Whole Genome Sequence of a Novel Bacteriophage APT65 Infecting Aeromonas hydrophila

Introduction

Intensive aquaculture increases the risk of infection by opportunistic pathogens, thus resulting in dependence on antimicrobial agents. ¹ Aeromonas hydrophila, an opportunistic aquatic pathogen, is the most prevalent cause of motile Aeromonas septicemia (MAS) in many fish species living in fresh and brackish water.^2,3 MAS has resulted in substantial economic losses for the aquaculture sector throughout the globe. ⁴ Also poikilothermy species, such as amphibians, fish, snakes, and turtles, are among the most susceptible to A. hydrophila infection. ⁵ It is a zoonotic pathogen that causes gastroenteritis and other systemic diseases in humans and other species. ⁶

When the water quality parameters are unsuitable for fish, A. hydrophila may induce mass mortality. ⁷ Antimicrobials are used to prevent mortality, which triggers the emergence of antimicrobial resistance bacteria and the accumulation of residues in fish tissue.^8,9 Antimicrobial resistant strains are able to reproduce and transmit their antibiotic resistance, resulting in the emergence of a large number of antibiotic-resistant bacteria. ¹⁰ To overcome these issues, bacteriophages can be used as an alternative to many antibiotics to control bacterial infections in aquaculture. ¹¹

The quick generation time, nontoxicity, high specificity to the bacterial host, and the fact that they do not negatively impact the viability of other flora in the environment make bacteriophages a promising, environmentally friendly, and sustainable antibiotic alternative in aquaculture. ¹² Considering the advantages of phages, a novel bacteriophage, Aeromonas phage T65 strain (APT65), specific to A. hydrophila, was isolated from domestic wastewater, ¹¹ and the whole genome of APT65 was mapped out in this study.

Materials and Methods

The phage specific to A. hydrophila was isolated from domestic wastewater in Trabzon, Türkiye. Thirty milliliters of filtered wastewater was mixed with 30 mL of tryptic soy broth that contained 1 mL of A. hydrophila and incubated at 25°C for 24 h. The sample was centrifuged for 15 min at 5000 × g, and the supernatant was filtered through a 0.22 μm syringe filter. After repeating this procedure three times, the double-layer agar technique was employed to detect phage activity.^11,13 The characteristics of the APT65 were described in the earlier work done by Ture et al. ¹¹ Initial concentration of the phage was 2 × 10⁹ PFU/mL.

The morphology of the phage was analyzed using transmission electron microscopy on negatively stained specimens. In brief, the phage solution was centrifuged for 2 h at 41,000g, and the pellet was resuspended at 10¹⁰ PFU/mL in the saline magnesium buffer (0.1 M NaCl, 8 mM MgSO₄, 7H₂O, 50 mM Tris-HCl pH 7.5, %1 gelatin). The 10 μL of the concentrated phage solution was adsorbed on the carbon-coated matrix and the sample was finally stained with 1% uranyl acetate for 15 s. The grid was examined with a transmission electron microscope after being rinsed twice with ddH2O and air dried before examination.

Genomic DNA of APT65 was extracted using the phenol-chloroform-isoamyl alcohol procedure. ¹¹ The sequencing library was prepared using the NEBNext DNA Library Prep Kit (New England Biolabs) and sequenced on the Illumina HiSeq4000 platform with a paired-end read length of 2 × 150 bp. The genome sequence was assembled using SPAdes 3.13.0 and annotated with Prokka 1.14.6, using the default settings.^14,15 Further annotation was conducted by BLASTp using the nonredundant protein database and hidden Markov models (https://www.ebi.ac.uk/Tools/hmmer/). Then tRNAs were predicted using Aragorn v. 1.2 software ¹⁶ and antimicrobial resistance or virulence genes were screened by ABRicate. ¹⁷ Furthermore, the phage lifestyle was determined using PhageLeads to predict its suitability for phage therapy. ¹⁸ The CGView Server was used to create a depiction of the phage APT65 genome. ¹⁹

Combining phylogenetic and clustering methodologies, the Genome Blast Distance Phylogeny web application of Virus Intergenomic Distance Calculator (VIRIDIC) was used to classify APT65. The VIRIDIC approach extends and improves on the standard BLASTN method, used by the International Committee on Taxonomy of Viruses (ICTV) to determine how closely related viral genomes are and how they are linked. VIRIDIC classifies phage genomes based on a prokaryotic virus classification algorithm. ²⁰ VirClust was used to cluster bacteriophages according to their protein profiles. ²⁰ The nucleotide similarity of the phages was determined by using the Orthologous Average Nucleotide Identity Tool (OAT; v0.93.1) that measures overall similarity between two genome sequences. ²¹ The cutoff for species demarcation is 95–96%. To demonstrate that the phage is novel.

Results

The APT65 genome was 85188 bp long and contained 39.41% G+C. A total of 116 open reading frames (ORFs) ranging from 303 to 4512 bases were predicted. Among the 116 ORFs, 32 showed similarities to known phage proteins, whereas 73 were labeled as hypothetical proteins, most likely due to a lack of knowledge regarding the functional genes of Aeromonas phage genomes. Although CRISPR repeats and rRNA were not found, 12 tRNAs [tRNA-Val(tac), tRNA-Lys(ttt), tRNA-Gln(ttg), tRNA-Arg(tct), tRNA-Leu(tag), tRNA-Leu(taa), tRNA-Asp(gtc), tRNA-Pro(tgg), tRNA-Gly(tcc), tRNA-Asn(gtt), tRNA-His(gtg), and tRNA-Thr(tgt)] were identified (Fig. 1). Based on the whole genome sequence, the APT65 phage belongs to the genus Lahexavirus (GenBank accession: OP491958.1), and it should be named Lahexavirus APT65.

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FIG. 1.

ORF map of the genome of Aeromonas phage, APT65. The genomic map was prepared by the CGView Server. The outer lane and the next lane display genes on the forward and reverse strands, respectively. APT65, Aeromonas phage T65 strain; ORF, open reading frame.

Protein profile-based phylogenetic analysis revealed that Aeromonas phage APT65 was closely related to Lahexavirus LAh6, Lahexavirus LAh8, Lahexavirus lv44572, and Lahexavirus LAh 9, but not to Siphoviridae, Haloferacalesvirus, Jhansiroadvirus, Przondovirus, Lafunavirus, or Lagaffevirus (Fig. 2). Pairwise intergenomic distances/similarities across viral genomes revealed that Lahexavirus LAh6, Lahexavirus LAh8, Lahexavirus LAh9, and Lahexavirus lv44572 had >88% similarity, whereas these phages shared between 45% and 47% similarity with the Lahexavirus APT65 phage (Fig. 3). Lahexavirus LAh9 and Lahexavirus lv44572 had 97.9% nucleotide similarity according to average nucleotide identity (ANI) results, whereas these phages shared 66.8% similarity with the APT65 phage (Fig. 4). These three phages bore no resemblance to the other phages.

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FIG. 2.

The clustering of viral bacteriophage proteins into two different levels. Proteins are first grouped into PCs based on their reciprocal BLASTP similarities; these PCs are then grouped into protein superclusters based on their HMM similarities. HMM, hidden Markov model; PCs, protein clusters.

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FIG. 3.

Distance Heatmap among bacteriophage genomes.

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FIG. 4.

ANI value of the APT65 phage in comparison with related and unrelated bacteriophages. ANI, average nucleotide identity.

Furthermore, no virulence factors, antibiotic resistance, or temperate lifecycle genes were found. The virions were icosahedral, 60 nm in diameter, with a lengthy contractile tail of 170 nm (Fig. 5). The results show that the Lahexavirus APT65 phage could be used safely to treat or prevent A. hydrophila infection in aquaculture.

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FIG. 5.

Transmission electron microscopy image of APT65 phage.

Discussion

Nowadays, a more holistic method is used in phage taxonomy, which is a significant departure from earlier morphology-based methods. ²² The APT65 phage belongs to the Heunggongvirae kingdom, Uroviricota phylum, Caudoviricetes class, and Lahexavirus genus, as confirmed by the ICTV (https://ictv.global) and whole genome sequence analysis (GenBank accession OP491958.1). There are four species in the Lahexavirus genus, including Lahexavirus LAh6, Lahexavirus LAh8, Lahexavirus LAh9, and Lahexavirus lv44572) (https://ictv.global). The ANI test revealed, however, that all of these species belong to the Lahexavirus genus and are the same species. They shared a nucleotide similarity of >97%, which is higher than the 96% cutoff value. In contrast, these four phages had a similarity of <66.8% with Lahexavirus APT65. Therefore, the other four species should be the same species, whereas Lahexavirus APT65 should be a distinct species.

Three phages known as N21, W3, and G65 were identified as being effective against virulent A. hydrophila. The phages obtained by TEM were classified as members of the Myoviridae family on the basis of their morphological characteristics. ²³ Nonetheless, this family is not present on the ICVT list. To classify bacteriophages, TEM images or other morphological characteristics are insufficient; WGS is necessary.

In conclusion, morphological classification of bacteriophages is not enough. The APT65 phage isolated and identified in this study belongs to the Lahexavirus genus. Therefore, the APT65 phage should be classified as a Lahexavirus APT65 species. Since the APT65 phage does not contain virulence factors, antibiotic resistance genes, or phage lifestyle genes, it has the potential to be used in phage therapy against A. hydrophila.