Risungbinella Massiliensis Sp. Nov., a New Member of Thermoactinomycetaceae Isolated from Human Gut
Total Page：16
File Type：pdf, Size：1020Kb
Antonie van Leeuwenhoek DOI 10.1007/s10482-016-0677-6 ORIGINAL PAPER Risungbinella massiliensis sp. nov., a new member of Thermoactinomycetaceae isolated from human gut Gre´gory Dubourg . Jean-Christophe Lagier . Catherine Robert . Nicholas Armstrong . Carine Couderc . Pierre-Edouard Fournier . Didier Raoult Received: 21 September 2015 / Accepted: 8 March 2016 Ó Springer International Publishing Switzerland 2016 Abstract A novel filamentous bacterium, desig- genome sequence and annotation. The G?C content of nated GD1T, was isolated from the gut microbiota of the genomic DNA was determined to be 40.1 mol %. a 38-year-old male who suffered from a Coxiella The major fatty acids of strain GD1T were identified as burnetii vascular for which he received multiple a iso-C15:0, iso-C17:0, anteiso-C15:0, iso-C14:0 and broad-spectrum antibiotic cocktail at the time of the C16:0. The 3,440,191 bp long genome contains 3540 stool collection. The strain was isolated as a part of protein-coding and 67 RNA genes, including three culturomics study by cultivation on 5 % sheep blood rRNA genes. Strain GD1T (= DSM 46691 = CSUR agar in aerobic condition at 28 °C, after 14 days of P1082) sp. nov. is here classified as the type strain of a incubation. Strain GD1T shows 16S rRNA gene new species, Risungbinella massiliensis, within the sequence similarities of 98.01 % to the type strain of family Thermoactinomycetaceae. To date, strain Risungbinella pyongyangensis. We describe here the GD1T is the first member of the family Thermoacti- features of this bacterium, together with the complete nomycetaceae isolated from human gut and the fourth from a human specimen. Electronic supplementary material The online version of Keywords Genome Á Culturomics Á Taxono- this article (doi:10.1007/s10482-016-0677-6) contains supple- genomics mentary material, which is available to authorized users. G. Dubourg Á J.-C. Lagier Á C. Robert Á D. Raoult N. Armstrong Á P.-E. Fournier Á D. Raoult (&) Special Infectious Agents Unit, King Fahd Medical Unite´ de Recherche sur les Maladies Infectieuses et Research Center, King Abdul Aziz University, Jeddah, Tropicales Emergentes, UM 63, CNRS 7278, IRD 198, Saudi Arabia Inserm 1095, Institut Hospitalo-Universitaire Me´diterrane´e-Infection, Faculte´ de me´decine, Aix- Marseille Universite´, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France e-mail: [email protected] G. Dubourg Á C. Couderc Á P.-E. Fournier Á D. Raoult Poˆle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fe´de´ration de Bacte´riologie–Hygie`ne– Virologie, University, Hospital Centre Timone, Institut Hospitalo-Universitaire (IHU) Me´diterrane´e Infection, Assistance Publique–Hoˆpitaux de Marseille, Marseille, France 123 Antonie van Leeuwenhoek Introduction Risungbinella Strain GD1T (= DSM 46691 = CSUR P1082) sp. nov. is here classified as the type strain of a A novel Gram-positive, aerobic bacterium designated T new species, Risungbinella massiliensis sp. nov., strain GD1 (= DSM 46691 = CSUR P1082) was together with the description of the complete genomic isolated as a part of a culturomics study (Lagier et al. sequencing and annotation. 2012) from the gut microbiota of a 38-year-old male who suffered from a Coxiella burnetii vascular infection complicated by an esophageal fistula, for Methods which he received a multiple broad-spectrum antibi- otic cocktail at the time of the stool collection Growth conditions and phenotypic tests (Dubourg et al. 2013, 2014). Currently, the classification of prokaryotes is A stool sample was collected from a 38-year-old male mainly founded on a combination of phenotypic and who suffered from a C. burnetii vascular infection genotypic characteristics (Stackebrandt and Ebers complicated by an esophageal fistula, for which he 2006; Tindall et al. 2010) including 16S rRNA gene received a multiple broad-spectrum antibiotic cocktail phylogeny, DNA–DNA hybridization (DDH) and at the time of the stool collection (Dubourg et al. G?C content. However, these methods suffer several 2014). The study was approved by the Ethics Com- pitfalls, even if they remain considered as a ‘‘gold mittee of the Institut Fe´de´ratif de Recherche IFR48, standard’’ (Moore et al. 1987; Rossello´-Mora 2006). Faculty of Medicine, Marseille, France, under agree- Thus, we recently proposed to add genomic informa- ment number 09-002. The faecal specimen was tion to phenotypic criteria for the description of new preserved at -80 °C after collection. Strain GD1T bacterial species, considering the declining cost of was isolated in March 2012 by cultivation on 5 % sequencing and the rapid growth in the number of sheep blood agar in aerobic conditions at 28 °C, after bacterial genomes being sequenced (Lagier et al. 14 days of incubation. 2015). Growth at different temperatures (22, 30, 37 and To date, the family Thermoactinomycetaceae accom- 56 °C) was tested. Growth of the strain was tested in modates 18 genera containing 31 species (http://www. 5 % sheep blood-enriched Columbia agar (BioMer- bacterio.net/thermoactinomycetaceae.html). Microor- ieux) and Tryptic Soy agar (Becton–Dickinson) under ganisms that belong to Thermoactinomycetaceae are anaerobic and microaerophilic conditions using the aerobic, yield filamentous growth and are Gram-pos- GENbag anaer and GENbag microaer systems, itive. Most of these strains have been isolated from respectively (BioMerieux), and under aerobic condi- environmental sources such as marine sediments, tions, with or without 5 % CO2. Growth was tested for sugar cane, soil or mushroom compost (Addou et al. salt tolerance, with 0–5, 50 and 100 % (w/v) NaCl. 2012, 2013; Chen et al. 2012; Han et al. 2013; The pH range for growth was tested at pH 6, 7, and 8 Hatayama et al. 2005; Kim et al. 2015; Li et al. 2012, using Tryptic Soy agar. Phenotypic tests were per- 2013; Matsuo et al. 2006; Park et al. 2007; Tsubouchi formed using an API ZYM strip (BioMerieux), an API et al. 2013; Wu et al. 2015; Yang et al. 2013, 2015; 50 CH strip (BioMe´rieux), and API 20 NE (BioMe´r- Yao et al. 2014; Yoon et al. 2005; Yu et al. 2012; ieux). Tests for hydrolysis of gelatin, starch were Zarparvar et al. 2014; Zhang et al. 2013; Zhang et al. performed as described by Gonzalez et al. (1978). 2015; Zhang et al. 2007; Zhou et al. 2014). However, In vitro susceptibility to antibiotics was determined Desmospora activia has been cultured from sputa from using the disk-diffusion method on Mueller–Hinton a patient for which tuberculosis was suspected (Yassin agar with 5 % blood. et al. 2009). In the same manner, Kroppenstedtia Electronic microscopy was performed with detec- eburnea or Hazenella coriaceae were isolated from tion Formvar coated grids which were deposited on a various clinical samples, most of which were blood 40 lL bacterial suspension drop and incubated at (Buss et al. 2013; von Jan et al. 2011). 37 °C for 30 min, followed by a 10 s incubation on Risungbinella pyongyangensis was recently iso- ammonium molybdate 1 %. Grids were then observed lated from an agricultural soil sample (Kim et al. 2015) using a Morgagni 268D transmission electron micro- and is to date the sole member of the genus scope (Philips) at an operating voltage of 60 kV. 123 Antonie van Leeuwenhoek MALDI-TOF analysis phylogenetic inferences were obtained using the max- imum-likelihood method within the MEGA6 pro- Sample preparation gramme. Actinomyces polynesiensis strain MS2T (HF952919) was used as an outgroup. Two or three freshly grown colonies were transferred with a plastic loop into a polypropylene microtube Fatty acid methyl ester (FAME) analysis by GC/ 1.5 ml containing 300 lL of ultra-pure water; the MS microtubes were then centrifuged at 13,0009g for 2 min and the supernatant was discarded. Formic acid Approximately 40 mg of bacterial biomass was har- (70 %) and acetonitrile (ACN) were added in a 1:1 (v/ vested from a 48 h pure culture on Columbia agar with v) ratio to the bacterial pellet. The mixture was 5 % sheep blood (BioMe´rieux). Cellular fatty acid vortexed for 30 s and centrifuged for 2 min. An methyl esters were prepared as previously described aliquot of 1 lL of the supernatant was transferred to a (Sasser 2006). GC/MS analyses were carried out on a spot onto a 96- well stainless steel MALDI target Clarus 500 gas chromatograph equipped with a SQ8S plate. Applied cell supernatants were air-dried for MS detector (Perkin Elmer, Courtaboeuf, France). 2 10 min following deposition of 1 lL of the a-cyano-4- lL of duplicate FAME extracts was volatised at hydroxycinnamic (CHCA, Sigma-Aldrich, Sa˜o Paulo, 250 °C (split 20 mL/min) in a Focus liner with wool Brazil) matrix prepared in an organic solvent mixture and separated on an Elite-5MS column (30 m, to a final saturation concentration in a 50 % acetoni- 0.25 mm i.d., 0.25 mm film thickness) using a linear trile/2.5 % trifluoroacetic acid (TFA) solution that was temperature gradient (70–290 °Cat6°C/min) overlaid and allowed to dry. Each sample was spotted enabling the detection of C4 to C24 fatty acid methyl 16 times. Mass spectra acquisition and data analysis esters. Helium flowing at 1.2 mL/min was used as MALDI-TOF MS analysis of all strains was per- carrier gas. MS inlet line was set at 250 °C and the EI formed on a MicroFlex mass spectrometer (Bruker source at 200 °C. Full scan monitoring was performed Daltonics, Bremen, Germany). The spectra were from 45 to 500 m/z. All data was collected and recorded in the linear positive mode at a laser processed using Turbomass 6.1 (Perkin Elmer, frequency of 60 Hz within a mass range from m/z Courtaboeuf, France). Fatty acid methyl esters were 2000 to 20,000.