Open Access

Genome sequence and description of Corynebacterium ihumii sp. nov.

  • Roshan Padmanabhan
  • , Grégory Dubourg
  • , Jean-Christophe Lagier
  • , Carine Couderc
  • , Caroline Michelle
  • , Didier Raoult,
  • and Pierre-Edouard Fournier
Corresponding author

DOI: 10.4056/sigs.5149006

Received: 15 March 2014

Accepted: 15 March 2014

Published: 15 June 2014

Abstract

Corynebacterium ihumii strain GD7T sp. nov. is proposed as the type strain of a new species, which belongs to the family Corynebacteriaceae of the class Actinobacteria. This strain was isolated from the fecal flora of a 62 year-old male patient, as a part of the culturomics study. Corynebacterium ihumii is a Gram positive, facultativly anaerobic, nonsporulating bacillus. Here, we describe the features of this organism, together with the high quality draft genome sequence, annotation and the comparison with other member of the genus Corynebacteria. C. ihumii genome is 2,232,265 bp long (one chromosome but no plasmid) containing 2,125 protein-coding and 53 RNA genes, including 4 rRNA genes. The whole-genome shotgun sequence of Corynebacterium ihumii strain GD7T sp. nov has been deposited in EMBL under accession number GCA_000403725.

Keywords:

Corynebacterium ihumiigenomeculturomicstaxono-genomics

Introduction

Corynebacterium ihumii strain GD7T sp. nov. (= CSUR P902, = DSM 45751) is the type strain of Corynebacterium ihumii strain GD7T sp. nov. This bacterium is a Gram-positive, facultativly anaerobic, non spore-forming, non-motile bacillus that was isolated from the stool of a 62 year-old French male who was admitted to the intensive care unit in the Timone Hospital, Marseille, France, for respiratory distress. This strain was isolated as a part of “culturomics” project whose scope is to cultivate all species within human feces [1,2].

The current classification of prokaryotes is based on a combination of phenotypic and genotypic characteristics [3,4] that include 16S rRNA gene phylogeny and nucleotide sequence similarity, G + C content and DNA–DNA hybridization (DDH). Despite being considered as a “gold standard” these genotypic tools exhibit several drawbacks that are overcome by newer sequencing methods [5,6]. Because of the rapidly declining cost of sequencing, the number of sequenced bacterial genomes rapidly increased (almost 7,000 to date [7]). Hence, we recently proposed to incorporate genomic information among criteria used for the description of new bacterial species [8-29].

Corynebacteria are Gram-positive bacteria that belong to the phylum Actinobacteria and have a high G+C content. They are found in diverse ecological niches such as soil, clinical specimens, cheese smear, vegetables, sewage etc. The genus Corynebacterium was created by Lehmann and Neumann in 1896 [30] which currently comprises 112 distinct species and 11 subspecies [31]. Many Corynebacterium species are involved in human and animal diseases and include C. diphtheriae [32], C. jeikeium, C. urealyticum, C. striatum, C. pseudotuberculosis, and C. ulcerans [33]. Others have industrial applications for amino acid production like C. glutamicum [34].

Here, we present a summary classification and a set of features for Corynebacterium ihumii strain GD7T sp. nov. (=CSUR P902, =DSM 45751) together with the description of the genome sequencing and annotation.

Classification and Features

A stool sample was collected from a 62 year-old male admitted to the intensive care unit of the Timone Hospital in Marseille, France. The patient gave a written informed consent for the study. The study was approved by the Ethics Committee of the Institut Fédératif de Recherche IFR48, Faculty of Medicine, Marseille, France, under agreement number 09-022. The fecal specimen was preserved at -80°C after collection. Strain GD7T (Table 1) was isolated in January 2012 by cultivation on PVX agar (BioMerieux, Marcy l’Etoile, France) in aerobic condition with 5% CO2 at 37°C, after 21 days of incubation.

Table 1

Classification and general features of Corynebacterium ihumii strain GD7T according to the MIGS recommendations [35]

MIGS ID

Property

  Term

  Evidence codesa

Current classification

  Domain Bacteria

  TAS [36]

  Phylum Actinobacteria

  TAS [37]

  Class Actinobacteria

  TAS [38]

  Order Actinomycetales

  TAS [38-41]

  Family Corynebacteriaceae

  TAS [38-40,42]

  Genus Corynebacterium

  TAS [39,43,44]

  Species Corynebacterium ihumii

  IDA

  Type strain GD7

  IDA

Gram stain

  positive

  IDA

Cell shape

  rod

  IDA

Motility

  non motile

  IDA

Sporulation

  non endospore forming

  IDA

Temperature range

  mesophilic

  IDA

Optimum temperature

  37°C

  IDA

MIGS-6.3

Salinity

  unknown

  IDA

MIGS-22

Oxygen requirement

  facultative anaerobic

  IDA

Carbon source

  unknown

  NAS

Energy source

  unknown

  NAS

MIGS-6

Habitat

  human gut

  IDA

MIGS-15

Biotic relationship

  free living

  IDA

MIGS-14

PathogenicityBiosafety levelIsolation

  unknown  2  human feces

  IDA

MIGS-4

Geographic location

  France

  IDA

MIGS-5

Sample collection time

  January 2012

  IDA

MIGS-4.1

Latitude

  43.296482

  IDA

MIGS-4.1

Longitude

  5.36978

  IDA

MIGS-4.3

Depth

  Surface

  IDA

MIGS-4.4

Altitude

  0 m above sea level

  IDA

a 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 anecdotal evidence). These evidence codes are from the Gene Ontology project [45]. If the evidence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.

To understand the phylogenetic relationships of C. ihumii GD7T, we constructed a 16S rRNA-based neighbor joining tree with 90 Corynebacterium species (Figure 1). The 16S rRNA sequence similarity among Corynebacterium species ranged from 82.9 to 99.60%. Strain GD7T exhibited a highest 16S rRNA sequence similarity of 99.1% with C. pilbarense. This value, although higher 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 [4], is in the range of values observed within the Corynebacterium genus.

Figure 1

Phylogenetic tree highlighting the position of Corynebacterium ihumii strain GD7T relative to other type strains within the Corynebacterium genus. GenBank accession numbers are indicated for each strain. Sequences were aligned using CLUSTALW, and phylogenetic inferences obtained using the neighbor-joining method within the MEGA software. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 1,000 times to generate a majority consensus tree. Mycobacterium tuberculosis was used as an outgroup. The scale bar represents a 2% nucleotide sequence divergence.

Various growth temperatures (25, 30, 37, 45 and 56°C) were tested. Growth occurred between 30 and 45°C on blood-enriched Columbia agar (BioMérieux), with the optimal growth being obtained at 37°C. Growth of the strain was tested under anaerobic and microaerophilic conditions using the GENbag Anaer and GENbag microaer systems, respectively (BioMérieux), and under aerobic conditions, with or without 5% CO2. Optimal growth was achieved aerobically, but cell growth was also observed under microaerophilic and anaerobic conditions. The motility test was negative and the cells were nonsporulating. Colonies were white and granular with a diameter of 0.5 mm on blood-enriched Columbia agar (BioMérieux). Gram staining showed short Gram-positive rods (Figure 2). By electron microscopy, cells grown on agar had a mean length and diameter of 1.26 µm (range 1.1 – 1.4) and 0.7 µm (range 0.6-0.85), respectively (Figure 3).

Figure 2

Gram staining of C. ihumii strain GD7T

Figure 3

Transmission electron microscopy of C. ihumii strain GD7T, using a Morgani 268D (Philips) at an operating voltage of 60kV. The scale bar represents 1 μm.

Strain GD7T was catalase positive and oxidase negative. Using the API ZYM system (BioMérieux), positive reactions were observed for alkaline phosphatase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. Negative reactions were observed for esterase (C4), esterase lipase (C8), lipase (C14), trypsin, α-chemotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, N actetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Using the API CORYNE system (BioMérieux), positive reactions were observed for pyrazinamidase, alkaline phosphatase, and glucose and ribose fermentation. Negative reactions were observed for reduction of nitrates, pyrolidonyl arylamidase; β-glucuronidase, β-galactosidase, α-glucosidase N-acetyl-β-glucosaminidase, β-glucosidase, urease, gelatin hydrolysis, fermentation of xylose, mannitol, maltose, lactose, saccharose and glycogen. Using an API 50CH strip (BioMérieux), positive reactions were observed for fermentation of L-arabinose, D-ribose, D-xylose, methyl-βD xylopranoside, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, D-mannitol, methyl-αD-xylopranoside, methyl-αD-glucopranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose, D-maltose, D-lactose, D-mellibiose, D-saccharose, D-trehalose, inulin, D-raffinose, amidon, glycogen and D-lyxose. Negative reactions were observed for fermentation of glycerol, erythritol, D-arabinose, L-xylose, D-adonitol, L-sorbose, dulcitol, inositol, D-sorbitol, esculin ferric citrate, D-melezitose, D- xylitol, gentiobiose, D-turanose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, and potassium 2-ketogluconate. Table 2 summarizes the differential phenotypic characteristics of C. ihumii, C. pilbarense, C. coylae, C. glaucum, and C. mucifaciens. C. ihumii strain GD7T was susceptible to amoxicillin, amoxicillin-clavulanic acid, ceftriaxone, imipenem, doxycycline, vancomycin, erythromycin, rifampicin, trimethoprim/sulfamethoxazole and ciprofloxacine whereas it was resistant to metronidazole.

Table 2

Differential characteristics of C. ihumii sp. nov. strain GD7T, C. pilbarense, C. coylae, C. glaucum and C. mucifaciens.

Properties

C. ihumii

C. pilbarense

C. coylae

C. glaucum

C. mucifaciens

Colony size (mm)

0.5

0.5 – 2.0

1.0

na

1.0 – 1.5

Oxygen requirement

facultative anaerobic

facultative anaerobic

facultative anaerobic

facultative anaerobic

facultative anaerobic

Gram stain

+

+

+

+

+

Motility

-

-

-

-

-

Endospore formation

-

-

-

-

-

Production of

Alkaline phosphatase

+

+

+

+

+

Acid phosphatase

+

+

+

-

+

Catalase

+

+

+

+

+

Oxidase

-

-

-

-

-

Nitrate reductase

-

-

-

-

-

Urease

-

-

-

-

-

α-galactosidase

-

-

-

-

-

β-galactosidase

-

-

-

-

-

β-glucuronidase

-

-

-

-

-

α -glucosidase

-

-

-

-

-

β-glucosidase

-

-

-

-

-

Esterase

-

-

+

-

+

Esterase lipase

-

-

+

+

+

naphthol-AS-BI-phosphohydrolase

+

+

na

+

na

N-acetyl-β-glucosaminidase

-

-

-

-

-

Pyrazinamidase

+

+

+

+

+

α-mannosidase

-

-

-

-

-

α-fucosidase

-

-

-

-

-

Leucine arylamidase

+

+

+

+

na

Valine arylamidase

+

-

-

-

-

Cystine arylamidase

-

-

+

-

+

α-chemotrypsin

-

-

-

-

-

Trypsin

-

-

-

-

-

Utilization of

5-keto-gluconate

-

na

+

na

-

D-xylose

+

-

-

-

-

D-fructose

+

na

+

na

+

D-glucose

+

+

+

+

+

D-mannose

+

na

+

na

+

Habitat

Human gut

Human joint fluid

Human blood

Cosmetic dye

Human blood

na = data not available

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was peformed as previously described [46] using a Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). The spectra from twelve isolated distinct GD7T colonies 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 validated Corynebacterium species, that were part of the reference data contained in the BioTyper database. The presumptive identification and discrimination of the tested species from those in the database was interpreted as follows: a score > 2 with a validly published species enabled the identification at the species level; a score > 1.7 but < 2 enabled the identification at the genus level; and a score < 1.7 did not enable any identification. For strain GD7T, no significant score was obtained, suggesting that GD7 isolate was not a member of any known species or genus (Figures 4 and 5).

Figure 4

Reference mass spectrum from C. ihumii strain GD7T. Spectra from 12 individual colonies were compared and a reference spectrum was generated.

Figure 5

Gel view comparing C. ihumii sp. nov. strain GD7T (= CSUR P902 = DSM 45751) to other members of the Corynebacterium genus. The gel view displays the raw spectra of all 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 Gray scale 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.

Genome sequencing information

Genome project history

As part of a 'culturomics' study of the human digestive flora, this organism was isolated and selected for sequencing on the basis of its phenotypic differences, phylogenetic position and 16S rRNA and rpoB sequence similarity to other members of the genus Corynebacterium [1,2]. It is the first sequenced genome of C. ihumii sp. nov. The GenBank Bioproject number is PRJEB646 and consists of 41 large contigs in 5 scaffolds. Table 3 shows the project information and its association with MIGS version 2.0 compliance [47].

Table 3

Project information

MIGS ID

Property

  Term

MIGS-31

Finishing quality

  High-quality draft

MIGS-28

Libraries used

  One 454 paired end 3-kb library

MIGS-29

Sequencing platforms

  454 GS FLX Titanium

MIGS-31.2

Fold coverage

  30×

MIGS-30

Assemblers

  Newbler version 2.5.3

MIGS-32

Gene calling method

  Prodigal

BioProject ID

  PRJEB646

Genbank Assembly ID

  GCA_000403725.1

Genbank Accession number

  CAVS000000000

Genbank Date of Release

  2013/05/29

MIGS-13

Project relevance

  Study of the human gut microbiome

Growth conditions and DNA isolation

C. ihumii sp. nov. strain GD7T strain was cultivated in Columbia broth (BioMérieux) at 37°C. Chromosomal DNA was extracted from 50mL of culture, following centrifugation at 4oC at 2000 xg for 20 min. Cell pellets were resuspended in 1 mL Tris/EDTA/NaCl [10mM Tris/HCl (pH7.0), 10 mM EDTA (pH8.0), and 300 mM NaCl] and re-centrifuged under the same conditions. The pellets were then re-suspended in 200µL TE buffer and proteinase K and kept overnight at 37°C for cell lysis. DNA purification with phenol/chloroform/isoamylalcohol (25:24:1) was followed by an overnight precipitation with ethanol at -200C. Then, the DNA was resuspended in 200 µL TE buffer. DNA concentration was 18.3ng/µl as measured using the Genios Tecan fluorometer and the Quant-it Picogreen kit (Invitrogen).

Genome sequencing and assembly

The 454 GS-FLX Titanium paired-end protocol (Roche, Meylan, France) was used for the library construction of C. ihumii strain GD7T which was then pyrosequenced. Briefly, 3.7µg of purified chromosomal DNA was mechanically fragmented on the Covaris device (KBioScience-LGC Genomics, Middlesex, UK) through miniTUBE-Red with an enrichment size at 5kb. The DNA fragmentation was visualized through the Agilent 2100 BioAnalyzer on a DNA labchip 7500 with an optimal size of 2.5 kb. Circularization and nebulization were performed on 100ng of the fragmented DNA and generated an optimal pattern of 443 bp. This was followed by 17 PCR amplification cycles followed by double size selection. The single stranded paired-end library was then quantified using Quant-it Ribogreen kit (Invitrogen) on the Genios_Tecan fluorometer at 207 pg/µL. The library concentration equivalence was calculated as 8.57E+08 molecules/µL. The library was stored at -20°C until further use. The shotgun library was clonally amplified with 0.5cpb and 1cpb in 2 emPCR reactions for each condition, using the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche).The yield of the shotgun emPCR reactions was 5.27 and 7.56% respectively for the two kinds of paired-end emPCR reactions according to the quality expected (range of 5 to 20%) from the Roche procedure. The library was loaded on the 1/4 region of a GS Titanium PicoTiterPlate (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 analyzed on the cluster through the gsRunBrowser and Newbler assembler (Roche). A total of 186,723 passed filter wells were obtained and generated 69.4Mb with a length average of 371 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40bp as overlap. The assembly lead to 5 scaffolds and 41 large contigs (>1500bp) and generated a genome size of 2,232,265 bp which corresponds to a coverage of 30.84× genome equivalent.

Genome annotation

Open Reading Frames (ORFs) prediction was performed using Prodigal [48] with default parameters. The predicted ORFs were excluded if they spanned a sequencing gap region. Functional assessment of protein sequences was carried out by comparing them with sequences in the GenBank [49] and Clusters of Orthologus Groups (COG) databases using BLASTP. tRNAs, rRNAs, signal peptides and transmembrane helices were identified using tRNAscan-SE 1.21 [50], RNAmmer [51], SignalP [52] and TMHMM [53], respectively. ORFans were identified if their BLASTP E- value was lower than 1e-3 for alignment lengths greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we used an E- value of 1e-5 [54]. PHAST was used to identify, annotate and graphically display prophage sequences within bacterial genomes or plasmids [55]. Artemis [56] was used for data management whereas DNA Plotter [57] was used for visualization of genomic features. In-house perl and bash scripts were used to automate these routine tasks.

To estimate the mean level of nucleotide sequence similarity at the genome level between C. ihumii and another 42 members of the genus Corynebacterium, we used the Average Genomic Identity of Orthologous gene Sequences (AGIOS) home-made pipeline. Briefly, this pipeline combines the Proteinortho software (with the following parameters: e-value 1e-5, 30% of identity, 50% coverage and algebraic connectivity of 50%) [58] for detecting orthologous proteins between genomes compared pairwise, then retrieves the corresponding genes and determines the mean percentage of nucleotide sequence identity among orthologous ORFs using the Needleman-Wunsch global alignment algorithm.

Genome properties

The genome of C. ihumii sp. nov. strain GD7T is 2,232,265 bp long (1 chromosome in 5 scaffolds, no plasmid) with a 65.1% GC content (Table 4, Figure 6). Of the 2,182 predicted genes, 2,125 were protein-coding genes and 57 were RNAs (53 tRNA and 4 rRNA genes). A total of 1,562 genes (71.58%) were assigned a putative function. Four hundred and twenty-two genes (19.8%) were annotated as hypothetical proteins, and 126 genes ORFans (5.9%). The distribution of genes into COGs functional categories is presented in Table 5. The properties and statistics of the genome are summarized in Tables 4 and 5. A quick search with PHAST revealed that C. ihumii harbors an incomplete bacteriophage.

Table 4

Nucleotide content and gene count levels of the genome

Attribute

  Value

   % of totala

Genome size (bp)

  2,232,265

DNA Coding region (bp)

  2,041,113

   91.43

DNA G+C content (bp)

  1,453,204

   65.1

Number of replicons

  1

Extrachromosomal elements

  0

Total genes

  2,182

   100

RNA genes

  57

   2.61

rRNA operons

  1

Predicted tRNA pseudogenes

  1

Protein-coding genes

  2,125

   97.38

Genes with function prediction

  1,562

   71.58

Genes assigned to COGs

  1,703

   78.04

Genes with peptide signals

  189

   8.66

Genes with transmembrane helices

  553

   25.34

CRISPR repeats

  1

a The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome

Figure 6

Graphical circular map of the chromosome. From the outside in, the outer two circles show open reading frames oriented in the forward and reverse directions (colored by COG categories), 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 the GC skew, purple and olive indicating negative and positive values, respectively.

Table 5

Number of genes associated with the 25 general COG functional categories

Code

Value

% of totala

  Description

J

142

6.68

  Translation

A

1

0.05

  RNA processing and modification

K

131

6.16

  Transcription

L

114

5.36

  Replication, recombination and repair

B

0

0.00

  Chromatin structure and dynamics

D

19

0.89

  Cell cycle control, mitosis and meiosis

Y

0

0.00

  Nuclear structure

V

31

1.46

  Defense mechanisms

T

60

2.82

  Signal transduction mechanisms

M

95

4.47

  Cell wall/membrane biogenesis

N

1

0.05

  Cell motility

Z

0

0.00

  Cytoskeleton

W

0

0.00

  Extracellular structures

U

22

1.04

  Intracellular trafficking and secretion

O

62

2.92

  Posttranslational modification, protein turnover, chaperones

C

83

3.91

  Energy production and conversion

G

100

4.71

  Carbohydrate transport and metabolism

E

158

7.44

  Amino acid transport and metabolism

F

63

2.96

  Nucleotide transport and metabolism

H

78

3.67

  Coenzyme transport and metabolism

I

46

2.16

  Lipid transport and metabolism

P

117

5.51

  Inorganic ion transport and metabolism

Q

35

1.64

  Secondary metabolites biosynthesis, transport and catabolism

R

204

9.60

  General function prediction only

S

141

6.63

  Function unknown

-

422

19.8

  Not in COGs

a The total is based on the total number of protein coding genes in the annotated genome.

Comparative genomics

Presently there are more than 75 genomic sequences (finished or draft) available for Corynebacterium species in GenBank. Here, we have compared C. ihumii sp. nov. strain GD7T with 41 finished or draft genome sequences from 25 Corynebacterium species. Table 6 shows a comparison of genome size, GC%, coding-density, and numbers of proteins for the compared Corynebacterium genomes. C. ihumii had a smaller genome than all other compared genomes except that of C. urealyticum strain DSM 7111. AGIOS values identities ranged from 65.23 to 80.59% among Corynebacterium species, and from 97.97 to 99.99% within Corynebacterium species (Supplementary Table). By comparison with other species, C. ihumii exhibited AGIOS values ranging from 67.15% with C. pseudotuberculosis to 76.30% with C. lipophiloflavum, thus confirming its new species status.

Table 6

Main characteristics of Corynebacterium genomes compared to that of C. ihumii strain GD7T.

Species

   Strain

  NCIBI ID

  Coding density

   Length (bp)

  GC%

   Proteins

Corynebacterium ihumii

   GD7T

  90.65

   2,232,265

  64.95

   2,125

Corynebacterium accolens

   ATCC 49726

  uid52361

  86.51

   2,465,976

  59.23

   2,360

Corynebacterium ammoniagenes

   DSM 20306

  uid48813

  90.3

   2,764,417

  55.56

   2,654

Corynebacterium amycolatum

   SK46

  uid55411

  85.4

   2,514,382

  58.58

   2,103

Corynebacterium casei

  uid78139

  85.95

   3,113,786

  55.34

   2,700

Corynebacterium aurimucosum

   ATCC 700975

  uid59409

  88.49

   2,790,189

  60.63

   2,531

Corynebacterium bovis

   DSM 20582

  uid67345

  85.72

   2,527,982

  72.55

   2,339

Corynebacterium diphtheriae

   VA01

  uid84305

  88.36

   2,395,441

  53.44

   2,191

Corynebacterium diphtheriae

   HC01

  uid84297

  88.03

   2,427,149

  53.43

   2,248

Corynebacterium diphtheriae

   HC02

  uid84317

  87.7

   2,468,612

  53.71

   2,230

Corynebacterium diphtheriae

   INCA 402

  uid83605

  87.72

   2,449,071

  53.65

   2,214

Corynebacterium diphtheriae

   NCTC 13129

  uid57691

  87.96

   2,488,635

  53.48

   2,272

Corynebacterium diphtheriae

   241

  uid83607

  87.87

   2,426,551

  53.43

   2,245

Corynebacterium durum

   F0235

  uid183766

  90.37

   2,809,766

  56.84

   2,823

Corynebacterium efficiens

   YS 314

  uid62905

  91.38

   3,147,090

  63.14

   2,938

Corynebacterium genitalium

   ATCC 33030

  uid52785

  90.81

   2,349,953

  62.73

   2,226

Corynebacterium glucuronolyticum

   ATCC 51867

  uid55397

  85.44

   2,809,779

  59.09

   2,645

Corynebacterium glutamicum

   R

  uid58897

  86.83

   3,314,179

  54.13

   3,052

Corynebacterium glutamicum

   ATCC 13032

  uid57905

  86.41

   3,309,401

  53.81

   2,993

Corynebacterium glutamicum

   ATCC 13032

  uid61611

  87.53

   3,282,708

  53.84

   3,057

Corynebacterium jeikeium

   K411

  uid58399

  89.41

   2,462,499

  61.4

   2,104

Corynebacterium kroppenstedtii

   DSM 44385

  uid59411

  86.73

   2,446,804

  57.46

   2,018

Corynebacterium lipophiloflavum

   DSM 44291

  uid55469

  87.87

   2,386,544

  64.26

   2,371

Corynebacterium matruchotii

   ATCC 14266

  uid51885

  86.43

   2,856,058

  57.09

   2,619

Corynebacterium nuruki

   S6 4

  uid77677

  89.61

   3,107,265

  69.49

   2,797

Corynebacterium pseudogenitalium

   ATCC 33035

  uid55395

  89.9

   2,601,506

  59.53

   2,493

Corynebacterium pseudotuberculosis

   FRC41

  uid50585

  87.91

   2,337,913

  52.19

   2,110

Corynebacterium pseudotuberculosis

   1002

  uid159677

  85.31

   2,337,913

  52.19

   2,090

Corynebacterium pseudotuberculosis

   267

  uid162175

  86.54

   2,337,628

  52.19

   2,148

Corynebacterium pseudotuberculosis

   42 02 A

  uid159669

  84.23

   2,337,606

  52.19

   2,051

Corynebacterium pseudotuberculosis

   P54B96

  uid157909

  84.93

   2,337,657

  52.19

   2,084

Corynebacterium resistens

   DSM 45100

  uid50555

  87.87

   2,601,311

  57.09

   2,171

Corynebacterium striatum

   ATCC 6940

  uid55471

  86.33

   2,829,831

  59.05

   2,677

Corynebacterium tuberculostearicum

   SK141

  uid55413

  89.57

   2,372,621

  60.01

   2,210

Corynebacterium ulcerans

   809

  uid159659

  87.66

   2,502,095

  53.3

   2,180

Corynebacterium ulcerans

   102

  uid169879

  87.66

   2,579,188

  53.36

   2,349

Corynebacterium ulcerans

   BR AD22

  uid68291

  87.72

   2,606,374

  53.4

   2,334

Corynebacterium urealyticum

   DSM 7109

  uid61639

  89.7

   2,369,219

  64.19

   2,022

Corynebacterium urealyticum

   DSM 7111

  uid188688

  88.17

   2,316,065

  64.24

   1,935

Corynebacterium variabile

   DSM 44702

  uid62003

  87.56

   3,433,007

  67.15

   3,039

Figure 7 shows the comparison of gene distribution into COG categories of C. ihumii with C. glutamicum strain ATCC 13032, C. efficiens YS 314, C. jeikeium K411, C. aurimucosum ATCC 700975, C. kroppenstedtii DSM 44385, C. resistens DSM 45100, C. variabile DSM 44702, C. diphtheriae BH8, C. pseudotuberculosis 1002, C. ulcerans 0102, C. halotolerans YIM 70093 and C. callunae DSM 20147. The overall COG distribution is similar, except C. variabile for category L genes.

Figure 7

Distribution of functional classes of predicted genes of Corynebacterium ihumii strain GD7T (colored in thick red line) along with other Corynebacterium genomes 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 Corynebacterium ihumii sp. nov. which contains strain GD7T (= CSUR P902 = DSM 45751). This bacterium was isolated from the fecal flora of a 62 year-old male admitted in intensive care unit for respiratory distress.

Description of Corynebacterium ihumii strain GD7T sp. nov

Corynebacterium 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 white and granular with a 0.5 mm diameter on blood-enriched Columbia agar. Cells are rod-shaped with a mean length and diameter of 1.26 µm (range 1.1 – 1.4) and 0.7 µm (range 0.6-0.85), respectively. Growth is observed between 30 and 45°C, with optimal growth obtained at 37°C on blood-enriched Columbia agar. Optimal growth is achieved aerobically, but cell growth is also observed under microaerophilic and anaerobic conditions. Cells stain Gram-positive, are nonmotile and nonsporulating. Catalase is positive, oxidase is negative. Using the API ZYM system, positive reactions are observed for alkaline phosphatase, leucine arylamidase, valine arylamidase, cystin arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. Negative reactions are observed for esterase (C4), esterase lipase (C8), lipase (C14), trypsin, α-chemotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, N actetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Using the API CORYNE system, positive reactions are observed for pyrazinamidase, alkaline phosphatase, and glucose and ribose fermentation. Negative reactions are observed for reduction of nitrates, pyrolidonyl arylamidase; β-glucuronidase, β-galactosidase, α-glucosidase N-acetyl-β-glucosaminidase, β-glucosidase, urease, gelatin hydrolysis, fermentation of xylose, mannitol, maltose, lactose, saccharose and glycogen. Using the API 50CH system, positive reactions are observed for fermentation of L-arabinose, D-ribose, D-xylose, methyl-βD xylopranoside, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, D-mannitol, methyl-αD-xylopranoside, methyl-αD-glucopranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose, D-maltose, D-lactose, D-mellibiose, D-saccharose, D-trehalose, inulin, D-raffinose, amidon, glycogen and D-lyxose. Negative reactions are observed for fermentation of glycerol, erythritol, D-arabinose, L-xylose, D-adonitol, L-sorbose, dulcitol, inositol, D-sorbitol, esculin ferric citrate, D-melezitose, D- xylitol, gentiobiose, D-turanose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, and potassium 2-ketogluconate. Cells are susceptible to amoxicillin, amoxicillin-clavulanic acid, ceftriaxone, imipenem, doxycycline, vancomycin, erythromycin, rifampicin, trimethoprim/sulfamethoxazole and ciprofloxacine but was resistant to metronidazole. The G+C content of the genome is 65.1%. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers JX424769 and CAVS000000000, respectively.

The type strain GD7T sp. nov. (= CSUR P902 = DSM 45751) was isolated from the feces of a patient admitted to intensive care in Marseille, France.

Declarations

Acknowledgements

The authors thank the Xegen Company for automating the genomic annotation process. This study was funded by the Mediterranee-Infection foundation.


This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

References

  1. Lagier JC, Armougom F, Million M, Hugon P, Pagnier I, Robert C, Bittar F, Fournous G, Gimenez G and Maraninchi M. Microbial culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect. 2012; 18:1185-1193PubMed
  2. Dubourg G, Lagier JC, Armougom F, Robert C, Hamad I, Brouqui P and Raoult D. The gut microbiota of a patient with resistant tuberculosis is more comprehensively studied by culturomics than by metagenomics. Eur J Clin Microbiol Infect Dis. 2013; 32:637-645 View ArticlePubMed
  3. Tindall BJ, Rosselló-Móra R, Busse HJ, Ludwig W and Kämpfer P. Notes on the characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol. 2010; 60:249-266 View ArticlePubMed
  4. Stackebrandt E and Ebers J. Taxonomic parameters revisited: tarnished gold standards. Microbiol Today. 2006; 33:152-155
  5. Wayne LG, Brenner DJ and Colwell RR. Report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics. Int J Syst Bacteriol. 1987; 37:463-464 View Article
  6. Rossello-Mora R. DNA-DNA reassociation methods applied to microbial taxonomy and their critical evaluation. In: Molecular identification, systematics, and population structure of prokaryotes 2006:23–50.
  7. Database GOLD. Web Site
  8. Kokcha S, Mishra AK, Lagier JC, Million M, Leroy Q, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of Bacillus timonensis sp. nov. Stand Genomic Sci. 2012; 6:346-355 View ArticlePubMed
  9. Lagier JC, El Karkouri K, Nguyen TT, Armougom F, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Anaerococcus senegalensis sp. nov. Stand Genomic Sci. 2012; 6:116-125 View ArticlePubMed
  10. Mishra AK, Gimenez G, Lagier JC, Robert C, Raoult D and Fournier PE. Genome sequence and description of Alistipes senegalensis sp. nov. Stand Genomic Sci. 2012; 6:1-16 View ArticlePubMed
  11. Lagier JC, Armougom F, Mishra AK, Nguyen TT, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Alistipes timonensis sp. nov. Stand Genomic Sci. 2012; 6:315-324 View ArticlePubMed
  12. Mishra AK, Lagier JC, Robert C, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Clostridium senegalense sp. nov. Stand Genomic Sci. 2012; 6:386-395PubMed
  13. Mishra AK, Lagier JC, Robert C, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of Peptoniphilus timonensis sp. nov. Stand Genomic Sci. 2012; 7:1-11 View ArticlePubMed
  14. Mishra AK, Lagier JC, Rivet R, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Paenibacillus senegalensis sp. nov. Stand Genomic Sci. 2012; 7:70-81 View ArticlePubMed
  15. Lagier JC, Gimenez G, Robert C, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Herbaspirillum massiliense sp. nov. Stand Genomic Sci. 2012; 7:200-209PubMed
  16. Roux V, El Karkouri K, Lagier JC, Robert C and Raoult D. Non-contiguous finished genome sequence and description of Kurthia massiliensis sp. nov. Stand Genomic Sci. 2012; 7:221-232 View ArticlePubMed
  17. Kokcha S, Ramasamy D, Lagier JC, Robert C, Raoult D and Fournier PE. Non-contiguous finished genome sequence and description of Brevibacterium senegalense sp. nov. Stand Genomic Sci. 2012; 7:233-245 View ArticlePubMed
  18. Ramasamy D, Kokcha S, Lagier JC, Nguyen TT, Raoult D and Fournier PE. Genome sequence and description of Aeromicrobium massiliense sp. nov. Stand Genomic Sci. 2012; 7:246-257 View ArticlePubMed
  19. Lagier JC, Ramasamy D, Rivet R, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of Cellulomonas massiliensis sp. nov. Stand Genomic Sci. 2012; 7:258-270 View ArticlePubMed
  20. Lagier JC, Elkarkouri K, Rivet R, Couderc C, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of Senegalemassilia anaerobia gen. nov., sp. nov. Stand Genomic Sci. 2013; 7:343-356 View ArticlePubMed
  21. Mishra AK, Hugon P, Lagier JC, Nguyen TT, Robert C, Couderc C, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of Peptoniphilus obesi sp. nov. Stand Genomic Sci. 2013; 7:357-369 View ArticlePubMed
  22. Mishra AK, Lagier JC, Nguyen TT, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of Peptoniphilus senegalensis sp. nov. Stand Genomic Sci. 2013; 7:370-381 View ArticlePubMed
  23. Lagier JC, El Karkouri K, Mishra AK, Robert C, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of Enterobacter massiliensis sp. nov. Stand Genomic Sci. 2013; 7:399-412 View ArticlePubMed
  24. Hugon P, Ramasamy D, Lagier JC, Rivet R, Couderc C, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of Alistipes obesi sp. nov. Stand Genomic Sci. 2013; 7:427-439 View ArticlePubMed
  25. Mishra AK, Hugon P, Robert C, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of Peptoniphilus grossensis sp. nov. Stand Genomic Sci. 2012; 7:320-330PubMed
  26. Mishra AK, Hugon P, Lagier JC, Nguyen TT, Couderc C, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of Enorma massiliensis gen. nov., sp. nov., a new member of the Family Coriobacteriaceae. Stand Genomic Sci. 2013; 8:290-305 View ArticlePubMed
  27. Ramasamy D, Lagier JC, Gorlas A, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of Bacillus massiliosenegalensis sp. nov. Stand Genomic Sci. 2013; 8:264-278 View ArticlePubMed
  28. Ramasamy D, Lagier JC, Nguyen TT, Raoult D and Fournier PE. Non contiguous-finished genome sequence and description of Dielma fastidiosa gen. nov., sp. nov., a new member of the Family Erysipelotrichaceae. Stand Genomic Sci. 2013; 8:336-351 View ArticlePubMed
  29. Mishra AK, Lagier JC, Robert C, Raoult D and Fournier PE. Genome sequence and description of Timonella senegalensis gen. nov., sp. nov., a new member of the suborder Micrococcinae. Stand Genomic Sci. 2013; 8:318-335 View ArticlePubMed
  30. Collins MD, Smida J and Stackebrandt E. Phylogenetic Evidence for the Transfer of Caseobacter polymorphus (Crombach) to the Genus Corynebacterium. Int J Syst Evol Microbiol. 1989; 39:7-9
  31. List of Prokaryotic names with Standing in Nomenclature. Web Site
  32. Wagner KS, White JM, Lucenko I, Mercer D, Crowcroft NS, Neal S and Efstratiou A. Diphtheria in the postepidemic period, Europe, 2000-2009. Emerg Infect Dis. 2012; 18:217-225 View ArticlePubMed
  33. Dias AA, Santos LS, Sabbadini PS, Santos CS, Silva Junior FC, Napoleão F, Nagao PE, Villas-Bôas MH, Hirata Junior R and Guaraldi AL. Corynebacterium ulcerans diphtheria: an emerging zoonosis in Brazil and worldwide. Rev Saude Publica. 2011; 45:1176-1191 View ArticlePubMed
  34. Gao B and Gupta RS. Phylogenetic framework and molecular signatures for the main clades of the phylum Actinobacteria. Microbiol Mol Biol Rev. 2012; 76:66-112 View ArticlePubMed
  35. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  36. Woese CR, Kandler O and Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA. 1990; 87:4576-4579 View ArticlePubMed
  37. Garrity GM, Holt JG. The road map to the manual. In Bergey’s Manual® of Systematic Bacteriology 2011; 119-166.
  38. Stackebrandt E, Rainey FA and Ward-Rainey NL. Proposal for a New Hierarchic Classification System, Actinobacteria classis nov. Int J Syst Bacteriol. 1997; 47:479-491 View Article
  39. Skerman VBD, McGowan V and Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol. 1980; 30:225-420 View Article
  40. Zhi XY, Li WJ and Stackebrandt E. An update of the structure and 16S rRNA gene sequence-based definition of higher ranks of the class Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher taxa. Int J Syst Evol Microbiol. 2009; 59:589-608 View ArticlePubMed
  41. Buchanan RE. Studies in the nomenclature and classification of bacteria. II. The primary subdivisions of the Schizomycetes. J Bacteriol. 1917; 2:155-164PubMed
  42. Lehmann KB, Neumann R. Lehmann's Medizin, Handatlanten. X Atlas und Grundriss der Bakteriologie und Lehrbuch der speziellen bakteriologischen Diagnostik., Fourth Edition, Volume 2, J.F. Lehmann, München, 1907, p. 270.
  43. Lehmann KB, Neumann R. Atlas und Grundriss der Bakteriologie und Lehrbuch der speziellen bakteriologischen Diagnostik, First Edition, J.F. Lehmann, München, 1896, p. 1-448.
  44. Bernard KA, Wiebe D, Burdz T, Reimer A, Ng B, Singh C, Schindle S and Pacheco AL. Assignment of Brevibacterium stationis (ZoBell and Upham 1944) Breed 1953 to the genus Corynebacterium, as Corynebacterium stationis comb. nov., and emended description of the genus Corynebacterium to include isolates that can alkalinize citrate. Int J Syst Evol Microbiol. 2010; 60:874-879 View ArticlePubMed
  45. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS and Eppig JT. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25:25-29 View ArticlePubMed
  46. Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM and Raoult D. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis. 2009; 49:543-551 View ArticlePubMed
  47. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  48. Prodigal. Web Site
  49. Benson DA, Karsch-Mizrachi I, Clark K, Lipman DJ, Ostell J and Sayers EW. GenBank. Nucleic Acids Res. 2012; 40:D48-D53 View ArticlePubMed
  50. Lowe TM and Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997; 25:955-964 View ArticlePubMed
  51. Lagesen K, Hallin P, Rødland EA, Staerfeldt HH, Rognes T and Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007; 35:3100-3108 View ArticlePubMed
  52. Bendtsen JD, Nielsen H, von Heijne G and Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol. 2004; 340:783-795 View ArticlePubMed
  53. Krogh A, Larsson B, von Heijne G and Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001; 305:567-580 View ArticlePubMed
  54. Fischer D and Eisenberg D. Finding families for genomic ORFans. Bioinformatics. 1999; 15:759-762 View ArticlePubMed
  55. Zhou Y, Liang Y, Lynch KH, Dennis JJ and Wishart DS. PHAST: a fast phage search tool. Nucleic Acids Res. 2011; 39:W347-W352 View ArticlePubMed
  56. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA and Barrell B. Artemis: sequence visualization and annotation. Bioinformatics. 2000; 16:944-945 View ArticlePubMed
  57. Carver T, Thomson N, Bleasby A, Berriman M and Parkhill J. DNA Plotter: circular and linear interactive genome visualization. Bioinformatics. 2009; 25:119-120 View ArticlePubMed
  58. Lechner M, Findeiss S, Steiner L, Marz M, Stadler PF and Prohaska SJ. Proteinortho: detection of (co-)orthologs in large-scale analysis. BMC Bioinformatics. 2011; 12:124 View ArticlePubMed