Complete genome sequence of Acinetobacter baumannii XH386 (ST208), a multi-drug resistant bacteria isolated from pediatric hospital in China.

Acinetobacter baumannii is an important bacterium that emerged as a significant nosocomial pathogen worldwide. The rise of A. baumannii was due to its multi-drug resistance (MDR), while it was difficult to treat multi-drug resistant A. baumannii with antibiotics, especially in pediatric patients for the therapeutic options with antibiotics were quite limited in pediatric patients. A. baumannii ST208 was identified as predominant sequence type of carbapenem resistant A. baumannii in the United States and China. As we knew, there was no complete genome sequence reproted for A. baumannii ST208, although several whole genome shotgun sequences had been reported. Here, we sequenced the 4087-kilobase (kb) chromosome and 112-kb plasmid of A. baumannii XH386 (ST208), which was isolated from a pediatric hospital in China. The genome of A. baumannii XH386 contained 3968 protein-coding genes and 94 RNA-only encoding genes. Genomic analysis and Minimum inhibitory concentration assay showed that A. baumannii XH386 was multi-drug resistant strain, which showed resistance to most of antibiotics, except for tigecycline. The data may be accessed via the GenBank accession number CP010779 and CP010780.

Direct link to deposited data

http://www.ncbi.nlm.nih.gov/nuccore/CP010779

http://www.ncbi.nlm.nih.gov/nuccore/CP010780

Experimental design, materials and methods

Introduction

Acinetobacter baumannii is an important bacterium which emerged as a significant nosocomial pathogen worldwide [1]. It caused bloodstream infection, pneumonia, endocarditis and so on [2]. The rise of A. baumannii was due to its multi-drug resistance, while it was difficult to treat multi-drug resistant A. baumannii with antibiotics [3], [4]. It caused by A. baumannii had a strong potential to develop antimicrobial resistance, which largely related to mobile genetic elements [5].

Carbapenme resistance in A. baumannii was mediated most by oxacillinases (OXAs) and less by metallo-β-lactamases (MBLs) [6]. Carbapenem resistance in A. baumannii was increasing worldwide, and was considered as a marker of emerging antibiotic resistance [7]. CRAB infection was also a growing problem in the pediatric population. The children were susceptible to infections while the therapeutic options with antibiotics were quite limited. However, the research focusing treatment options on CRAB infections in children was limited. The physicians were forced to use the data extrapolated from the adult literature [8]. For CRAB, sequence types (STs) belonging to the clonal complex 92 (CC92) and the pan-European clonal lineage II (EUII) were predominant in the United States. Of them, A. baumannii ST208 was one of the two most common STs of carbapenem-non-susceptible isolates [9]. Recently, ST 208 had been identified as predominant ST of Carbapenem Resistant A. baumannii (CRAB) in China [10], [11]. These high prevalence of ST208 carrying bla indicated that ST 208 was an emerging lineage mediating the spread of carbapenem resistance via bla [10].

The mobility of the resistance genes was mainly mediated by insertions sequences and transposons. The complete genome would be very useful to study the horizontal transferred resistance genes. Most of A. baumannii strains that harbored complete genome were isolated from adult patients. A. baumannii strain XH386 reported in the paper was isolated from a pediatric patient. This would be helpful to understand whether there was difference between A. baumannii strains isolated from adult patient and pediatric patient. As we knew, there was no complete genome sequence of ST208, although several whole genome shotgun sequences had been reported [12]. Here, we present the complete genome sequence of A. baumannii XH386 (ST208), which was isolated from a pediatric hospital in China, together with a summary classification and a set of features.

Organism information

Classification and features

A. baumannii XH386 is a non-fermentative, strictly aerobic, non-motile, non-pigmented, catalase-positive and oxidase-negative Gram-negative coccobacilli (Fig. 1). The strain grew on simple microbiological media optimally at ~ 37 °C, forming smooth colonies of ~ 2 mm diameter. To evaluate the phylogenomic relationships between A. baumannii XH386 and other strain in this genus, Phylogenetic tree was generated with MEGA 6.0 using neighbor-joining method with 500 bootstraps and standard settings. 16S rRNA gene sequences of Acinetobacter spp. were derived from NCBI GenBank. The phylogenetic neighborhood of A. baumannii XH386 in a 16S rDNA gene sequence based tree was showed in Fig. 2.

Fig. 1

Cellular and colonial morphology of A. baumannii XH386 Gram stained (A) (1000 ×) and grown on LB agar (B).

Fig. 2

(A) Phylogenetic tree of Acinetobacter spp. 16S rRNA gene sequences were derived from NCBI GenBank. The tree was generated with MEGA 6.0 using Neighbor-Joining method with 500 bootstraps and standard settings.

To evaluate the phylogenomic relationships between A. baumannii XH386 and other strains in this species A. baumannii, comparisons between all the strains were calculated as percentages of similarity using Gegennes (version 2.2.1). Then, the percentage of similarity was used to generate a phylogenomic tree with SplitsTree (version 4.13.1). The phylogenomic relationship in A. baumannii was shown in Fig 4A.

Fig. 4

(A) Phylogenetic tree of phylogenetic tree displaying the relationship between A. baumannii and selected strains of the same species. Comparisons between the strains were calculated as percentages of similarity using Gegennes. Then, the percentage of similarity was used to generate a phylogenomic tree with SplitsTree and MEGA. The centre of the figure showed the MLST of these A. baumannii strains. The left of the figures showed the abundance of the resistance genes among these A. baumannii strains. The resistance genes were detected by ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/), and the heat map of the resistance genes was plotted by the R package “ggplot2”. (B) The abundance of the resistance genes among plasmids of these A. baumannii strains. The resistance genes were detected by ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/), and the heat map of the resistance genes was plotted by the R package “ggplot2”.

Genome sequencing information

Genome project history

The genome was selected based on the isolation site of the strain XH386. A. baumannii XH386 was a multi-drug resistant bacteria isolated from a female patient, 10Y3M, with acute bronchopneumonia in a pediatric hospital in Hangzhu, China on May 29, 2014. The genome sequence was completed on 25 Jan., 2015. Annotation was performed by the NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP).

Growth conditions and genomic DNA preparation

A. baumannii XH386 was cultured to mid logarithmic phase in 50 ml of LB medium at 37 °C. DNA for sequencing was extracted via a QIAamp DNA minikit (Qiagen Valencia, CA) followed the protocol of the manufacturer. The quality of DNA was determined by gel electrophoresis and NanoDrop 2000 spectrophotometer (Nano-drop Technologies, Wilmington, DE).

Genome sequencing and assembly

The genome of A. baumannii XH386 was sequenced at Meiji Biotechnology Company (Shanghai, China) using a hybrid of the Illumina and Pacific Biosciences (PacBio) technologies. An Illumina standard shotgun library was constructed, and then was sequenced using the Illumina HiSeq 2000 platform. 3,798,266 reads totaling 953 Mb were generated from the standard shotgun library. A PacBio SMRTbell™ was constructed and sequenced on the PacBio RS platform. 150,292 raw PacBio reads yielded 76,398 adapter trimmed and quality filtered subreads totalling 355 Mb. De novo assembly of the read sequences was performed using continuous long reads following the Hierarchical Genome Assembly Process (HGAP) workflow (PacBio DevNet; Pacific Biosciences) as available in SMRT Analysis v2.3.0, and then Breseq v0.25b with Illumina short reads. The final assembly is based on 953 Mb of Illumina standard PE and 355 Mb of PacBio post filtered data, which provides an average 232 × Illumina coverage and 54.76 × PacBio coverage of the genome, respectively (Table 1).

Table 1

Summary of genome: one chromosome and one plasmid.

Label	Size (Mb)	Topology	INSDC identifier	RefSeq ID
Chromosome 1	4.08	Circular	PRJNA273343	CP010779.1
Plasmid 1	0.11	Circular	PRJNA273343	CP010780.1

Genome annotation

Annotation of A. baumannii XH386 was finished using the NCBI PGAAP annotation pipeline and manually checked. The pipeline uses Genemark to predict open reading frames (ORF) and searches against Proteins Clusters. Protein coding genes were searched against the NCBI RefSeq database using BLASTp. COG functional categories assignment of the ORFs were archived by BLAST against the COG database. InterPro searches were also done to identify conserved domains in each ORF.

Genome properties

The genome of A. baumannii XH386 is 4,199,500 nucleotides 39.1% GC content and contain one 4,087,343 bp circular chromosome and one 112,157 bp circular plasmid (Fig. 3). Among of the 4062 genes, predicted 3968 were protein-coding genes, and 94 RNAs; 26 pseudogenes were also identified. The genome summary and distribution of genes into COG functional categories are listed in Table 2, Table 3.

Fig. 3

Graphical map of the chromosome (A) and the plasmid pAB386 (B) of A. baumannii XH386. From outside to the centre: Genes on forward strand, genes on reverse strand, GC content (Black), GC skew (purple/olive).

Table 2

Nucleotide content and gene count levels of the genome.

Attribute	Genome (total)
Value	% of totala
Genome size (bp)	4,087,343	100
DNA coding (bp)	3,627,022	88.7
DNA G + C (bp)	1,596,791	39.1
DNA scaffolds	1	100
Total genes	4062	100
Protein coding genes	3968	97.7
RNA genes	94	2.3
Pseudo genes	26	0.6
Genes in internal clusters	Not determined	Not determined
Genes with function prediction	3887	95.7
Genes assigned to COGs	3039	74.9
Genes assigned Pfam domains	3268	80.4
Genes with signal peptides	322	21.3
Genes with transmembrane helices	864	2.3
CRISPR repeats	2

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. Also includes 26 pseudogenes and 6 frameshifted genes.

Table 3

Number of genes associated with the 25 general COG functional categories.

Code	Value	% of totala	Description
J	235	5.79	Translation
A	1	0.02	RNA processing and modification
K	269	6.62	Transcription
L	131	3.23	Replication, recombination and repair
B	0	0.00	Chromatin structure and dynamics
D	39	0.96	Cell cycle control, mitosis and meiosis
Y	0	0.00	Nuclear structure
V	66	1.62	Defense mechanisms
T	117	2.88	Signal transduction mechanisms
M	186	4.58	Cell wall/membrane biogenesis
N	55	1.35	Cell motility
Z	0	0.00	Cytoskeleton
W	3	0.07	Extracellular structures
U	55	1.35	Intracellular trafficking and secretion
O	121	2.98	Posttranslational modification, protein turnover, chaperones
C	201	4.95	Energy production and conversion
G	153	3.77	Carbohydrate transport and metabolism
E	263	6.47	Amino acid transport and metabolism
F	82	2.02	Nucleotide transport and metabolism
H	143	3.52	Coenzyme transport and metabolism
I	221	5.44	Lipid transport and metabolism
P	183	4.51	Inorganic ion transport and metabolism
Q	67	1.65	Secondary metabolites biosynthesis, transport and catabolism
R	238	5.86	General function prediction only
S	210	5.17	Function unknown
-	1023	25.18	Not in COGs

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

The abundance of the resistance genes among A. baumannii strains XH386 and other strains in this species were detected by ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/). The phylogenetic tree, MLST and resistance genes of A. baumannii strains was combined showed in Fig. 4A. The distribution of antibiotic resistance genes in A. baumannii XH386 was also shown in Table 4. Fig. 4B showed the distribution of resistance genes in the plasmids harbored by the A. baumannii strains. The difference of the distribution of antibiotic resistance genes between chromosome and plasmid demonstrate that the antibiotic resistance genes more often appeared in chromosome. A. baumannii XH386 was showed resistance to all antibiotics tested except tigcycline, namely tobramycin, gentamicin, levofloxacin, ciprofloxacin, cefoperazone-sulbactam, amoxicillin–clavulanic acid, piperacillin–tazobactam, ampicillin, ceftriaxone, cefepime, cefoxitin, imipenem, aztreonam, cefazolin, nitrofurantoin, sulfamethoxazole–trimethoprim (Table 5).

Table 4

Antibiotic resistance profiles of A. baumannii XH386.

Antibiotic class	Resistance gene	Predicted phenotype	Accession number
Aminoglycoside	aph(3′)-Ic	Aminoglycoside resistance	X62115
aacA4		M60321
aadA1	Aminoglycoside resistance	JQ414041
armA	Aminoglycoside resistance	AY220558
aph(3′)-Ic	Aminoglycoside resistance	X62115
strA	Aminoglycoside resistance	M96392
strB	Aminoglycoside resistance	M96392
Beta-lactam	blaADC-25	Beta-lactam resistance	EF016355
blaOXA-66	Beta-lactam resistance	FJ360530
blaTEM-1D	Beta-lactam resistance	AF188200
blaOXA-23	Beta-lactam resistance	HQ700358
Fluoroquinolone	aac(6′)Ib-cr	Fluoroquinolone and aminoglycoside resistance	EF636461
MLS — macrolide, lincosamide and streptogramin B	msr(E)	Macrolide, Lincosamide and Streptogramin B resistance	EU294228
mph(E)	Macrolide resistance	EU294228
Phenicol	catB8	Phenicol resistance	AF227506
Sulphonamide	sul1	Sulphonamide resistance	CP002151
sul2	Sulphonamide resistance	GQ421466
Tetracycline	tet(B)	Tetracycline resistance	AP000342

Table 5

The susceptibility profile of A. baumannii XH386.

Antimicrobial drug	MIC (mg/L)
Tobramycin	> = 128
Gentamicin	> = 16
Levofloxacin	> = 8
Ciprofloxacin	> = 4
Cefoperazone-sulbactam	14
Amoxicillin-clavulanic acid	> = 32
Piperacillin-tazobactam	> = 16
Ampicillin	> = 32
Ceftriaxone	> = 64
Cefepime	> = 64
Cefoxitin	> = 64
Imipenem	> = 16
Aztreonam	> = 64
Cefazolin	> = 64
Nitrofurantoin	> = 512
Sulfamethoxazole-trimethoprim	> = 320
Tigecycline	2

Insights from the genome sequence

The detection of bla explained the resistance to carbapenem. The existence of aac(6)lb-cr, aacA4, aadA1, aph(3)-lc and armA showed good correlation to the resistance of tobramycin and gentamicin. A. baumannii XH386 demonstrated more resistance genes than sensitive strains, but not the other resistance ST strains, that indicated the emergence of ST208 had affected by other factors, e.g. show high fitness in clinical environment, more virulence.

Conclusions

A. baumannii ST208 was identified predominant ST of Carbapenem Resistant A. baumannii in the United States and China. Although several whole genome shotgun sequences of A. baumannii ST208 had been reported, there was not complete genome sequence of ST208 so far. In current study, a complete genome of A. baumannii ST208 was reported. And the genomic analysis showed that multiple antibiotic resistance genes were detected in the genome, including resistance to aminoglycoside, beta-lactam, fluorequinolone, macrolide, sulphonamide and tetracycline. The genome sequence of A. baumannii XH386 would provide deeper insight into the molecular resistance mechanisms and it might facilitate the development of clinical research to control the antibiotic resistance in A. baumannii.

Nucleotide sequence accession number

This complete genome sequence of A. baumannii XH386 has been deposited at DDBJ/EMBL/GenBank under the accession number CP010779 and CP010780.

Competing interests

The authors declare that they have no competing interests.

Specifications
Organism/cell line/tissue	Acinetobacter baumannii
Sex	n/a
Sequencer or array type	Hiseq and PacBio
Data format	Analyzed
Experimental factors	Genome sequencing of an antimicrobial resistant strain
Experimental features	The complete genome sequence of a clinical strain of A. baumannii was sequenced and annotated to show the multidrug resistant genes.
Consent	n/a
Sample source location	Hangzhou, China