Open Access

High quality draft genome sequence of Staphylococcus cohnii subsp. cohnii strain hu-01

  • XinJun Hu1, 2,
  • Ang Li1, 2,
  • LongXian Lv1, 2,
  • Chunhui Yuan1, 2,
  • Lihua Guo1, 2,
  • Xiawei Jiang1, 2,
  • Haiyin Jiang1, 2,
  • GuiRong Qian1, 2,
  • BeiWen Zheng1, 2,
  • Jing Guo1, 2 and
  • LanJuan Li1, 2Email author
Standards in Genomic Sciences20149:9030755

DOI: 10.4056/sigs.5429581

Published: 15 June 2014

Abstract

Staphylococcus cohnii subsp. cohnii belongs to the family Staphylococcaceae in the order Bacillales, class Bacilli and phylum Firmicutes. The increasing relevance of S. cohnii to human health prompted us to determine the genomic sequence of Staphylococcus cohnii subsp. cohnii strain hu-01, a multidrug-resistant isolate from a hospital in China. Here we describe the features of S. cohnii subsp. cohnii strain hu-01, together with the genome sequence and its annotation. This is the first genome sequence of the species Staphylococcus cohnii.

Keywords

Staphylococcus cohnii subsp. cohnii genome Hiseq2000

Introduction

Staphylococcus cohnii belongs to the Coagulase-Negative Staphylococci group. It was described by Schleifer and Kloos (1975) and was named for Ferdinand Cohn, a German botanist and bacteriologist [1]. Recently, more cases of Staphylococcus cohnii infection have been reported in the literature. This organism may be responsible for brain abscess, pneumonia, acute cholecystitis, endocarditis, bacteremia, urinary tract infection and septic arthritis [2]. S. cohnii is comprised of two subspecies that are defined on the basis of their phenotypic characteristics: Staphylococcus cohnii subsp. cohnii and Staphylococcus cohnii subsp. urealyticus [3]. S. cohnii subsp. cohnii is a Gram-positive coccus, coagulase negative and catalase positive, that behaves like a commensal mucocutaneous bacterium [4]. It has more frequently been isolated in hospital than in non-hospital environments [2]. Here we report this draft genome of S. cohnii subsp. cohnii strain hu-01, the first genome of this species to be sequenced.

Classification and features

Strain hu-01 was isolated from a hospital environment in Zhejiang province, China, in October 2012. It is a Gram-positive, coccus-shaped bacterium that can grow on 5% sheep blood enriched Columbia agar (BioMérieux, Marcyl’Etoile, France) at 37°C. Growth occurs under either aerobic or anaerobic conditions. The optimum temperature for growth is 37 °C, with a temperature range of 15–45 °C (Table 1). Cell morphology, motility and sporulation were examined by using transmission electron (H-600, Hitachi) microscopy. Cells of strain hu-01 are coccoidal, 0.6 to 1.2 µm in diameter, occurring predominantly singly or in pairs (Figure 1 and Figure 2).
Figure 1.

Gram staining of S. cohnii subsp. cohnii strain hu-01

Figure 2.

Transmission electron micrograph of cells of strain hu-01. Bar: 0.5 µm

Table 1.

Classification and general features of S. cohnii subsp. cohnii strain hu-01 according to the MIGS recommendations [9].

MIGS ID

Property

Term

Evidence codea

 

Current classification

Domain Bacteria

TAS [20]

 

Phylum Firmicutes

TAS [2123]

 

Class Bacilli

TAS [24,25]

 

Order Bacillales

TAS [26,27]

 

Family Staphylococcaceae

TAS [24,28]

 

Genus Staphylococcus

TAS [26,2931]

 

Species Staphylococcus cohnii subsp. cohnii

TAS [1,3]

 

Strain hu-01

IDA

 

Gram stain

Positive

IDA

 

Cell shape

coccus

IDA

 

Motility

Nonmotile

IDA

 

Sporulation

Nonsporulating

IDA

 

Temperature range

15–45°C

IDA

 

Optimum temperature

37°C

IDA

MIGS-6.3

Salinity

Tolerates 10% NaCl

IDA

MIGS-22

Oxygen

Facultatively anaerobic

IDA

 

Carbon source

D-mannitol, fructose, trehalose

IDA

 

Energy source

fructose, trehalose

IDA

MIGS-6

Habitat

Hospital environment

IDA

MIGS-15

Biotic relationship

Free living

IDA

MIGS-14

Pathogenicity

Opportunistic pathogen

IDA

 

Isolation

Hospital

IDA

MIGS-4

Geographic location

Hangzhou, China

IDA

MIGS-5

Sample collection time

October, 2012

IDA

MIGS-4.1

Latitude

30°16′N

IDA

MIGS-4.2

Longitude

120°12′E

IDA

MIGS-4.3

Depth

unknown

IDA

MIGS-4.4

Altitude

50 (meters)

IDA

aEvidence 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 [32]. If the evidence code is IDA, then the property should have been directly observed, for the purpose of this specific publication, for a live isolate by one of the authors, or an expert or reputable institution mentioned in the acknowledgements.

Comparative 16S rRNA gene sequence analysis by BLASTN [5,6] using the NCBI-NR/NT database revealed 94–99% sequence similarity to members of genus Staphylococcus. Neighbor-Joining phylogenetic analysis based on Kimura 2-parameter model indicated the strain hu-01 is most closely related the strain Staphylococcus cohnii subsp. urealyticus (AB009936.1) (Figure 3).
Figure 3.

Phylogenetic tree depicting the relationship between S. cohnii subsp. cohnii strain hu-01 and other members of the genus Staphylococcus. The strains and their corresponding Genbank accession numbers are shown following the organism name and indicated in parentheses. The phylogenetic tree uses 16S rRNA gene sequences aligned by the CLUSTALW [7], and phylogenetic inferences were made using Neighbor-joining method based on Kimura 2-parameter model within the MEGA5 software [8] and rooted with Bacillus subtilis subsp. subtilis. Bootstrap consensus trees were inferred from 100 replicates, only bootstrap values > 50% were indicated.

Biochemical features were tested by using two automated systems, the Vitek2 Compact (bioMérieux, Marcy l’Etoile, France) and Phoenix 100 ID/AST system (Becton Dickinson Company [BD], Sparks, Maryland, USA). Positive reactions were obtained for D-fructose, trehalose, D-gluconic acid and D-mannitol. Negative reactions were observed for glucose, D-trehalose, D-sucrose, maltose, urea, cellobiose, glucoside, D-tagatose and maltotriose. This strain was susceptible to gentamicin, ciprofloxacin, levofloxacin, moxifloxacin, quinupristin, linezolid, vancomycin, tetracycline, tigecycline, nitrofurantoin, rifampicin, trimethoprim and resistant to cefoxitin, benzylpenicillin, oxacillin, erythromycin, clindamycin.

Genome sequencing information

Genome project history

S. conhii subsp. cohnii strain hu-01 was selected for sequencing because of its increasing relevance to human health. The strain was isolated from a hospital environment in China. This whole genome shotgun project of S. conhii subsp. cohnii strain hu-01 was deposited at DDBJ/EMBL/GenBank under the accession AYOS00000000. Table 2 presents the project information and its association with MIGS version 2.0 compliance [9].
Table 2.

Project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

High-quality draft

MIGS-28

Libraries used

One pair-end 500 bp library

MIGS-29

Sequencing platforms

Illumina HiSeq 2000

MIGS-31.2

Fold coverage

150×(based on 500 bp library)

MIGS-30

Assemblers

Velvet 1.2.07

MIGS-32

Gene calling method

Glimmer 3.0

 

Genbank ID

AYOS00000000

 

Genbank Date of Release

Jan 06, 2014

 

GOLD ID

Gi0062613

MIGS-13

Project relevance

Biotechnology, Pathway, Pathogenic

Growth conditions and DNA isolation

S. conhii subsp. cohnii strain hu-01 was grown aerobically on Columbia blood agar base, at 37°C for 24h. Genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Germany), according to the manufacturer’s recommended protocol. The quantity of DNA was measured by the NanoDrop Spectrophotometer and Cubit. Then 10µg of DNA was sent to the State Key Laboratory for Diagnosis and Treatment of Infectious Disease at Zhejiang University for sequencing on a Hiseq2000 (Illumina, CA) sequencer.

Genome sequencing and assembly

One DNA library was generated (500 bp insert size, with the Illumina adapter at both ends, detected by Agilent DNA analyzer 2100), then sequencing was performed by using an Illumina Hieseq 2000 genomic sequencer, with a 2×100 pair end sequencing strategy. A total of 1,103 M bp of sequence data was produced which was assessed for quality by the following criteria: 1) Reads linked to adapters at both end were considered as sequencing artifacts then removed. 2) Bases with a quality index lower than Q20 at both ends were trimmed. 3) Reads with ambiguous bases (N) were removed. 4) Single qualified reads were discarded (In this situation, one read is qualified but its mate is not). A total of 867.94 M clean filtered reads were assembled into scaffolds using the Velvet version 1.2.07 with parameters “-scaffolds no” [10], then we used a PAGIT flow [11] to prolong the initial contigs and correct sequencing errors. to arrive at a set of improved scaffolds.

Genome annotation

Predict genes were identified using Glimmer version 3.0 [12], tRNAscan-SE version 1.21 [13] was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer version 1.2 [14]. To annotate predicted genes, we used HMMER version 3.0 [15], with parameters ‘hmmscan -E 0.01 -domainE 0.01’ to align genes against Pfam version 27.0 [16] (only pfam-A was used) to find genes with conserved domains. The KAAS server [17] was used to assign translated amino acids (with genetic code table 11) into KEGG Orthology with SBH (single-directional best hit) method. Translated genes were aligned with the COG database using NCBI blastp (hits should have scores no less than 60, e-value is no more than 1e-6). To find genes with hypothetical or putative function, we aligned genes against NCBI nucleotide sequence database (nt database was downloaded at Sep 20, 2013) by using NCBI blastn, only if hits have an identity of no less than 0.95, coverage no less than 0.9, and the reference gene had an annotation of putative or hypothetical. To define genes with signal peptide, we use signalp version 4.1 [18] to identify genes with signal peptide with default parameters except “ -t gram+”. TMHMM2.0 [19] was used to identify genes with transmembrane helices.

Genome properties

The draft genome sequence of S. conhii subsp. cohnii strain hu-01 revealed a genome size of 5,761,489 bp and a G+C content of 34.85% (521 scaffolds with N50 is 39,926 bp). These scaffolds contain 5,820 coding sequences (CDSs), 61 tRNAs (excluding 6 Pseudo tRNAs) and incomplete rRNA operons (10 small subunit rRNA and 3 large subunit rRNAs). A total of 1,840 protein-coding genes were assigned as putative function or hypothetical proteins. 3,734 genes were categorized into COGs functional groups. The properties and the statistics of the genome are summarized in Table 3 and Table 4.
Table 3.

Genome statistics of S. cohnii subsp. cohnii strain hu-01

Attribute

Value

% of totala

Genome size (bp)

5,761,489

DNA coding region (bp)

4,751,472

82.469

DNA G+C content (bp)

1,697,984

29.471

Total genes

5,833

RNA genes

13

0.221

Protein-coding genes

5,820

99.777

Genes with function prediction

1,840

31.544

Genes assigned to COGs

3,734

64.015

Genes assigned to Pfam domains

4,943

84.741

Genes with signal peptides

431

7.388

Genes with transmembrane helices

1,629

27.927

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

Table 4.

Number of genes associated with the general COG functional categories

Code

Valuea

%ageb

Description

J

230

3.95

Translation, ribosomal structure and biogenesis

K

452

7.77

Transcription

L

184

3.16

Replication, recombination and repair

B

3

0.05

Chromatin structure and dynamics

D

72

1.24

Cell cycle control, cell division, chromosome partitioning

V

187

3.21

Defense mechanisms

T

238

4.09

Signal transduction mechanisms

M

254

4.36

Cell wall/membrane/envelope biogenesis

N

70

1.20

Cell motility

Z

1

0.02

Cytoskeleton

W

1

0.02

Extracellular structures

U

57

0.98

Intracellular trafficking, secretion, and vesicular transport

O

147

2.53

Posttranslational modification, protein turnover, chaperones

C

292

5.02

Energy production and conversion

G

384

6.60

Carbohydrate transport and metabolism

E

640

11.0

Amino acid transport and metabolism

F

140

2.41

Nucleotide transport and metabolism

H

234

4.02

Coenzyme transport and metabolism

I

165

2.84

Lipid transport and metabolism

P

389

6.68

Inorganic ion transport and metabolism

Q

197

3.38

Secondary metabolites biosynthesis, transport and catabolism

R

841

14.45

General function prediction only

S

403

6.92

Function unknown

c

483

8.30

Not archived in COGs

d

1603

27.54

No hits

a) For some genes, qualified alignments can occur with several genes belonging to different COG categories. In such cases only the best match to a single COG category is considered. b) The total is based on the total number of protein coding genes(5,820) in the annotated genome. c) These genes have alignments with reference genes archived in COG, but these reference genes do not have COG categories. d) Genes without a qualified hit to a reference genes.

Conclusion

Staphylococcus cohnii ssp. cohnii are part of the normal flora of human skin and mucous membranes which, in particular conditions, may become opportunistic pathogens [4]. The genome sequence of Staphylococcus cohnii subsp. cohnii strain hu-01 will provide the basis to elucidate the molecular principles of host colonization and insight into the genetic background of this organism’s pathogenesis.

Declarations

Acknowledgements

We thank Qiang Ye and Li Liang, Chinese Center of Medical Culture Collections/National Institutes for Food and Drug Control for providing the Biochemical features. This study was supported by the National Basic Research Program of China (973 program) (No. 2013CB531401) and the key Program of the National Natural Science Foundation of China (No. 81330011).

Authors’ Affiliations

(1)
State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, Zhejiang University
(2)
Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases

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Copyright

© The Author(s) 2014