The clinical MDR A. baumanii strain, named MDR-SHH02, was isolated from the blood obtained from a 65-year-old woman with terminal-stage breast cancer at Shanghai Sixth People's Hospital Affiliated to Shanghai Jiaotong University, Shanghai, China. This patient had received a double mastectomy and nearby lymph node excision. After being discharged from the hospital, this patient was hospitalized again due to symptoms of fever, cough (lasting for days) and shortness of breath. During her second hospital administration, she received several antimicrobial therapies, including maxipime, impenem, methylprednisolone, levofloxacin and cefoperazone-sulbactam sodium. A. baumanii was positive in the blood culture and sputum culture. The results of the antimicrobial susceptibility test revealed that the A. baumanii strain was resistant to multiple commonly used antibiotics. This study was approved by the Shanghai Health and Family Planning Commission Foundation (Shanghai, China), and informed consent was obtained from the patient.

The isolated strain was inoculated onto blood agar plates and then incubated in an atmosphere of 5% CO₂ at 35°C for 48 h. Afterwards, this strain was identified using morphological and biochemical tests according to standard methods (14). Colonies with typical morphological and biochemical characteristics of Acinetobacter were cultivated on 5% sheep blood agar and identified using an automated Microscan^® system (Dade Behring, Inc., West Sacramento, CA, USA). The A. baumanii strain was stored at −70°C in skim milk for further analyses.

According to the Clinical and Laboratory Standards Institute (CLSI) guidelines (15), disc diffusion assay (DDA) with dry wafers saturated by 19 types of antibiotics, including gentamicin (10 µg/wafer), tobramycin (30 µg/wafer), amikacin (30 µg/wafer), ampicillin-sulbactam (10/10 µg/wafer), ceftazidime (30 µg/wafer), ciprofloxacin (5 µg/wafer), levofloxacin (5 µg/wafer), imipenem (10 µg/wafer), meropenem (10 µg/wafer), piperacillin/tazobactam (100/10 µg/wafer), ticarcillin/clavulanic acid (75/10 µg/wafer), cefepime (30 µg/wafer), cefotaxime (30 µg/wafer), ceftriaxone (30 µg/wafer), doxycycline (30 µg/wafer), minocyline (30 µg/wafer), tetracycline (30 µg/wafer), piperacillin (100 µg/wafer) and trimethoprim-sulfamethoxazole (1.25/23.75 µg/wafer) (all from Oxoid, Ltd., Basingstoke, UK), were carried out using the Kirby-Bauer (KB) method, as previously described (16). Briefly, Mueller-Hinton agar (Oxoid, Ltd.) plates were overlaid with the inocula of the clinical A. baumanii strain, and the turbidity of the inocula was equivalent to the 0.5 McFarland standard. Subsequently, dry wafers saturated by antibiotics were placed on the surface of the agar, and plates were placed in an atmosphere of 5% CO₂ at 35°C. Following 24 h of culture, the diameter of the inhibition zone around each wafer was measured according to the CLSI criteria (15). In this test, Escherichia coli ATCC 25922, ATCC 35218 and Pseudomonas aeruginosa ATCC 27853 obtained from the Clinical Laboratory Center of the Ministry of Health were used as reference strains.

The genomic DNA of A. baumanii MDR-SHH02 was extracted using a bacterial genomic DNA purification kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions. The Illumina sequencing library was then prepared using the Nextera™ DNA Sample Preparation kit (Illumina^®-Compatible). Paired-end dual index 2×90 bp sequencing was fulfilled following the Illumina HiSeq 2000. Sequencing was performed by Beijing Genomics Institute (BGI; Shenzhen, China). The sequencing data were uploaded to the public database the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/) under the BioProject PRJNA256112 with BioSample accession no. SAMN02991371.

For the raw sequencing data, the reads were cleaned by removing the empty reads, adapter sequences and reads with n≥10% using the SeqPrep program (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle). In addition, the reads were trimmed by discarding the reads containing >30% bases with a Q-value ≤20 in the 3′ terminal, as well as reads with adaptor sequences (the length of overlapping sequences between adaptor and read was at least >15 bp, and the number of mismatch bases was <3 bp).

The clean reads were assembled using the short oligonucleotide analysis package SOAPdenovo (version 2.04; http://soap.genomics.org.cn/). To determine whether the GC content has a significant effect on sequencing randomness or not, the GC content and average depth of the genomic sequence were calculated without repetition as a unit of 500 bp.

Genes in the assembled genomic sequence were predicted using Glimmer 3.0 (http://www.cbcb.umd.edu/software/glimmer/) (17), which is a system for identifying genes in DNA sequences of microorganism, particularly bacteria, archaea and viruses.

Furthermore, tRNA and rRNA (5S, 16S, and 23S rRNA) in the genomic sequence were searched using tRNAscan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/) (18) and RNAmmer 1.2 (http://www.cbs.dtu.dk/services/RNAmmer/) (19), respectively.

Additionally, tandem repeat sequences and clustered regularly interspaced short palindromic repeats (CRISPR) in the genomic sequence were predicted using the Tandem Repeat Finder (http://tandem.bu.edu/trf/trf.html) and CRISPR Finder (http://crispr.u-psud.fr/Server/) software, respectively. Insertion sequences (ISs) were characterized using the IS Finder database (https://www-is.biotoul.fr//), and the parameter -e was set as 1e-5, identity set as 35%. Besides, protein domains associated with the genomic sequence were predicted using the InterPro database (https://www.ebi.ac.uk/interpro/), and the parameter was set as -appl PfamA.

Sequence alignment of the amino acid sequences of genes to the Cluster of Orthologous Groups (COGs) of proteins database (http://www.ncbi.nlm.nih.gov/COG/) (20) was performed using the Basic Local Alignment Search Tool (BLASTP; version 2.0) program from NCBI (E-value ≤10–4) (21). We also performed sequence alignment of the amino acid sequences of genes to the NCBI non-redundant (NR) database (E-value ≤10–10, identity score ≥35%, and coverage length ≥80%). If the amino acid sequence of a gene was aligned to multiple sequences in the databases, the optimal result was retained.

Based on the NCBI 16S rRNA gene database, 16S rRNA gene sequences of 5 A. baumanii strains, including ATCC 17978, ATCC 19606, CIP 70.34, DSM 30007 and A. baumanii JCM 68415, as well as 2 species belonging to Acinetobacter (A. haemolyticus ATCC 17906 and A. bereziniae ATCC 17924), were used to construct the phylogenetic tree, along with MDR-SHH02. Briefly, multiple sequence alignment was performed using ClustalW-2.1 (22). Subsequently, the software package PHYLIP 3.695 (http://evolution.genetics.washington.edu/phylip.html), along with the bootstrap algorithm, was used to construct the maximum likelihood phylogenetic tree, and the phylogenetic tree was visualized by FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).

The Pathogenicity Island database (PAIDB; http://www.paidb.re.kr/about_paidb.php), which is a web-based user-friendly resource and widely used for detecting PAIs in newly sequenced genomes (23), was utilized to predict PAIs in the genomic sequence of MDR-SHH02.

To identify potential antibiotic resistance genes in the genomic sequence of MDR-SHH02, sequence alignment of the protein sequences of antibiotic resistance genes in the Antibiotic Resistance Genes database (ARDB; http://ardb.cbcb.umd.edu/) (24) and MDR-SHH02 genomic sequence was conducted using BLASTP (E-value ≤10–10, identity score ≥90%, and coverage length ≥80%).

The antibiotic-resistance assay revealed that the diameter of the inhibition zone for 17 types of antibiotics on MDR-SHH02 plates was 6 mm, apart from levofloxacin and minocyline (diameter, 10 mm) (Table I). According to the CLSI criteria, MDR-SHH02 was resistant to all of the tested antibiotics.

During the genome assembly, a total of 85 scaffolds were generated, and the scaffold N50 length was 131,822 bp. The total draft genome length of MDR-SHH02 was 4,003,808 bp, with 38.99% of GC content. There were 3,787 coding sequences, 62 tRNA sequences and 3 rRNA sequences in the genomic sequence. Moreover, 2 CRISPR and 36 tandem repeat sequences, as well as 29 ISs were predicted in the genomic sequence (Table II).

Furthermore, numerous protein domains were predicted in the genomic sequence of MDR-SHH02, such as the AcrB/AcrD/AcrF family (Table III).

According to the COG annotation, 74.25% (2,812/3,787) of coding sequences were annotated into 21 COG terms, which were divided into 3 categories: information storage of processing, cellular processes and signaling, and metabolism. Apart from the category of poorly characterized, most o the coding sequences were annotated into 'transcription' (number of coding sequences, 261) and 'amino acid transport and metabolism' (number of coding sequences, 250) (Fig. 1A). The distribution of COG categories in the genomic sequence of A. baumannii MDR-SHH02 is shown in Fig. 1B.

Based on the 16S rRNA gene sequences of A. baumanii in NCBI, the phylogenetic tree revealed that the genomic sequence of MDR-SHH02 was most similar to the 16S rRNA gene sequence of A. baumanii ATCC 17978 (Fig. 2).

During the process of bacterial infection, PAIs play pivotal roles in the evolution of pathogens and the development of diseases. In the genomic sequence of MDR-SHH02, a total of 45 PAIs homologous to the sequence MDRSHH02000806 were detected (Table IV). Most of the PAIs were previously identified from Escherichia coli [e.g., locus of enterocyte effacement (LEE)] and Pseudomonas (e.g., PAPI-1 and T-PAI).

To reveal the genes relevant to the antibiotic resistance of MDR-SHH02, sequence alignment of the protein sequences of antibiotic resistance genes in ARDB and MDR-SHH02 genomic sequence was performed. Based on the selection criteria, a total of 12 gene sequences (e.g., MDRSHH02002408, MDRSHH02000600 and MDRSHH02000597) in MDR-SHH02 were highly similar to the sequences of antibiotic resistance genes in ARDB, such as aac(3)-Ia, aac(6′)-Ib, ant(2″)-Ia and aph(3′)-Ia. According to the antibiotics that were resistant by the 12 gene sequences, MDR-SHH02 was resistant to multiple antibiotics, such as gentamicin, amikacin, tobramycin, spectinomycin, streptomycin and neomycin (Table V), which was partly consistent with the aforementioned results of antibiotic-resistance assay.