Open Access

Draft genome sequence of Acinetobacter baumannii strain NCTC 13423, a multidrug-resistant clinical isolate

  • Joran E. Michiels1,
  • Bram Van den Bergh1,
  • Maarten Fauvart1, 2 and
  • Jan Michiels1Email author
Standards in Genomic Sciences201611:57

https://doi.org/10.1186/s40793-016-0181-7

Received: 21 March 2016

Accepted: 19 August 2016

Published: 1 September 2016

Abstract

Acinetobacter baumannii is a pathogen that is becoming increasingly important and causes serious hospital-acquired infections. We sequenced the genome of A. baumannii NCTC 13423, a multidrug-resistant strain belonging to the international clone II group, isolated from a human infection in the United Kingdom in 2003. The 3,937,944 bp draft genome has a GC-content of 39.0 % and a total of 3672 predicted protein-coding sequences. The availability of genome sequences of multidrug-resistant A. baumannii isolates will fuel comparative genomic studies to help understand the worrying spread of multidrug resistance in this pathogen.

Keywords

Draft genome Acinetobacter baumannii Nosocomial pathogen Multidrug resistance Human isolate

Introduction

Acinetobacter baumannii recently emerged as an increasingly important pathogen causing healthcare-associated bloodstream, urinary tract, pulmonary, and device-related infections [1]. A. baumannii strains are often resistant against multiple antibiotics, owing to their high intrinsic resistance and a variety of acquired resistance mechanisms [2]. Carbapenem is usually an effective treatment choice, but carbapenem-resistant strains are globally on the rise, and alternative treatment options are limited [3].

Here, we present the draft genome sequence of A. baumannii NCTC 13423, a strain belonging to international clone lineage II isolated from a patient in a UK hospital in December 2003 [4]. NCTC 13423 shows resistance to ampicillin, amoxicillin-clavulanic acid, aztreonam, cefepime, cefotaxime, ceftazidime, cefoxitin, piperacillin, piperacillin-tazobactam, ciprofloxacin, gentamicin, and sulbactam [4]. Although originally reported as carbapenem-sensitive, a later report classified it to be also carbapenem-resistant [5]. Additionally, this strain is highly virulent and a strong biofilm producer [6].

Organism information

Classification and features

Bacteria in the genus Acinetobacter are Gram-negative, strictly aerobic, nonfermenting, nonmotile, catalase-positive, oxidase-negative coccobacilli [7] (Table 1). The genus Acinetobacter has gone through many taxonomic changes over the years, and the species A. baumannii has only been officially recognized since 1986 [8, 9]. A. baumannii belongs to the family Moraxellaceae , order Pseudomonadales , class Gammaproteobacteria , and phylum Proteobacteria . Acinetobacter species are ubiquitous organisms, widely distributed in nature, and can be recovered from virtually any soil or water sample. However, A. baumannii seems to be an exception to this rule, as it currently has no known habitats except the hospital [10]. Microscopically, they are often observed as pairs of cells (Fig. 1). A. baumannii can withstand prolonged desiccation, allowing it to survive on dry surfaces and probably contributing to its persistent residence in hospital settings [11]. A phylogenetic tree based on 16S rDNA sequences showed strong clustering with other A. baumannii strains (Fig. 2).
Table 1

Classification and general features of Acinetobacter baumannii strain NCTC 13423 according to the MIGS recommendations [12]

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [29]

  

Phylum Proteobacteria

TAS [30]

  

Class Gammaproteobacteria

TAS [31, 32]

  

Order Pseudomonadales

TAS [33, 34]

  

Family Moraxellaceae

TAS [35]

  

Genus Acinetobacter

TAS [34, 36]

  

Species Acinetobacter baumannii

TAS [8]

  

Strain NCTC 13423

NAS

 

Gram stain

Negative

TAS [8]

 

Cell shape

Coccobacillus

TAS [8]

 

Motility

Non-motile

TAS [37]

 

Sporulation

Non-sporulating

TAS [8]

 

Temperature range

Mesophilic

TAS [38]

 

Optimum temperature

37 °C

TAS [38]

 

pH range; Optimum

Unknown

NAS

 

Carbon source

Chemoorganoheterotrophic; citrate, lactate, ethanol, glutarate, malate, aspartate, tyrosine, 2,3-butanediol, 4-aminobutyrate

TAS [8]

MIGS-6

Habitat

Hospital

NAS

MIGS-6.3

Salinity

Unknown

NAS

MIGS-22

Oxygen requirement

Strictly aerobic

TAS [8]

MIGS-15

Biotic relationship

Free-living

TAS [8]

MIGS-14

Pathogenicity

Pathogenic

TAS [4]

MIGS-4

Geographic location

United Kingdom

TAS [4]

MIGS-5

Sample collection

12/2003

TAS [4]

MIGS-4.1

Latitude

Unknown

NAS

MIGS-4.2

Longitude

Unknown

NAS

MIGS-4.4

Altitude

Unknown

NAS

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 [39]

Fig. 1

Phase-contrast micrograph of A. baumannii NCTC 13423

Fig. 2

16S rRNA phylogenetic analysis showing the evolutionary relationship between A. baumannii NCTC 13423 and related type (T) and non-type A. baumannii strains and Acinetobacter species. Moraxella catarrhalis was used as an outgroup. Genbank accession numbers of the aligned sequences are indicated between brackets. Sequence alignment was performed using MUSCLE [27], and a neighbour-joining algorithm using the Kimura 2-parameter distance model was used to construct a phylogenetic tree in MEGA (version 7) [28]. The rate variation among sites was modelled with a gamma distribution (shape parameter = 1). The optimal tree with the sum of branch lengths = 0.1583 is shown, and bootstrap support values above 60 % (1000 replicates) are indicated next to the branches

Genome sequencing information

Genome project history

The strain NCTC 13423 was isolated in 2003 in the United Kingdom from a repatriated casualty of the Iraq conflict [4], and was selected for sequencing because of its multidrug-resistant and virulence characteristics. Sequencing was carried out at the EMBL GeneCore facility (Heidelberg, Germany). Sequences were assembled using CLC Genomics Workbench (version 7.5.1) and annotated using NCBI’s Prokaryotic Genome Annotation Pipeline (PGAP). This draft whole-genome sequence has been deposited at DDBJ/ENA/GenBank under the accession LOHD00000000. The project information, and its association with MIGS version 2.0 [12], is summarised in Table 2.
Table 2

Project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

High-quality draft

MIGS-28

Libraries used

One paired-end Illumina library (Nextera)

MIGS-29

Sequencing platforms

Illumina HiSeq 2000

MIGS-31.2

Fold coverage

203

MIGS-30

Assemblers

CLC NGS Cell 7.5.1

MIGS-32

Gene calling method

GeneMarkS+

 

Locus Tag

AUC58

 

Genbank ID

LOHD00000000

 

GenBank Date of Release

2016/02/26

 

GOLD ID

-

 

BIOPROJECT

PRJNA305394

MIGS-13

Source Material Identifier

NCTC 13423

 

Project relevance

Medical

Growth conditions and genomic DNA preparation

Cultures for DNA isolation were inoculated from a single colony on LB agar in 5 ml lysogeny broth and grown overnight at 37 °C with orbital shaking (200 rpm). DNA was isolated using the DNeasy Blood&Tissue Kit (Qiagen) following the manufacturer’s instructions and pre-treatment protocol for Gram-negative bacteria. DNA concentration and purity were assessed using the Nanodrop ND-1000 spectrophotometer and Qubit fluorometer (ThermoFisher Scientific).

Genome sequencing and assembly

Sequencing was performed using the Nextera DNA Library Preparation Kit with the Illumina HiSeq 2000 platform (100 bp, paired-end) at the EMBL GeneCore facility (Heidelberg, Germany). The read library contained a total of 8,765,016 sequences in pairs. Sequence data was analysed using Qiagen’s CLC Genomics Workbench (version 7.5.1). First, reads were trimmed for quality (score limit 0.05) and ambiguous nucleotides (maximum 2 ambiguities). Next, de novo assembly was performed (mismatch cost: 2, deletion cost: 3, insertion cost: 3, length fraction: 0.5, similarity fraction: 0.8), yielding 196 contigs (minimum length 200 bp) with an average coverage of 203x. Contigs averaged 20,092 bp in length (N50 of 111,328 bp). The total length of the draft genome is 3,937,944 bp with a GC-content of 39.0 %.

Genome annotation

All contigs were annotated using NCBI’s Prokaryotic Genome Annotation Pipeline (PGAP). The Batch Web CD-Search Tool from NCBI [13] was used to identify Pfam domains [14] in the predicted protein sequences. Classification of predicted proteins in Clusters of Orthologous Groups (COG) functional categories [15] was done with the WebMGA web server for metagenomic analysis [16]. Signal peptides, transmembrane domains, and CRISPR repeats were predicted using the SignalP 4.1 server [17], the TMHMM server [18], and the CRISPRFinder tool [19], respectively. Only confirmed and not questionable CRISPR hits were taken into account.

Genome properties

Table 3 summarises the properties of the draft genome. Reads were assembled into 196 contigs, totalling 3,937,944 bp with a 39.0 % GC-content. PGAP predicted a total number of 3875 genes, including 3672 protein coding genes (totalling 3,384,768 base pairs), 135 pseudo genes, and 68 RNA genes (64 tRNA, 3 rRNA, and 1 ncRNA). 75.17 % of the protein-coding genes had a putative function assigned, the remainder was annotated as a hypothetical protein. Additional characteristics of the predicted genes are given in Table 3, and Table 4 shows their distribution amongst the different functional COG categories.
Table 3

Genome statistics

Attribute

Value

% of Total

Genome size (bp)

3,937,944

100

DNA coding (bp)

3,384,768

85.95

DNA G + C (bp)

1,537,664

39.05

DNA scaffolds

196

100

Total genes

3875

100

Protein coding genes

3672

94.76

RNA genes

68

1.75

Pseudo genes

135

3.48

Genes in internal clusters

-

-

Genes with function prediction

2913

75.17

Genes assigned to COGs

3174

81.91

Genes with Pfam domains

3,002

77.47

Genes with signal peptides

313

8.08

Genes with transmembrane helices

882

22.76

CRISPR repeats

0

-

Table 4

Number of genes associated with general COG functional categories

Code

Value

%age

Description

J

177

4.82

Translation, ribosomal structure and biogenesis

A

1

0.03

RNA processing and modification

K

272

7.41

Transcription

L

125

3.40

Replication, recombination and repair

B

0

0.00

Chromatin structure and dynamics

D

32

0.87

Cell cycle control, Cell division, chromosome partitioning

V

40

1.09

Defense mechanisms

T

97

2.64

Signal transduction mechanisms

M

193

5.26

Cell wall/membrane biogenesis

N

42

1.14

Cell motility

U

88

2.40

Intracellular trafficking and secretion

O

112

3.05

Posttranslational modification, protein turnover, chaperones

C

202

5.50

Energy production and conversion

G

138

3.76

Carbohydrate transport and metabolism

E

288

7.84

Amino acid transport and metabolism

F

81

2.21

Nucleotide transport and metabolism

H

131

3.57

Coenzyme transport and metabolism

I

182

4.96

Lipid transport and metabolism

P

185

5.04

Inorganic ion transport and metabolism

Q

97

2.64

Secondary metabolites biosynthesis, transport and catabolism

R

406

11.06

General function prediction only

S

285

7.76

Function unknown

-

498

13.56

Not in COGs

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

Insights from the genome sequence

Functional analysis of the genome sequence by RAST annotation [20] revealed A. baumannii ACICU as the closest related sequenced neighbor. A. baumannii ACICU is an epidemic, multidrug-resistant strain isolated from a hospital outbreak in Rome [21]. The high genetic relatedness between A. baumannii ACICU and A. baumannii NCTC 13423 was confirmed by calculating their two-way average amino acid identity (AAI), which was 99.30 % based on 3360 protein sequences [22]. Indicative for the multidrug-resistant phenotype, annotations by RAST included six different β-lactamase enzymes, among which two AmpC-type β-lactamases (class C), a metallo-β-lactamase (class B), two class A β-lactamases (of which one TEM-type broad-spectrum β-lactamase) and an oxa-51 like carbapenemase (class D). Using TAfinder, a web-based tool to identify type II toxin-antitoxin (TA) loci in bacterial genomes [23], we predicted the presence of 12 type II TA modules in the A. baumannii NCTC 13423 draft genome. Considering only TAfinder hits with normalized homology scores (H-value) > 0.5, five putative TA modules remain, three of which are also present in the genome of A. baumannii ACICU. Interestingly, A. baumannii has been reported to form antibiotic-tolerant persister cells [24, 25], and these TA modules might play a role in their formation [26].

Conclusions

We determined the draft genome sequence of the highly virulent, multidrug-resistant A. baumannii NCTC 13423 clinical isolate. The availability of genomic sequences of clinical A. baumannii isolates from a variety of locations and sources will benefit comparative genomic studies to better understand the worrying spread of multidrug resistance in this pathogen.

Abbreviations

COG: 

Clusters of orthologous groups

PGAP: 

Prokaryotic genome annotation pipeline

Declarations

Acknowledgements

JEM and BVDB are recipients of a fellowship from the Agency for Innovation by Science and Technology (IWT) and the Research Foundation Flanders (FWO), respectively. This work was supported by grants from the KU Leuven Research Council (PF/10/010 “NATAR”, IDO/09/01), the Interuniversity Attraction Poles program initiated by the Belgian Science Policy Office (IAP P7/28) and the FWO (grants G.0413.10, G.0471.12 N, G.0B25.15 N). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Authors’ contributions

JEM performed the experiments, analysed the data, and wrote the manuscript. BVDB and MF helped analysing the data and edited the manuscript. JM initiated and supervised the study, and edited the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Centre of Microbial and Plant Genetics, KU Leuven
(2)
Smart Systems and Emerging Technologies Unit, Department of Life Science Technologies, imec

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Copyright

© The Author(s). 2016