Curr Microbiol DOI 10.1007/s00284-016-1137-2 Description of Gabonibacter massiliensis gen. nov., sp. nov., a New Member of the Family Porphyromonadaceae Isolated from the Human Gut Microbiota 1,2 1 3,4 Gae¨l Mourembou • Jaishriram Rathored • Jean Bernard Lekana-Douki • 5 1 1 Ange´lique Ndjoyi-Mbiguino • Saber Khelaifia • Catherine Robert • 1 1,6 1 Nicholas Armstrong • Didier Raoult • Pierre-Edouard Fournier Received: 9 June 2016 / Accepted: 8 September 2016 Ó Springer Science+Business Media New York 2016 Abstract The identification of human-associated bacteria Gabonibacter gen. nov. and the new species G. mas- is very important to control infectious diseases. In recent siliensis gen. nov., sp. nov. years, we diversified culture conditions in a strategy named culturomics, and isolated more than 100 new bacterial Keywords Gabonibacter massiliensis Á Taxonogenomics Á species and/or genera. Using this strategy, strain GM7, a Culturomics Á Gabon Á Gut microbiota strictly anaerobic gram-negative bacterium was recently isolated from a stool specimen of a healthy Gabonese Abbreviations patient. It is a motile coccobacillus without catalase and CSUR Collection de Souches de l’Unite´ des oxidase activities. The genome of Gabonibacter mas- Rickettsies siliensis is 3,397,022 bp long with 2880 ORFs and a G?C DSM Deutsche Sammlung von content of 42.09 %. Of the predicted genes, 2,819 are Mikroorganismen protein-coding genes, and 61 are RNAs. Strain GM7 differs MALDI-TOF Matrix-assisted laser desorption/ from the closest genera within the family Porphyromon- MS ionization time-of-flight mass adaceae both genotypically and in shape and motility. spectrometry Thus, we propose that strain GM7T (=CSUR URMITE Unite´ de Recherche sur les Maladies P2336 = DSM 101039) is the type strain of the new genus Infectieuses et Tropicales Emergentes FAME Fatty acid methyl ester GC/MS gas chromatography/mass spectrometry & Pierre-Edouard Fournier ORF Open reading Frame [email protected] BMI Body mass index 1 URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095, IHU Me´diterrane´e-Infection, Faculte´ de Me´decine, Aix- Introduction Marseille Universite´, 27 Bd Jean Moulin, Marseille, France 2 ´ Ecole Doctorale Regionale d’Afrique Centrale, B.P. 876, The human gut microbiota is abundant and diversified, and Franceville, Gabon 3 has a great influence on human health. Therefore, charac- Unite´ de Parasitologie Me´dicale (UPARAM), CIRMF B.P. terizing its various members is of great interest for 769, Franceville, Gabon understanding its role in health and diseases. It was pre- 4 De´partement de Parasitologie Mycologie et de Me´decine viously demonstrated that patients infected with Plasmod- Tropicale, Universite´ des Sciences de la Sante´, B.P. 4009 Libreville, Gabon ium falciparum may present an increased risk of bacteremia caused by gut bacteria [35]. In Africa, studies 5 De´partement de Microbiologie, Laboratoire national de re´fe´rence IST/sida, Faculte´ de Me´decine, Universite´ des of febrile patients have demonstrated that the proportion of Sciences de la Sante´, B.P. 4009 Libreville, Gabon cases without identified etiology remains elevated [2]. This 6 Special Infectious Agents Unit, King Fahd Medical Research may be due to emerging or as-yet unknown pathogens, or Center, King Abdulaziz University, Jeddah, Saudi Arabia to the lack of adequate diagnostic tools as recently reported 123 G. Mourembou et al.:Description of Gabonibacter massiliensis gen. nov., sp. nov., a new member… in Gabon [23]. In this country, the prevalence of Rickettsia inoculum on 5 % sheep blood-enriched Columbia agar felis infections was demonstrated to be elevated in febrile (bioMe´rieux, Marcy l’Etoile, France) under anaerobic patients [23], although many of them had been treated conditions at 37 °C[17]; and (d) identification of all dif- empirically for malaria [2]. As a consequence, it is ferent bacterial colonies using MALDI-TOF MS as previ- important to search new bacteria using efficient tools such ously reported [24–26, 33]. Reference spectra are available as culturomics [13]. in our online database (http://www.mediterranee-infection. The concept of culturomics recently developed in our com/article.php?laref=256&titre=urms-database). Protein laboratory is based on the use of diversified culture con- profiles of the new bacterium were compared to those of ditions (medium, atmosphere, and temperature). This the closest bacteria (Fig. 1). The gel view displays the raw strategy enabled us to isolate an important number of spectra of loaded spectrum files arranged in a pseudo-gel- previously undetected or unknown bacteria in clinical like look. The x-axis records the m/z value. The left y-axis samples [13]. Initially, culturomics was developed using displays the running spectrum number originating from more than 200 combinations of culture conditions [13]. subsequent spectra loading. The peak intensity is expressed Recently, our team demonstrated that the number of opti- by a Grayscale scheme code. The color bar and the right mal culture conditions could be reduced to 18 [17]. Cou- y-axis indicate the relation between the color a peak is pled to taxonogenomics, a new combination of genomic displayed with and the peak intensity in arbitrary units. and phenotypic analyses for the taxonomic description of Displayed species are indicated on the left. new bacteria [27], we proposed the creation of more than 50 new species and genera to date [7, 14–21, 24–26, 28]. 16S rRNA Sequencing and Phylogeny Here, we report the biochemical features, complete geno- mic sequencing, and annotation of Gabonibacter mas- DNA from strain GM7 was extracted using the BioRobot siliensis gen. nov., sp. nov., strain GM7T (= CSUR EZ1 Advanced XL (Qiagen, Courtaboeuf, France). The P2336 = DSM 101039), a new bacterium isolated from the 16S rRNA was then amplified using three sets of primers gut microbiota of a healthy Gabonese male. (fD1/800R, 536F/1050R, and 800F/rP2) [24–26]. Briefly, each PCR reaction was performed using the 2720 thermal cycler (Applied Biosystems, Bedford, MA, USA). Materials and Methods Sequencing was performed using an ABI Prism 3130xl Genetic Analyzer sequencer (Applied Biosystems) as pre- Ethics and Sample Collection viously described [22]. The obtained 16S rRNA sequence was compared to those in GenBank using BLASTn (http:// A culturomics study of the human gut microflora aiming at blast.ncbi.nlm.nih.gov.gate1.inist.fr/Blast.cgi) to determine exploring its composition was conducted in Gabon. The the percentage of sequence similarity with the closest new species described here was isolated from a stool bacteria. specimen of a healthy 16 year-old male (BMI = 19.03) To construct a phylogenic tree, 16S rRNA sequences from Lebamba, a city located in the Ngounie´ province of were aligned using the CLUSTALW software, inferences south Gabon. Written informed consent was obtained from were obtained using the neighbor joining and maximum- the patient. This study was validated by the ethics com- likelihood methods, and the MEGA software with 1000 mittees of the institute fe´de´ratif de recherche 48 (Marseille, bootstrap repetitions [24–26]. France) and the National Ethics Committee of Gabon under registration numbers 09–022 and 0023/2013/SG/CNE, Growth Conditions respectively. The stool sample was collected in January 2015 and stored at -80 °C until being shipped to the To test the range of growth temperatures of strain GM7, the URMITE laboratory in dry ice. bacterium was cultivated on 5 % sheep blood-enriched Columbia agar (BioMe´rieux) in anaerobic atmosphere at Strain Isolation and MALDI-TOF MS Identification 28, 37, 45, and 55 °C. The pH tolerance was also tested from 6.0 to 9.0. The atmosphere preference was evaluated Strain isolation consisted of four main steps: (a) dilution of using 5 % CO2, GENbag anaer and GENbag microaer the stool sample in phosphate-buffered saline (PBS) (Life systems (bioMe´rieux) at 37 °C to assess the ability of strain technologies, Paisley, UK);(b) preincubation of diluted GM7 to grow in aerobic, anaerobic, or microaerophilic stool in an anaerobic blood culture bottle (Becton–Dick- conditions. Additionally, the salinity test was performed inson, Pont de Claix, France); (c) subculture of the [24–26]. 123 G. Mourembou et al.:Description of Gabonibacter massiliensis gen. nov., sp. nov., a new member… Fig. 1 Gel view of MALDI- TOF MS spectra comparing Gabonibacter massiliensis strain GM7T to other closely related bacteria. The gel view displays the raw spectra of loaded spectrum files arranged in a pseudo-gel-like look Biochemical Properties and Motility Assays Antibiotic Susceptibility Biochemical assays were performed using the API ZYM, Antibiotics used to test the susceptibility of strain GM7 API 20A, and API 50CH strips (bioMe´rieux). The catalase included amoxicillin, amoxicillin/clavulanic acid, imipe- (bioMe´rieux) and oxidase (Becton, Dickinson and Com- nem, doxycycline, rifampicin, nitrofurantoin, vancomycin, pany, Le Pont de Claix, France) activities were also tested. clindamycin, gentamicin, erythromycin, metronidazole, Fatty acid composition of outer membrane was performed trimethoprim/sulfamethoxazole, ciprofloxacin, amikacin, using fatty acid methyl ester (FAME) analysis by GC/MS. and tobramycin (i2a, Montpellier, France). Inhibition Approximately, 40 mg of bacterial biomass was harvested diameters were measured using the Scan1200Ò scanner from three different culture plates. Cellular FAMEs were (Interscience, Saint-Nom-La Breteˆche, France) [24–26]. prepared