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Biological Characteristics and Genomic Analysis of Aeromonas Hydrophila Phage BUCT551 Isolated From Aquatic Sewage

Aeromonas hydrophila is a common opportunistic pathogen in aquaculture and is ubiquitous in aquatic environment. Whereby its accessibility, variety and host specicity, phage is increasingly considered as a promising complementary medicine for antibiotics. However, a small amount of A. hydrophila phages have been characterized, which suggests the signicance to isolate and characterize novel A. hydrophila phages. In this study, we isolated a novel Aeromonas hydrophila phage using A. hydrophila strain A18 as an indicator and designated it as BUCT551, and it was identied as Myoviridae phage by transmission electron microscopy (TEM). The whole genome sequencing of the phage BUCT551 revealed that it has a linear DNA genome of 613,82 bp. BLASTn analysis showed that phage BUCT551 shared 86.75% homology with A. hydrophila phage LAh_7 (Genebank ID: MK838113.1). The one-step growth curve demonstrated that phage BUCT551 had a latent period of 20 min and the burst size of 32 pfu/cell at its optimal MOI of 0.1. The phage BUCT551 had a survival pH range from 5 to 10 and tolerant temperature from 0 ℃ to 40 ℃ . Host range analysis shown that the phage was able to lyse not only A. hydrophila, but also A. veronii.


Introduction
Aeromonas hydrophila is a gram-negative, non-spore forming, rod-shaped and facultative anaerobic organism belonging to genus Aeromonas family Aeromonadaceae [1]. It is ubiquitous in natural environment because of its wide range of survival and reproductive temperature from 4℃ to 40℃, usually found in aquatic environment like river fresh water, and it is recognized as an opportunistic pathogenic of poikilothermic and homoeothermic animals including human [2,3]. A. hydrophila is regarded as a causative agents pathogen of many sh species involved in multiple diseases, including ulcerative disease, hemorrhagic disease, red sore disease, and septicemia in sh, which leads to the severe economic loss to the aquaculture industry [4][5][6]. In addition, A. hydrophila is pathogenic to human especially immunocompromised people by means of colonizing in human intestinal tract, and it is linked to gastroenteritis, skin diseases and septicemia [4,6,7,[8][9][10]. A.hydrophila produces virulence factors including aerolysin, hemolysin, adhesion factor, enterotoxin and extracellular protease that results in tissue damage, which act individually or in synergy, contributing to the pathogenicity of A.hydrophila [8]. Aeromonas infections have been reported in recent decades, and a statistic from Japan indicated Aeromonas species were isolated from 5% travelers (23,215 persons in total) who returned from the developing countries [9]. In 2018 to 2019, Hilt et al. isolated several multi-drug resistant A. hydrophila isolates from two solid organ transplant patients [11]. In another case, Hasan et al. recently reported the Aeromonas hydrophila infection in an immunocompromised 69 years old female, with surgical site sepsis/infection and several comorbidities [12].
The bacteria antibiotics resistance is a great challenge and threat to the human life [13,14], and antibiotics resistance of aeromonas has attracted more and more attention in recent years. Elbehiry et al. isolated A. hydrophila from 150 meat and water samples and to screen their antibiotics resistance, and found that some A. hydrophila strains were resistanct to ampicillin, cefotaxime, pe oxacin, ceftazidime and cipro oxacin [15]. In another study, A. hydrophila was detected to carry an acquired gene encoding the carbapenemase GES-24, indicating its property of carbapenem-resistant [16]. Given that A. hydrophila exhibits pathogenicity to both humans and animals, and its outbreak may lead to economic loss in agriculture and aquaculture or public health incident, the potential antibiotic resistance of A. hydrophila should be paid to the attention.
Under the circumstance of antibiotic drug resistance, phage therapy was found more and more important in the antibacterial theory either using alone or in combination with antibiotics [17][18][19]. Phage is exclusively virulent to prokaryotic microbe and do not lyse animal and human cells. Phages are classi ed into lytic phage and lysogenic phage depending on their life cycle. Lytic phage lyses host bacteria immediately after accomplishing replication and assembly process in the host, consequently leading to the elimination of host bacteria. The characterstic of lytic phage endows therapeutic potential of phage as a treatment for bacterial infection [10]. Some phages demonstrate high speci city to a single species or even a certain strain of bacteria [20]. To enhance the e cacy when using phage as antibacterial agents, a strategy that formulate phages into cocktails comes out [6].
However, only few A. hydrophila phages are isolated and characterized [21]. Here our study described a novel A. hydrophila phage with 86.75% genome coverage to unpublished A. hydrophila phage LAh_7, and found that the phage could lyse aeromonas veronii. Our study provides a new option for the treatment of aeromonas infections.

Bacterial Strain Isolation and Identi cation
The host strain A18 was isolated from sewage water of aquatic product market (Nanjing, China). The molecular identi cation of A18 was performed through sequence analysis of 16S rRNA gene. Microbial DNA was extracted and puri ed from 1 mL individual bacterial colonies culture using extraction kit according to manufacture's instructions. Subsequently, the puri ed DNA was applied to amplify the V3-V4 region of the 16S rRNA gene using primers reported previously [23]. According to the method described by Pan [23], we con rmed that A18 belonged to species Aeromonas hydrophila. The strain A18 was cultured at 28 ~ 30℃ and 220×g in Tryptic Soy Broth (TSB) medium (BD Difco) [23].

Phage isolation and puri cation
The sewage water sample collected from Nanjing aquatic market was centrifugated at 12000×g for 10 min, followed by supernatant ltration using 0.22 µm membrane. Phage ltrate was pipetted into culture solution with A. hydrophila strain A18 of logarithmic phase. The mixture was incubated overnight at 28℃ in the shaker to allow phages to propagate in a large scale, and was centrifuged at 12000×g for 3 min to get rid of bacterial cells and debris. In order to further remove residual cells and debris, the supernatant was ltered by 0.22 µm membrane. The phage stock was obtained after centrifugation and ltration. Veri cation of phage was warranted to pipetted phage stock onto double-layer agar plates with the upper agar mixing with A18 and incubated until the appearance of plaque. Phage isolate puri cation was performed subsequently when the phage was veri ed. After serial 10-fold dilution of the stock solution, diluents with different concentrations were mixed with host bacteria, and the mixture was added to TSB medium to make double-layer agar plates. After incubation, single plaque was selected and cultured in TSB medium using A18 as an indicator. Phages were re-harvested by centrifugation and ltration. The puri cation processes were conducted for three times [24].

Transmission Electron Microscopy (TEM)
Transmission electron microscopy (TEM) was performed to determine the morphological characteristics of phage after concentration using cesium chloride (CsCl) gradient centrifugation [25]. 20 µL of puri ed phages suspension was pipetted onto carbon-coated support membrane copper mesh and retained 20 min, followed by negative staining with 2% phosphotungstic acid solution for 1 min retention. After further drying, phage particles were observed under an electron microscope(JEM-1200EX, Japan) at an accelerating voltage of 100 kV.

Phage genomic DNA extraction
The genome of the isolated phage was extracted for sequencing. According to the method described by Tang et al. and with slight modi cation, genomic DNA of the phage was extracted by proteinase K /SDS method [26]. DNase I and RNase A (Thermo Scienti c, USA) were introduced to puri ed phage to the nal concentration of 1 mg/L, and was incubated overnight at 37 ℃. Then the solution was heated at 80 ℃ for 15 min for enzyme inactivation. EDTA, proteinase K and SDS were added to nal concentration of 0.02 mol/L, 50 mg/L and 0.5%, respectively, and the solution was incubated at 56 ℃ for 1 h. After incubation, balanced phenol was added at the same volume and the mixture was centrifuged at 12000×g for 10 min. The supernatant was collected and the same volume of phenol/chloroform/isoamyl alcohol (25:24:1, by volume) was added, then centrifuged at 12000×g for 10 min. Mixed the supernatant with 400 µL of isoamyl alcohol (Sinopharm Chemical Reagent Co., Ltd.) and retained at -20 ℃ for more than 1 h. The mixture was centrifuged at 12000×g for 20 min at 4℃, then the liquid was drained to harvest the precipitation. The precipitation was washed with 75% pre-cooled ethanol (-20℃) and centrifuged at 12000×g for 10 min at 4℃. Removed the ethanol and collected the the DNA precipitation, the precipitation was resuspended in nucleic acid-free water.

Determination of the Multiplicity of Infection (MOI)
Multiplicity of infection (MOI) signi es the ratio of the number phages in a system to the total amount of bacteria and it can be interpreted as the number of phages added to each cell during the infection. Dilute phage stock solution and bacterial suspension in 10-fold serially to various concentrations, mixed then proportionally to acquire infectious systems with different MOI. Phage titers were determined by double layer agar method after incubated overnight at 28℃ [27].
In order to investigate the vigor of phage BUCT551 in multiple environmental condition, we measured the phage titers in diverse temperatures and values of pH with the method previously described [27]. In thermal stability assay, phages (approximately 10 9 pfu/mL) were incubated in water bath at different temperatures (4℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃) for an hour, and then the phage titers were determined. Similarly, phages were incubated at 28℃ in the TSB medium with different pH values for an hour, then phage titers were calculated [3]. All experiments were performed in triplicate and results were averaged.

One-Step Growth Curve Assay
The one-step growth curve assay was implemented with the purpose of determining the eclipse period and burst size of phage BUCT551. The phage BUCT551 was introduced to A. hydrophila strain A18 in exponential phase at the optimal MOI and was allowed for 30 min absorption at 28℃. Mixture was centrifugated at 12000×g for 1 min to separate free phage particles with host bacteria immediately after the absorption procedure. Resuspended the sediment and cultured at 28℃ in the shaker for 205 min and samples were collected at 0, 10,20,30,40,55,70,85,100,115,130,145,160,175,190 and 205 min during incubation, and phage titers were determined by double-layer agar method as described above. Taking the infection time (minute) as the abscissa and the phage titer (pfu/mL) as the ordinate, and the one-step growth curve was drawn, clearly the eclipse and lysis periods of the phage were obtained, and the amount of phage released was calculated. The assay was conducted in triplicate and results were averaged [28].

Host range analysis
The bacterial susceptibility level was inspected using spotting methods as previously described [24]. About 20 strains of Vibrio Parahemolyticus that were stored in Biosafety Technology Research Center, Beijing University of Chemical Technology and 4 strains of Aeromonas hydrophila were selected randomly for host range determination. Transfered 500 µL of the A. hydrophila culture to 5 mL of TSB medium with 0.75 % agar and promptly poured on a plate paved with tryptic soy agar (TSA). After solidi cation, pipetted the phage BUCT551 onto double-layer agar, then the plates were cultured overnight to observe the plaque formation.

Drug sensitivity test of host strain A18
In order to further prevent the contamination caused by Aeromonas, we tested the sensibility of A18 to some common antibiotics. Drug sensitivity test was performed using inhibition zone method on the grounds of "Antimicrobial Sensitivity Test Standard". 12 kinds of common antibiotics in clinic were involved in the test. Bacterial suspension was evenly spreaded in the plate, then various disks with antibiotics were placed on the plates to allow for 24 h incubation at 28℃, the diameter of the inhibition zone was measured for drug sensitivity evaluation [29].

Whole-genome Sequencing Analysis and Bioinformatic Analysis
The phage BUCT551 DNA library was constructed in accordance to NEBNext Fast DNA Library Prep Set for Ion Torrent protocol kit. The concentration of nucleic acid was determined by Qubit 2.0 (Invitrogen, USA). Phage whole-genome sequencing for the phage BUCT551 was performed using MiSeq (Illumina, USA) platform. Newbler V3.0 software (Roche 454) and CLC software (CLC Bio) were used to genomic sequence assembling [30]. Low-quality sequences were ltered out using the Trimmomatic (V0. 32) program.
Phylogenetic tree was constructed based on the amino acid sequene of conserved major capsid protein in order to analyze the relationship between the new isolate phage BUCT551 and other phages of the same subfamily recorded by the International Committee on Taxonomy of Viruses database (ICTV). MEGA version 7.0 was employed to constructed phylogenetic tree.

Phage Morphology
Aeromonas hydrophila phage was isolated from aquacultural waste water and was named as BUCT551. Transmission electron microscope revealed that the phage BUCT551 had a polyhedral capsid(50 ± 3 nm in diameter)and a long tail 180 ± 5 nm (Fig. 1).
Baesd on the latest ICTV classi cation system, it belonged to the family Myoviridae.
The thermal stability assay showed that the phage BUCT551 demonstrated vitality from the temperature range of 4℃~40℃. The survival rate decreased signi cantly over 50℃, and the phage was inactivated completely after incubating at 70℃ for 60 min (Fig. 2a). The pH sensitivity test showed that the phage exhibited good vitality in the pH range of 5 ~ 8. Viral titers drastically dropped when the phage BUCT551 was incubated for an hour at the pH of 4 and 11, indicating that the growth of the phage BUCT551 was inhibited in extreme pH environment (Fig. 2b).

Optimal MOI and One-Step Growth Curve
The virion productively of the phage BUCT551 using different MOI uctuated slightly. Based on the maximum titer obtained from phage BUCT551 infection of A. hydrophila strain A18, the optimal MOI of the phage BUCT551 was 0.1 (Fig. 3a). One-step growth curve assay exhibited that the phage BUCT551 had a latent period of 20 minutes and a growth period of 85 minutes, and reached the plateau at the 105 minutes, when the viral titer was 10 9 pfu/mL. The burst size of the phage BUCT551 was 32 pfu/cell (Fig. 3b).

Host Range analysis
In order to identify the host strain of BUCT551, all the 9 strains of A. hydrophila (Ah 154, ATCC 419140, AS1. 18031, Ah 7, Ah 9, Ah 10, Ah 17, Ah 18, No. 201308071) and 8 strains of A. veronii (Av 4, Av 6, Av 15, Av 26, Av 25, Av 13, Av 21, Av 3) in our lab and 20 strains of vibrio parahemolyticus (for control) were selected as candidate hosts for the reason that A. hydrophila and vibrio parahemolyticus shared an identical family vibrionaceae. Result exhibited that 1 strain of A. hydrophila and 4 strains of A. veronii were susceptible to the phage BUCT551, suggesting that BUCT551 was potential to be employed as a treatment to Aeromonas infection (Table 1). Drug sensitivity analysis of Aeromonas hydrophila A18 The host bacteria A18 was highly sensitive to cefalexin, ceftriaxone, cefalotin, cefaclor, o oxacin and nor oxacin while developed resistance to erythromycin, penicillin, roxithromycin and amoxicillin (Table 2).

Whole-genome analysis
The whole genome sequencing revealed that BUCT551 had a linear genome of 61,382 bp with the C + G content of 61.77% (accession number MT952005) (Fig. 4). According to the homology comparison of the whole genome sequence, we found that BUCT551 exhibited the highest homology with Aeromonas phage LAh_7 (Fig. 5). Based on the peculiarity that restriction endonucleases speci cally function on double-strand DNA, restriction endonucleases digestion then electrophoresis were able to determine the characteristic of nucleic acid. The phage BUCT551 genome enzyme-digested product exhibited multiple electrophoretic bands, which preliminary veri ed that BUCT551 belonged to double-stranded DNA phage (Fig. 6).

Functional ORFs analysis
BLASTn analysis indicated that BUCT551 shared 86.75% homology (87% genome coverage) with unpublished Aeromonas hydrophila phage LAh_7. RAST gene annotation identi ed that the phage genome contained 74 open reading frames (ORFs), none of which corresponded to tRNA gene, among which ATG was the initiation codon of most ORFs. Among the predicted protein coding genes, 27 of the 74 ORFs were identi ed as functional coding sequences, and the rest were annotated as functional unknown proteins or hypothetical proteins (Table 3). We classi ed the 27 proteins with known functions into four functional divisions: replication division, modi cation and regulation division, structural and packaging division and lytic division. Both BLASTp and RAST analysis demonstrated that the ORF65 and ORF65 encoded the large subunit and mall subunit of terminase, respectively, which played a signi cant role in phage DNA packaging. With regard to the phage replication division, ORF68 (DNA polymerase), ORF32 (putative 3'-5' exoribonuclease), ORF34 (putative 3'-5' exoribonuclease), and ORF39 (putative 3'-phosphatase, 5'-polynucleotide kinase) encoded by phage BUCT551 participated in the regulation of phage DNA replication. In addition, DNA helicase encoded by ORF66 and HNH endonuclease by ORF1 were speculated to play a crucial role in DNA replication and regulation, and the subsequent modi cation [31]. ORF35 (N-6adenine-methyltransferase) was likely to participate in phage DNA modi cation, whose aim was to circumvent the protection system of host bacteria [32].
Phage structual proteins were encoded by multiple ORFs including ORF47 (putative tail protein), ORF51 (putative tape measure protein), ORF54 (putative major tail protein), ORF55 (putative minor tail protein), ORF53 (putative tail assembly chaperone), ORF59 (putative major capsid protein) and ORF62 (putative portal protein). Portal protein encoded by ORF62 was considered to function in the channel formation of capsid and phage genome injection into host cells [33]. The protein encoded by ORF51 could assist and regulate phage tail assembly and was identi ed as an essential protein that assisted phage attachment to host cells during infection period [34].
ORF18 (DNRLRE domain-containing protein), ORF20 (GntR family transcriptional regulator), ORF25 (RES domain-containing protein), ORF49 (DUF2163 domain-containing protein), ORF53 (putative tail assembly chaperone), ORF60 (decorator protein) and ORF61 (putative S49 family peptidase) encoded proteins that were considered to play roles in phage modi cation and regulation. ORF53 encoding tail assembly chaperone widely existed in the genomes of Siphoviridae and Myococcal phages, and generally located between tail protein and tape measure protein, participating in the regulation of frameshift [35]. ORF60 encoded decorator proteins, a class of proteins that located on the surface of certain phage capsids especially the phages with double-strand DNA, which able to protect the phage from extreme changs such as environmental changes by enhancing and promoting the robustness of the capsid. Moreover, many decorator proteins possessed other function, such as facilitating target cell recognition, involving in phage assembling or regulating host genes expression, etc. Previous researches had demonstrated that GntR family of transcription factors encoded by ORF20 generally existed in prokaryotes and certain virus, which regulatd various kinds of important metabolism pathways. GnTR family of transcription factors were recognized to play an irreplaceable role in the growth and reproduction in prokaryotes, and were inferred to protect phages from extreme temperature and pH, etc [36].
ORF45 (putative lysis protein A) and ORF74 (putative AlpA family regulatory protein) belonged to phage lytic division, they probably encoded lytic peptidases and enabled phage to lyse host cells effectively [37].
It was worth noting that 47 ORFs were classi ed as functional unknown proteins, which accounted for the majority of the overall ORFs. Future researches are warranted to further analyze peoteins encoded by ORFs of unkonwn function so as to gain an insight into phages.
Phylogenetic tree analysis for phage BUCT551 The evolutionary relationship between the phage BUCT551 and other phages was determined on the basic of the sequence of a conserved major capsid protein (ORF59) to determine the phylogeny of BUCT551, which was commonly used as a marker gene and to study phage diversity [38,39]. Phylogenetic analysis revealed that BUCT551 (MT952005.1) was mostly related to the phage LAh_7 (MK838113.1), which belonged to the family Siphoviridae (Fig. 7) .

Discussion
Aeromonas hydrophila is considered as a opportunistic pathogenic bacteria, infecting dozens kinds of cultured shes. Aeromonas hydrophila are pathogenic to shes such as atlantic salmon, trout, turbot, etc, and the cardinal symptoms of A. hydrophila infection in sh are tail bleeding and the epidermis fester [4]. At present, the main prevention and treatment of A. hydrophila infection still depend on disinfectants and antibiotics. However, the long-term use of antibiotics may lead to drug resistance. Given this inevitable situation, novel antibacterial therapies are deserved to be researched and exploited. As a novel type of antibacterial agent, phage possesses a series of strengths such as no obvious side effects,, easily acquired, and no residue. Phage therapy provides a new alternative to control diseases caused by Aeromonas hydrophila.
BUCT551 had a double-strand of DNA genome with the length of 61,382 bp, and the C + G content was 61.77%. In the genome of the phage BUCT551, we found no genes (such as gene encoding integrase) was associated with the viral genome integration (Table 2). Through the predicted protein, we found that the phage BUCT551 encoded N-6-adenine-methyltransferase (ORF35), which played a vital role in escaping the protective system of bacteria. In addition, in the analysis of other proteins that may be expressed by phages, we did not identify any genes related to toxins or virulence factors, which indicated that phage BUCT551 was promising to use as a therapeutic agent for Aeromonas hydrophila A18.
In this study, we succesfully isolated a novel phage BUCT551 against Aeromonas hydrophila from sewage samples in an aquatic producs wholesale market. Morphological and genetic characteristics indicated that the phage BUCT551 belonged to Myoviridae. The one-step growth curve of the phage BUCT551 at the optimal multiplicity of infection (MOI) showed that it possessed characteristics of short incubation period and fast growth rate, and further con rmed that the phage BUCT551 was highly lytic.
The phage BUCT551 showed a good viability below 40℃, indicating that it has the similar growth temperature to its host, and pH stability test shown that phage activity remianed relatively stable in the pH range from 5 to 10. In conclusion, the above phage biological characteristics indicated that the phage BUCT551 had robust and stable bacteriolytic competence, which is an important basis for phage therapy. In the host range analysis assay, we detected the susceptibility of 16 strains of aeromonas, and found that 5 of them were susceptible to the phage BUCT551. In view of the small amount of A. hydrophila phages that have been isoated and characterized, the phage BUCT551 has the potential to remedy the A. hydrophila and A. veronii infection.

Conclusions
In conclusion, due to the large-scale use of antibiotics, the application of phage to control bacterial infection of sh in the aquatic environment exhibited more sustainability and security. In this study, the Aeromonas hydrophila phage BUCT551 had been identi ed and characterized, and results indicated that the phage BUCT551, along with other A. hydrophila phages, had a great potential to use in combination with antibiotics. Besides, given the lack of identi ed Aeromonas hydrophila phages, exploring the large amounts of hypothetical proteins encoded in its genome will be the priority of our future research.

Author Contributions
Hongbo Qin and Shiting He drafted the manuscript and carried out experiments. Hongbo Qin, Shiting He, Ke Liu, Fuxing Lou, Shuqi Wang and Xiaoping An analyzed the data, Lihua Song,Huahao Fan and Yigang Tong designed the experiment and revised the manuscript.
3 . Hua C, Wang T, Shao X et al (2020) Pseudomonas syringae dual-function protein Lon switches between virulence and metabolism by acting as both DNA-binding transcriptional regulator and protease in different environments. Environ  Figure 1 The morphology of phage BUCT55 as seen in an transmission electron microscope (TEM).

Figure 2
a. The survival rate decreased drastically when treated with temperature over 30℃. b. The survival rates maintain stability with pH from 5 to 8.  The homology comparison between BUCT551 and its proximal phages. The gradual red vertical bar in the lower right corner of the gure indicates the genetic homology between the phage BUCT551 and the other phage in the picture.
Page 17/18   Phylogenetic analysis of BUCT551.An evolutionary tree was constructed using capsid proteins. Phylogenetic trees were constructed using the neighbor joining method of 1000 bootstrap copies in MEGA7.