F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.144007.1 Genome Note Articles Isolation and complete genome sequence of Aeromonas bacteriophage Gekk3-15

[version 1; peer review: 2 approved, 1 approved with reservations]

 Golomidova A.K. Investigation Writing – Original Draft Preparation Writing – Review & Editing 1 Kulikov E.E. Data Curation Visualization Writing – Review & Editing https://orcid.org/0000-0002-9101-1543 1 Kuznetsov A.S. Investigation Writing – Review & Editing https://orcid.org/0000-0002-8016-3760 1 Pechenov P. Yu. Investigation Writing – Review & Editing https://orcid.org/0000-0002-3600-7170 1 Belalov I.S. Data Curation Formal Analysis Writing – Review & Editing 1 Letarov A.V. Conceptualization Funding Acquisition Project Administration Supervision Writing – Original Draft Preparation Writing – Review & Editing 1 Galyov E.E. Conceptualization Funding Acquisition Project Administration Writing – Review & Editing https://orcid.org/0000-0003-2609-0748 a 2 Winogradsky Institute of Microbiology, Research Center “Fundamentals of Biotechnology” RAS, Moscow, Russian Federation Genetics and Genome Biology, University of Leicester, Leicester, UK eg98@le.ac.uk

No competing interests were disclosed.

 24 4 2024 2024 13 380 9 4 2024 Copyright: © 2024 Golomidova AK et al. 2024 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Bacteria of the genus Aeromonas, especially A. hydrophila and A. veronii are recognized as important fish pathogens that cause significant economic losses in aquaculture. Environmentally friendly bacteriophage-based solutions for the treatment of fish and for the reduction of colonization by pathogenic bacteria in production facilities are currently in high demand. The bacteriophage Gekk3-15 was isolated during a search for novel phage strains potentially suitable for Aeromonas biocontrol applications. Genome sequencing revealed that this virus is a relatively small myovirus with a 64847 bp long dsDNA genome, which is consistent with virion electron microscopy data. Bacteriophage Gekk3-15 is distinct in its nucleotide and encoded aa sequences from all other sequenced bacteriophage genomes, and may represent a new viral taxon at the genus or subfamily level.

 bacteriophage Aeromonas veronii aquaculture phage therapy Biotechnology and Biological Sciences Research Council BB/R012822/1 Ministry of Science and Higher Education of the Russian Federation #075-15-2022-318 This work was supported by the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement #075-15-2022-318 dated April 20, 2022, on the provision of a grant in the form of subsidies from the federal budget of the Russian Federation for the implementation of the state support for the creation and development of a world-class scientific center “Agrotechnologies for the Future” and, in part, by funding from the University of Leicester and BBSRC grant BB/R012822/1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction

Currently, aquaculture is the primary producer of nutritional fish in most developed countries. 1 The cultivation of hydrobionts in production ponds is associated with very high densities of animal populations, which far exceed the typical population densities in wild habitats. Such conditions make cultivated fish specifically vulnerable to bacterial infections, of which aeromonoses comprise a significant fraction. 2 , 3 The use of antibiotics to reduce economic losses associated with bacterial infections is restricted or prohibited in many countries; therefore, using phages as a tool for environmentally friendly biocontrol of the iсhtyopathogenic Aeromonas has attracted significant interest in scientific and business communities. Therefore, the search for new bacteriophage strains that are potentially suitable for use as phage therapy agents is important. Here, we present the isolation and sequencing of a novel A. veronii bacteriophage that represents a new group of bacteriophages that infect the genus Aeromonas.

Methods Phage isolation and cultivation

The bacteriophage Gekk3-15 was isolated as part of a project to establish a panel of bacteriophages to target the collection of A. hydrophila and A. veronii strains that cause infections in aquaculture facilities in Russia. The phage was isolated from a water sample collected from the Norishka Stream in Mikhailovsky Park in Moscow, Russia, on September 14, 2021. The bacteriophage was isolated using the enrichment culture setup as follows: A non-sterilized water sample (0.5 l) was placed in a 2 l sterile plastic bottle and supplemented with 50 ml of sterile 10x LB medium (for 1× medium: 10 g Bacto Tryptone (Amresco, Am-J859-0.25), 5 g yeast extract (Amresco, J850-500G), 10 g NaCl (Amresco, J869-500G), distilled H 2O up to 1 l). The culture was supplemented with 0.5 ml of an overnight liquid culture of A. veronii AV-3 strain and incubated at 28 °C in a thermostat without agitation. Following incubation, 1 mL of the enrichment culture was centrifuged at 12 000 rpm in a tabletop microcentrifuge, filter-sterilized using a 0.22 μm syringe membrane filter (Millex-GP, Millipore), and plated onto the lawn of A. veronii strain 4 using the conventional double-layer technique. 4

Phage plaques were formed after an overnight incubation at 28°C. The phage was purified using two consecutive single-plaque isolations. To obtain a high-titer lysate, five 90 mm Petri dishes were filled with solid LB medium (15 g of Bacto Agar (BD214030, BD Difco) per 1 l). Without drying the plates, they were overlaid with 5 ml of soft LB agar (6 g Bacto Agar (BD214030, BD Difco) per 1 l) per plate. Soft agar was inoculated with 300 μl of a 4h log-phase liquid culture of the host strain and approximately 1×10 5 PFU of the phage per plate. The plates were incubated overnight at 28 °C. To extract the phage, the soft agar layer was gently destroyed with a spreader, transferred into 50 ml plastic centrifuge tubes, and layered with 10 ml of LB medium. Chloroform (Fisher Scientific, C298-4)(100 μL) was added to each tube, followed by vigorous vortexing for 1 min, and allowed to stand at room temperature for 2 h. After incubation, the agar fragments and bacterial cells were pelleted by centrifugation at 10 000 g for 5 min, and the supernatant was collected and centrifuged again under the same conditions. The phage was purified using a sucrose gradient as it described elsewhere. 5 The purified phage sample was used for DNA extraction and transmission electron microscopy (TEM), as previously described. 6

DNA extraction, sequencing, assembly and annotation

To extract DNA, the phage stock with a titer of approximately 10 11 PFU ml −1 was treated with DNAse (Thermo Scientific, EN0521)(0,01 mg ml −1) for 30 min at room temperature, and the phage particles were then collected by ultracentrifugation in an angle rotor at room temperature (Beckman 45Ti, 1 h, 75000 g). Genomic DNA was extracted from the precipitates with CTAB cetyltrimethylammonium bromide (CTAB) extraction as described previously. 7 DNA quality and quantity were assessed using agarose gel electrophoresis and a Qubit dsDNA HS fluorometer assay (Qubit, USA). Phage genomic DNA libraries were prepared and sequenced using an Ion Proton sequencer (Applied Biosystems, Foster City, CA, USA) with the standard chemistry according to the manufacturer’s instructions. The raw reads from the run were combined and filtered using the error correction tool Pollux ( https://github.com/emarinier/pollux). Contigs were assembled using Newbler version 3.0 (RRID:SCR_011916) (Roche Diagnostics, USA). A single contig with a phage genome of 64 847 b.p. with an average coverage of 200 bp was obtained.

Annotation was performed using Prokka (RRID:SCR_014732) 8 with subsequent manual curation. Potential open reading frames (ORFs) were detected using GeneMarkS ( https://genemark.bme.gatech.edu/genemarks.cgi)(RRID:SCR_011930) and subsequently analyzed using HMMER (RRID:SCR_005305), 9 HHPRED (RRID:SCR_010276) 10 (MPI Bioinformatics Toolkit), NCBI BLAST (RRID:SCR_004870), 11 and tRNAscan-SE (RRID:SCR_008637). 12

Results

The bacteriophage Gekk3-15 was found to be a relatively small myovirus with an isometric head ( Figure 1). The genome consisted of 64 847 b.p. and encoded 101 ORF, including putative virion structural proteins. A set of proteins for the contractile phage tail was detected, confirming the bacteriophage TEM examination data. The Gekk3-15 genome also contains genes for DNA metabolism enzymes, a cell lysis system, and two potential auxiliary metabolic genes (AMGs), encoding 3-oxoacyl-[acyl-carrier-protein] reductase FabG and glyoxylase/dioxygenase superfamily proteins. No tRNA genes were identified. Phage Gekk3-15 does not contain any markers potentially associated with a lysogenic lifestyle or virulence factors. A BLASTN (RRID:SCR_001598) search against the NCBI nucleotide collection did not produce any relevant hits for related bacteriophage genomes. However, the BLASTX (RRID:SCR_001653) search identified a number of distantly related viruses, among which the closest relative by the large terminase a.a. sequence was the Pseudomonas phage EPa61 (GenBank NC_048744), sharing 57% aa identity with the Gekk3-15 protein. The levels of a. a. identity between the structural proteins of bacteriophages Gekk3-15 and EPa61 varied between 38% and 63%. This degree of similarity indicates that taxonomically, these phages belong to different genera and, probably, different subfamilies within the Caudoviricetes kingdom. Therefore, we conclude that phage Gekk3-15 represents a novel genus and, probably, a novel bacteriophage taxon of a higher rank.

Morphology of the Gekk3-15 phage virions with extended (left) and contracted (right) tail. Ethics and consent

Ethical approval and consent were not required.

 Data availability

NCBI GenBank: Aeromonas phage Gekk3-15, complete genome. Accession number OR661252; https://www.ncbi.nlm.nih.gov/nuccore/OR661252.1. 13

References Bondad-Reantaso MG MacKinnon B Karunasagar I : Review of alternatives to antibiotic use in aquaculture. Rev. Aquac. 2023;15(4):1421–1451. 10.1111/raq.12786 Dien LT Ngo TPH Nguyen TV : Non-antibiotic approaches to combat motile Aeromonas infections in aquaculture: Current state of knowledge and future perspectives. Rev. Aquac. 2022;15(1):333–366. 10.1111/raq.12721 Rai S Kaur B Singh P : Perspectives on phage therapy for health management in aquaculture. Aquac. Int. 2023;1–45. 10.1007/s10499-023-01220-6 Letarov AV Kulikov EE : Determination of the bacteriophage host range: culture-based approach. Methods Mol. Biol. 2018;1693:75–84. 29119433 10.1007/978-1-4939-7395-8_7 Kornienko M Kuptsov N Gorodnichev R : Contribution of Podoviridae and Myoviridae bacteriophages to the effectiveness of anti-staphylococcal therapeutic cocktails. Sci. Rep. 2020 Oct 29;10(1):18612. Epub 2020/10/31. 33122703 10.1038/s41598-020-75637-x PMC7596081 Efimov A Kulikov E Golomidova A : Isolation and sequencing of three RB49-like bacteriophages infecting O antigen-producing E. coli strains. F1000Res. 2021;10:1113. 10.12688/f1000research.74169.1 Kulikov E Golomidova A Babenko V : A simple method for extraction of the horse feces virome DNA, suitable for oxford nanopore sequencing. Microbiology. 2020;89:246–249. 10.1134/S002626172002006X Seemann T : Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014 Jul 15;30(14):2068–2069. Epub 2014/03/20. 24642063 10.1093/bioinformatics/btu153 Eddy SR : Accelerated Profile HMM Searches. PLoS Comput. Biol. 2011 Oct;7(10):e1002195. Epub 2011/11/01. 22039361 10.1371/journal.pcbi.1002195 PMC3197634 Soding J Biegert A Lupas AN : The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W244–W248. 15980461 PMC1160169 Altschul SF Gish W Miller W : Basic local alignment search tool. J. Mol. Biol. 1990 Oct 5;215(3):403–410. Epub 1990/10/05. 2231712 10.1016/S0022-2836(05)80360-2 Chan PP Lin BY Mak AJ : tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Res. 2021 Sep 20;49(16):9077–9096. Epub 2021/08/22. 34417604 10.1093/nar/gkab688 PMC8450103 Golomidov AK Kulikov EE Letarov AV : Isolation and complete genome sequences of Aeromonas bacteriophage.[Dataset]. NCBI GenBank. 2023. Reference Source 10.5256/f1000research.157743.r278896 Reviewer response for version 1 Tarabili Reham M El 1 Referee Bacteriology, Immunology and Mycology Department, Suez Canal University, Ismailia, Ismailia Governorate, Egypt

Competing interests: No competing interests were disclosed.

 3 9 2024 Copyright: © 2024 Tarabili RME 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

The authors declared, “Isolation and complete genome sequence of Aeromonas bacteriophage Gekk3-15”. Despite the importance of the study, the article lacks a good presentation. It has many grammar and language mistakes.

The order of event writing should be the same either in the abstract, introduction, material, ……….so on.

A title should be modified

- Please write the scientific names of bacterial pathogens and genes in the correct form (italic form) all over the manuscript and the references section.

Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Yes

Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Yes

Are the rationale for sequencing the genome and the species significance clearly described?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

microbiology ,immunology , mycology , molecular biology and food safety

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.5256/f1000research.157743.r278893 Reviewer response for version 1 Temperton Ben 1 Referee Boot-Handford Liam 1 Co-referee https://orcid.org/0009-0006-9015-9657 University of Exeter, Exeter, England, UK

Competing interests: No competing interests were disclosed.

 17 6 2024 Copyright: © 2024 Temperton B and Boot-Handford L 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

Editorial Note from the F1000Research Editorial Team: This peer-review report was originally published on 17th June 2024, and on 1st July 2024 the report was edited to include an additional paragraph provided by the reviewer relating to Reviewer 1's (Ruben Avendano-Herrera's) report for this article.

Review for: Isolation and complete genome sequence of Aeromonas bacteriophage Gekk3-15

This work details the method of isolation and sequencing of a novel bacteriophage that infects Aeromonas veronii, as well as translations of the genome. This is a useful finding as the isolated phage shares a low degree of similarity to previously sequenced phages, and the most closely related phage infects a host belonging to a different genus.

Major comments:

The framing of the study may be too broad for the content. The abstract discusses the use of phage in Aeromonas hydrophila, but this is not tested in the main text, and it is unclear if this particular phage would infect this host. We recommend either re-framing the abstract to specify Aeromonas veronii only, as this is the only isolate tested, or to test the phage infectivity on A. hydrophila also. This is especially important if the aim of the phage isolation is to be used in phage therapy.

Minor comments:

Page 3, under Methods, 'Phage isolation and cultivation', paragraph 1: "at 28 deg. Celsius in a thermostat without agitation". "at 28 deg. Celsius in a thermostatic incubator without agitation" would be clearer.

Same paragraph: please add how many minutes the enrichment culture was centrifuged for. The x g should be listed rather than rpm

Same paragraph: it is unclear what A. veronii strain was used to isolate this phage, initially it is listed as AV-3 strain, and later as strain 4. Either these are different strains or one was mis-named in the manuscript, but regardless it would be pertinent to introduce any strains used and include information about the origin(s).

Page 3, under Methods, 'Phage isolation and cultivation', paragraph 2: what method was used for single-plaque isolations? Please provide a reference.

Same paragraph: should read 'as described elsewhere' not 'as it described'.

Page 3, under Methods, 'DNA extraction, sequencing, assembly and annotation', paragraph 1: should read '0.01 mg ml -1' not '0,01 mg ml -1'

Please use 'L' rather than 'l' for litres as this removes possible confusion between 'l', 'I', and 1.'L' is the approved symbol for litre ( https://www.nist.gov/pml/owm/si-units-volume).

The authors attempt to classify their phage into an appropriate taxonomy based on amino acid identity of structural proteins. While this is not the gold standard as recommended by the ICTV (nucleotide similarity across the genome as specified by VIRIDIC), it is probably as close as is possible, given the paucity of suitably similar reference genomes. For instance we attempted to identify the closest phage genome in both INPHARED (Cook et al., 2021 1 ) and PhageClouds (Rangel-Pineros et al., 2021 2 ) (at both 0.15 and 0.2 distance), which yielded no hits. Therefore, the cautious interpretation of the placement by the authors is appropriate and should be revisited when more closely related genomes are deposited.

The reviewer Ruben Avendano-Herrera is incorrect in requesting that the authors use the term Myoviridae. This term was abandoned by the ICTV in their latest guidance which now requires the use of 'myovirus' to describe morphology, due to a lack of evidence for a shared common ancestor (Turner et al., 2023[ref-3).

Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Yes

Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Partly

Are the rationale for sequencing the genome and the species significance clearly described?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

phage biology; phage bioinformatics

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

References : INfrastructure for a PHAge REference Database: Identification of Large-Scale Biases in the Current Collection of Cultured Phage Genomes. Phage (New Rochelle) .2021;2(4) : 10.1089/phage.2021.0007 214-223 36159887 10.1089/phage.2021.0007 : From Trees to Clouds: PhageClouds for Fast Comparison of ∼640,000 Phage Genomic Sequences and Host-Centric Visualization Using Genomic Network Graphs. Phage (New Rochelle) .2021;2(4) : 10.1089/phage.2021.0008 194-203 36147515 10.1089/phage.2021.0008 : Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee. Arch Virol .2023;168(2) : 10.1007/s00705-022-05694-2 74 36683075 10.1007/s00705-022-05694-2 10.5256/f1000research.157743.r283363 Reviewer response for version 1 Avendaño-Herrera Ruben 1 Referee Universidad Andres Bello, Santiago, Santiago Metropolitan Region, Chile

Competing interests: No competing interests were disclosed.

 10 6 2024 Copyright: © 2024 Avendaño-Herrera R 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The manuscript is well written and requires the modifications indicated below. On the other hand, the title is too generic, considering that it only has activity against one species of Aeromonas, especially one isolate. The title needs to be more specific. It is suggested to modify it. The main issue is that the conclusion is too speculative, and further evidence is needed to confirm whether it corresponds to a new genus. Therefore, this situation should be verified and confirmed, or else it should be concluded that the bacteriophage has activity against an Aeromonas isolate. There are results that are not provided and are indicated at the end of this evaluation.

Abstract

Change to 64,847 bp

Amino acids (aa)

Keywords

Italicize Aeromonas veronii

Introduction Reference No. 1 could be replaced by the FAO report or another article more relevant to the statement.

"Aeromonoses" seems to be a term not globally used. Replace it with " Aeromonas infections" or " Aeromonas diseases."

Bacteriophage isolates

 How many are the isolates from the collection of the two Aeromonas species?

LB, the full name of the medium should be provided before the abbreviation.

How was it identified that AV-3 corresponds to the species A. veronii?

This should be assured, as the taxonomy of this group is not easy and requires molecular analysis. Make this clarification for all Aeromonas used in this study.

"Strain" or "isolates," I think the latter. Review throughout the article.

It is not necessary to give the LB recipe twice; just indicate the agar percentage in the last case.

What concentration of DNA was sent for sequencing? This data is very important.

Specify what "bp" is and correct the abbreviation, removing the periods.

Results

Indicate that it belongs to the family Myoviridae instead of "myovirus" in italics.

The last lines where the conclusion is drawn, I believe it is too speculative to state that it corresponds to a new genus and further tests are required to confirm it.

The article should modify the conclusion and reduce the enthusiasm, unless there are other tests that prove it. I suggest modifying it. If it was tested with a collection of Aeromonas, I would like to know if the activity is broad-spectrum or if it's only an isolate that was analyzed. This situation is very important.

Additionally, was any physiological characterization of the virus conducted, considering that the introduction suggests they are an alternative for use in fish aquaculture?

Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Partly

Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Partly

Are the rationale for sequencing the genome and the species significance clearly described?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Some of my main research topics include pathology in aquaculture; diseases of aquatic organisms; biotechnological tools applied to aquaculture; standardization of procedures to control and validate the appropriate use of antibiotics; and the development of vaccines against aquatic pathogens.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.