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Non-contiguous finished genome sequence and description of Alistipes ihumii sp. nov.

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Abstract

Alistipes ihumii strain AP11T sp. nov. is the type strain of A. ihumii sp. nov., a new species within the genus Alistipes. This strain, whose genome is described here, was isolated from the fecal flora of a 21-year-old French Caucasian female, suffering from a severe restrictive form of anorexia nervosa since the age of 12 years. A. ihumii is a Gram-negative anaerobic bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,753,264 bp long genome (one chromosome but no plasmid) contains 2,254 protein-coding and 47 RNA genes, including 3 rRNA genes.

Introduction

Alistipes ihumii strain AP11T (= CSUR P204 = DSM 26107) is the type strain of A. ihumii sp. nov. This bacterium is a Gram-negative, non-spore-forming, anaerobic and non-motile bacillus that was isolated from the stool of a 21-year-old French female suffering from anorexia nervosa, and is part of a “culturomics” study aiming at cultivating individually all species within human feces [13].

Prokaryotic taxonomy is episodically confronted with the advancement of methodological and conceptual innovations. The current classification methodology for prokaryotes is known as polyphasic taxonomy, and relies on a combination of phenotypic and genotypic characteristics [4]. The number of completely sequenced genomes is geometrically increasing with time, concurrently with the decrease in cost of such techniques. To date, more than 6,000 bacterial genomes have been published and approximately 25,000 genome sequencing projects have been announced [5]. We recently proposed to integrate genomic information in the taxonomic framework for the description of new bacterial species [627].

The genus Alistipes (Rautio et al. 2003) was created in 2003 [28] and is composed of strictly anaerobic Gram-negative rods that resemble the Bacteroides fragilis group in that most species are bile-resistant and indole-positive [29]. This genus is currently comprised of five species with validly published names, including A. finegoldii, A. putredinis [28], A. indistinctus [30], A. onderdonkii and A. shahii [31], to which we added three proposed new species, A. senegalensis [8], A. timonensis [9] and A. obesi [22].

Here we present a summary classification and a set of features for a new Alistipes species, A. ihumii sp. nov. strain AP11T (= CSUR P204 = DSM 26107), together with the description of the complete genomic sequence and its annotation.

Classification and features

A stool sample was collected from a 21-year-old French Caucasian female suffering from severe restrictive form of anorexia nervosa since the age of 12 years. At the time of sample collection, she was hospitalized in our hospital for recent aggravation of her medical condition (BMI: 10.4 kg/m2). The patient gave an informed and signed consent. Both this study and the assent procedure were approved by the Ethics Committee of the Institut Fédératif de Recherche IFR48, Faculty of Medicine, Marseille, France under reference 09-022. Ten other potentially new bacterial species were isolated from this patient’s stool, all of which are currently being described. Microbial culturomics also enabled the isolation of several other new bacterial species from other stool specimens [627]. The fecal specimen was stored at −80°C immediately after collection. Strain AP11T was isolated in November 2011 after 2 days of inoculation in anaerobic blood culture bottle with the addition of 5mL of thioglycolate and further inoculation on Columbia agar (BioMerieux, Marcy l’Etoile, France).

This strain exhibited a 95% 16S rRNA sequence similarity with A. indistinctus [30], the phylogenetically closest Alistipes species with a validly published name (Table 1, Figure 1), and 92% with A. onderdonkii [28] and A. putredinis [31]. This value was in the range of 16S rRNA sequence identities among species within the genus Alistipes that range from 90 to 95%, and lower than the 98.7% 16S rRNA gene sequence threshold recommended by Stackebrandt and Ebers to delineate a new species without carrying out DNA-DNA hybridization [41].

Figure 1.
figure1

Phylogenetic tree highlighting the position of Alistipes ihumii strain AP11T relative to other type strains within the genus Alistipes. 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. Bacteroides splanchnicus was used as the outgroup. The scale bar represents a 2% nucleotide sequence divergence.

Table 1. Classification and general features of Alistipes ihumii strain AP11T according to the MIGS recommendations [32]

Different growth temperatures (25, 30, 37, 45°C) were tested. Growth was observed between 25 and 45°C, with optimal growth at 37°C after 24 hours of inoculation. Colonies were about 0.2 mm in diameter, transparent, and exhibited a ß-hemolytic activity on blood-enriched Columbia agar. Growth of the strain was tested on 5% sheep blood agar, under anaerobic and microaerophilic conditions using the GENbag anaer and GENbag microaer systems, respectively (BioMerieux), and under aerobic conditions with or without 5% CO2. Optimal growth of this strain was obtained anaerobically, weak growth was observed under microaerophilic conditions, and no growth was observed under aerobic atmosphere. The motility test was negative. Cells grown on agar are Gram-negative rods (Figure 2) and have mean diameter and length of 0.72 and 1.69 µm, respectively, as determined using electron microscopy (Figure 3). Strain AP11T exhibited oxidase but no catalase activities. Using API 50CH (BioMérieux), we observed that strain AP11T was asaccharolytic. Using API 32A (BioMérieux), positive reactions were obtained for α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, mannose and raffinose fermentation, alkaline phosphatase, leucyl glycine arylamidase, alanine arylamidase, and glutamyl glutamic acid arylamidase. Weak reactions were observed for α-galactosidase and glutamic acid decarboxilase. Negative reactions were obtained for urease, arginine dihydrolase, β-galactosidase, 6 phospho-β-galactosidase, α-arabinosidase, β-glucuronidase, α-fucosidase, nitrate reduction, indole production, arginine arylamidase, proline arylamidase, phenylalanine arylamidase, leucine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, glycine arylamidase, histidine arylamidase, and serine arylamidase. A. ihumii is susceptible to amoxicillin, imipenem, and clindamycin, but resistant to vancomycin. When compared with representative species from the genus Alistipes, strain AP11T exhibited the phenotypic differences detailed in Table 2.

Figure 2.
figure2

Gram stain of A. ihumii strain AP11T

Figure 3.
figure3

Transmission electron microscopy of A. ihumii strain AP11T, using a Morgani 268D (Philips) at an operating voltage of 60kV. The scale bar represents 500 nm.

Table 2. Differential characteristics of Alistipes strains

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was carried out as previously described [42] using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). Twelve individual colonies were deposited on a MTP 384 MALDI-TOF target plate (Bruker). The twelve AP11T spectra were imported into the MALDI BioTyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against the main spectra of 4,706 bacteria, including spectra from A. finegoldii, A. onderdonkii, A. shahii, A. senegalensis, A. obesi and A. timonensis, used as reference data in the BioTyper database. The output score enabled the presumptive identification and discrimination of the tested species from those in the database: a score ≥ 2 with a validated species identifies a strain at the species level; and a score < 1.7 indicates a species-level match was not made. For strain AP11T, no significant score was obtained, suggesting that our isolate was not a member of any known species (Figures 4 and 5). We added the spectrum from strain AP11T to our database.

Figure 4.
figure4

Reference mass spectrum from A. ihumii strain AP11T. Spectra from 12 individual colonies were compared and a reference spectrum was generated.

Figure 5.
figure5

Gel view comparing spectra from Alistipes ihumii strain AP11T and other members of the genus Alistipes (A. obesi, A. timonensis, A. senegalensis, A. shahii, A. onderdonkii and A. finegoldii). The Gel View displays the raw spectra of all loaded spectrum files arranged in a pseudo-gel like look, with each peak displayed as a band or bar. The peak intensity is reflected by the intensity of the gray color. The right y-axis shows the relationship between the shades of gray and the peak intensity in arbitrary units. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading.

Genome sequencing information

Genome project history

The organism was selected for sequencing on the basis of its phylogenetic position and 16S rRNA similarity to other members of the Alistipes genus, and is part of a “culturomics” study of the human digestive flora aiming at isolating all bacterial species within human feces. It was the eighth sequenced genome from an Alistipes species and the first from Alistipes ihumii sp. nov. A summary of the project information is shown in Table 3. The Genbank accession number is CAPH00000000 and consists of 60 contigs. Table 3 shows the project information and its association with MIGS version 2.0 compliance [43].

Table 3. Project information

Growth conditions and DNA isolation

A. ihumii sp. nov. strain AP11T, (= CSURP204 = DSM 26107), was grown aerobically on 5% sheep blood agar medium at 37°C. Five Petri dishes were spread and resuspended in 3x100µl of G2 buffer (EZ1 DNA Tissue kit, Qiagen). A first mechanical lysis was performed by glass powder on the Fastprep-24 device (Sample Preparation system, MP Biomedicals, USA) for 2×20 seconds. DNA was treated with 2.5 µg/µL of lysozyme (30 minutes at 37°C) and extracted using the BioRobot EZ 1 Advanced XL (Qiagen). The DNA was concentrated and purified on a Qiamp kit (Qiagen). The yield and the concentration of DNA was 70.7 ng/µl as measured by using Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer.

Genome sequencing and assembly

A 3kb paired-end sequencing strategy (Roche, Meylan, France) was used. DNA (5 µg) was mechanically fragmented for the paired-end sequencing, using a Covaris device (Covaris Inc., Woburn, MA, USA) with an enrichment size of 3–4 kb. The DNA fragmentation was visualized through an Agilent 2100 BioAnalyzer on a DNA Labchip 7500 which yielded an optimal size of 2.3 kb. The library was constructed using the 454 GS FLX Titanium paired-end rapid library protocol. Circularization and nebulization were performed which generated a pattern of optimal size of 457 bp. PCR amplification was performed for 17 cycles followed by double size selection. The single-stranded paired-end library was quantified using a Quant-it Ribogreen Kit (Invitrogen) and the Genios Tecan fluorometer. The library concentration equivalence was calculated as 1.94× 1010 molecules/µL. The library was stored at −20°C until further use.

The paired-end library was clonally amplified with 0.5 and 1 cpb in 2 emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yield of the shotgun emPCR reactions was 6.24 and 16.24% respectively for the two kinds of paired-end emPCR reactions according to the quality expected (range of 5 to 20%) from the Roche procedure. Two libraries were loaded on the GS Titanium PicoTiterPlates (PTP Kit 70x75, Roche) and pyrosequenced with the GS Titanium Sequencing Kit XLR70 and the GS FLX Titanium sequencer (Roche). The run was performed overnight and then analyzed on the cluster through the gsRunBrowser and Newbler assembler (Roche). A total of 260,838 passed filter wells were obtained and generated 96.3 Mb with an average length of 369 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 9 scaffolds and 60 contigs (> 1,500 bp) and generated a genome size of 2.75 Mb which corresponds to a coverage of 35× genome equivalent.

Genome annotation

Open Reading Frames (ORFs) were predicted using Prodigal [44] with default parameters but the predicted ORFs were excluded if they were spanning a sequencing gap region. The predicted bacterial protein sequences were searched against the GenBank database [45] and the Clusters of Orthologous Groups (COG) databases using BLASTP. The tRNAScan-SE tool [46] was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer [47] and BLASTn against the GenBank database. Lipoprotein signal peptides and numbers of transmembrane helices were predicted using SignalP [48] and TMHMM [49] respectively. ORFans were identified if their BLASTP E-value was lower than 1e−03 for alignment length greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we used an E-value of 1e−05. Such parameter thresholds have already been used in previous works to define ORFans.

Orthologous gene sets composed of one gene from A. ihumii compared to each of A. obesi strain ph8T (GenBank accession number CAHA00000000), A. finegoldii strain AHN 2437 (CP003274), A. indistinctus strain YIT 12060 (ADLD00000000), A. putredinis strain DSM 17216 (ABFK00000000), A. senegalensis strain JC50T (CAHI00000000), A. shahii strain WAL 8301 (FP929032), and A. timonensis strain JC136T (CAEG00000000) were identified using the Proteinortho software (version 1.4) [50] using a 30% protein identity and an E-value of 1e−05. The average percentage of nucleotide sequence identity of each orthologous set was determined using the Needleman-Wunsch algorithm global alignment technique. Artemis [51] was used for data management and DNA Plotter [52] was used for visualization of genomic features. The Mauve alignment tool was used for multiple genomic sequence alignment and visualization [53].

Genome properties

The genome of A. ihumii strain AP11T is 2,753,264 bp long (1 chromosome, but no plasmid) with a 57.90% G + C content (Figure 6 and Table 4). Of the 2,301 predicted genes, 2,254 were protein-coding genes, and 47 were RNAs. One rRNA operon (one 16S rRNA, one 23S rRNA and one 5S rRNA) and 44 predicted tRNA genes were identified in the genome. A total of 1,465 genes (63.66%) were assigned a putative function. Two hundred thirty-seven genes were identified as ORFans (10.29%). The remaining genes were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Tables 4 and 5. The distribution of genes into COGs functional categories is presented in Table 5.

Figure 6.
figure6

Graphical circular map of the chromosome. From the outside in, the outer two circles show open reading frames oriented in the forward and reverse (colored by COG categories) directions, respectively. The third circle marks the rRNA gene operon (red) and tRNA genes (green). The fourth circle shows the G+C% content plot. The inner-most circle shows GC skew, purple and olive indicating negative and positive values, respectively.

Table 4. Nucleotide content and gene count levels of the genome
Table 5. Number of genes associated with the 25 general COG functional categories

Genome comparison with other Alistipes species

Here, we compared the genome of A. ihumii strain AP11T to those of A. obesi strain ph8T (GenBank accession number CAHA00000000), A. finegoldii strain AHN 2437 (CP003274), A. indistinctus strain YIT 12060 (ADLD00000000), A. putredinis strain DSM 17216 (ABFK00000000), A. senegalensis strain JC50T (CAHI00000000), A. shahii strain WAL 8301 (FP929032), and A. timonensis strain JC136T (CAEG00000000). The draft genome of A. ihumii is larger than that of A. putredinis (2.75 and 2.55 Mb, respectively) but smaller than those of A. indistinctus, A. obesi, A. timonensis, A. finegoldii, A. shahii and A. senegalensis (2.85, 3.16, 3.49, 3.73, 3.76, and 4.01 Mb, respectively). The G+C content of A. ihumii is comparable to that of A. shahii (57.90 and 57.60%, respectively), lower than those of A. timonensis and A. senegalensis (58.8 and 58.4%, respectively) and higher than those of A. putredinis, A. indistinctus and A. finegoldii (53.30, 54.80 and 56.60%, respectively). A. ihumii has a smaller gene content than those of A. putredinis, A. indistinctus, A. obesi, A. timonensis, A. shahii, A. senegalensis, and A. finegoldii (2,301, 2,335, 2,342, 2,619, 2,709, 3,132, 3,161, and 3,231, respectively). The ratio of genes per MB of A. ihumii is higher than those of A. timonensis, A. senegalensis, A. indistinctus, and A. obesi (836, 776, 788, 821, and 828, respectively), comparable to that of A. shahii (833) and smaller than those of A. finegoldii and A. putredinis (866 and 915, respectively).

The average genomic nucleotide sequence identity between A. ihumii and other Alistipes species ranged from 70.23 to 74.37%, whereas values ranged from 69.70 to 90.98% among other Alistipes species (Table 6).

Table 6. Numbers of orthologous proteins shared between genomes

However, the distribution of genes into COG categories was not entirely similar in all eight compared genomes (Figure 7).

Figure 7.
figure7

Distribution of functional classes of predicted genes in Alistipes ihumii (colored in green), A. senegalensis (pink), A. timonensis (yellow), A. shahii (brown), A. finegoldii (blue), A. putredinis (red), A. obesi (orange) and A. indistinctus (black) chromosomes according to the clusters of orthologous groups of proteins.

Conclusion

On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Alistipes ihumii sp. nov. that contains strain AP11T. This bacterial strain has been isolated from the fecal flora of a patient suffering from anorexia nervosa living in Marseille, France. Several other new bacterial species were also cultivated from this patient as well as fecal samples from other patients using microbial culturomics [627], thus suggesting that the human fecal flora from human remains partially unknown.

Description of Alistipes ihumii sp. nov.

Alistipes ihumii (i.hum.i’i. N.L. gen. n. ihumii, based on the acronym IHUMI, the Institut Hospitalo-Universitaire Méditerranée-Infection, where the type strain was isolated).

Colonies are 0.2 mm in diameter and are translucent on blood-enriched Columbia agar. Cells are rod-shaped with a mean diameter of 0.72 µm and a mean length of 1.69 µm. Optimal growth is achieved anaerobically. No growth is obtained aerobically but weak growth is observed in microaerophilic conditions. Growth occurs between 25°C and 45°C, with an optimal growth observed at 37°C.

Cells stain Gram-negative, are non motile and are asaccharolytic. Activities present are α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, mannose and rafinnose fermentation, alkaline phosphatase, leucyl glycine arylamidase, alanine arylamidase, and glutamyl glutamic acid arylamidase. Cells are negative for urease, arginine dihydrolase, β-galactosidase, 6-phospho-β-galactosidase, α-arabinosidase, β-glucuronidase, α-fucosidase, nitrate reduction, indole production, arginine arylamidase, proline arylamidase, phenylalanine arylamidase, leucine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, glycine arylamidase, histidine arylamidase, and serine arylamidase. Cells are susceptible to amoxicillin, imipenem, and clindamycin, but resistant to vancomycin. The G+C content of the genome is 57.90%. The 16S rRNA and genome sequences are deposited in Genbank under accession numbers JX101692 and CAPH00000000, respectively.

The type strain AP11T (= CSUR P204 = DSM 26107) was isolated from the fecal flora of a 21-year-old French Caucasian female suffering from severe anorexia nervosa.

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Acknowledgements

The authors thank the Xegen Company (www.xegen.fr) for automating the genomic annotation process. This study was funded by the Mediterranee-Infection Foundation.

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Correspondence to Pierre-Edouard Fournier.

Additional information

These two authors contributed equally to this work

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Keywords

  • Alistipes ihumii
  • genome
  • culturomics
  • taxono-genomics