Isolation and genome-wide analysis of new Acinetobacter baumannii bacteriophage vB_AbaM_AB3P2

doi:10.21203/rs.3.rs-3340412/v1

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Isolation and genome-wide analysis of new Acinetobacter baumannii bacteriophage vB_AbaM_AB3P2

https://doi.org/10.21203/rs.3.rs-3340412/v1

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New lytic Acinetobacter baumannii phage, vB_AbaM_AB3P2, was isolated from sewage treatment plant of Luoyang, China. The genome of vB_AbaM_AB3P2 is composed of double-strand DNA that is 44,824bp in length with G + C content of 37.75%. 96 open reading frames were identified, genes for antibiotic resistance and virulence factors were not found. The genomic and phylogenetic analyses of this phage revealed that it represents a new species in genus Obolenskvirus. Phage vB_AbaM_AB3P2, which has short latent period (10 min) and high stability in 30–70 ◦C and pH 2–10, is potentially useful for controlling multi-drug resistant A. baumannii in the future.

Acinetobacter baumannii

phage

sewage treatment plant

genome

Obolenskvirus

multi-drug resistant

As one of the six super-resistant bacteria of "ESKAPE", A. baumannii is an important opportunistic human pathogen, causing nosocomial infections with mortality rates of up to 35%[4]. Due to its ability to quickly acquire resistance and the difficulties in the development of new effective antibiotics, phage therapy as an important alternative treatment has once again become a focus of research again, and this area has made substantial progress in recent years[7].

As the richest entity in the biosphere, bacteriophages are highly effective antibacterial weapons[3]. In comparison with antibiotics, bacteriophages are highly specific to their targets and can effectively kill host bacteria without affecting the commensal flora [21]. Although bacteriophages are generally considered safe, guidelines for the safety assessment of pre-clinical phages lack guidelines for the safety of therapeutic phages, including morphological, biological, and genomic analysis etc, which should be considered according to the purpose of application[15]. Despite the diversity of bacteriophages in the earth, the number of isolated A. baumannii phages are still relatively limited[21, 23]. It is important to isolate and characterize more and more effective A. baumannii phages from the environment that can be used for phage therapy[21, 23]. In this study, a new A. baumannii phage was isolated from a sewage treatment plant in Luoyang city, China. Genetic analysis and biological experiments results proved that it was a new A. baumannii phage with excellent lysing ability and may be potential useful for clinical treatment in the future.

2.1 Bacterial strain and phage isolation

The A. baumannii strains used in the present study were isolated from clinical samples in Guangzhou, China (Supplemental Table 1). The water samples were obtained from a sewage treatment plant in Luoyang city, China. A. baumannii strain AB3 was used as the host for phage isolation. CaCl2 was added into 500ml of sewage at a final concentration to 1mmol/L, and the mixture were centrifuged at 4 ◦C and 10000rpm for 10min to remove large particulates and bacteria, then filtered through a membrane with 0.45-µm pores (Youbo Experimental Equipment Ltd, Guangzhou, China) [6]. 1ml of filtrate and 4ml of host bacterial culture were added into 200ml of LB broth. After overnight shaken at 37◦C with 200 rpm, the culture was filtered by a membrane with 0.22-µm pores (Youbo Experimental Equipment Ltd, Guangzhou, China). Then, 100µL of the diluted phage was mixed with 100µL of the host strain in the early log phase. The mixture was added to 5 mL of top agar (LB with 0.7% agar), and then poured onto a plate (LB with 1.5% agar)—i.e. the double-layer method[24]. The plaques were obtained after incubation for 10–12 h at 37◦C. The pure bacteriophage vB_AbaM_AB3P2 was obtained by at least four single-plaque isolations, and then stored at 4◦C in LB broth for further use and − 80◦C in 30% glycerol or SM preservation solution for long-term preservation.

2.2 Electron microscopy observation

The phage was immobilized by 25% glutaraldehyde. The mixture was applied to the surface of carbon-coated copper grids, and the excess liquid was drawn off with filter paper. The phage was negatively stained with 2% uranyl acetate, which was removed after 1 min[14]. The prepared samples were examined with a transmission electron microscope(TEM; Hitachi HT7700, Japan).

2.3 Host scope

The host range of the phage was tested by plaque assay on a panel of strains (Supplemental Table 1). Briefly, 100µL of bacteria in the log phase with serially diluted phages and 5 mL of LB containing 0.7% agar were mixed and poured onto the LB plate (15 mL). The plates were incubated at 37◦C for 10–12 h.

2.4 Multiplicity of infection (MOI) and one-step growth experiment

The MOI and one-step growth experiment was used with some modifications[24]. The phages were purified by One-step salting-out extraction[5]. The host bacteria in the early log phase were diluted to 1🞨10⁸CFU/mL [10]. Based on the ratios of 10, 1.0.1, 0.01, 0.001, 0.0001 (bacteriophages to host bacteria), plus 1mL of host strain cells and 1mL of serially diluted phages were mixed and shaken at 37◦C for 4h at 200rpm and centrifuged at 12,000 rpm for 10 min. The supertant was filtered through a membrane with 0.45-µm pores.The one with the highest phage titers was the optimal MOI. Three parallel experiments were performed.

According to MOI, after incubated at 37 ◦C for 5 min, the culture was centrifuged at 12,000 rpm for 30 s to remove unabsorbed free phage. The precipitate was washed with LB broth, transferred to 10 mL of LB broth, and incubated at 37◦C. The time-points were: t = 0 s, 5 min, 10 min, 15 min, 20 min, and intervals of 10 min thereafter up to 90 min, with a final time-point of 120 min. The phage particles were titrated using the double-layer agar method. The experiment was repeated three times.

2.5 Thermal and pH stability

To assess its thermal stability, the phage preparation (1 × 109 PFU/mL) was incubated at 25, 37, 40, 45, 50, 55, 60, 65, and 70◦C for 1 h. To assess its pH stability, the phage preparation (1 × 109 pfu/mL) was incubated at pH 2–12 for 1 h. Three parallel experiments were performed to determine the stabilities of the phage preparations, and phage titres were determined using the double-layer agar technique.[9].

2.6 DNA extraction

1mL of the purified vB_AbaM_AB3P2 phage was treated with DNase I and RNase A (Hunan Accurate Biotechnology, China) at final concentrations of 10 mg/L and 5 mg/L, respectively. The mixture was incubated for 60 min at 37^◦C. Next, 20 mmol/L of EDTA (p H8.0), 50 µg/mL of protease K and 0.5% SDS were add to the mixture, which was further incubated at 56◦C for 1 h. An equal volume of phenol was added to extract the DNA. After centrifugation at 5000g for 10 min, the aqueous layer was transferred to equal volume of chloroform, after centrifugation at 5000g for 10 min, 1/10 volume of 3 M NaAc (pH 5.2) and twice the volume of absolute ethanol was added to aqueous layer, precipitated the nucleic acids at -20°C[19]. The next day, the precipitate was washed with 70% ethanol and absolute ethanol, respectively. After drying, 100 µL double distilled water was used for dissolution, stored at -20°C.

2.7 Bioinformatics analysis

The genome was compared with other nucleotide sequences using NCBI BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Genome annotation was performed using Phaprokka[2]. The classification prediction is done by PhaGCN2.0[8]. The calculation of average nucleotide sequences (ANI) and their heatmap was completed with pyani (https://github.com/widdowquinn/pyani). The whole genome alignment was performed by clustalw[22] and the phylogenetic tree was constructed by FastTree[16]. Colinear comparison of phages was completed by Easyfig 2.23[20].

3.1 Phage isolation, morphology, and host-range

Phage vB_AbaM_AB3P2 was able to form small pieces of round plaque ranging in size from 1 to 2 mm in diameter (Fig. 1A).The morphological characteristics of phage vB_AbaM_AB3P2 particles reveal that it has an icosahedral head with a diameter of 70 ± 3 nm and a contractible muscle tail with a length of 140 ± 2 nm and a width of 20 ± 0.9 nm (Fig. 1B)[1]. The possible host range of this phage was tested using 12 strains including 5 A. baumannii, 5 E. coli and 2 P. aeruginosa (Supplemental Table 1). Phage vB_AbaM_AB3P2 was able to lyse A. baumannii AB3 and AB9. In conclusion, phage vB_AbaM_AB3P2 is a specific A. baumannii phage that belongs to myoviridae.

3.2 Multiplicity of infection (MOI) and one-step growth

The MOI test revealed that 0.1 was the optimal MOI for the phage titre, and there were no obvious differences between the various MOIs (Fig. 1C). One-step growth experiment showed that the latent period of vB_AbaM_AB3P2 was about 15 min, and then entered the rise period including exponential growth phase for 15 min and slow growth phase approximately 60 minutes. The growth of phages was finally reached plateau phase after 90 minutes (Fig. 1D).

3.3 Stability of bacteriophages

Phage vB_AbaM_AB3P2 retained high levels of activity after the water incubation from 30 to 70°C (Fig. 1E). The activity of bacteriophages only decreases 0.6 log at 50°C-70°C, compared with its strongest infectivity at 40°C In previous studies, the activity of A. baumannii phages BUCT628 and pIsf-AB02, sharply decreased at 50°C and inactivates at 70°C[18, 26]. It indicates that phage vB_AbaM_AB3P2 has a thermal tolerance advantage in the application.

At the same time, vB_AbaM_AB3P2 remained stable at pH 2–10, and inactive at pH 11–13 (Fig. 1F). A. baumannii phage Phab24 was stable at pH values at pH 4–12, but lost activity at a pH value of lower than 3[25]. A. baumannii phage TCUP2199 was lost of activity at pH 3, with decreased activity at pH 5 and 11[13]. Compared with these two phages, vB_AbaM_AB3P2 has a better stability at acidic environment but less activity at alkalis conditions. The pH of the human stomach and intestine is 1–2 and 5.5[12]. vB_AbaM_AB3P2 may be more suitable to use in gastrointestinal infections by oral.

3.4 Genomic analysis

Phage vB_AbaM_AB3P2 genome was composed of a 44,824-bp linear dsDNA molecule with 37.5% G + C content. According to the International Committee on Taxonomy of Viruses (ICTV), the current main species classification criteria for defining new species of bacteria and archaeal viruses are set at 95% genome sequence identity[17]. Comparison of this phage genome with nucleotide sequences already in NCBI non-redundant dababase, phage vB_AbaM_AB3P2 showed a high sequence identity toA. baumannii phage Abp2(identity 95.7%, coverage 66%), A. baumannii phage NJ02(identity 91.23%, coverage 60%), vB_AbaM_WUPSU(identity 91.53%, Coverage 54%) and WCHABP12 (89.98% 69%). The heatmap and ANI analysis (Fig. 2A) showed that phage vB_AbaM_AB3P2 is similar to t A. baumannii phage Brutus (92.6%), A. baumannii phage Abp2(92.5%), A. baumannii phage Cato (92.5%), A. baumannii phage Scipio (92.3%) and A. baumannii phage WCHABP12(92%). According to the prediction results by PhaGCN2.0, vB_AbaM_AB3P2 was classified as genus Obolenskvirus. In the phylogenetic tree based on whole genome nucleotide sequences (Fig. 2B), all the phages were belonged to class Caudoviricetes. Phage vB_AbaM_AB3P2 was gathered with other 14 phages belonging to the genus Obolenskvirus into a big cluster, but it was distant from other three phages belonging to the family Autographiviridae. Every phage in the cluster of genus Obolenskvirus represented one species, phage vB_AbaM_AB3P2 had a relatively close phylogenetic relationship with phage WCHABP12 (low bootstrap value) but was still distinct from these phages. These results indicated that phage vB_AbaM_AB3P2 not belonged to existed species.

There was a total of 96 open reading frames (ORFs) in the genome of phage vB_AbaM_AB3P2, and 36.4% (35/96) of the annotated genes were associated with the general functions of the phage, including head protein, tail protein and baseplate protein etc. Among these functional genes, 14 were predicted to be responsible for transcriptional, 19 for phage structure, and 2 for endolysin. No rRNA, tRNA, antibiotic resistance genes and virulence factors were detected [11]. To compare the genomic difference of A. baumannii. phage vB_AbaM_AB3P2 with the highest similar phage WCHABP12, a genome colinear comparison was made using Easyfig software. As shown in Fig. 3, the phage structure proteins like head and tail proteins etc were highly similar, but most of the transcriptional, metabolism and hypothetical proteins had very low similarity, some of these are even completely different.

According to the current criterion for defining new species of bacteria and archaeal viruses[17], the highest identity of 95.7% (but low coverage 66%) with A. baumannii phage Abp2 according to BLASTn, less than 93% similarity with all homologous phages by ANI analysis, the distinct phylogenetic relationship, and the genome difference by colinear comparison between highly similar phage, all these results suggested that phage vB_AbaM_AB3P2 was a new species of Obolenskvirus genus.

A new A. baumannii. phage vB_AbaM_AB3P2 was isolated from a sewage treatment plant in Luoyang city, China. It belongs to a new species of the genus Obolenskvirus. it is a acid resistant phage,with short incubation period period. Phage vB_AbaM_AB3P2 has great potential for clinical application in the control of A. baumannii infection. The discover of vB_AbaM_AB3P2 enriches the existing A. baumannii phage repertoire. We will continue to isolate more useful phages for the control of A. baumannii infection in the future.

Acknowledgments This work was supported by the National Natural Science Foundation of China [grant number 32072327] and the Guangdong Basic and Applied Basic Research Foundation [grant number 2021A1515010865].

Author contributions Yujing Tan and Jianhui Su performed the experiments, analyzed the data, and prepared the initial draft of the manuscript. Haiyan Zeng conceived and designed the study, revised the final manuscript. Dandan Luo and Bingshao Liang provided important experimental materials and analyzed the data. All authors read and proved the manuscript.

Data Availability The GenBank accession number of the whole genome sequence of A. baumannii. phage vB_AbaM_AB3P2 reported in this article is OR526523.

Competing Interests The authors declare that there are no conflicts of interests.

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Isolation and genome-wide analysis of new Acinetobacter baumannii bacteriophage vB_AbaM_AB3P2

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Bacterial strain and phage isolation

2.2 Electron microscopy observation

2.3 Host scope

2.4 Multiplicity of infection (MOI) and one-step growth experiment

2.5 Thermal and pH stability

2.6 DNA extraction

2.7 Bioinformatics analysis

3. Results

3.1 Phage isolation, morphology, and host-range

3.2 Multiplicity of infection (MOI) and one-step growth

3.3 Stability of bacteriophages

3.4 Genomic analysis

4. Conclusions

Declarations

References

Supplementary Files

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