA–DNA relatedness values between strain CR3272T and the related type strains Nocardia goodfellowii DSM 45516T, Nocardia caishijiensis JCM 11508T and Nocardia alba DSM 44684T were determined by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zelkulturen (DSMZ), Braunschweig, Germany. DNA was isolated using a French pressure cell (Thermo Spectronic) and was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA–DNA hybridization was carried out as described by De Ley et al. (1970) and incorporating the modifications described by Huss et al. (1983), using a model Cary 100 Bio UV/VISspectrophotometer equipped with a Peltier-thermostatted 666 multicell changer and a temperature controller with in situ temperature probe (Varian). Biomass for chemotaxonomic studies was prepared by growing strain CR3272T in ISP 2 broth at 160 r.p.m. for 10 days at 28 uC; cells were harvested by centrifugation, washed twice in distilled water, recentrifuged and freezedried. Whole-cell amino acids and sugars were prepared according to Lechevalier & Lechevalier (1970) and analysed by thin layer chromatography (Staneck & Roberts, 1974). The N-acyl type of muramic acid in the cell-wall peptidoglycan of strain CR3272T was determined with the glycolate test described by Uchida & Seino (1997). Mycolic acids were detected with TLC as described by Minnikin et al. (1980). Polar lipid and respiratory quinone analyses were carried out by the Identification Service of the DSMZ. Respiratory quinones were extracted from 100 mg of freeze-dried cells based on the two-stage method described by Tindall (1990a, 1990b). Respiratory quinones were separated into their different classes by thin layer chromatography on silica gel (Macherey-Nagel Art. no. 805 023), using hexane/tert-butylmethylether (9 : 1, v/v) as the solvent. UV-absorbing bands corresponding to menaquinones were removed from the plate and further analysed by HPLC. This step was carried out on a LDC Analytical HPLC (Thermo Separation Products) fitted with a reversephase column (Macherey-Nagel, 2 mm6125 mm, 3 mm, RP18) using methanol as the eluant; respiratory quinones were detected at 269 nm. Polar lipids were extracted and analysed by the method of Minnikin et al. (1984) as modified by Kroppenstedt & Goodfellow (2006). For the extraction of whole-cell fatty acids, cells were grown in 1630
20 ml Trypticase Soy Broth (TSB) at 28 uC with shaking at 150 r.p.m. After 5 days incubation, 5 ml seed culture was inoculated into 50 ml TSB. The inoculated flask was incubated as before for 5 days. After harvesting by cellulose membrane filtration (0.45 mm), 200 mg wet cells were placed in an extraction tube. Cellular fatty acids were extracted, derivatized to their fatty acid methyl esters (FAME) and separated by the Microbial Identification System (MIDI; Microbial ID), utilizing an Agilent Technologies 6890N gas chromatograph with a G2614A autosampler and a 6783 injector (Sasser, 1990; Ka¨mpfer & Kroppenstedt, 1996). FAME peaks were analysed using the TSBA 5.0 database. The DNA G+C content of strain CR3272T was determined following the procedure developed by Gonzalez & Saiz-Jimenez (2005). The morphological and physiological features of strain CR3272T were determined in comparison with the phylogenetically closely related type strains, N. goodfellowii DSM 45516T, N. caishijiensis JCM 11508T and N. alba DSM 44684T Cultural characteristics of strain CR3272T were determined after incubation at 28 uC for 14 days on various media as described by Shirling & Gottlieb (1966): yeast extract-malt extract agar (ISP 2), oatmeal agar (ISP 3), inorganic salt-starch agar (ISP 4), glycerol-asparagine agar (ISP 5), peptone-yeast extract-iron agar (ISP 6), tyrosine agar (ISP 7), modified Bennett’s agar (MBA; Jones, 1949) and Czapek’s Agar (Waksman, 1967). National Bureau of Standards (NBS) Colour Name Charts (Kelly, 1964) were used for determining colour designation and names. Growth tolerance was determined for temperatures (10–45 uC), pH (5.0–10.0) and NaCl concentrations (0–5 %; w/v) using ISP 2 media. Decomposition of various compounds was examined using glucose-yeast extract agar (GYEA) recommended by Athalye et al. (1985). In addition, degradation of Tweens 20, 40 and 80 (1.0 %, w/v) was examined using Sierra medium (Sierra, 1957). Utilization of sole carbon sources was determined as described by Goodfellow (1971) using Stevenson’s basal medium (Stevenson, 1967) supplemented with a final concentration of 1.0 % (w/v) of the tested carbon sources and 0.1 % (w/v) propionic acids/succinic acids. Nitrogen source utilization was examined using the basal medium recommended by Williams et al. (1983) supplemented with a final concentration of 0.1 % (w/v) of the tested nitrogen sources. Antimicrobial susceptibility testing was performed using antimicrobial susceptibility test discs (Bioanalyse) according to the manufacturer’s instructions. Colony morphology and micromorphological properties of isolate CR3272T were determined by examining gold-coated dehydrated specimens of 14-day cultures from ISP 2 medium using a JSM 6060 (JEOL) scanning electron microscope. The almost-complete 16S rRNA gene sequence (1462 nt) of strain CR3272T determined in this study was compared with the corresponding sequences of members of the genus Nocardia. The phylogenetic tree based on the neighbourjoining algorithm showed that strain CR3272T formed a distinct branch from other species of the genus Nocardia, International Journal of Systematic and Evolutionary Microbiology 64
Nocardia sungurluensis sp. nov.,
DSM 44484T (98.2 %; 27 nt differences at 1457 locations), Nocardia exalbida IFM 0803T (98.1 %; 27 nt differences at 1413 locations), Nocardia shimofusensis IFM 10311T (98.1 %; 28 nt differences at 1462 locations), Nocardia thailandica IFM 10145T (98.0 %; 28 nt differences at 1430 locations), Nocardia anaemiae IFM 0323T (97.95 %; 30 nt differences at 1462 locations), Nocardia neocaledoniensis SBHR OA6T (97.91 %; 30 nt differences at 1437 locations) and N. caishijiensis F829T (97.90 %; 30 nt differences at 1426 locations). Lower sequence similarities (,97.90 %) were found with all other species of the genus Nocardia with validly published names.
notably from its nearest neighbours of the genus, N. goodfellowii A2012T, N. caishijiensis F829T and N. alba YIM 30243T (Fig. 1). The relationship between strain CR3272T and its closest neighbours, N. goodfellowii A2012T, N. caishijiensis F829T and N. alba YIM 30243T, was supported by the maximum-likelihood algorithm but not a high bootstrap value, however, this relationship was not supported by maximum-parsimony or by high bootstrap values (Supplementary Figs S1 and S2). Sequence similarity of identity calculations indicated that the closest relatives of strain CR3272T were N. goodfellowii A2012T (98.6 %; 20 nt differences at 1462 locations), N. alba YIM 30243T (98.5 %; 21 nt differences at 1442 locations), Nocardia asteroides ATCC 19247T (98.3 %; 25 nt differences at 1455 locations), Nocardia abscessus IMMIB D-1592T (98.2 %; 26 nt differences at 1447 locations), Nocardia lijiangensis YIM 33378T (98.2 %; 27 nt differences at 1462 locations), Nocardia cyriacigeorgica
The similarity of the gyrB gene (1265 nt) between strain CR3272T and N. goodfellowii A2012T, N. asteroides ATCC 19247T, N. neocaledoniensis DSM 44717T, N. thailandica JCM 12356T and N. niwae W9241T was 95.07 %, 90.86 %, 90.62 %, 90.62 % and 90.22 %, respectively. The novel strain showed gyrB sequence similarity values of ,90.0 %
N. goodfellowii A2012T (HQ157183)
93
N. caishijiensis F829T (AF459443)
0.01
N. alba YIM 30243T (AY222321) Nocardia sungurluensis CR3272T (JN989289) 59
N. nova JCM 6044T (Z36930) N. anaemiae IFM 0323T (AB162801) 69
N. vinacea MK703-102F1T (AB024312)
N. paucivorans IMMIB D-1632T (AF179865) N. niwae W9241T (FJ765056) N. exalbida IFM 0803T (AB187522) 99 N. gamkensis CZH20T (DQ235272) N. takedensis MS1-3T (AB158277) N. lijiangensis YIM 33378T (AY779043) N. xishanensis AS 4.1860T (AY333115) N. shimofusensis IFM 10311T (AB108775)
86 59
N. farcinica ATCC 3318T (Z36936) N. thailandica IFM 10145T (AB126874) N. asteroides ATCC 19247T (Z36934)
76
N. neocaledoniensis SBHR OA6T (AY282603) N. abscessus IMMIB D-1592T (AF218292) N. cyriacigeorgica DSM 44484T (AF430027) N. grenadensis GW5-5797T (FR729900) 94 99
N. carnea DSM 43397T (Z36929) N. testacea JCM 12235T (AB192415) N. flavorosea JCM 3332T (Z46754) N. rhamnosiphila 202GMOT (EF418604)
N. transvalensis DSM 43405T (Z36926) N. altamirensis OFN S17T (EU006090) 79 54
N. brasiliensis ATCC 19296T (Z36935) N. iowensis UI 122540T (DQ925490) Streptomyces somaliensis DSM 40738T (AJ007403)
Fig. 1. Neighbour-joining phylogenetic tree based on a comparative analysis of 16S rRNA gene sequences, showing the relationships between strain CR3272T and closely related species of the genus Nocardia. Numbers at nodes indicate the levels of bootstrap support (% of 1000 replications); only values ¢50 % are shown. GenBank accession numbers are given in parentheses. Streptomyces somaliensis DSM 40738T was used as an outgroup. Bar, 0.01 substitutions per site. http://ijs.sgmjournals.org
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M. Camas, A. Veyisoglu and N. Sahin
with the type strains of other species of the genus Nocardia (Fig. S3). DNA–DNA relatedness values between strain CR3272T and its closest phylogenetic neighbours were 37.8±1.06 % with N. goodfellowii DSM 45516T, 24.4±4.8 % with N. alba DSM 44684T and 23.7±4.12 % with N. caishijiensis JCM 11508T (values are mean of duplicate determinations). These values are clearly well below the 70 % cut-off point recommended for the assignment of strains to the same genomic species (Wayne et al., 1987). Strain CR3272T grew well on all of the media tested. Whitish/yellowish aerial mycelium was formed on ISP 2, ISP 3, ISP 4, ISP 5, ISP 7, Czapek’s, modified Bennett’s and nutrient agars. The colour of the substrate mycelium was orange–yellow. Diffusible pigments were not produced. Melanoid pigments were not produced on ISP 6 or ISP 7 medium. Strain CR3272T produced extensively branched, irregular rod-shaped elements (Goodfellow & Lechevalier, 1989) (Fig. 2). The physiological properties that distinguish strain CR3272T from closely related species of the genus Nocardia are listed in Table 1. Strain CR3272T also indicated typical markers of members of the genus Nocardia. It contained meso-diaminopimelic acid as the cell-wall diamino acid and whole-cell sugars included arabinose, galactose, glucose and xylose (Type IV; Lechevalier & Lechevalier, 1970). The acyl type of strain CR3272T was N-glycolated. The polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unknown glycolipid (Fig. S4). Onedimensional TLC of whole-cell acid methanolysates of strain CR3272T revealed the presence of mycolic acids as identified by its RF value (0.41; Fig. S5). The predominant menaquinone of strain CR3272T was MK-8(H4cyc) (85 %); MK-8 (H4) (2.0 %) and two minor unidentified components were also detected. The major cellular fatty acids were C16 : 0 (34.2 %), C18 : 1cis9 (19.7 %), C18 : 0 10-methyl (TBSA)
Table 1. Phenotypic properties of strain CR3272T and type strains of the most closely related species of the genus Nocardia Strains: 1, CR3272T; 2, N. goodfellowii DSM 45516T; 3, N. alba DSM 44684T; 4, N. caishijiensis JCM 11508T. All strains were positive for nitrate reduction; growth with D-fructose (1.0 %), maltose (1.0 %) and propionic acid (0.1 %) as sole carbon sources; utilization of Lproline, L-serine and L-valine (all 0.1 %) as sole nitrogen sources; and growth at pH 10 and with 1–3 % NaCl. All strains were negative for degradation of adenine (0.5 %), casein (1.0 %), chitin (0.5 %), elastin (0.3 %), guanine (0.05 %), RNA (0.5 %), Tween 80 (1.0 %) and xylan (0.4 %); utilization of D-arabinose, cellobiose, melezitose, melibiose, dextran, inulin, L-sorbose, L-rhamnose, lactose, Lglutamate, myo-inositol, raffinose, starch, sucrose and xylitol (all 1.0 %) as sole carbon sources; utilization of DL-phenylalanine, Larginine, L-methionine and L-threonine (all 0.1 %) as sole nitrogen sources; and for growth at 45 uC. All data were obtained in this study. Characteristic Hydrolysis of: Aesculin Arbutin Allantoin Urea Degradation of: DNA (0.5 %) Gelatin (0.5 %) Hypoxanthine (0.4 %) Tween 20 (1.0 %) Growth on sole carbon sources (1.0 %, w/v) Methyl a-D-glycoside Adonitol D-Salicin D-Sorbitol D-Galactose D-Mannose D-Mannitol Dextrin L-Arabinose Succinic acid (0.1 %) Xylose Growth on sole nitrogen sources (0.1 %, w/v) a-Isoleucine L-Alanine L-Cysteine L-Histidine L-Hydroxyproline Growth at/with: pH 5 10 uC 4 % (w/v) NaCl 5 % (w/v) NaCl
1
2
3 4
+ + + +
+ + 2 +
+ + 2 2
2 2 2 +
+ 2 + +
2 + + 2
2 + 2 2
2 + 2 2
+ 2 + 2 + + 2 2 + + +
2 + 2 + + + + 2 2 + 2
2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 + + + 2 + 2
+ + + + +
+ + + + 2
2 + 2 2 2
2 2 + 2 2
2 + + 2
+ + + +
2 2 2 2
+ + + +
Fig. 2. Scanning electron micrograph of strain CR3272T grown on ISP 2 agar at 28 6C for 14 days. Bar, 1 mm.
(19.3 %), C16 : 1cis9 (13.4 %); minor amounts of C18 : 0 (7.5 %), C14 : 0 (1.8 %) and C15 : 0 (1.4 %) were also present (Table S1). The G+C content of the DNA was 69.8 mol%.
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International Journal of Systematic and Evolutionary Microbiology 64
Nocardia sungurluensis sp. nov.,
It is evident from the genotypic and phenotypic data that isolate CR3272T represents a novel species within the genus Nocardia, for which the name Nocardia sungurluensis sp. nov. is proposed.
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Aerobic, Gram-reaction-positive, non-acid-fast, non-motile actinomycete which forms an extensively branched, orange– yellow substrate mycelia that bear white or pale yellow aerial mycelia on several tested media. Aerial mycelia fragment into irregular rod-shaped elements. Diffusible pigments are not produced. Melanoid pigments are not produced on ISP 6 or ISP 7 agars. Growth occurs at pH 6.0–10.0 and 10– 37 uC, but not pH 5.0 or 45 uC. Optimal growth occurs at 28 uC and pH 7.0. Growth is observed in the presence of 0–4 % (w/v) NaCl. Aesculin, allantoin, arbutin and urea hydrolysis and nitrate reduction are positive. DNA, hypoxanthine and Tween 20 are degraded, but adenine, casein, elastin, chitin, gelatin, guanine, RNA, Tween 80 and xylan are not. Methyl a-D-glycoside, L-arabinose, D-fructose, D-galactose, maltose, D-mannose, D-salicin, xylose, propionic acid and succinic acid are utilized as sole carbon and energy sources, but not adonitol, cellobiose, D-sorbitol, starch, melezitose, melibiose, D-mannitol, dextrin, dextran, inulin, L-sorbose, L-rhamnose, lactose, myo-inositol, sucrose, L-glutamate or xylitol. Utilizes a-isoleucine, L-alanine, L-cysteine, L-histidine, L-hydroxyproline, L-proline, L-serine, L-tyrosine and L-valine as sole nitrogen sources, but not D-phenylalanine, L-phenylalanine, L-arginine, L-methionine or L-threonine. Growth does not occur in the presence of filter paper discs soaked in amikacin (30 mg), amoxicilin/ clavulanate (30 mg), ceftriaxone (30 mg), ciprofloxacin (5 mg), imipenem (10 mg), linezolid (30 mg), tigecycline (15 mg), trimethoprim/sulfamethoxazole (25 mg), gentamicin (10 mg), streptomycin (10 mg) and penicillin (10 U). MK-8(H4cyc) is the major menaquinone. The phospholipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and an unknown glycolipid. Major fatty acids are C16 : 0, C18 : 1cis9, C18 : 0 10-methyl (TBSA) and C16 : 1cis9.
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This research was supported by Ondokuz Mayis University (OMU), project no. PYO. FEN. 1901.09.003.
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