Characterization, sequencing and comparative genomic analysis of vB_AbaM-IME-AB2, a novel lytic bacteriophage that infects multidrug-resistant Acinetobacter baumannii clinical isolates
- Fan Peng†1, 2, 3,
- Zhiqiang Mi†1,
- Yong Huang†1,
- Xin Yuan2,
- Wenkai Niu2,
- Yahui Wang1,
- Yuhui Hua1,
- Huahao Fan1,
- Changqing Bai2Email author and
- Yigang Tong1Email author
© Peng et al.; licensee BioMed Central Ltd. 2014
Received: 14 July 2013
Accepted: 25 June 2014
Published: 5 July 2014
With the use of broad-spectrum antibiotics, immunosuppressive drugs, and glucocorticoids, multidrug-resistant Acinetobacter baumannii (MDR-AB) has become a major nosocomial pathogen species. The recent renaissance of bacteriophage therapy may provide new treatment strategies for combatting drug-resistant bacterial infections. In this study, we isolated a lytic bacteriophage vB_AbaM-IME-AB2 has a short latent period and a small burst size, which clear its host’s suspension quickly, was selected for characterization and a complete genomic comparative study.
The isolated bacteriophage vB_AbaM-IME-AB2 has an icosahedral head and displays morphology resembling Myoviridae family. Gel separation assays showed that the phage particle contains at least nine protein bands with molecular weights ranging 15–100 kDa. vB_AbaM-IME-AB2 could adsorb its host cells in 9 min with an adsorption rate more than 99% and showed a short latent period (20 min) and a small burst size (62 pfu/cell). It could form clear plaques in the double-layer assay and clear its host’s suspension in just 4 hours. Whole genome of vB_AbaM-IME-AB2 was sequenced and annotated and the results showed that its genome is a double-stranded DNA molecule consisting of 43,665 nucleotides. The genome has a G + C content of 37.5% and 82 putative coding sequences (CDSs). We compared the characteristics and complete genome sequence of all known Acinetobacter baumannii bacteriophages. There are only three that have been sequenced Acinetobacter baumannii phages AB1, AP22, and phiAC-1, which have a relatively high similarity and own a coverage of 65%, 50%, 8% respectively when compared with our phage vB_AbaM-IME-AB2. A nucleotide alignment of the four Acinetobacter baumannii phages showed that some CDSs are similar, with no significant rearrangements observed. Yet some sections of these strains of phage are nonhomologous.
vB_AbaM-IME-AB2 was a novel and unique A. baumannii bacteriophage. These findings suggest a common ancestry and microbial diversity and evolution. A clear understanding of its characteristics and genes is conducive to the treatment of multidrug-resistant A. baumannii in the future.
Acinetobacter baumanni is a non-fermentative, aerobic, gram-negative bacillus, and is an opportunistic pathogen with global distribution. It is frequently found in elderly patients and cancer patients with compromised immune function, especially in intensive care units. With the use of broad-spectrum antibiotics, immunosuppressive drugs, and glucocorticoids, A. baumannii (AB) has become a major nosocomial pathogen species. Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan drug-resistant (PDR) A. baumannii strains are increasingly prevalent. MDR-AB refers to A. baumannii strains that are resistant to at least three of the following five types of antimicrobial agents: cephalosporins, carbapenems, β-lactamase inhibitors (including piperacillin/tazobactam, cefoperazone/sulbactam, ampicillin/sulbactam), fluoroquinolones, and aminoglycosides[2–4].
Bacteriophage therapy is a potential alternative treatment for multidrug-resistant bacterial infections. A bacteriophage is a bacterial virus that can lyse and kill the host cell. Phage-related studies have gone through three stages. Félix d’Herelle discovered bacteriophage for the treatment of bacterial infections in 1917. After the emergence of antibiotics in the 1940s, phages were seldom used for therapeutic purposes, and mainly functioned as molecular and genetic research tools. With the recent emergence of multidrug-resistant bacteria, however, there has been renewed interest in methods of phage therapy. In this study we isolated a lytic bacteriophage IME-AB2, and compared biological characteristics and genomic sequence with other Acinetobacter baumannii phages. The genomes of A. baumannii phages IME-AB2, A. baumannii AB1, A. baumannii AP22, and A. baumannii phiAC-1 were compared thoroughly in this study. To our knowledge this is the first report of comparison of the characteristics and complete genome sequence of Acinetobacter baumannii bacteriophages. A clear understanding of its genes is conducive to the treatment of multidrug-resistant A. baumannii in the future.
Isolation of a lytic bacteriophage against multidrug-resistant A. baumannii
Antibiotic resistance profile of A. baumannii strain MDR-AB2
Growth and lytic characteristics of IME-AB2
For one-step growth curve analysis, MDR-AB2 cells (OD600 = 0.3) were infected with phage IME-AB2 at a MOI of 0.1. The bacteriophage was allowed to adsorb for 15 min at 37°C. The mixture was then centrifuged at 12,000 × g for 30 s to remove unadsorbed phage particles, and the resultant pellet was re-suspended in 5 ml of LB medium. Samples were incubated at 37°C and collected every 10 min during 0–60 min, as well as at 90 and 120 min. As shown in Figure 3b, the latent period of phage IME-AB2 lasted for 20 min, the burst period reached a peak at 30 min, and the phage multiplication reached the final plateau phase at 50 min. The burst size of phage IME-AB2 was determined to be 62 pfu/cell (burst size = the total number of phages liberated at the end of one cycle of growth /the number of infected bacteria).
Analysis of the phage proteins and genome
The genome analysis indicated that phage IME-AB2 has a double-stranded DNA genome, approximately 40 kb in size. The genome of phage IME-AB2 could be digested with endonuclease Nde I, HincII and HindIII (Figure 4b). It was found that endonuclease enzymes, HindIII and HincII, have the 35 and 16 cutting sites on the genome of phage IME-AB2 respectively by Vector NTI. Compared to other A. baumannii complete genome , the two endonucleases also have most restriction enzyme cutting sites on them.
Functional classification of the 82 CDSs in the IME-AB2 genome
CDSs and putative functions
CDS.12 putative capsid protein.
CDS.13 putative structural protein.
CDS.24 putative phage head protein.
CDS.71 putative tail fiber.
CDS.72 similar to the N-terminal region of tail fiber protein.
CDS.74 putative baseplate J-like protein.
CDS.77 putative phage baseplate assembly protein.
CDS.06 putative cobalt transport protein.
CDS.08 putative RNA polymerase.
CDS.33 putative binding HTH domain or homeodomain-like.
CDS.47 putative bacteriophage-associated immunity protein.
CDS.52 putative HNH endonuclease domain protein.
CDS.66 putative nucleoside triphosphate pyrophosphohydrolase.
CDS.68 putative lysozyme family protein.
CDS.81 putative lysozyme protein.
CDS.26 putative phage head portal protein.
CDS.27 putative phage terminase, large subunit.
CDS.28 putative phage terminase,small subunit.
CDS.50 putative replicative DNA helicase.
CDS.51 putative primosomal protein.
CDS.58 putative transcriptional regulator.
CDS.62 putative recombinational DNA repair protein.
Other hypothetical proteins
Comparative genomic analysis of A. baumannii phage IME-AB2, A. baumannii phage AB1, A. baumanni i phage AP22, and A3 baumannii phage phiAC-1
Seven CDSs encoding structural proteins were identified in the phage IME-AB2. The putative capsid protein (CDS.12) is similar to that of phage AB1 (gp15), phage AP22 (gp30), phage phiAC-1 (0043). Phage AB1 (gp27), phage AP22 (gp18), phage phiAC-1 (0031) also share homology to the putative phage head protein encoded by IME-AB2 CDS.24. CDS.71 and CDS.72 of phage IME-AB2 are identified to be associated with tail fiber protein. CDS.74 and CDS.77 of phage IME-AB2 are predicted to encode proteins responsible for baseplate. These two related proteins can be found similar area in the other three phages. The results also demonstrate the phage tail related proteins generally cluster together (Table 3).
A unique feature of the IME-AB2 genome is that it encodes cobalt transport protein (CDS.6). Notably, cobalt is a cofactor and is required by enzymes from bacteria. It is possible that these metabolic enzymes benefit phage by enhancing the metabolism of the infected bacterial cell, which could in turn increase phage proliferation. No similar cobalt proteins were found in the other three phages sharing homology with IME-AB2. CDS.8 encodes a putative RNA polymerase protein. It is necessary for constructing RNA chains using DNA genes as templates, a process called transcription. Transcription of most double-stranded DNA bacteriophages rely on their host bacteria. The putative CDS.33 of IME-AB2 is predicted to encode HTH domains which have been recruited to a wide range of functions beyond transcription regulation, such as DNA repairing and replication, RNA metabolism and protein-protein interactions in diverse signaling contexts. Beyond their basic role in mediating macromolecular interactions, the HTH domains have also been incorporated into the catalytic domains of diverse enzymes. In functional terms, the HNH endonuclease domain (CDS.52) is found in CRISPR-related proteins. CRISPR functions as a prokaryotic immune system, in that it confers resistance to exogenous genetic elements. CDS.47 is predicted to be a putative bacteriophage-associated immunity protein, which was considered to be responsible for phage superinfection immunity. CDS.68 and CDS.81 are putative lysozyme family protein. Unlike, CDS.0057, 0058, 0059 encoded by phiAC-1, which are putative lysozyme-like domain protein and adjacent to each other, the two lysozyme-like proteins from IME-AB2 are not clustered.
In the four similar Acinetobacter baumannii phages, the genetic elements encoding the products involved in the packaging system are commonly found adjacent to one another in the phage genomes. Packaging system is generally composed of big subunit (CDS.27) and small subunit (CDS.28). Usually, the two subunits of the terminase and head portal protein (CDS.26) are closely connected in the packaging system, while the portal protein is a bacteriophage component that forms a hole, or portal, enabling DNA passage during packaging and ejection. It also forms the junction between the phage head (capsid) and the tail proteins.
With the emergence of a growing number of drug-resistant bacterial species, and the difficulties surrounding the development of novel antibiotics, exploring novel or alternative therapeutic methods is imperative. The recent renaissance of bacteriophage therapy may provide new treatment strategies for combatting drug-resistant bacterial infections. Although a large number of work on phage therapy in human disease had been done[22–24], the host-specific infection and the relatively narrow lytic spectrum of phage is one of the obstacles to their further application. Individualized phage therapy may represent the future of phage therapy, where bacterial infections will be treated with phage combinations that have already been shown as effective for that particular bacterium. To overcome these limitations, strategies such as screening more lytic phages, combining phages with antibiotics, or administrating phages cocktails should be investigated[25, 26]. Therefore, it is very important to isolate novel and sensitive phages to enrich the phage arsenal.
Comparative analysis of all known Acinetobacter baumannii bacteriophages
G C content (%)
Major protein sizes
Adsorption time (>99%)
Burst size (PFU/cell)
38 kDa (35–130 kDa)
3 of 22
hospital sewage, Beijing,China
33.1 kDa (14.4–97.4 kDa)
32 kDa (18–87 kDa)
89 of 130
Clinical material, Russia
First phage infect A. soli,South Korea
19 of 39
Inhibit host biofilm formation
23 of 39
Inhibit host biofilm formation
3 of 23
fishpond water, Zhenzhou,
29 kDa–116 kDa
35 kDa (35–264 kDa)
YMC/09/ 02/B1251 ABA BP
Cell survival test
Podoviridae, phiKMV-like phages
45.2 kb to 46.9 kb
14 to 80 kilo-dalton
Marine sediment, Taiwan
Podoviridae, phiKMV-like phages
A lytic A. baumannii bacteriophage IME-AB2 was isolated and characterized in this research. The complete genome of IME-AB2 was sequenced and compared to those of A. baumannii phage AB1, A. baumannii phage AP22, and A. baumannii phage phiAC-1 in detail. The genome of IME-AB2 was replete with novel genes without known relatives, which indicated that IME-AB2 was a novel and unique A. baumannii bacteriophage. Although the resistant A. baumannii appeared finally after infection with IME-AB2, the comprehensive understanding of the phage’s characteristics is conducive to the treatment of multidrug-resistant A. baumannii in the future.
Bacterial strains, Phage isolation, propagation, and titration
This study included 22 clinical strains of A. baumannii (MDR-AB1139, MDR-AB1, MDR-AB2, MDR-AB3, MDR-AB4, MDR-AB5, … , MDR-AB19,MDR-AB20, MDR-AB21). All the clinical samples were taken as part of standard patient care at the PLA Hospital 307, Beijing, China. The patients were orally informed that the specimens would be used for screening bacteria and the tests were optional on laboratory sheet. Blood, sputum and skin swabs were collected from patients with consent under the Ethics Committee of the PLA Hospital 307. The protocol of screening bacteria was approved by the Ethics Committee of the PLA Hospital 307 and Beijing Institute of Microbiology and Epidemiology Ethics Committee.
Multidrug-resistant A. baumannii strain MDR-AB2 was used as an indicator for bacteriophage screening of raw sewage samples collected from PLA Hospital 307. Sewage samples were separated by centrifugation at 12,000 × g for 20 min. Following removal of the solid impurities by centrifugation, the supernatants were filtered through a 0.45 μm pore-size membrane filter to remove bacterial debris. Filtrate (4 ml) was added to 2 ml of 3× Luria-Bertani (LB) broth medium and mixed with 0.1 ml of A. baumannii overnight culture (OD600 = 0.6) to enrich the phage at 37°C overnight. Following enrichment, the culture was centrifuged at 12,000 × g for 10 min, and then the supernatant was filtered with a 0.45 μm pore-size membrane filter to remove the residual bacterial cells. The filtrate (0.1 ml) was mixed with 0.5 ml of A. baumannii in LB culture (OD600 = 0.6) and 5 ml of molten top soft nutrient agar (0.75% agar), which was then overlaid onto solidified base nutrient agar (1.5% agar). Following incubation for 6 h at 37°C, the clear phage plaques were picked from the plate. The phage titer was determined using the double-layered method previously described by Adams.
Phage concentration , purification and storage
A single plaque was picked into 5 ml of LB medium containing MDR-AB2 (OD600 = 0.6) and cultured at 37°C for 6 h. A 5 ml aliquot of suspension was transferred into 500 ml of LB medium for culture at 37°C overnight. Chloroform was then added to the 500 ml of culture to a final concentration 0.1% before being mixed gently and allowed to stand at room temperature for about 30 min. Solid NaCl was added to the culture to a final concentration of 1 M, which was then incubated in an ice water bath for 1 h. The culture was centrifuged at 11,000 × g for 10 min to remove cell debris, and polyethylene glycol 6000 (PEG6000) was added to the supernatant to a final concentration of 10% (w/v) while slowly stirring with a magnetic stirrer at room temperature. This solution was transferred to a polypropylene centrifuge tube in an ice water bath and incubated at least 1 h to precipitate the phage particles. Following centrifugation (11,000 × g for 10 min at 4°C), the phage-containing precipitate was resuspended in 5 ml of SM buffer (50 mM Tris-Cl, 100 mM NaCl, 8 mM MgSO4, pH 7.5). An equal volume of chloroform was then added to separate the phage particles from PEG6000. Following centrifugation at 3,000 × g for 10 min, the aqueous phase was recovered and filtered through a 0.22 μm pore-size membrane filter to remove debris. The concentrated 1.0 ml of phage suspensions were layered on the top of a cesium chloride gradient solutions (density of 1.3 g/ml-0.45 g of cesium chloride in 1.0 ml of water; density of 1.5 g/ml-0.83 g of cesium chloride in 1.0 ml of water; density of 1.7 g/ml-1.28 g of cesium chloride in 1.0 ml of water) in 5.0 ml cellulose nitrate centrifuge tube. After centrifugation in a Beckman Coulter Swinging Bucket Rotor (SW41, Ti) for 40 min at 100,000 × g, the concentrated phages at the visible band were collected by means of a capillary pipette. The purified phage was stored at 4°C.
Determination of lytic spectrum
The host range was determined by spot test. Briefly, 0.5 ml of bacterial overnight culture was mixed with 5 ml of molten top soft nutrient agar (0.75% agar) and then overlaid on the surface of solidified base nutrient agar (1.5% agar). Once the top layer also solidified, 2 μl of the phage preparation(1 × 109 pfu/ml) was spotted onto the plate, which was incubated for 6 h at 37°C.
Phage stock solution was directly stained with phosphotungstic acid (PTA) for 2 min. After being dried at room temperature, the grid was examined using a Philips TECNAI-10 transmission electron microscope (TEM) to observe and record the morphology of the phage particles.
Extraction of phage genomic DNA
Purified phage particles were treated with DNase I (1 μg/ml) (Takara) and RNase A (1 μg/ml) (Takara) for 30 min, and then the nucleases were inactivated at 80°C for 15 min. Ethylene diamine tetraacetic acid (EDTA) (20 mM), proteinase K (50 μg/ml) and sodium dodecyl sulfate (SDS) (0.5%) were then added and the mixture was incubated at 56°C for 1 h. Phage lysate was extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). Chloroform extraction was repeated until there was no phenol odor. An equal volume of isopropanol (AR grade) was added and the sample was incubated overnight at -20°C to precipitate the phage genomic DNA. The pellet was washed with 75% ethanol, and then deionized water was used to dissolve the precipitated genomic DNA.
Whole genome sequence and bioinformatics analysis
The genomic DNA of IME-AB2 was subjected to high-throughput sequencing using a Life Technologies Ion Personal Genome Machine Ion Torrent sequencer (San Francisco, CA) according to the manufacturer’s instructions. The complete genome sequence of phage IME-AB2 was assembled using Velvet and CLC Bio (Aarhus, Denmark), and annotated using RAST and InterPro. Sequence similarity analysis and comparison were performed using NCBI packages.
This research was supported by a grant from the National Natural Science Foundation of China (No. 81072350), the National Hi-Tech Research and Development (863) Program of China (No. 2012AA022-003), the China Mega-Project on Major Drug Development (No. 2011ZX09401-023), the China Mega-Project on Infectious Disease Prevention (No. 2013ZX10004-605 and No. 2011ZX10004-001), the State Key Laboratory of Pathogen and BioSecurity Program (No. SKLPBS1113), and the Particular Clinical Applied Research of the Capital of China (No.Z121107001012127).
Nucleotide sequence accession number: The whole-genome sequence of phage vB_AbaM-IME-AB2 has been deposited in the NCBI nucleotide sequence database under GenBank assession number: JX976549.
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