Characterization and genome analysis of two novel Aeromonas hydrophila phages PZL-Ah1and PZL-Ah8

doi:10.21203/rs.3.rs-933679/v1

Aeromonas hydrophila (A.hydrophila) is an opportunistic pathogen of fish-human-livestock, which poses a seriously affects to the development of aquaculture. Phage therapy is considered as a process to alternatively control bacterial infections and contaminations. In this study, the genomes of two Aeromonas hydrophila- specific phages PZL-Ah1 and PZL-Ah8 were isolated, characterized and genomic sequence analyzed. Transmission electron microscopy showed that the two phages had been classified as the member of the Podoviridae family. Both the two phages in this study had relatively narrow host range with lytic activity against Aeromonas spp. strains. However, they could lyse 3 common A.hydrophila strain. As revealed from the whole genomic sequence analysis, PZL-Ah1 and PZL-Ah8 coverd the double-stranded genome of 38,641 bp and 40,855 bp in length, with the GC content of 53.68% and 51.89%, respectively. Through gene comparison in NCBI database revealed that PZL-Ah1 and PZL-Ah8 were 97.67% − 95.51% identical to Stenotrophomonas phage IME15 and Aeromonas Phage T7-Ah. Phylogenetic analysis showed that PZL-Ah8, PZL-Ah1 and other two phages belonged to the same genus. A total of 44 and 52 open reading frames (ORFs) were predicted in the PZL-Ah1 and PZL-Ah8 genome, respectively. In the process of gene annotation, 28 (63.6%) ORFs in PZL-Ah1 and 29 (55.8%) ORFs in PZL-Ah8 were known to functional proteins in NCBI database, while the remaining ORFs were classified as “hypothetical proteins”, whose functions were yet unknown. By comparing, ORF 02, ORF 29 and ORF 04 in PZL-Ah1, ORF24 in PZL-Ah8 were responsible for the host cell lysis. In conclusion, genomic studies of these two novel phages would lay the foundation for expanding the phage genome database and providing good candidates for phage typing applications.

Virology

A.hydrophila

phage characterized

genome analysis

Aeromonas hydrophila, pertaining to the genus Aeromonas of Vibrio family, refers to a type of gram-negative short Bacilli. Over the past few years, it had imposed significant economic losses to aquaculture industry, thereby caused fish, frogs and soft-shelled turtles to bacterial septicemia or hemorrhagic disease [1]. Moreover, A.hydrophila, as a fish-human-livestock symbiotic bacterium, it could also cause septicemia and acute gastroenteritis in humans [2, 3]. The existing treatment of A.hydrophila was the dependence of antibiotics as well as some chemical drugs. In contrast, continued use of antibiotics could lead to the development of resistant bacteria. The problem of bacterial drug resistance was increasing seriously [4, 5]. Thus, novel drugs capable of effectively treating the infection attributed by resistant A.hydrophila should be urgently developed.

Bacteriophage as a bacterial virus, could lyse and kill bacteria. Bacteriophage therapy exhibited significant advantages over antibiotics when treated bacterial infection. It was capable of killing bacteria specifically with lower cost and easy-to-use characteristics [6, 7]. As a suitable substitute for antibiotics, bacteriophage had aroused extensive attention from industry experts and relevant enterprises [8]. Sequencing and analysis of phage genomes were also an important part of phage research[9]. Each gene encoded different protein and thus had a different function. For example, some gene encoded proteins worked as lytic enzymes or lysins, which directly affected the ability of phages to lyse bacteria [10]. The analysis of phage genome was conducive to better understanding the function of phage, and provided a basis for further development and utilization of phage. In our study, we successfully isolated two bacteriophages of Aeromonas hydrophila (PZL-Ah1 and PZL-Ah8) and analyzed their biological characteristics and genomes. We found that these two phages had unique characteristics. Sequencing and analysis with the NCBI database showed that these were two completely new phages. It laid the foundation for the combination of phage cocktail therapy and construction of engineering phage in the future clinical application.

PZL-Ah1 and PZL-Ah8 were isolated from the sewerage systems in Changchun, China. Two distinct plaque morphologies were identified, PZL-Ah1 and PZL-Ah8 phages formed plaques with 2 to 4 mm diameters surrounded by a thin area of secondary lysis of 1 mm. TEM image revealed that virion of PZL-Ah1 and PZL-Ah8 had capsid diameter ≈ 51 ± 1 nm and tail ≈ 21 ± 1 nm (Fig. 1A). The morphological characteristics of PZL-Ah1 and PZL-Ah8 suggested that they both belonged to the Podoviridae family (Fig. 1B). The infection of Aeromonas bacteria had obvious host specificity, and they could rarely infect extraspecific bacteria. However, there was a certain cross infection rate for intraspecific bacteria. We determined the host ranges of PZL-Ah1 and PZL-Ah8 against a panel of 41 Aeromonas spp. strains at a multiplicity of infection (MOI) of 0.1 (PZL-Ah1) and (MOI) of 0.01 (PZL-Ah8) to detect visible plaques. Phage PZL-Ah1 could lyse 7 Aeromonas strains, including (4/23) strains of A.hydrophila (AH-119, AH-150, AH-87, AH-138), 3/18 strain of Aeromonas veronii (AV-6, AV-11, AV-77) (Table 1). For the phage PZL-Ah8, it could also lyse (7/23) A.hydrophila strains (AH -119, AH -138, AH -103, AH -68, AH -142, AH -64 and AH -87). By testing, we found that the PZL-Ah1 and PZL-Ah8 could lyse a common A.hydrophila strain A.hydrophila AH-138.

Table 1. Bactericidal spectrum of PZL-Ah1 and PZL-Ah8

Organism	Bacterial strain	PZL-Ah1	PZL-Ah8	Organism	Bacterial strain	PZL-Ah1	PZL-Ah8
Organism	Bacterial strain	Efficiency	Efficiency	Organism	Bacterial strain	Efficiency	Efficiency
A.hydrophila	147^C			A.veronii	1^C
A.hydrophila	119^C			A.veronii	3^C
A.hydrophila	138^C			A.veronii	4^C
A.hydrophila	1021^C			A.veronii	5^C
A.hydrophila	150^C			A.veronii	6^C
A.hydrophila	107^C			A.veronii	7^C
A.hydrophila	166^C			A.veronii	9^C
A.hydrophila	69^C			A.veronii	11^C
A.hydrophila	TPS^C			A.veronii	20^C
A.hydrophila	36^C			A.veronii	21^C
A.hydrophila	103^C			A.veronii	47^C
A.hydrophila	68^C			A.veronii	77^C
A.hydrophila	6^C			A.veronii	85^C
A.hydrophila	142^C			A.veronii	115^C
A.hydrophila	143^C			A.veronii	155^C
A.hydrophila	152^C			A.veronii	TH0426^C
A.hydrophila	BSK^C			A.veronii	QXF0711B^C
A.hydrophila	54^C			A.veronii	520^C
A.hydrophila	183^C
A.hydrophila	93^C
A.hydrophila	87^C
A.hydrophila	148^C
A.hydrophila	64^C

: strains that can be lysed by phages；

: strains that cannot be lysed by phages;

C : isolated from the carp kept in our lab (Changchun, Jilin province, China).

The complete genome sequences and annotated information of PZL-Ah1 (MT681669.1) and PZL-Ah8 (MZ851152.1) have been submitted to GenBank. The PZL-Ah1 and PZL-Ah8 phage genome were 38,641 bp and 40,855bp contiguous sequence of double-stranded DNA, linear, respectively. The overall G་C content was 53.68% including composition of G (26.11%), C (27.57%), A (22.03%) and T (24.29%) in the gene of phage PZL-Ah1. For the gene of PZL-Ah8, the overall G་C content was 51.89% including composition of G (24.94%), C (26.95%), A (23.23%) and T (24.87%), respectively (Fig. 2A).

BLASTn was used to determine the degree of sequence identity of the two phage genomes and also to other phage genomes in the NCBI nucleotide (nt) database. Through gene comparison in NCBI database showed that PZL-Ah1 and PZL-Ah8 were 97.67% − 95.51% identical to Stenotrophomonas phage IME15 and Aeromonas Phage T7-Ah. We also used the Emboss Stretcher database to analysed and made the comparison. The result showed that PZL-Ah1 and PZL-Ah8 were 48.2% -48.2% identical to Stenotrophomonas phage IME15 and Aeromonas Phage T7-Ah. We compared the whole genome of phage PZL-Ah1 with phage PZL-Ah8 and found that although both phages belonged to Aeromonas hydrophila phage, only in the range of 16000 − 2400 bp and 32000–40000 bp having relatively similarities (Fig. 2B). The results indicating that the two phages had their own unique sequences.

Open reading frame (ORF) prediction in assembled contigs was carried out in the GeneMark suite. The functional annotation of proteins encoded by these ORFs were performed using BLASTp, NCBI Conserved Domain Search, HHpred Interactive Server and Inter-ProScan. The phage genomes were also analyzed by tRNAscan-SE to detect tRNA genes. Phage PZL-Ah1 and Phage PZL-Ah8 included with 44 ORFs and 52 ORFs, respectively. The size of proteins encoded by 44 ORFs of PZL-Ah1 ranged from 4.39 kDa (ORF 19) to 143.40 kDa (ORF 06). For the phage PZL-Ah8, the protein sizes ranged from 4.50 kDa (ORF 08) to 150.07 kDa (ORF 01). During the annotation process, 28 (63.6%) ORFs in PZL-Ah1 and 29 (55.8%) ORFs in PZL-Ah8 were homologous to the sequences of known functional proteins in NCBI database. In addition, 16 (36.4 %) ORFs in PZL-Ah1 and 23 (44.2%) ORFs in PZL-Ah8 were classified as “hypothetical proteins” with yet unknown functions (Table S1 and S2).

Like most phages, the coding region of Aeromonas phage genome was closely arranged, and the genes related to the function were clustered. In terms of gene functions, except for genes with unknown functions, the genome of Aeromonas phage could be divided into three functional categories, so as to the PZL-Ah1 and PZL-Ah8: nucleic acid metabolism and replication module; phage structure and packaging; host lysis. The gene sequence of each module was relatively conservative. PZL-Ah1 and PZL-Ah8 belonged to the Podoviridae family. A major difference between Podoviridae and Siphoviridae was the small number of ORFs, from about 20 to 50. But none of them contain lysogenic modules [11]. The lysogenic module consists of integrase and repressor proteins that were key to getting the phage into the lysogenic or lytic cycle, no ORFs were associated with drug resistance or lysogeny (e.g., site-specific integrases and repressors) in both genomes (Fig. 3). The nucleic acid metabolism and replication module contains genes related to DNA replication and gene regulation respectively. There were 8 ORFs and 12 ORFs related to nucleic acid metabolism and replication module in PZL-Ah1 and PZL-Ah8. The phage structure and packaging module contains terminal enzymes related to DNA packaging and genes in head and tail morphogenesis. 15 ORFs and 14 ORFs related to the structure and packaging module existed in the PZL-Ah1 and PZL-Ah8 genomes. Endolysin (lysin) proteins, which were found to be encoded in the two phage genomes (ORF 02, ORF 29 and ORF 04) in PZL-Ah1, ORF 24 in PZL-Ah8), were responsible for host cell lysis and release of phage progeny. Due to their ability to kill bacteria by degrading their cell wall, lysins had recently emerged as novel anti-microbial molecules, that could potentially be used to tackle emerging antimicrobial resistance challenges in bacteria [12, 13]. The receptor binding protein (RBP) interaction determined the range and specificity of the phage host. By comparing the ORFs of the two phages, we found the two phages encoded host range proteins (ORF16 in PZL-Ah1, ORF12 in PZL-Ah8). ORF16 in PZL-Ah1 encoded 89 amino acids with a protein size of about 9.15kDa, while ORF12 in PZL-Ah8 encoded 95 amino acids with a protein size of about 10.10 kDa. As for the receptor binding protein, we could use genetic engineering technology to expand the host range. So as to effectively solve the problems of strong specificity and narrow host range of phage application. Bardyt [14] found that when used genetic engineering techniques to modify the host decision region genes of phages, which could broaden the host range of phages. Yoichi [15] exchanged the GP37 and GP38 regions of T2 phage with PP01 phage to form the new recombinant T2 phage, which could infect the heterogeneic Escherichia coli, thus effectively expanded the host spectrum of T2 phage.

Horizontal gene transfer was thought to play an important role in phage evolution, especially in phage genomic diversity. A total of 34 phages with high homology to these two phages were compared and MEGA7 was used to map the phylogenetic tree (Fig. 4A). As shown in Fig. 4A, the two bacteriophages were on the same large branch with the phage PZL-Ah152 and phage Rostov-1, and the bootstrap in this branch was 100, indicating that the four bacteriophages were closely related. PZL-Ah1, PZL-Ah8 and PZL-Ah152 belonged to the same Aeromonas genus, and they were in the same Vibrio family as the phage Rostov-1. We performed collinearity analysis of PZL-Ah1 and PZL-Ah8 with two other phages from the same and different branches, respectively (Fig. 4B). As revealed from a comparative analysis, phage PZL-Ah1 and PZL-Ah8 genome were highly similar to that of the Aeromonas phage PZL-Ah152 and Vibrio phage Rostov-1.

Bacteriophages could recognize bacterial cell walls, transfer genetic information, and specifically killed their host strain [16]. In the present study, the genomes of two A. hydrophila-specific phages PZL-Ah1 and PZL-Ah8 were isolated, characterized and sequenced. According to gene sequences comparition, their genomes did not exhibit 100% identical sequence homologies to currently known phage strains in GenBank, suggesting that they were two novel phage strains. A total of 114 Aeromonas phage genome sequences have been released in GeenBank. The discovery of two novel phages could help us better understand the A. hydrophila phage genome characters, which expanded the diversity of Aeromonas phage gene on NCBI. As well as provide reference for application of phage in the future.

Accession numbers of nucleotide sequences

The genome sequences of PZL-Ah1 and PZL-Ah8 are available in the NCBI GenBank database under the accession numbers MT681669.1 and MZ851152.1, respectively.

Acknowledgements

All the authors acknowledge and thank their respective institutes and universities.

Funding

This project the coordinated research of the 13th Five-Year Science and Technology Research and Planning Project of Education Department of Jilin province (JJKH20200353KJ); the Youth talent promotion project of Jilin Province (201905);

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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Characterization and genome analysis of two novel Aeromonas hydrophila phages PZL-Ah1and PZL-Ah8

Status:

Version 1

Abstract

Figures

1. Introduction

2 Phage Purification And Characteristics

3 The Whole Genome Analysis

4 The Function Of Open Reading Frames (Orfs)

5 Phylogenetic Tree And Genome Collinearity Analysis

6 Conclusion

Declarations

References

Supplementary Files

Status:

Version 1