Introduction

The human gut microbiota is abundant and diversified, and has a great influence on human health. Therefore, characterizing its various members is of great interest for understanding its role in health and diseases. It was previously demonstrated that patients infected with Plasmodium falciparum may present an increased risk of bacteremia caused by gut bacteria [35]. In Africa, studies of febrile patients have demonstrated that the proportion of cases without identified etiology remains elevated [2]. This may be due to emerging or as-yet unknown pathogens, or to the lack of adequate diagnostic tools as recently reported in Gabon [23]. In this country, the prevalence of Rickettsia felis infections was demonstrated to be elevated in febrile patients [23], although many of them had been treated empirically for malaria [2]. As a consequence, it is important to search new bacteria using efficient tools such as culturomics [13].

The concept of culturomics recently developed in our laboratory is based on the use of diversified culture conditions (medium, atmosphere, and temperature). This strategy enabled us to isolate an important number of previously undetected or unknown bacteria in clinical samples [13]. Initially, culturomics was developed using more than 200 combinations of culture conditions [13]. Recently, our team demonstrated that the number of optimal culture conditions could be reduced to 18 [17]. Coupled to taxonogenomics, a new combination of genomic and phenotypic analyses for the taxonomic description of new bacteria [27], we proposed the creation of more than 50 new species and genera to date [7, 14–21, 24–26, 28]. Here, we report the biochemical features, complete genomic sequencing, and annotation of Gabonibacter massiliensis gen. nov., sp. nov., strain GM7T (= CSUR P2336 = DSM 101039), a new bacterium isolated from the gut microbiota of a healthy Gabonese male.

Materials and Methods

Ethics and Sample Collection

A culturomics study of the human gut microflora aiming at exploring its composition was conducted in Gabon. The new species described here was isolated from a stool specimen of a healthy 16 year-old male (BMI = 19.03) from Lebamba, a city located in the Ngounié province of south Gabon. Written informed consent was obtained from the patient. This study was validated by the ethics committees of the institute fédératif de recherche 48 (Marseille, France) and the National Ethics Committee of Gabon under registration numbers 09–022 and 0023/2013/SG/CNE, respectively. The stool sample was collected in January 2015 and stored at −80 °C until being shipped to the URMITE laboratory in dry ice.

Strain Isolation and MALDI-TOF MS Identification

Strain isolation consisted of four main steps: (a) dilution of the stool sample in phosphate-buffered saline (PBS) (Life technologies, Paisley, UK);(b) preincubation of diluted stool in an anaerobic blood culture bottle (Becton–Dickinson, Pont de Claix, France); (c) subculture of the inoculum on 5 % sheep blood-enriched Columbia agar (bioMérieux, Marcy l’Etoile, France) under anaerobic conditions at 37 °C [17]; and (d) identification of all different bacterial colonies using MALDI-TOF MS as previously reported [24–26, 33]. Reference spectra are available in our online database (http://www.mediterranee-infection.com/article.php?laref=256&titre=urms-database). Protein profiles of the new bacterium were compared to those of the closest bacteria (Fig. 1). The gel view displays the raw spectra of loaded spectrum files arranged in a pseudo-gel-like look. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed by a Grayscale scheme code. The color bar and the right y-axis indicate the relation between the color a peak is displayed with and the peak intensity in arbitrary units. Displayed species are indicated on the left.

Fig. 1
figure 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

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16S rRNA Sequencing and Phylogeny

DNA from strain GM7 was extracted using the BioRobot EZ1 Advanced XL (Qiagen, Courtaboeuf, France). The 16S rRNA was then amplified using three sets of primers (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). Sequencing was performed using an ABI Prism 3130xl Genetic Analyzer sequencer (Applied Biosystems) as previously described [22]. The obtained 16S rRNA sequence was compared to those in GenBank using BLASTn (http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/Blast.cgi) to determine the percentage of sequence similarity with the closest bacteria.

To construct a phylogenic tree, 16S rRNA sequences were aligned using the CLUSTALW software, inferences were obtained using the neighbor joining and maximum-likelihood methods, and the MEGA software with 1000 bootstrap repetitions [24–26].

Growth Conditions

To test the range of growth temperatures of strain GM7, the bacterium was cultivated on 5 % sheep blood-enriched Columbia agar (BioMérieux) in anaerobic atmosphere at 28, 37, 45, and 55 °C. The pH tolerance was also tested from 6.0 to 9.0. The atmosphere preference was evaluated using 5 % CO2, GENbag anaer and GENbag microaer systems (bioMérieux) at 37 °C to assess the ability of strain GM7 to grow in aerobic, anaerobic, or microaerophilic conditions. Additionally, the salinity test was performed [24–26].

Biochemical Properties and Motility Assays

Biochemical assays were performed using the API ZYM, API 20A, and API 50CH strips (bioMérieux). The catalase (bioMérieux) and oxidase (Becton, Dickinson and Company, Le Pont de Claix, France) activities were also tested. Fatty acid composition of outer membrane was performed using fatty acid methyl ester (FAME) analysis by GC/MS. Approximately, 40 mg of bacterial biomass was harvested from three different culture plates. Cellular FAMEs were prepared as previously described [32]. GC/MS analyses were carried out on a Clarus 500 gas chromatograph equipped with a SQ8S MS detector (Perkin Elmer, Courtaboeuf, France). Two µL of FAME extracts were volatized at 250 °C (split 20 mL/min) in a Focus liner with wool and separated on an Elite-5MS column (30 m, 0.25 mm i.d., 0.25 mm film thickness) using a linear temperature gradient (70–290 °C at 6 °C/min) enabling the detection of C4–C24 FAMEs. Helium flowing at 1.2 mL/min was used as carrier gas. MS inlet line was set at 250 °C and EI source at 200 °C. Full-scan monitoring was performed from 45 to 500 m/z. All data were collected and processed using Turbomass 6.1 (Perkin Elmer). FAMEs were identified by comparison with the FAME mass spectral database (Wiley, Chichester, UK) and the MS search using MS 2.0 operated with the standard reference database 1A (NIST, Gaithersburg, USA). A 37-component FAME mix (Supelco, Sigma-Aldrich, Saint-Quentin Fallavier, France) was used for retention time correlations with estimated non-polar retention indexes from the NIST database; FAME identifications were confirmed using this index.

Antibiotic Susceptibility

Antibiotics used to test the susceptibility of strain GM7 included amoxicillin, amoxicillin/clavulanic acid, imipenem, doxycycline, rifampicin, nitrofurantoin, vancomycin, clindamycin, gentamicin, erythromycin, metronidazole, trimethoprim/sulfamethoxazole, ciprofloxacin, amikacin, and tobramycin (i2a, Montpellier, France). Inhibition diameters were measured using the Scan1200® scanner (Interscience, Saint-Nom-La Bretêche, France) [24–26].

Microscopy

Individual cells of strain GM7 were visualized using a Tecnai G20 electron microscope (FEI company, Limeil-Brevannes, France) [24–26]. The Color Gram 2 Kit (BioMérieux) was used for the gram staining observed at a 1000× magnification using the DM1000 photonic microscope (Leica Microsystems) [24–26].

Genome Sequencing and Assembly

The genome sequencing of strain GM7 was performed using the MiSeq sequencer (Illumina, San Diego, CA, USA) with the Mate Pair strategy [24–26]. To be mixed with 11 other bacterial DNAs using the Nextera Mate Pair sample prep kit (Illumina), the genomic DNA of strain GM7 was barcoded. Overall, all genome sequencing and assembly steps were performed as previously reported [26]. The reads were trimmed and assembled using the CLC genomics Workbench v4.7.2 software (CLC bio, Aarhus, Denmark) [24–26].

Genome Annotation and Comparison

Genome annotation and comparison were performed using the Multi-Agent Software System DAGOBAH [5] that includes Figenix [6]. The genomic features of strain GM7 were compared to those of Butyricimonas virosa strain MT12T [31], Butyricimonas synergistica strain MT01T [31], Parabacteroides goldsteinii strain WAL 12034T [29], Parabacteroides distasonis strain JCM5825T [29], Parabacteroides merdae strain ATCC 43184T [29], and Parabacteroides johnsonii strain M-165T [30]. Two parameters, dDDH which was highly correlated with DDH [1, 18], and AGIOS [27] which was designed to be independent from DDH, were determined in order to evaluate the genomic similarity among studied strains.

Results

MALDI-TOF and Phylogenic analysis

MALDI-TOF MS failed to identify strain GM7 (score <1.7). Therefore, its spectrum was added to our database to improve its content (Fig. 1). The 16S rRNA sequencing showed that strain GM7 was 90 % similar to Butyricimonas virosa, the closest phylogenetic neighbor with standing in nomenclature (Fig. 2). This percentage of similarity is lower than the 16S rRNA gene sequence threshold recommended to delineate a genus [12]. Therefore, we propose that strain GM7 be a member of a new genus in the family Porphyromonadaceae for which we propose the name Gabonibacter gen. nov. In addition, this strain exhibited a degree of 16S rRNA similarity of 97.85 % to “Sanguibacteroides justesenii” strain OUH 308042 that has no standing in nomenclature but may belong to the same genus [9, 29, 34] (Table 1). The 16S rRNA sequence of strain GM7 was deposited in GenBank under accession number LN881588.

Fig. 2
figure 2

Neighbor Joining-based phylogenetic tree highlighting the position of Gabonibacter massiliensis strain GM7T compared to other members of the family Porphyromonadaceae. The respective GenBank accession numbers for 16S rRNA genes are indicated in parentheses. Numbers at the nodes are bootstrap values (>70 %) obtained by repeating the analysis 1000 times. The scale bar represents a 2 % nucleotide sequence divergence

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Table 1 Classification and general features of Gabonibacter massiliensis strain GM7T
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Phenotypic and Biochemical Characterization

Strain GM7 is a gram-negative coccobacillus. Cells are motile. Their mean length and diameter are 1.2 µm (range 0.92–1.38 µm) and 0.48 µm (range 0.4–0.56 µm), respectively (Fig. 3). Catalase and oxidase activities were negative (Table 2). Colonies of strain GM7 grown on 5 % sheep blood-enriched agar are white with a diameter of 2 mm. Strain GM7 was able to grow at 28 °C but its optimal growth temperature is 37 °C. No growth was observed at 45 and 55 °C. Strain GM7 is strictly anaerobic and unable to grow under microaerophilic or aerobic conditions. Its salinity tolerance varies from 0 to 5 g/L and pH tolerance ranges from 6.0 to 8.5. Results of API 50CH, API ZYM, and API 20A strips are detailed in Table 2 and in the species description below.

Fig. 3
figure 3

Electron microscopy of Gabonibacter massiliensis strain GM7 T.Transmission electron microscopy of Gabonibacter massiliensis strain GM7 T, using a Morgagni 268D (Philips) at an operating voltage of 60 kV. The scale bar represents 200 nm

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Table 2 Differential characteristics of Gabonibacter massiliensis strain GM7T (data from this study), Butyricimonas synergistica [31], Butyricimonas virosa [31], Parabacteroides merdae [29], Parabacteroides distasonis [29], Parabacteroides goldsteinii [29], and Parabacteroides johnsonii [30]
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Among tested antibiotics, cells were susceptible to imipenem, metronidazole, and nitrofurantoin but resistant to amoxicillin, amoxicillin/clavulanic acid, doxycycline, rifampicin, vancomycin, clindamycin, gentamicin, erythromycin, trimethoprim/sulfamethoxazole, ciprofloxacin, amikacin, and tobramycin.

The predominant cellular fatty acids of strain GM7 and the closest species are summarized in Table 3.

Table 3 Differential main membrane fatty acids compositions of Gabonibacter massiliensis strain GM7T (data from this study), Butyricimonas synergistica [31], Butyricimonas virosa [31], Parabacteroides merdae [29], Parabacteroides distasonis [29], Parabacteroides goldsteinii [29], and Parabacteroides johnsonii [30] in  %
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Genome Properties

The genome of strain GM7 is 3,397,022 bp long with a G+C content of 42.09 % (Table 4). It is composed of seven scaffolds and eight contigs (Fig. 4). On the 2880 predicted genes, 2819 were protein-coding genes, and 61 were RNAs (1 is 16S rRNA, 2 23S rRNA, 4 5S rRNA, 54 tRNAs). A total of 1858 genes (65.91 %) were assigned a putative function (by comparison to the COG or NR databases). Another 777 genes (27.56 %) were annotated as hypothetical proteins and 124 genes were identified as ORFans (4.40 %). Table 3 details the properties and statistics of the genome. The distribution of genes into COG functional categories is summarized in Table 5. The genome from strain GM7 was deposited in Genbank under accession number FAVK00000000.

Table 4 Nucleotide content and gene count levels of the genome
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Fig. 4
figure 4

Graphical circular map of the genome of Gabonibacter massiliensis strain GM7 T. From outside to the center: Contigs (red/gray), COG category of genes on the forward strand (three circles), genes on forward strand (blue circle), genes on reverse strand (red circle), COG category on the reverse strand (three circles), GC content

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Table 5 Number of genes associated with the 25 general COG functional categories
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Genome Comparison

The genome size of strain GM7 (3.39 Mb) is smaller than those of all compared species (B. virosa [4.72 Mb], B. synergistica [4.77 Mb], P. goldsteinii [7.09], P. distasonis [4.81 Mb], P. merdae [4.43], and P. johnsonii [4.63]) (Table 5). The G+C content of strain GM7 (42.09 %) is lower than those of B. virosa (42.3 %), B. synergistica (44.8 %), P. goldsteinii (43.5 %), P. distasonis (45.1 %), P. merdae (45.3 %), and P. johnsonii (45.1 %). The total number of genes of strain GM7 (2880) is smaller than those of B. virosa (3895), P. distasonis (3980), B. synergistica (3851) P. goldsteinii (5593), P. merdae (3653), and P. johnsonii (3731). The number of protein-coding genes of strain GM7 (2819) is smaller than those of B. virosa (3739), P. distasonis (3829), B. synergistica (3737), P. goldsteinii (5386), P. merdae (3414), and P. johnsonii (3599). The degrees of 16S rRNA similarity and orthologous protein content between these bacteria are summarized in Table 6. The AGIOS value ranged from 62.91 % between B. virosa and P. merdae to 90.95 between P. merdae and P. johnsonii among compared species except strain GM7. When strain GM7 was compared to other species, AGIOS values ranged from 62.71 (P. merdae and P. johnsonii) to 72.04 (B. virosa) (Table 7). The dDDH value ranged from 12.6 ± 0.99 to 49.4 ± 0.31 among compared species except strain GM7 (Table 8). When strain GM7 was compared to other species, dDDH values ranged from 12.5 ± 0.99 (P. merdae, P. johnsonii, and P. distasonis) to 13.2 ± 0.96 (B. synergistica) (Table 8). The distribution of functional classes of predicted genes into COG categories was similar in all compared genomes (Fig. 5).

Table 6 Genome comparison of closely related species to Gabonibacter massiliensis strain GM7
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Table 7 Numbers of orthologous protein (upper right)* and AGIOS values (lower left) observed between genomes
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Table 8 Pairwise comparison of Gabonibacter massiliensis with other species using GGDC, formula 2 (dDDH estimates based on identities/HSP length)* upper right
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Fig. 5
figure 5

Distribution of functional classes of predicted genes according to the clusters of orthologous groups of proteins

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Phenotypic Comparison

All compared species (G. massiliensis, B. synergistica, B. virosa, P. merdae, P. distasonis, P. goldsteinii, and P. johnsonii) are gram-negative, strictly anaerobic, without urease activity. However, only G. massiliensis is motile. Indole is produced only by strain GM7, B. virosa, and B. synergistica. Except for G. massiliensis, other compared species exhibit alkaline phosphatase activity. Catalase activity varies among studied species (Table 2). The most common cellular fatty acids shared by G. massiliensis, B. synergistica, B. virosa, P. merdae, P. distasonis, P. goldsteinii, and P. johnsonii are summarized in Table 3.

Discussion

Assessment of the human gut microbiota is of great interest in human health. Indeed, it was reported that the human gut microbiota composition (relative abundance of the distinct phyla) is linked to various diseases including, among others, obesity, type II diabetes, kwashiorkor, and marasmus [13, 17, 37]. However, the exhaustive assessment of complex microfloras is difficult. Recent efforts such as metagenomics and culturomics have permitted to increase the known repertory of bacteria thanks to the discovery of new families, genera, and species [4, 13, 17]. In our laboratory, over the past 5 years, we isolated representative strains from 124 putative new bacterial species including 39 strict anaerobes [4]. Of these, the names of 17 species have officially been validated [4, 7, 16, 19, 28]. The new bacterium described in this article, G. massiliensis gen. nov., sp. nov., strain GM7 was isolated from the same stool specimen as another two recently described new species, Gabonia massiliensis gen. nov., sp. nov., strain GM3 and Kallipyga gabonensis sp. nov., strain GM4 [24, 25].

As a large fraction of febrile infections remain undiagnosed in Gabon [2], due to the lack of adequate diagnostic tools [23] and/or the existence of as-yet undetected agents, our study participates in a better knowledge of human-associated microorganisms.

Conclusion

On the basis, the observed phenotypic and genomic differences with phylogenetically close species, we formally propose the creation of Gabonibacter massiliensis gen. nov., sp. nov. that contains strain GM7T. Strain GM7T was isolated from a stool specimen from a healthy Gabonese male and is the type strain of G. massiliensis gen. nov., sp.nov.

Taxonomic and Nomenclatural Proposals

Description of Gabonibacter gen. nov

Gabonibacter (Ga.bo.ni.bac.ter N. L. masc. n. Combination of Gaboni, from Gabon, the Sub-saharan African Country where stool specimen was collected and bacter, of bacteria the domain to which this microorganism belongs) cells are gram-negative and motile coccobacilli exhibiting a mean length and diameter of 1.2 µm (range 0.92–1.38 µm) and 0.48 µm (range 0.4–0.56 µm), respectively. Gabonibacter bacteria are strictly anaerobic and their optimal growth is obtained at 37 °C and their pH tolerance ranges from 6 to 8.5. Colonies grown on 5 % sheep blood-enriched Columbia agar are white with a diameter of 2 mm. Cells do not produce catalase and oxidase. Based upon 16S rRNA gene sequence analysis, Gabonibacter is a member of the family Porphyromonadaceae. Gabonibacter massiliensis sp. nov. and strain GM7T are the type species and type strain, respectively, of Gabonibacter gen. nov.

Description of Gabonibacter massiliensis gen nov., sp nov

Gabonibacter massiliensis strain GM7T (ma.si.li.e’n.sis. L. adj. masc. massiliensis, of Massilia, the Latin name of Marseille, where strain GM7T was isolated) is a gram-negative and motile coccobacillus whose individual cells measure 0.86 µm in length and 0.5 µm in diameter. It is strictly anaerobic and optimal growth is obtained at 37 °C. Colonies grown on 5 % sheep blood-enriched Columbia agar (bioMérieux) are white and exhibit a diameter of 2 mm. Strain GM7T produces indole but has no catalase and oxidase activities. Using an API 50 CH strip, cells use d-ribose, d-glucose, d-trehalose, d-tagatose, and potassium 5-ketogluconate. Using API ZYM, strain GM7 exhibits esterase (C4), esterase lipase (C8), acid phosphatase, and naphthol-AS-BI-phosphohydrolase activities. Using API 20A, positive reactions are only observed for D-mannitol, salicin, gelatin, and D-trehalose assays. Strain GM7 exhibits no alkaline phosphatase, lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, α and β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase and α-fucosidase activities. Additionally, strain GM7 does not metabolize L-tryptophane, urea, d-glucose, d-lactose, sucrose, d-maltose, d-xylose, l-arabinose, esculin ferric citrate, glycerol, d-cellobiose, d-mannose, d-melezitose, d-raffinose, d-sorbitol, and l-rhamnose. The bacterium is susceptible to imipenem, metronidazole, and nitrofurantoin. Its predominant cellular fatty acids are 13-methyl-tetradecanoic acid (15:0 iso), hexadecanoic acid (16:0), 9-octadecenoic acid (18:1 n9), 3-methyl-butanoic acid (5:0 iso), 3-hydroxy-heptadecanoic acid (17:0 3-OH), 3-hydroxy-hexadecanoic acid (16:0 3-OH), 12-methyl-tetradecanoic acid (15:0 anteiso), tetradecanoic acid (14:0), and 9,12-octadecadienoic acid (18:2 n6). The genome of G. massiliensis is 3,397,022 bp long with a G+C content of 42.09 %. The genome and 16S rRNA sequences from strain GM7T were deposited in Genbank under accession numbers FAVK00000000 and LN881588, respectively. The type strain GM7T, deposited in the CSUR and DSMZ collections under numbers CSUR P2336 and DSM 101039, respectively, was cultivated from the feces of a healthy 16-year-old Gabonese male.