Introduction

Corynebacterium jeddahense strain JCBT (= CSUR P778 = DSM 45997) is the type strain of C. jeddahense sp. nov. This bacterium is a Gram-positive bacillus, non-spore-forming, strictly aerobic and non-motile that was isolated from the feces of a 24 year-old man living in Jeddah, Saudi Arabia, who suffered from morbid obesity. This isolation was part of a “culturomics” study aiming at cultivating the maximum number of bacterial species from human feces [1,2].

The current classification of bacteria remains a matter of debate and relies on a combination of phenotypic and genomic characteristics [3]. Currently, more than 12,000 bacterial genomes have been sequenced [4], and we recently proposed an innovative concept for the taxonomic description of new bacterial species that integrates their genomic characteristics [5–35] as well as proteomic information obtained by MALDI-TOF-MS analysis [36].

In the present study, we present a summary classification and a set of features for Corynebacterium jeddahense sp. nov., strain JCBT (CSUR P778 = DSM 45997), including the description of its complete genome sequence and annotation. These characteristics support the circumscription of the species Corynebacterium jeddahense. The genus Corynebacterium was created in 1896 by Lehmann and Neumann and currently consists of mainly Gram-positive, non-spore-forming, rod-shaped bacteria with a high DNA G+C content [37]. This genus belongs to the phylum Actinobacteria and currently includes more than 100 species with standing in nomenclature [38]. Members of the genus Corynebacterium are found in various environments including water, soil, sewage, and plants as well as in human normal skin flora and human or animals clinical samples. Some Corynebacterium species are well-established human pathogens while others are only considered as opportunistic pathogens. Corynebacterium diphteriae, causing diphtheria, is the most significant pathogen in this genus [39]. However, many Corynebacterium species including, among others, C. jeikeium, C. urealyticum, C. striatum, C. ulcerans and C. pseudotuberculosis, are recognized agents of bacteremias, endocarditis, urinary tract infections, and respiratory or wound infections [40].

Classification and features

A stool sample was collected from a 24-year-old man living in Jeddah, Saudi Arabia, who suffered from morbid obesity (BMI=52). The patient gave a signed informed consent. The study and the assent procedure were approved by the Ethics Committees of the King Abdulaziz University, King Fahd medical Research Center, Saudi Arabia, under agreement number 014-CEGMR-2-ETH-P, and of the Institut Fédératif de Recherche 48, Faculty of Medicine, Marseille, France, under agreement number 09-022. The patient was not taking any antibiotics at the time of stool sample collection and the fecal sample was kept at −80°C after collection. Strain JCBT (Table 1) was first isolated in July 2013 by cultivation on 5% sheep blood-enriched Columbia agar (BioMerieux, Marcy l’Etoile, France) in aerobic atmosphere with 5% CO2 at 37°C after a 14-day preincubation of the stool sample in an aerobic blood culture bottle that also contained sterile rumen sheep fluid. Several other new bacterial species were isolated from this stool specimen using various culture conditions.

Table 1. Classification and general features of C. jeddahense strain JCBT according to the MIGS recommendations [41].
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This strain exhibited a 96.8% nucleotide sequence similarity with C. coyleae, the phylogenetically most closely related Corynebacterium species with a validly published name (Figure 1). The similarity value was 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 [52], and was in the 82.9 to 99.60% range observed among members of the genus Corynebacterium with standing in the nomenclature [53].

Figure 1.
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Phylogenetic tree highlighting the position of Corynebacterium jeddahense strain JCBT relative to other type strains within the genus Corynebacterium. GenBank accession numbers are indicated in parentheses. Sequences were aligned using CLUSTALW, and phylogenetic inferences obtained using the maximum-likelihood method in the MEGA software package. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 500 times to generate a majority consensus tree. Mycobacterium avium was used as outgroup. The scale bar represents 1% nucleotide sequence divergence.

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Four growth temperatures (25, 30, 37, 45°C) were tested. Growth occurred between 30 and 45°C on blood-enriched Columbia agar (BioMerieux), with the optimal growth being obtained at 37°C after 48 hours of incubation. Growth of the strain was tested under anaerobic and microaerophilic conditions using GENbag Anaer and GENbag microaer systems, respectively (BioMerieux), and under aerobic conditions, with or without 5% CO2. Optimal growth was achieved aerobically. Weak cell growth was observed under microaerophilic and anaerobic conditions. The motility test was negative and the cells were not sporulating. Colonies were translucent and 1 mm in diameter on blood-enriched Columbia agar. Cells were Gram-positive rods (Figure 2). In electron microscopy, the bacteria grown on agar had a mean diameter and length of 0.63 and 1.22 µm, respectively (Figure 3).

Figure 2.
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Gram-stain of C. jeddahense strain JCBT

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Figure 3.
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Transmission electron micrograph of C. jeddahense strain JCBT, taken using a Morgani 268D (Philips) at an operating voltage of 60kV. The scale bar represents 1 µm.

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Strain JCBT was catalase positive and oxidase negative. Using an API CORYNE strip, a positive reaction was observed only for alkaline phosphatase and for catalase. Negative reactions were observed for reduction of nitrates, pyrolidonyl arylamidase, pyrazinamidase, β-glucuronidase, β-galactosidase, α-glucosidase N-acetyl-β-glucosaminidase, β-glucosidase, urease, gelatin hydrolysis and fermentation of glucose, ribose xylose, mannitol, maltose, lactose, saccharose and glycogen. Using the Api Zym system (BioMerieux), alkaline and acid phosphatases and Naphtol-AS-BI phosphohydrolase activities were positive, but esterase (C4), esterase lipase (C8), lipase (C14), trypsin, α-chemotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, N actetyl-β-glucosaminidase, leucine arylamidase, valine arylamidase, cystin arylamidase, α-mannosidase and α-fucosidase activities were negative.

Substrate oxidation and assimilation were examined with an API 50CH strip (BioMerieux) at 37°C. All reactions were negative, including fermentation of starch, glycogen, glycerol, erythritol, esculin ferric citrate, amygdalin, arbutin, salicin, L-arabinose, D-ribose, D-xylose, methyl β-D-xylopyranoside, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, D-mannitol, methyl α-D-xylopyranoside, methyl α-D-glucopyranoside, N-acetylglucosamine, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-saccharose, D-trehalose, inulin, D-raffinose, D-lyxose, D-arabinose, L-xylose, D-adonitol, L-sorbose, dulcitol, inositol, D-sorbitol, D-melezitose, D-xylitol, gentiobiose, D-turanose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, and potassium 2-ketogluconate.

C. jeddahense is susceptible to amoxicillin, ceftriaxone, imipenem, rifampin, gentamicin, doxycycline and vancomycin, but resistant to ciprofloxacin, trimethoprim/sulfamethoxazole, eyrthromycin and metronidazole. When compared with representative species from the genus Corynebacterium, C. jeddahense strain JCBT exhibited the phenotypic differences detailed in Table 2.

Table 2. Differential characteristics of C. jeddahense strain JCBT and closely related strains.
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(MALDI-TOF) MS protein analysis was carried out as previously described [36] using a Microflex spectrometer (Brüker Daltonics, Leipzig, Germany). Twelve individual colonies were deposited on a MTP 384 MALDI-TOF target plate (Brüker). The twelve spectra were imported into the MALDI BioTyper software (version 2.0, Brüker) and analyzed by standard pattern matching (with default parameter settings) against the main spectra of 4,706 bacteria, including 169 spectra from 69 validly named Corynebacterium species used as reference data in the BioTyper database. The score generated enabled the presumptive identification and discrimination of the tested species from those in a database: a score > 2 with a validated species enabled the identification at the species level; and a score < 1.7 did not enable any identification. For strain JCBT, no significant score was obtained, suggesting that our isolate was not a member of any known species (Figures 4 and 5).

Figure 4.
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Reference mass spectrum from C. jeddahense strain JCBT. Spectra from 12 individual colonies were compared and a reference spectrum was generated.

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Figure 5.
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Gel view comparing C. jeddahense strain JCBT (= CSUR P778 = DSM 45997) to other species from the genus Corynebacterium. The gel view displays the raw spectra of loaded spectrum files as a pseudo-electrophoretic gel. 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 grey scale scheme code. The grey scale bar on the right y-axis indicates the relation between the shade of grey of the “band” and the peak intensity, in arbitrary units. Displayed species are indicated on the left.

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Genome sequencing information

Genome project history

The organism was selected for sequencing on the basis of its phylogenetic position, 16S rDNA similarity and phenotypic differences with members of the genus Corynebacterium and is part of a culturomics study of the human digestive flora aiming at isolating all bacterial species within human feces [2]. It was the 96th genome from a Corynebacterium species. The EMBL accession number is CBYN00000000and consists of 244 contigs. Table 3 shows the project information and its association with MIGS version 2.0 compliance [41].

Table 3. Project information
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Growth conditions and DNA isolation

C. jeddahense sp. nov strain JCBT (= CSUR P778 = DSM 45997) was grown aerobically on sheep blood-enriched Columbia agar medium at 37°C. Two petri dishes were spread and resuspended in 6x100µl of G2 buffer (EZI DNA Tissue Kit, Qiagen). A first mechanical lysis was performed using glass powder on the Fastprep-24 device (Sample Preparation System, MP Biomedicals, USA) using 2x20 second bursts. DNA was treated with 2.5µg/µL of lysozyme for 30 minutes at 37°C) and extracted using the BioRobot EZ 1 Advanced XL (Qiagen). The DNA was then concentrated and purified on a Qiamp kit (Qiagen). The DNA concentration, as measured by the Qubit assay with the high sensitivity kit (Life Technologies, Carlsbad, CA, USA), was 3.1ng/µl.

Genome sequencing and assembly

Genomic DNA of C. jeddahense was sequenced on a MiSeq sequencer (Illumina Inc, San Diego, CA, USA) using both paired-end and mate-pair sequencing with the Nextera XT DNA sample and Nextera Mate Pair sample prep kits, respectively (Illumina).

To prepare the paired-end library, Genomic DNA was diluted 1:3 to obtain a 1ng/µl concentration. The “tagmentation” step fragmented and tagged the DNA with a mean size of 1.4kb. Then, a limited PCR amplification (12 cycles) completed the tag adapters and introduced dual-index barcodes. After purification on AMPure XP beads (Beckman Coulter Inc, Fullerton, CA, USA), the library was then normalized on specific beads according to the Nextera XT protocol (Illumina). The pooled single strand library was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and paired end sequencing with dual index reads were performed in a single 39-hours run in 2x250-bp. Total information of 5.3Gb was obtained from a 574 K/mm2 cluster density with a cluster passing quality control filters of 95.4% (11,188,000 clusters). Within this run, the index representation for Corynebacterium jeddahense was determined to 6.2%. The 641,099 reads were filtered according to the read qualities.

The mate-pair library was prepared with 1µg of genomic DNA using the Nextera mate-pair Illumina guide. The genomic DNA sample was simultaneously fragmented and tagged with a mate-pair junction adapter. The profile of the fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies Inc, Santa Clara, CA, USA) with a DNA 7500 labchip. The DNA fragments ranged in size from 1kb up to 10kb with a mean size of 2.6kb. No size selection was performed and 105ng of tagmented fragments were circularized. The circularized DNA was mechanically sheared to small fragments with an optimal at 409bp on the Covaris device S2 in microtubes (Covaris, Woburn, MA, USA). The library profile was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent Technologies Inc, Santa Clara, CA, USA). After a denaturation step and dilution at 10pM, the library was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and sequencing run were performed in a single 42-hour run in a 2x250-bp. Total information of 3.9Gb was obtained from a 399 K/mm2 cluster density with a cluster passing quality control filters of 97.9% (7,840,000 clusters). Within this run, the index representation for Corynebacterium jeddahense was determined to 8.17%. The 626,585 reads were filtered according to the read qualities. Genome assembly was performed using Newbler (Roche).

Genome annotation

Open Reading Frames (ORFs) were predicted using Prodigal [60] with default parameters. However, the predicted ORFs were excluded if they spanned a sequencing gap region. The predicted bacterial protein sequences were searched against GenBank [61] and Clusters of Orthologous Groups (COG) databases using BLASTP. The tRNAs and rRNAs were predicted using the tRNAScanSE [62] and RNAmmer [63] tools, respectively. Lipoprotein signal peptides and numbers of transmembrane helices were predicted using SignalP [64] and TMHMM [65], respectively. ORFans were identified if their BLASTP E-value was lower than 1e−3 for alignment length greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we use an E-value of 1e−5. Such parameter thresholds have already been used in previous works to define ORFans.

Artemis [66] and DNA Plotter [67] were used for data management and visualization of genomic features, respectively. The Mauve alignment tool (version 2.3.1) was used for multiple genomic sequence alignments [68]. To estimate the mean level of nucleotide sequence similarity at the genome level between C. jeddahense and another 4 members of the Corynebacterium genus (Tables 6A and 6B), we used the Average Genomic Identity Of gene Sequences (AGIOS) home-made software [35]. Briefly, this software combines the Proteinortho software [69] for detecting orthologous proteins between genomes compared two by two, 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 C. jeddahense strain JCBT is 2,472,125 bp long (one chromosome, no plasmid) with a G+C content of 67.2% (Figure 6, Table 4). Of the 2,412 predicted chromosomal genes, 2,359 were protein-coding genes and 53 were RNAs. A total of 1,462 genes (60.61%) were assigned a putative function. Sixty-seven genes were identified as ORFans (2.77%) and the remaining genes were annotated as hypothetical proteins. The properties and statistics of the genome are summarized in Table 4. The distribution of genes into COGs functional categories is presented in Table 5.

Figure 6.
figure 6

Graphical circular map of the C. jeddahense strain JCBT genome. From the outside in, the outer two circles shows open reading frames oriented in the forward (colored by COG categories) 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 indicating negative values whereas olive for positive values.

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Table 4. Nucleotide content and gene count levels of the Chromosome
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Table 5. Number of genes associated with the 25 general COG functional categories
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Genome comparison of C. jeddahense with other Corynebacterium genomes

We compared the genome of C. jeddahense strain JCBT with those of C. efficiens YS-314T, C. lipophiloflavum strain DSM 44291T, C. glutamicum strain ATCC 13032T and C. pseudotuberculosis strain CIP 102968T (Table 6 and 7). The draft genome sequence of C. jeddahiense strain JCBT is larger than those of C. efficiens, C. lipophiloflavum and C. glutamicum (2.47, 2.26, 2.43 and 2.11 Mb, respectively), but smaller than that of C. pseudotuberculosis (2.48 Mb). The G+C content of C. jeddahense is larger than those of C. efficiens , C. lipophiloflavum, C. glutamicum and C. pseudotuberculosis (67.2, 62.9, 64.8, 53.8, and 52.1%, respectively). The gene content of C. jeddahense (2,359) is smaller than those of C. efficiens, C. lipophiloflavum and C. glutamicum (2,398, 2,371 and 2,993, respectively) but larger that of C. pseudotuberculosis (2,060). The distribution of genes into COG categories was similar but not identical in all four compared genomes (Figure 7).

Figure 7.
figure 7

Distribution of functional classes of predicted genes in the genomes from C. jeddahense JCBT (colored in sea blue), C. efficiens YS-314T (blue), C. lipophiloflavum strain DSM 44291T (green), C. glutamicum strain ATCC 13032T (yellow) and C. pseudotuberculosis strain CIP 102968T (red) chromosomes, according to the clusters of orthologous groups of proteins.

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Table 6. Genomic comparison of C. jeddahense and 4 other Corynebacterium species.†
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Table 7. Genomic comparison of C. jeddahense and 4 other Corynebacterium species. †
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In addition, C. jeddahense shared 1,369, 1,345, 1,385 and 1,230 orthologous genes with C. efficiens, C. lipophiloflavum, C. glutamicum and C. pseudotuberculosis, respectively. The AGIOS value ranged from 66.7 to 75.04 among compared Corynebacterium species except C. jeddahense. When compared to other species, the AGIOS value ranged from 66.44% with C. pseudotuberculosis to 77.26% with C. lipoflavum, thus confirming its new species status (Table 7).

Conclusion

On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Corynebacterium jeddahense sp. nov., that contains the strain JCBT. The strain has been isolated from the fecal flora of a Saudi man suffering from morbid obesity. Several other as yet undescribed bacterial species were also cultivated from different fecal samples through diversification of culture conditions [5–35], thus suggesting that the human fecal flora of humans remains partially unknown.

Description of Corynebacterium jeddahense sp. nov.

Corynebacterium jeddahense (jed.dah.en’se N.L. neut. adj. Jeddah the name of the town in Saudi Arabia where the specimen was obtained).

Grows occurred between 30 and 45°C on blood-enriched Columbia agar (BioMerieux). Optimal growth obtained at 37°C in aerobic atmosphere. Weak growth obtained in microaerophilic and anaerobic conditions. Colonies are translucent and 1 mm in diameter. Not motile, not endospore-forming. Cells are Gram-positive rods and have a mean diameter and length of 0.63 and 1.22 µm, respectively. Catalase positive, oxidase negative. Using the API Coryne (BioMerieux) system, cells are alkaline phosphatase positive but negative for reduction of nitrates, pyrolidonyl arylamidase, pyrazinamidase, β-glucuronidase, β-galactosidase, α-glucosidase N-acetyl-β-glucosaminidase, β-glucosidase, urease, gelatin hydrolysis and fermentation of glucose, ribose xylose, mannitol, maltose, lactose, saccharose and glycogen. Using the Api Zym (BioMerieux) system, alkaline and acid phosphatases and Naphtol-AS-BI phosphohydrolase activities are positive, but esterase (C4), esterase lipase (C8), lipase (C14), trypsin, α-chemotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, N actetyl-β-glucosaminidase, leucine arylamidase, valine arylamidase, cystin arylamidase, α-mannosidase and α-fucosidase activities are negative. Using the API 50CH system (BioMerieux), fermentation of starch, glycogen, glycerol, erythritol, esculin ferric citrate, amygdalin, arbutin, salicin, L-arabinose, D-ribose, D-xylose, methyl β-D-xylopyranoside, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, D-mannitol, methyl α-D-xylopyranoside, methyl α-D-glucopyranoside, N-acetylglucosamine, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-saccharose, D-trehalose, inulin, D-raffinose, D-lyxose, D-arabinose, L-xylose, D-adonitol, L-sorbose, dulcitol, inositol, D-sorbitol, D-melezitose, D-xylitol, gentiobiose, D-turanose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, and potassium 2-ketogluconate are negative. Cells are susceptible to amoxicillin, ceftriaxone, imipenem, rifampicin, gentamicin, doxycycline and vancomycin but resistant to ciprofloxacin, trimethoprim/sulfamethoxazole, eyrthromycin and metronidazole.

The G+C content of the genome is 67.2%. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers HG726038 andCBYN00000000, respectively. The habitat of the microorganism is the human digestive tract. The type strain JCBT (= CSUR P778 = DSM 45997) was isolated from the fecal flora of a Saudi male who suffered from morbid obesity.