Standards in Genomic Sciences (2012) 6:315-324 DOI:10.4056/sigs.2685971 Non-contiguous finished genome sequence and description of Alistipes timonensis sp. nov. Jean-Christophe Lagier1, Fabrice Armougom1, Ajay Kumar Mishra1, Thi-Tien Nguyen 1, Didier Raoult1 and Pierre-Edouard Fournier1* 1Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UMR, Aix-Marseille Université, Marseille, France *Corresponding author: Pierre-Edouard Fournier ([email protected]) Keywords: Alistipes timonensis, genome Alistipes timonensis strain JC136T sp. nov. is the type strain of A. timonensis sp. nov., a new species within the genus Alistipes. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. A. timonensis is an obligate anaerobic rod. Here we describe the features of this organism, together with the complete genome sequence and an- notation. The 3,497,779 bp long genome (one chromosome but no plasmid) contains 2,742 protein-coding and 50 RNA genes, including three rRNA genes. Introduction Alistipes timonensis strain JC136T (= CSUR P148 = Here we present a summary classification and a DSM 25383) is the type strain of A. timonensis sp. set of features for A. timonensis sp. nov. strain nov. This bacterium is a Gram-negative, anaerobic, JC136T together with the description of the com- indole-positive bacillus and was isolated from the plete genomic sequencing and annotation. These stool of a healthy Senegalese patient as part of a characteristics support the circumscription of the “culturomics” study aiming at cultivating individ- species A. timonensis. ually all species within human feces. The genus Alistipes (Rautio et al. 2003) was creat- With more than 3,000 genome sequences availa- ed in 2003 [5]. To date, this genus, composed of ble, bacterial genomics has revolutionized several bile-resistant, strictly anaerobic and Gram- aspects of microbiology. To date, taxonomy has negative bacilli, contains five species including A. remained unaffected by this progress, despite the finegoldii (Rautio et al. 2003) [5], A.indistinctus debate around the definition of bacterial species. (Nagai et al. 2010) [6], A. onderdonkii (Song et al. Despite its elevated cost, poor reproducibility and 2006) [7], A. putredinis (Weinberg et al. 1937) inter-laboratory comparability, DNA-DNA hybrid- Rautio et al. 2003 [5], and A. shahii (Song et al. ization remains the “gold standard” criterion [1]. 2006) [7]. Pigment production, initially consid- Even the application of internationally validated ered as characteristic of Alistipes species, was re- cutoff values in 16S rRNA sequence similarity that cently demonstrated to be inconstant [8]. Mem- enabled the taxonomic classification or reclassifi- bers of the genus Alistipes are members of the cation of hundreds of taxa, is debated [2]. High normal human intestinal microbiota, but have also throughput genome sequencing and mass spec- been reported in urine and the mouth [7], and trometric analyses of bacteria provide access to a have occasionally been isolated from abdominal, wealth of genetic and proteomic information [3]. appendiceal and rectal abscesses, blood cultures We propose to use a polyphasic approach [4] to from colon cancer patients [9], and feces from describe new bacterial taxa that includes their ge- children with irritable bowel syndrome [10]. A. nome sequence, MALDI-TOF spectrum and main putredinis was also demonstrated to be associated phenotypic characteristics (habitat, Gram-stain to cruciferous vegetable intake [11]. In addition, A. reaction, culture and metabolic characteristics, finegoldii has been suspected to play the role of and when applicable, pathogenicity). growth promoter in chickens [12]. The Genomic Standards Consortium Alistipes timonensis sp. nov. Classification and features blood-enriched Columbia agar (BioMerieux, Marcy A stool sample was collected from a healthy 16- l’Etoile, France). This strain exhibited 96.98% and year-old male Senegalese volunteer patient living 98.13% nucleotide sequence similarities with A. in Dielmo (a rural village in the Guinean-Sudanian shahii (Song et al. 2006) and A. senegalensis (Mish- zone in Senegal), who was included in a research ra et al. 2012), respectively, the phylogenetically protocol. The patient gave an informed and signed closest validated Alistipes species (Figure 1) [7]. consent, and the agreement of the National Ethics This value was lower than the 98.7% 16S rRNA Committee of Senegal and the local ethics commit- gene sequence threshold recommended by tee of the IFR48 (Marseille, France) were obtained Stackebrandt and Ebers to delineate a new species under agreement 09-022. The fecal specimen was without carrying out DNA-DNA hybridization [2]. preserved at -80°C after collection and sent to It should be noted that both A. senegalensis strain Marseille. Strain JC136 (Table 1) was isolated in JC50T and strain JC136 were cultivated from the June 2011 by anaerobic cultivation on 5% sheep same individual. Table 1. Classification and general features of Alistipes timonensis strain JC136T MIGS ID Property Term Evidence codea Domain Bacteria TAS [13] Phylum Bacteroidetes TAS [14,15] Class Bacteroidia TAS [14,16] Current classification Order Bacteroidales TAS [14,17] Family Rikenellaceae TAS [14,18] Genus Alistipes TAS [5,19] Species Alistipes timonensis IDA Type strain JC136T IDA Gram stain Negative IDA Cell shape Bacilli IDA Motility Nonmotile IDA Sporulation Nonsporulating IDA Temperature range Mesophile IDA Optimum temperature 37°C IDA MIGS-6.3 Salinity Growth in BHI medium + 1% NaCl IDA MIGS-22 Oxygen requirement Anaerobic IDA Carbon source Unknown NAS Energy source Unknown NAS MIGS-6 Habitat Human gut IDA MIGS-15 Biotic relationship Free living IDA Pathogenicity Unknown Biosafety level 2 MIGS-14 Isolation Human feces NAS MIGS-4 Geographic location Senegal IDA MIGS-5 Sample collection time September 2010 IDA MIGS-4.1 Latitude 13.7167 IDA MIGS-4.1 Longitude 16.4167 IDA MIGS-4.3 Depth Surface IDA MIGS-4.4 Altitude 51 m above sea level IDA Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or an- ecdotal evidence). These evidence codes are from the Gene Ontology project [20]. If the evi- dence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements. 316 Standards in Genomic Sciences Lagier et al. Figure 1. Phylogenetic tree highlighting the position of Alistipes timonensis strain JC136T relative to other type strains within the Alistipes genus. GenBank accession numbers are indicated in parentheses. Sequences were aligned using CLUSTALW, and phylogenetic inferences obtained using the maximum-likelihood method within the MEGA software. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 500 times to generate a majority consensus tree. Porphyromonas asaccharolytica was used as an outgroup. The scale bar represents a 2% nucleotide sequence divergence. Different growth temperatures (25, 30, 37, 45°C) (BioMerieux), and in the presence of air, with or were tested; no growth occurred at 25°C and 45°C, without of 5% CO2, and in aerobic conditions. Op- growth occurred at 30°C, and optimal growth was timal growth was achieved anaerobically. No observed at 37°C. Colonies were 0.2 mm to 0.3 growth was observed in aerobic, microaerophilic mm in diameter on blood-enriched Columbia agar and 5% CO2 atmospheres. Gram staining showed and Brain Heart Infusion (BHI) agar. Growth of the Gram negative rods (Figure 2). A motility test was strain was tested under anaerobic and negative. Cells grown on agar have a mean diame- microaerophilic conditions using GENbag anaer ter of 0.62 µm (Figure 3) and produce brown pig- and GENbag microaer systems, respectively ment. Figure 2. Gram staining of A. timonensis strain JC136T http://standardsingenomics.org 317 Alistipes timonensis sp. nov. Figure 3. Transmission electron microscopy of A. timonensis strain JC136T, using a Morgani 268D (Philips) at an operating voltage of 60kV. The scale bar represents 900 nm. Strain 136T exhibited catalase activity but no oxi- dry for five minutes. Measurements were per- dase activity, and was resistant to 20% bile. Using formed with a Microflex spectrometer (Bruker). API Rapid ID 32A, a positive reaction was obtained Spectra were recorded in the positive linear mode - - - for the mass range of 2,000 to 20,000 Da (parame- glucuronidase, glutamic acid decarboxylase, leucyl ter settings: ion source 1 (IS1), 20 kV; IS2, 18.5 kV; glycinefor αarylamidasegalactosidase, and βalaninegalactosidase, arylamidase. β lens, 7 kV). A spectrum was obtained after 675 Weak reactions were obtained for indole produc- shots at a variable laser power. The time of acqui- tion and N-acetyl- -glucosaminidase. No mannose sition was between 30 seconds and 1 minute per and raffinose fermentation were observed. A. spot. The four JC136 spectra were imported into timonensis is susceptibleβ to penicillin G, amoxicil- the MALDI BioTyper software (version 2.0, lin + clavulanic acid, imipeneme, clindamycin, Bruker) and analyzed by standard pattern match- metronidazole and resistant to vancomycin. By ing (with default parameter settings) against the comparison with A. senegalensis, strain