Identification of antibiotic resistance genes in the multidrug-resistant Acinetobacter baumannii strain, MDR-SHH02, using whole-genome sequencing
- Authors:
- Published online on: December 30, 2016 https://doi.org/10.3892/ijmm.2016.2844
- Pages: 364-372
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Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Acinetobacter baumannii (A. baumanii) is a notable pathogen that causes hospital-acquired infections among immune-compromised patients, accounting for 5% of Gram-negative infections (1). Due to a strong resistance to desiccation and multiple antibacterial agents, the widespread dissemination of multidrug-resistant (MDR) A. baumanii strains has been a threat to hospitalized patients in recent years (2).
Antibiotic resistance determinants play pivotal roles in whether or not A. baumannii will flourish in the host (3). For instance, the expression of β-lactamase genes [e.g., oxacillinase (OXA)-235 gene, blaOXA-51 and blaTEM-1] has been shown to be involved in antibiotic resistance (4–6). An 86-kb region AbaR resistant to heavy metal and antibiotics has been found in a MDR isolate AYE (7), indicating the important role of AbaR in the spread of A. baumannii in hospitals (8,9). Furthermore, other resistance determinants, such as macrolide (msrA/msrB), aminoglycoside (e.g., aacC1, armA, and aphA1) and tetracycline [e.g., tet (39), tet (A), and tet (B)] have been identified in various A. baumannii isolates (10).
Bacterial whole-genome sequencing (WGS) has enhanced our ability to evaluate antibiotic resistance determinants. For example, a AbaR-type genomic resistance island, AbaR22, has been identified in the MDR A. baumannii strain, MDR-ZJ06, via WGS (11). A whole-genome comparison detected 18 putative single nucleotide polymorphisms (SNPs) between 2 pre- and post-therapy A. baumannii isolates (12). Furthermore, 10 types of AbaR resistance islands were identified in 2 A. baumannii isolates using WGS (13). Despite increased research on antibiotic resistance determinants in A. baumannii, the molecular mechanisms of antibiotic resistance in MDR A. baumannii are not yet fully understood, and various antibiotic resistance genes have not been detected.
In the present study, we applied WGS to obtain the whole genomic sequence of the MDR A. baumannii strain, MDR-SHH02, isolated from a patient with breast cancer. Furthermore, the antibiotic resistance of MDR-SHH02 to multiple antibiotics was determined, and potential antibiotic resistance genes in MDR-SHH02 were predicted. The results of our study may enhance our understanding of the molecular mechanisms of antibiotic resistance in MDR A. baumannii, and provide a clinical guidance for the therapy of A. baumannii-infected patients.
Materials and methods
Isolation and identification of A. baumanii strain
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.
Antibiotic resistance test for A. baumanii MDR-SHH02
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.
DNA preparation, library construction and sequencing
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.
Quality control
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).
Genome assembly
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.
Genome annotation
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.
Functional annotation of genes
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.
Construction of phylogenetic tree
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/).
Prediction of pathogenicity islands (PAIs)
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.
Identification of antibiotic resistance genes
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%).
Results
Antibiotic-resistance of A. baumanii MDR-SHH02
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.
Assembly and annotation of the genomic sequence of A. baumanii MDR-SHH02
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).
Functional annotation of the genomic sequence of A. baumanii MDR-SHH02
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.
Analysis of the phylogenetic tree
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).
Analysis of PAIs
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).
 | Table IVPathogenicity islands homologous to a region in the genomic sequence of A. baumannii MDR-SHH02. |
Screening of antibiotic resistance genes
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.
Discussion
In the present study, we reported the draft genomic sequence of the clinical MDR A. baumannii strain, MDR-SHH02, and predicted one gene seuqence homologous to 45 PAIs and 12 potential gene sequences relevant to antibiotic resistance. The antibiotic-resistance assay and the high similarity between the 12 gene sequences in MDR-SHH02 and the sequences of antibiotic resistance genes in ARDB, revealed that MDR-SHH02 was resistant to multiple antibiotics.
According to the prediction of PAIs, the gene sequence MDRSHH02000806 was homologous to 45 PAIs, such as LEE and PAPI-1. LEE PAIs were previously identified from multiple enteropathogenic Escherichia coli strains, and they are highly conserved in gene order and nucleotide sequence (25,26). PAPI-1 was previously identified from the P. aeruginosa strain, PA14, and it contributes directly and synergistically along with PAPI-2 to the virulence of PA14 (27). Therefore, the virulence of MDR-SHH02 may be due to the presence of MDRSHH02000806 homologous to these PAIs.
In this study, we discovered a set of gene sequences that were highly similar to the sequences of multiple genes encoding aminoglycoside-modifying enzymes (AMEs), including 2 aminoglycoside N-acetyltransferase genes [aac(3)-Ia and aac(6′)-Ib], 2 aminoglycoside O-nucleotidylyltransferase genes [ant(2″)-Ia and ant(3″)-Ia], and 3 aminoglycoside O-phosphotransferase genes [aph33ib, aph(3′)-Ia and aph(6)-Id]. The expression of AMEs enables bacteria to catalyze the modification of amino and hydroxyl groups on sugar moieties, such as aminoglycosides (28), which is a major cause of aminoglycoside resistance in many bacteria (29). The majority of aminoglycoside-resistant Acinetobacter isolates have the ability of enzymatic modification of aminoglycosides by acetyltransferases, nucleotidyltransferases and/or phosphotransferases (30). Previous studies have reported the prevalence of multiple AME genes [e.g., aac(3)-Ia, and aac(6′)-Ib] in a set of A. baumannii isolates that are resistant to various aminogl ycosides (e.g., amikacin, gentamicin and tobramycin) (31,32). Besides, Aph(6)-Id and ant(3″)-Ia have been detected in the A. baumannii strain, MRSN 12227, which is resistant to various antibiotics, such as amikacin, tobramycin and cefotaxime (33). Another study reported that ant(2″)-Ia present in a group of A. baumannii isolates (62.6%) was associated with resistance to the tested aminoglycosides (amikacin, tobramycin and gentamicin) (34). Furthermore, in this study, we found that MDR-SHH02 was resistant to 19 antibiotics, such as several types of aminoglycosides (amikacin, gentamicin and tobramycin), indicating that the resistance of MDR-SHH02 to aminoglycosides likely resulted from the coding sequences highly similar to AME genes. However, aph33ib has not been previously detected in A. baumannii isolates, and it is worthy of further study. For example, following knockout and complementation of the gene sequence that is highly similar to aph33ib, the resistance of MDR-SHH02 to aminoglycosides is determined.
In this study, several gene sequences in MDR-SHH02 had a high similarity to class A β-lactamase genes (bl2b_tem and bl2b_tem1), group B chloramphenicol acetyltransferase gene (catb3), sulfonamide-resistant dihydropteroate synthase genes (sul1 and sul2) and tetracycline efflux pump gene (tetb). The gene bl2b_tem has been detected in Staphylococcus aureus (35), and bl2b_tem1 was detected in a series of marine bacteria, such as Pelagibacter, Polaribacter and Roseobacter (36) . However, there is no evidence to support that bl2b_tem and bl2b_tem1 are carried by A. baumannii isolates. All other genes (catb3, sul1, sul2 and tetb) have been found in A. baumannii isolates (37–40).
Despite the aforementioned results, there were still several limitations to this study. The association of MDRSHH02000806 with the virulence of MDR-SHH02 needs to be validated in further studies. Besides, the associations between the 12 gene sequences similar to AME genes and the antibiotic resistance of MDR-SHH02 are also needs to be confirmed in further studies. We aim to conduct such experiments in our future studies.
In conclusion, this study fulfilled the draft genomic sequence of the clinical MDR A. baumannii strain, MDR-SHH02, and 12 gene sequences in MDR-SHH02 had a highly similarity to the sequences of genes encoding AMEs [e.g., aac(3)-Ia, ant(2″)-Ia, aph33ib and aph(3′)-Ia], β-lactamase genes (bl2b_ tem and bl2b_tem1), sulfonamide-resistant dihydropteroate synthase genes (sul1 and sul2), catb3 and tetb. Of these genes, aph33ib, bl2b_tem and bl2b_tem1 were potential new antibiotic resistance genes. Furthermore, the antibiotic-resistance assay revealed that MDR-SHH02 was resistant to multiple antibiotics, such as amikacin, gentamicin and tobramycin. These findings were expected to enrich the data of antibiotic resistance genes in MDR A. baumannii, and provide a clinical guidance for the personalized therapy of A. baumannii-infected patients.
Acknowledgments
This study was supported by a project supported by Shanghai Health and Family Planning Commission Foundation, China (grant no. 20134010) and a project supported by the Natural Science Foundation of Shanghai, China (grant no. 15ZR1436100).
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