Corynebacterium phoceense sp. nov., strain MC1T a new bacterial species isolated from human urine.

Corynebacterium phoceense strain MC1 (= CSUR P1905 = DSM 100570) is a novel Corynebacterium species isolated from the urine of a kidney transplant recipient as a part of a culturomics study. Corynebacterium phoceense is a Gram-positive, sporogenous, strictly aerobic, and nonmotile coccobacillus. Here we describe strain MC1 and provide its complete annotated genome sequence according to the taxonogenomics concept. Its genome is 2 793 568 bp long and contains 2575 protein-coding genes and 67 RNA genes, including eight rRNA genes.

Introduction

The genus Corynebacterium was described in 1896 by Lehmann and Neumann and belongs to the Actinobacteria class [1]. Currently it consists of a heterogeneous group of Gram-positive, non-spore-forming, rod-shaped bacteria with a high DNA G+C content [2]. In the genus Corynebacterium many species are involved in human and animal diseases, whereas many others are normal flora on skin and mucous membranes [3], [4], [5]. Corynebacteria are found in different environments such as water, soil, plants and human samples [6], [7], [8], [9], [10]. Among corynebacteria, the most significant human pathogen is Corynebacterium diphtheriae, which causes diphtheria worldwide [11]. However, most corynebacteria are opportunistic pathogens [6]. Bacteria found in urine are occasionally associated with urinary infection [12], [13].

Currently bacterial classification is focused on a polyphasic approach with phenotypic and genotypic characteristics such as DNA-DNA hybridization, G+C content and 16S rRNA sequence similarity [14], [15], [16]. This classification system has its limitations, such as the high cost of the DNA-DNA hybridization technique and its low reproducibility [14], [17]. A new bacterial description concept was developed in our laboratory [18], [19], [20], [21], [22] with the recent development of genome sequencing technology [23]. This concept, taxonogenomics [24], is a combination of proteomic description and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profile [25] associated with a phenotypic description and the sequencing, annotation and comparison of the complete genome of the new bacteria species [26].

Here we describe Corynebacterium phoceense sp. nov., strain MC1 (= CSUR P1905 = DSM 100570), according the taxonogenomics concept.

Material and methods

Organism information

Corynebacterium phoceense was isolated from culture of a midstream urine specimen from a 25-year-old kidney transplant recipient from Comoros. He underwent transplantation to treat faecal and segmental glomerulosclerosis in Marseille, France. A urine sample was collected 2 years after his kidney transplantation without clinical signs of urinary tract infection. The patient did not receive antibiotics at the time of urine collection. Informed consent was obtained from the patient, and the study was approved by the Institut Fédératif de Recherche 48, Faculty of Medicine, Marseille, France, under agreement 09-022.

Strain identification by MALDI-TOF MS and 16S rRNA sequencing

Among the 18 culture conditions previously selected by culturomics [27], strain MC1 grew on sheep's blood–enriched Colombia agar (bioMérieux, Marcy l’Etoile, France). The colonies were obtained by spreading samples on a solid medium. They were then purified by subculture and identified by MALDI-TOF MS [28], [29]. Colonies were deposited in duplicate on a MTP 96 MALDI-TOF MS target plate (Bruker Daltonics, Leipzig, Germany), which was analysed with a Microflex spectrometer (Bruker). The 12 spectra obtained were matched against the references of the 7567 bacteria contained in the database by standard pattern matching (with default parameter settings), with MALDI BioTyper database software 2.0 (Bruker). An identification score over 1.9 with a validated species allows identification at the species level, and a score under 1.7 does not enable any identification. When identification by MALDI-TOF MS failed, the 16S rRNA was sequenced [30]. Stackebrandt and Ebers [31] suggest similarity levels of 98.7% with the 16S rRNA sequence as a threshold to define a new species without performing DNA-DNA hybridization.

Growth conditions

To establish our strain's optimal growth conditions, different temperatures (25, 28, 37, 45 and 56°C) and atmospheres (aerobic, microaerophilic and anaerobic) were tested. GENbag anaer and GENbag microaer systems (bioMérieux) were used respectively to test anaerobic and microaerophilic growth. Aerobic growth was carried out with and without 5% CO2.

Morphologic, biochemical and antibiotic susceptibility testing

Gram staining, motility, catalase, oxidase and sporulation were tested as previously described [27]. To perform a biochemical description, according to the manufacturer's instructions, we use API Coryne (bioMérieux) to identify coryneiforms, API ZYM (bioMérieux) to search enzymatic activities and API 50CH (bioMérieux) to estimate capacity to ferment different carbohydrates. Cellular fatty acid methyl ester (FAME) analysis was performed by gas chromatography/mass spectrometry (GC/MS). Two samples were prepared with approximately 60 mg of bacterial biomass per tube collected from several culture plates. FAMEs were prepared as previously described (http://www.midi-inc.com/pdf/MIS_Technote_101.pdf), and GC/MS analyses were carried out as previously described [32]. Briefly, FAMEs were separated using an Elite 5-MS column and monitored by mass spectrometry (Clarus 500-SQ 8 S; Perkin Elmer, Courtaboeuf, France). A spectral database search was performed using MS Search 2.0 operated with the Standard Reference Database 1A (NIST, Gaithersburg, MD, USA) and the FAMEs mass spectral database (Wiley, Chichester, UK).

Antibiotic susceptibility testing was performed using the disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2015 recommendations (http://www.eucast.org/). Corynebacterium phoceense resistance and susceptibility are estimated with 22 antibiotic treatments (vancomycin 30 μg, rifampicin 30 μg, doxycycline 30 IU, erythromycin 15 IU, amoxicillin 25 μg, nitrofurantoin 300 μg, gentamicin 15 μg, ciprofloxacin 5 μg, ceftriaxone 30 μg, amoxicillin 20 μg + clavulanic acid 10 μg, penicillin G 10 μg, gentamicin 500 μg, trimethoprim 1.25 μg + sulfamethoxazole 23.75 μg, oxacillin 5 μg, imipenem 10 μg, tobramycin 10 g, metronidazole 4 μg, amikacin 30 μg, linezolid 30 μg, clindamycin 15 μg, daptomycin in stripe 0.016–256 μg (bioMérieux) and chloramphenicol 5 mg, (Sigma-Aldrich, St. Louis, MO, USA)). The bacterial suspension (0.5 McFarland standard) is made in 2 mL NaCl 0.85% medium. Petri dishes with Mueller-Hinton + 5% sheep's blood (Becton Dickinson, San Diego, CA, USA) are seeded by swabbing with bacteria suspension. Different antibiotic dishes (SirScan) are dispensed on petri dishes. Electron microscopy figure was obtained by performing a negative staining of strain MC1. Detection Formvar-coated grids were deposited on a 40 μL bacterial suspension drop, then incubated at 37°C for 30 minutes and on ammonium molybdate 1% for 10 seconds. The dried grids on blotted paper were observed with a Tecnai G20 transmission electron microscope (FEI Company, Limeil-Brevannes, France).

Growth conditions and genomic DNA preparation

Corynebacterium phoceense strain MC1 (= CSUR P1905 = DSM 100570) was grown on 5% sheep's blood–enriched Columbia agar (bioMérieux) at 37°C in aerobic atmosphere. Bacteria grown on three petri dishes were collected and resuspended in 4 × 100 μL of Tris-EDTA (TE) buffer. Then 200 μL of this suspension was diluted in 1 mL TE buffer for lysis treatment that included a 30-minute incubation with 2.5 μg/μL lysozyme at 37°C, followed by an overnight incubation with 20 μg/μL proteinase K at 37°C. Extracted DNA was then purified using three successive phenol–chloroform extractions and ethanol precipitations at −20°C overnight. After centrifugation, the DNA was resuspended in 160 μL TE buffer.

Genome sequencing and assembly

Genomic DNA (gDNA) of Corynebacterium phoceense was sequenced on the MiSeq Technology (Illumina, San Diego, CA, USA) with the mate pair strategy. The gDNA was barcoded in order to be mixed with nine other projects with the Nextera Mate Pair sample prep kit (Illumina).

gDNA was quantified by a Qubit assay with the high sensitivity kit (Life Technologies, Carlsbad, CA, USA) to 59.1 ng/μL. The mate pair library was prepared with 1.5 μg 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 fragmentation pattern was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) with a DNA 7500 lab chip. The DNA fragments ranged in size from 1.5 to 11 kb, with an optimal size at 7.364 kb. No size selection was performed, and 600 ng of tagmented fragments were circularized. The circularized DNA was mechanically sheared to small fragments with an optimal at 589 bp on the Covaris S2 device in T6 tubes (Covaris, Woburn, MA, USA). The library profile was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent Technologies), and the final concentration library was measured at 25.29 nmol/L.

The libraries were normalized at 2 nM and pooled. After a denaturation step and dilution at 15 pM, the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and sequencing runs were performed in a single 39-hour run in a 2 × 151 bp read length.

Total information of 0.7 Gb was obtained from a 129K/mm2 cluster density with a cluster passing quality control filters of 80.3% (2 435 000 passing filter paired reads). Within this run, the index representation for Corynebacterium phoceense was determined to 11.16%. The 271 698 paired reads were trimmed, then assembled in 14 scaffolds.

Genome annotation and comparison

Genome annotation and comparison open reading frames (ORFs) were predicted using Prodigal [33] with default parameters, but the predicted ORFs were excluded if they spanned a sequencing gap region (contains N). The predicted bacterial protein sequences were searched against the Clusters of Orthologous Groups (COGs) database using BLASTP (E value 1e-03, coverage 70%, identity percentage 30%). If no hit was found, it searched against the NR database using BLASTP with an E value of 1e-03 coverage 70% and identity percentage of 30%. If sequence lengths were smaller than 80 amino acids, we used an E value of 1e-05. The tRNAScanSE tool [34] was used to find tRNA genes, whereas rRNAs were found using RNAmmer [35]. Lipoprotein signal peptides and the number of transmembrane helices were predicted using Phobius [36]. ORFans were identified if all the performed BLASTP procedures did not give positive results (E value smaller than 1e-03 for ORFs with sequence size higher than 80 aa or E value smaller than 1e-05 for ORFs with a sequence length smaller than 80 aa). Such parameter thresholds have already been used in previous work to define ORFans.

Genomes were automatically retrieved from the 16s RNA tree using Xegen's PhyloPattern software [37]. For each selected genome, complete genome sequence, proteome and ORFeome genome sequence were retrieved from the National Center for Biotechnology Information FTP site. All proteomes were analysed with proteinOrtho [38]. Then for each couple of genomes a similarity score was computed. This score is the mean value of nucleotide similarity between all orthologue couples between the two genomes studied (AGIOS) [26].

The entire proteome was annotated to define the distribution of functional classes of predicted genes according to the clusters of orthologous protein groups (using the same method as for the genome annotation). To evaluate the genomic similarity among the compared strains, we determined two parameters: digital DNA-DNA hybridization, which exhibits a high correlation with DNA-DNA hybridization (DDH) [39], [40], and AGIOS [26], which was designed to be independent from DDH.

Results

Strain identification and phylogenetic analyses

Strain MC1 (Table 1) was first isolated in February 2015 by spreading a urine sample on 5% sheep's blood–enriched Colombia agar (bioMérieux) in an aerobic atmosphere at 37°C after 48 hours of incubation.

Table 1

Classification and general features of Corynebacterium phoceense strain MC1T

Property	Term
Current classification	Domain: Bacteria
	Phylum: Actinobacteria
	Class: Actinobacteria
	Order: Actinomycetales
	Family: Corynebacteriaceae
	Genus: Corynebacterium
	Species: Corynebacterium phoceense
	Type strain: MC1
Gram stain	Positive
Cell shape	Rod
Motility	Nonmotile
Sporulation	Non–spore forming
Temperature range	Mesophilic
Optimum temperature	37°C

Using MALDI-TOF MS for identification, no significant score was obtained for strain MC1, suggesting that this isolate's spectrum did not match any spectra in our MALDI-TOF MS database. The nucleotide sequences of the 16S rRNA and the rpoB genes of strain MC1 (GenBank accession nos. LN849777 and LN849778 respectively) showed 96.3% similarity with Corynebacterium simulans, the phylogenetically closest species with a validly published name (Fig. 1), therefore defining it as a new species within the genus Corynebacterium. C. phoceense spectra (Fig. 2) were incremented in our database. The reference spectrum for C. phoceense was then compared to the spectra of phylogenetically close species within the genus Corynebacterium, and the differences were exhibited in a gel view photo (Fig. 3).

Fig. 1

Phylogenetic trees highlighting position of Corynebacterium phoceense strain MC1 (=CSUR P1905 = DSM 100570) relative to other strains within genus Corynebacterium. Sequences of 16S rRNA gene (A) and rpoB genes (B) were aligned by CLUSTALW. Scale bar represents 1 and 2% nucleotide sequence divergence for (A) and (B) respectively.

Fig. 2

Reference mass spectrum from Corynebacterium phoceense strain MC1. Spectra from 12 individual colonies were compared and reference spectrum was generated.

Fig. 3

Gel view comparing Corynebacterium phoceense strain MC1 (= CSUR P1905 = DSM 100570) to other species within Corynebacteriaceae family. Gel view displays raw spectra of loaded spectrum files arranged in pseudo-gel-like look. x-axis records m/z value. Left y-axis displays running spectrum number originating from subsequent spectra loading. Peak intensity is expressed by greyscale scheme code. Colour bar and right y-axis indicate relation between colour peak displayed and peak intensity in arbitrary units. Displayed species are indicated at left.

Phenotypic description

Growth was observed from 25 to 56°C on 5% sheep's blood–enriched Columbia agar (bioMérieux), with optimal growth at 37°C in aerobic conditions after 48 hours of incubation.

Weak cell growth was observed under microaerophilic and anaerobic conditions. The motility test was negative, and after thermal shock, we observed bacterial growth, which means Corynebacterium phoceense is sporogenous. Cells were Gram-positive coccobacillus (Fig. 4). On Columbian blood agar, colonies are circular with entire margin, up to 1.0 mm in diameter after 48 hours' growth at 37°C. Under electron microscopy, cells had a mean diameter of 0.5 μm and a length of 3 μm (Fig. 5).

Fig. 4

Gram staining of Corynebacterium phoceense strain MC1.

Fig. 5

Transmission electron microscopy of Corynebacterium phoceense strain MC1T with Morgani 268D (Philips, Amsterdam, the Netherlands) at operating voltage of 60 kV. Scale bar = 500 nm.

The major fatty acids were 9-octadecenoic acid (51%) and hexadecanoic acid (46%). The other fatty acids described were below 3% (Table 2).

Table 2

Cellular fatty acid composition (%)

Fatty acid	Name	Mean relative %a
18:1n9	9-Octadecenoic acid	51.1 ± 0.5
16:0	Hexadecanoic acid	46.0 ± 0.3
18:0	Octadecanoic acid	2.1 ± 0.2
15:0	Pentadecanoic acid	TR
14:0	Tetradecanoic acid	TR
18:2n6	9,12-Octadecadienoic acid	TR
16:1n7	9-Hexadecenoic acid	TR
17:0	Heptadecanoic acid	TR
16:1n5	11-Hexadecenoic acid	TR

Mean peak area percentage; TR = trace amounts <1%.

The presence of catalase was tested using 3% (v/v) H2O2 and gave a positive result. The oxidase test was also negative for strain MC1. Corynebacterium phoceense strain MC1 was capable of fermenting glucose, ribose, maltose, saccharose, d-ribose, d-galactose, d-glucose, d-fructose, d-mannose, arbutin, ferric citrate esculin, d-maltose and d-saccharose. Nitrates were reduced into nitrites. Alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, β-glucosidase, pyrazinamidase, pyrrolidonyl arylamidase and naphthol-AS-BI-phosphohydrolase activities were highlighted.

Corynebacterium phoceense strain MC1 was susceptible to all antibiotic treatments tested except nitrofurantoin and metronidazole, to which it was resistant.

The biochemical and phenotypic features of strain MC1 were compared to the corresponding features of other close representatives of the genus Corynebacterium (Table 3). We particularly observed the absence of endospores forming in the closest Corynebacterium spp.

Table 3

Differential characteristics of Corynebacterium phoceense strain MC1T, Corynebacterium freiburgense strain 1045T, Corynebacterium mastitidis strain CECT 4843T, Corynebacterium terpenotabidum strain Y-1 IT, Corynebacterium lactis strain RW2-5T, Corynebacterium aurimucosum strain IMMIB D-1488T and Corynebacterium mustelae strain 3105T[41], [42], [43], [44], [45], [46]

Property	C. phoceense	C. freiburgense	C. mastitidis	C. terpenotabidum	C. lactis	C. aurimucosum	C. mustelae
Cell diameter (μm)	0.5	0.5	0.5	0.7	0.6	0.5	0.5
Oxygen requirement	+	+	+	+	+	+	+
Gram stain	+	+	+	+	+	+	+
Salt requirement	−	−	−	−	−	−	−
Motility	−	−	−	−	−	−	−
Endospore formation	+	−	−	−	−	−	−
Production of:Alkaline phosphatase	+	−	+	NA	+	+	−
Catalase	+	+	+	+	+	+	+
Oxidase	−	NA	−	−	−	NA	−
Nitrate reductase	+	+	−	−	NA	−	−
Urease	−	−	v	+	−	−	−
β-Galactosidase	−	+	−	NA	−	−	−
N-acetyl-glucosamine	−	−	−	+	−	−	+
Ribose	+	+	−	−	+	−	+
Pyrazinamidase	+	−	+	NA	+	+	+
Pyrrolidonyl arylamidase	+	−	−	NA	−	−	−
Mannose	+	+	NA	+	+	−	+
Mannitol	−	+	−	−	−	−	−
Sucrose	−	+	−	−	NA	+	+
d-Glucose	+	+	−	−	+	+	+
d-Fructose	+	+	NA	−	+	+	+
d-Maltose	+	+	−	−	−	+	+
d-Lactose	+	+	−	−	−	−	−
Habitat	Human	Human	Sheep's milk	Soil	Cow's milk	Human	Human

+, positive result; −, negative result; NA, data not available.

Genome properties

The genome is 2 793 868 bp long with 63.23% GC content (Table 4, Fig. 6). It is composed of 14 scaffolds (composed of 71 contigs). Of the 2575 predicted genes, 2508 were protein-coding genes and 67 were RNAs (four genes are 5S rRNA, three genes are 16S rRNA, three genes are 23S rRNA and 57 genes are tRNA genes). A total of 1804 genes (71.93%) were assigned as putative function (by COGs or by NR BLAST). One hundred fifty-one genes were identified as ORFans (6.02%). The remaining 475 genes were annotated as hypothetical proteins (18.94%). The National Center for Biotechnology Information ID is PRJEB14666, and the genome is deposited under accession number FLTI01000001. The distribution of genes into COGs functional categories is presented in Table 5.

Table 4

Nucleotide content and gene count levels of genome

Attribute	Genome (total)
	Value	% of totala
Size (bp)	2 793 868	100.0
G+C content (bp)	1 753 790	63.2
Coding region (bp)	2 265 533	81.1
Total of genes	2575	100.0
RNA genes	67	2.6
Protein-coding genes	2508	100.0
Protein with function prediction	1804	71.9
Protein assigned to COGs	1511	60.2
Genes with peptid signals	313	12.4
Genes with transmembrane helices	612	24.4

COGs, Clusters of Orthologous Groups database.

Total is based on either size of genome in base pairs or total number of protein coding genes in annotated genome.

Fig. 6

Graphical circular map of chromosome. From outside to centre: genes on forward strain coloured by COGs categories (only gene assigned to COGs), RNA genes (tRNAs green, rRNAs red), GC content and GC skew.

Table 5

Number of genes associated with 25 general COGs functional categories

Code	Value	% of totala	Description
J	153	6.10	Translation
A	1	0.04	RNA processing and modification
K	106	4.22	Transcription
L	68	2.71	Replication, recombination and repair
B	0	0	Chromatin structure and dynamics
D	18	0.71	Cell cycle control, mitosis and meiosis
Y	0	0	Nuclear structure
V	56	2.23	Defense mechanisms
T	55	2.19	Signal transduction mechanisms
M	57	2.27	Cell wall/membrane biogenesis
N	3	0.11	Cell motility
Z	0	0	Cytoskeleton
W	3	0.11	Extracellular structures
U	17	0.67	Intracellular trafficking and secretion
O	66	2.63	Posttranslational modification, protein turnover, chaperones
X	53	2.11	Mobilome: prophages, transposons
C	94	3.74	Energy production and conversion
G	129	5.14	Carbohydrate transport and metabolism
E	172	6.85	Amino acid transport and metabolism
F	74	2.95	Nucleotide transport and metabolism
H	105	4.18	Coenzyme transport and metabolism
I	73	2.91	Lipid transport and metabolism
P	123	4.90	Inorganic ion transport and metabolism
Q	44	1.75	Secondary metabolites biosynthesis, transport and catabolism
R	131	5.22	General function prediction only
S	78	3.11	Function unknown
—	997	39.75	Not in COGs

COGs, Clusters of Orthologous Groups database.

Total is based on total number of protein-coding genes in annotated genome.

Genome comparison

Table 6 compares Corynebacterium phoceense's genomic characteristics to other close species. The draft genome sequence of C. phoceense is smaller than those of Corynebacterium mustelae, Corynebacterium freiburgense and Corynebacterium aurimucosum (2.794, 3.474, 2.91 and 2.905 Mb respectively), but larger than those of Corynebacterium lactis, Corynebacterium ulcerans, Corynebacterium mastitidis and Corynebacterium terpenotabidum (2.77, 2.61, 2.37 and 2.75 Mb respectively).

Table 6

Genome comparison of closely related species to Corynebacterium phoceense strain MC1T

Species	Size (Mb)	G+C (%)	Total genes
Corynebacterium lactis strain RW2-5	2.76	60.5	2364
Corynebacterium ulcerans strain NCTC_7910	2.61	53.3	2296
Corynebacterium mustelae strain 3105	3.47	52.5	3146
Corynebacterium freiburgense strain 1045	2.91	49.8	2667
Corynebacterium mastitidis strain S-8 CECT_4843	2.37	68.9	2241
Corynebacterium aurimucosum strain IMMIB D-1488	2.90	59.2	2769
Corynebacterium phoceense strain MC1	2.79	63.2	2508
Corynebacterium terpenotabidum strain Y-11 IFO 14764	2.75	67	2369

The G+C content of Corynebacterium phoceense is smaller than those of Corynebacterium mastitidis and Corynebacterium terpenotabidum (63.23, 68.9 and 67.02% respectively), but larger than those of Corynebacterium lactis, Corynebacterium ulcerans, Corynebacterium mustelae, Corynebacterium freiburgense and Corynebacterium aurimucosum (60.53, 53.39, 52.57, 49.82 and 59.21% respectively).

The gene content of Corynebacterium phoceense is smaller than those of Corynebacterium mustelae, Corynebacterium freiburgense and Corynebacterium aurimucosum (2508, 3146, 2667 and 2769 respectively), but larger than those of Corynebacterium lactis, Corynebacterium ulcerans, Corynebacterium mastitidis and Corynebacterium terpenotabidum (2364, 2296, 2241 and 2369 respectively).

Finally, the distribution of genes into COGs categories was similar in all compared genomes except for those corresponding to the cell cycle control, mitosis and meiosis category, which were only present in C. mastitidis, C. mustelae and C. ulcerans (Fig. 7). C. phoceense strain MC1 shared 2667, 1358, 1332, 1248, 1128, 1211 and 1442 orthologous genes with C. freiburgense, C. aurimucosum, C. ulcerans, C. lactis, C. terpenotabidum, C. mastitidis and C. mustelae respectively (Table 7). Among species with standing in nomenclature, AGIOS values ranged from 54.12% between C. terpenotabidum and C. freiburgense to 74.86% between C. aurimucosum and C. phoceense. When C. phoceense was compared to the other species, AGIOS values ranged from 58.13% with C. ulcerans to 74.86% with C. aurimucosum.

Fig. 7

Distribution of functional classes of predicted genes according to COGs of proteins.

Table 7

Numbers of orthologous protein shared between genomes (upper right) a

	C. freiburgense	C. aurimucosum	C. ulcerans	C. lactis	C. phoceense	C. terpenotabidum	C. mastitidis	C. mustelae
Corynebacterium freiburgensestrain 1045	2667	1358	1332	1248	1232	1128	1211	1442
Corynebacterium aurimucosumstrain IMMIB	63.97	2769	1336	1252	1407	1189	1315	1392
Corynebacterium ulceransstrain NCTC 7910	57.66	58.26	2296	1197	1221	1091	1213	1354
Corynebacterium lactisstrain RW2-5	57.63	60.50	58.47	2364	1116	1105	1149	1249
Corynebacterium phoceensestrain MC1	63.56	74.86	58.13	61.48	2508	1079	1194	1264
Corynebacterium terpenotabidumstrain Y-11 IFO 14764	54.12	58.32	54.92	56.16	59.86	2369	1123	1146
Corynebacterium mastitidisstrain S-8 CECT 4843	63.90	69.01	57.33	61.39	70.81	60.55	2241	1252
Corynebacterium mustelaestrain 3105	65.46	64.85	58.55	58.25	64.48	55.39	64.16	3146

Average percentage similarity of nucleotides corresponding to orthologous protein shared between genomes (lower left) and numbers of proteins per genome (bold).

Conclusion

On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Corynebacterium phoceense which contains the type strain MC1T. This bacterial strain has been isolated from the urine of a kidney transplant recipient.

Description of Corynebacterium phoceense type strain MC1T sp. nov.

Corynebacterium phoceense (pho.ce'en'se, L. gen. masc., phoceense, “of Phoceen,” the old Latin name of the city of Marseille, where strain MC1T was isolated). Cells have mean diameter of 0.5 μm and a mean length of 3 μm. Colonies are round and 1.0 mm in diameter on 5% sheep's blood–enriched Columbia agar (bioMérieux). Positive reactions are observed for glucose, ribose, maltose, saccharose, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, β-glucosidase, pyrazinamidase, pyrrolidonyl arylamidase and naphthol-AS-BI-phosphohydrolase. d-Ribose, d-galactose, d-glucose, d-fructose, d-mannose, arbutin, d-maltose and d-saccharose were metabolized. Cells were susceptible to vancomycin, rifampicin, doxycycline, erythromycin, amoxicillin, nitrofurantoin, gentamicin, ciprofloxacin, ceftriaxone, amoxicillin + clavulanic acid, penicillin G, gentamicin, trimethoprim + sulfamethoxazole, oxacillin, imipenem, tobramycin, metronidazole, amikacin, linezolid, clindamycin, daptomycin and chloramphenicol.

The G+C content of the genome is 63.23%. The 16S rRNA gene sequence and whole-genome shotgun sequence of C. phoceense strain MC1T are deposited in GenBank under accession numbers LN849777 and FLTI01000001, respectively. The type strain MC1T (= CSUR P1905 = DSM 100570) was isolated from the urine of a kidney transplant recipient.