Background

Aeromonas and Pseudomonas are considered one of the most important fish pathogens among the etiological agents of bacterial fish diseases with capacity of hemolysis and biofilm formation [1,2,3]. These aquatic bacteria are responsible for ulcer type diseases including ulcerative syndrome, bacteria haemorrhagic septicaemia, tail and fin rot, bacteria gill rot and dropsy [4,5,6,7,8,9,10]. The increasing prevalence of bacterial infections leads to the indiscriminate use of antimicrobials that are the most common solution in combating pathogenic microorganisms. However, in the case of aquaculture, the range of authorized antibiotics is very narrow, e.g. the Food and Drug Administration (FDA) in 2014 approved only oxytetracycline, florfenicol, and sulfadimethoxine/ormetoprim [4]. In addition, the vast majority of bacteria pathogenic to aquaculture are resistant to multiple antibiotics [11, 12]. That forces fish farmers to look for alternative solutions that allow effective protection of breeding populations. Bacteriophages (phages) are one alternative solution that could be considered as a prospective anti-microbial strategy in aquaculture [1, 13,14,15,16,17,18,19,20,21,22,23,24]. Bacteriophages are the most abundant biological entities on Earth. In almost all ecosystems that so far have been subjected to in-depth studies, it has been estimated that there are around ten phages for every microbial cell giving approximately1030–1031 phages globally [25, 26]. However, their genomic sequences represent a small fraction in public databases. Only 1956 bacteriophage genomes are currently available in the NCBI genome database [National Center for Biotechnology Information (NCBI) Genome database https://www.ncbi.nlm.nih.gov/genome . Accessed 27 Nov 2018], of which 19 are genome sequences of phages infecting Aeromonas (phages belonging to Myoviridae, Podoviridae, and Siphoviridae families) and 144 infecting Pseudomonas (phages belonging to Myoviridae, Podoviridae, Siphoviridae, Cystoviridae, Leviviridae, Inoviridae families and 5 unclassified). Phages useful for therapeutic purposes must meet numerous criteria, from which the most important is their lytic nature. Only whole genome sequencing (WGS) enable an in-depth characterization of phages and the choice of the phages suitable for phage therapy. In this study, we present 7 new phages infecting Aeromonas and 4 new phages infecting Pseudomonas that significantly increase the genomic information of Aeromonas and Pseudomonas phages. Furthermore, we present results of bioinformatics analysis of genomic data assessing the likelihood of these phages to enter the lysogenic cycle and experimental data on their specificity towards a large number of bacterial field isolates representing different location. Taken together these data provide an essential basis for rational selection of bacteriophages for application in phage therapy of affected populations.

Methods

Bacterial strains isolation

Bacterial strains were isolated from diseased rainbow trout (Oncorhynchus mykiss, Walbaum 1972) from 12 different freshwater farms in Poland. Samples were collected from March 2013 to August 2014 and at least 10 fish from each farm were examined. For further research samples of damaged tissues (spleen, kidney, gills) were taken. Prepared samples were diluted with sterile phosphate-buffered saline (PBS) at 1:1 (w/v). The suspensions were incubated at 27 °C for 48 h on Aeromonas selective medium (AM) (Oxoid, Basingstoke, UK) and King B Agar (Biolab, Polska). The presumed Aeromonas and Pseudomonas colonies were isolated and identified by Gram-stain, oxidase and catalase tests, standard biochemical characteristics using the API 20NE system (bio Merieux, France) and genetically by restriction fragment length polymorphism analysis (RFLP) of the 16S rRNA gene for Aeromonas spp. [27] and by species-specific PCR with DNA primers against a sequence of the 16S rRNA gene for P. fluorescens described by Scarpellini et al. [28].

Forty five strains were isolated during this study and 13 were provided by the Adam Mickiewicz University in Poznan as reference strains (both available in public repositories and clinical human isolates, Table 1.). The strains were kept at − 80 °C in LB broth supplemented with 25% glycerol. Strains were grown at 25 °C.

Table 1 Bacterial strains
Full size table

Bacteriophage isolation

Bacteriophages were isolated from samples taken from the intake manifolds, representing an initial stage of the wastewater treatment process, received from the Main Sewage Treatment Plant (GOS) in Lodz or from samples of fish pond water obtained from The Stanisław Sakowicz Inland Fisheries Institute (IRS) in Olsztyn (Table 2.). The enrichment protocol was used following the procedure given by Van Twest and Kropinski [29]. Briefly, wastewater or pond water samples were filtered through a sterile filter with a pore diameter of 0.2 μm (Sartorius). Appropriate volume of purified water sample was mixed with the same volume of 2x concentrated LB broth (LabEmpire) and bacterial culture to be used in the enrichment. The enrichment cultures were incubated for 20 h at 25 °C with agitation to allow amplification of bacteriophages active against strain used in the enrichment. Following incubation, the culture was centrifuged at 4000×g, at 4 °C for 30 min and supernatant was filtered through a sterile filter (0.2 μm). The presence of lytic bacteriophages in supernatant was detected by a modified version of the double-layer method [30]. One hundred microliters of bacteriophages was mixed with 100 μl of host cells and added to four milliliters of 48 °C top agar (LB with 0.7% agar). Then, the mixture was poured onto bottom agar plate (LB with 2% agar) and incubated for 24 h. The presence of bacteriophages in the form of plaques was detected. All enrichments and phage titrations were carried out at 25 °C.

Table 2 Bacteriophage strains
Full size table

For purification of single bacteriophages, a single plaque was picked with a sterile Pasteur pipette and the phages were eluted with shaking for a minimum of 1.5 h in SM buffer (50 mM Tris-Cl, pH 7.5, 99 mM NaCl, 8 mM MgSO4, 0.01% gelatin). After chloroform (50 μl/ml) extraction and centrifugation (9000 x g, 5 min, room temp.), the supernatant was transferred to a new tube. Five successive plaque purifications were carried out on each phage isolate.

Phages were annotated by giving a number and abbreviation coming from the host strain name. The phage samples were stored at 4 °C.

Host range

The host range was determined via spot test against 49 Aeromonas spp. and 9 Pseudomonas sp. strains. Bacterial lawns of each strain were made in triplicates using the double agar overlay method, on which 20 μl droplets of the phage stocks (diluted to 106 plaque-forming units (PFU)/ml) were applied. After overnight incubation, the degree of lysis of the lawns was determined. The spot test was repeated three times for each phage. The following spot evaluation system was used: completely clear spot – complete bacterial lysis in the spot, turbid spot-weak bacterial lysis in the spot, no clearing – no bacterial lysis in the spot.

Transmission electron microscopy

Visualization of bacteriophages by transmission electron microscopy were based on the method described by Maszewska et al. [31]. The high titer bacteriophage lysates were centrifuged at 24500 g for 3 h at 4 °C. Then the phages were washed twice with 5% ammonium molybdate solution (Sigma-Aldrich) pH 6.0 using the above spin conditions. The final sediments were suspended in 5% ammonium molybdate to obtain the titer of 1011 pfu ml− 1. Subsequently, one drop of the phage suspension was placed onto the formvar and carbon coated 200-mesh copper grid (Polysciences, Inc., Warrington, USA) and drained for 3 min. Then samples were negatively stained for 45 s. with 2% (w/v) phosphotungstic acid (PTA) in darkness. The ultrastructure of bacteriophages was visualized by transmission electron microscopy (TEM) with the JEM 1010 electron microscope (JOEL Ltd., Tokyo, Japan) at 80 kV in the Laboratory of Microscopic Imaging and Specialized Biological Techniques of the Faculty of Biology and Environmental Protection, University of Lodz. To examine bacteriophages samples the magnification of 60,000 to 100,000 was used.

DNA extraction and purification

Genomic DNA were extracted using the modified method of Su et al. [32]. Briefly, bacteriophage lysates obtained after propagation on host strain was subjected to DNase I for disrupting debris of bacterial DNA. Then, for pelleting the phage particles 2 M ZnCl2 solution in 1:50 (v:v) was used. Next, the phage pellet was dissolved in TENS buffer (50 mM Tris-HCl, pH 8.0, 100 mM EDTA, 100 mM NaCl, 0.3% SDS) supplemented with proteinase K, which disrupted phage capsids. Deproteinated phage DNA was subjected to the extraction by the solution of phenol/chloroform/isoamyl alcohol (25:24:1).

Eluted DNA concentrations were measured using a BioSpectrometer® (Eppendorf, Hamburg, Germany) and stored at − 20 °C for further analysis.

RFLP analysis

Digestion reaction was performed by incubating 1 μg of isolated DNA with 2.5 U of enzyme (ThermoScientific) in a final reaction volume of 20 μl at 37 °C for 2 h. The restriction fragments were separated by 1.5% agarose gel electrophoresis in TAE buffer for 2 h at 30 V and stained by the nucleic acid stain (SimplySafe™, Eurx).

DNA sequencing

Bacteriophage genomes were sequenced by whole genome sequencing (WGS). Whole-genome shotgun sequencing was performed on the Illumina platform. DNA was sequenced using MiSeq with 2 × 300 bp reads and assumed coverage 1000 times. The actual coverage and average contig length for each bacteriophage is presented in (Additional file 1: Table S1.). The draft genomes were de novo assembled by CLC Genomic Workbench 7.5 in Genomed, Poland.

Bioinformatic analysis

Bioinformatic analysis started with annotation of assembled genomes which was carried out automatically using DNA Master v 5.23.2 based on GeneMarks and Glimmer algorithms (tool written by Dr. Jeffrey Lawrence, the University of Pittsburgh). Then, reference sequences were found using the Basic Local Alignment Search Tool (BLASTn), NCBI which allowed to classify analyzed bacteriophages into taxonomic groups. Circular genomic maps were obtained in GenomeVx, a tool for circular chromosome visualisation (http://wolfe.ucd.ie/GenomeVx/, accessed September 2018) while linear maps were prepared in Biopython 1.72. Determination of lytic or lysogenic lifecycle was performed on the basis of PHACTS [33] as well as on careful analysis of each ORF (open reading frame) determined by DNA Master. It was performed both in BLASTp and in HHPred at web service MPI Bioinformatics Toolkit (toolkit.tuebingen.mpg.de/#/tools/hhpred; accessed February 2018) which finds remote homologs of query amino acid sequences. tRNA genes were searched using ARAGORN, a program to detect tRNA and tmRNA genes [34]. A phylogenetic tree was created based on the sequences of terminase large subunit. The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model [35]. The tree with the highest log likelihood (− 11,846.74) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 17 amino acid sequences. There were a total of 870 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [36]. Identification of antibiotic resistance genes and virulence factors was performed with help of online tools from CGE server: ResFinder 3.0 [37] and VirulenceFinder 1.5 [38]. The genome sequences of phages described in this study were deposited in GenBank under accession numbers: MH179470 – MH179480.

Results

Thirty six Aeromonas spp. and nine Pseudomonas sp. bacterial strains were isolated from infected fish (Table 1.) and eleven bacteriophage strains were isolated from environment: seven active against Aeromonas spp. and four against Pseudomonas sp., for which 5 Aeromonas hydrophila and 2 Pseudomonas fluorescens strains were used routinely as the hosts (Table 2.).

For all of the phages we assessed the host range: in the case of the Aeromonas phages with a panel of 49 Aeromonas spp. isolates (A. hydrophila, A. salmonicida, A. sobria) and in the case of Pseudomonas phages with 9 P. fluorescens isolates (Table 3.). The resulting host range patterns were different for each tested phage. The broadest host range had 13AhydR10PP, 14AhydR10PP, 85AhydR10PP, 22PfluR64PP and 67PfluR64PP (42–51% of bacterial isolates were sensitive to these phages). Phage 25AhydR2PP had the narrowest host range with only 4/49 (8%) of bacterial isolates being sensitive to it. Overall, these phages showed different but complementary host ranges.

Table 3 Bacteriophages’ specificity
Full size table

Visualization of bacteriophages by transmission electron microscopy showed that the tested bacteriophages 13AhydR10PP, 14AhydR10PP, 50AhydR13PP, 60AhydR15PP, 85AhydR10PP consisted of a polyhedral head and tail, which allowed classifying them in the order Caudovirales. In addition those phages were found to have a contracted tail characteristic for viruses belonging to the family Myoviridae. In contrary, phages 22PfluR64PP, 25AhydR2PP, 62AhydR11PP, 67PfluR64PP, 71PfluR64PP, 98PfluR60PP consisted of a polyhedral head and very short tail characteristic for viruses belonging to the family Podoviridae (Fig. 1).

Fig. 1
figure 1

TEM micrographs of Aeromonas phages: 13AhydR10PP (a, magnification 100,000x), 14AhydR10PP (b, magnification 60,000x), 25AhydR2PP (c, magnification 60,000x), 50AhydR13PP (d, magnification 60,000x), 60AhydR15PP (e, magnification 60,000x), 62AhydR11PP (f, magnification 100,000x), 85AhydR10PP (g, magnification 60,000x), and Pseudomonas phages: 22PfluR64PP (h, magnification 100,000x), 67PfluR64PP (i, magnification 100,000x), 71PfluR64PP (j, magnification 60,000x), 98PfluR60PP (k, magnification 60,000x)

Full size image

Afterwards, isolation of DNA and restrictive analysis with enzymes: SspI and EcoRI were carried out. Obtained restriction profiles (Additional file 2: Figure S1.) allowed for the definition of initial genetic characteristics of the bacteriophages. Subsequently, after NGS sequencing (Additional file 1: Table S1.), a detailed genetic analysis of bacteriophages was performed (Table 4.). It was found that phages 13AhydR10PP, 14AhydR10PP and 85AhydR10PP possess genome sizes about 47–48 kbp and belong to double-stranded DNA viruses of Myoviridae family with circular genomes. They are homologues of bacterial viruses: Aeromonas phage 32, Aeromonas phage Asp37, Aeromonas phage 3, Aeromonas phage Ahp2 and Aeromonas phage 59.1. Moreover, their lifestyles were classified as lysogenic after conducting bioinformatic analysis, i.e. analysis of presence of genes coding for certain characteristic proteins (such as integrases or resolvases) among their ORFs and analysis of their amino acid sequences in PHACTS. They are highly similar to each other, with 96% query cover and 96% identity between 13AhydR10PP and 14AhydR10PP and 78% query cover and 89% identity between 13AhydR10PP or 14AhydR10PP and 85AhydR10PP. Phages 50AhydR13PP and 60AhydR15PP were also classified to Myoviridae family (Caudovirales order), containing linear double-stranded DNA (with circularly permuted genome) in size of approximately 165 kbp, but showing high similarity to the lytic Myoviridae bacteriophages specific against many bacteria from Aeromonas sp. They are highly similar to each other, with 94% query cover and 97% identity. PHACTS classified both of them as confidently lytic. Unclassified phage 62AhydR11PP with genome size of about 44 kbp showed low similarity with Aeromonas-infecting phages of Myoviridae family. It is also similar to the group of viruses with PLPE-like virion morphology. However TEM analysis allowed to classify this phage to Podoviridae family. Careful analysis of its ORFs allowed for the classification of this phage as probably lysogenic. Phages 25AhydR2PP, 22PfluR64PP, 67PfluR64PP and 71PfluR64PP belong to Caudovirales, Podoviridae, Autographivirinae with short, unshrinkable tails and icosaedral capsid containing linear double-stranded DNA of approximately 40 kbp in size. They showed a high similarity to lytic bacteriophages of T7 group specific to bacteria of the Aeromonas and Pseudomonas sp. Genome representations of these phages are linear with direct terminal repeats (DTRs). Among their ORFs no proteins responsible for lysogeny were found. Therefore, it was assumed that they exhibit lytic lifestyle. Phage 98PfluR60PP with genome size about 74 kbp has one reference genome in the NCBI database, i.e. Pseudomonas phage Littlefix active against Pseudomonas sp. It is classified into Podoviridae family, however with ORFs showing no or very little similarity to any known phage proteins therefore it was impossible to classify the genome of 98PfluR60PP as lytic or lysogenic on the basis of current knowledge. Labeled genetic maps (linear or circular depending on the genome) of all analyzed phages are presented in (Additional file 3: Figure S2, Additional file 4: Figure S3, Additional file 5: Figure S4, Additional file 6: Figure S5, Additional file 7: Figure S6, Additional file 8: Figure S7, Additional file 9: Figure S8, Additional file 10: Figure S9, Additional file 11: Figure S10, Additional file 12: Figure S11 and Additional file 13: Figure S12.). Afterwards, all genomes were subjected to phylogenetic analysis (Fig. 2.). The related phages can be divided into the following groups, along with the systematic classification given by GeneBank: Gr.1: 22PfluR63PP, 67PfluR64PP, 71PfluR64PP with the reference strain Pseudomonas phage PFP1, belong to the genus T7virus, Gr.2: 13AhydR10PP, 14AhydR10PP, 85AhydR10PP with the reference strain Aeromonas phage 32 are most likely to belong to the Myoviridae family, Gr.3: 25AhydR2PP together with the reference strain Aeromonas phage phiAS7 belong to the sub-family Autographivirinae, Gr.4: 98PfluR60PP with the reference strain Pseudomonas phage Littlefix belong to the Podoviridae family, Gr.5: 50AhydR13PP, 60AhydR15PP with the reference strain Aeromonas phage phiAS4 belong to the genus unclassified Secunda5virus.

Table 4 Genomic features of bacteriophages
Full size table
Fig. 2
figure 2

Phylogenetic tree of phage genomes (phages described in this study together with the reference strains)

Full size image

The 62AhydR11PP phage appears to be unique. It has low similarity to phage Aeromonas 56 which is reflected on the phylogenetic tree, and they do not occur in the same clade. 62AhydR11PP bacteriophage due to the lack of similar sequences in the NCBI database and separation on the phylogenetic tree may belong to the group of phages that has not been recognized yet. Finally, all of the studied phages are deprived of any antibiotic resistance or virulence genes according to conducted bioinformatics analysis.

Discussion

Among the entire population of phages only a few have the potential for use in phage therapy. It was previously estimated that only 50% of phages isolated from the environment are useful for therapeutic purposes [39]. However, the development of new analytical methods, including WGS, can change this proportion. Whole genome sequencing is an indispensable tool used in the study of phage biology [40,41,42,43]. WGS facilitates a detailed characterisation of phages that allows them to be classified as useful for therapeutic purposes. Phages that are promising for phage therapy should be excluded of toxin genes, virulence factor genes, and genes responsible for lysogeny [44,45,46,47]. In this study, we demonstrated 11 new bacteriophages among which 6 (25AhydR2PP, 50AhydR13PP, 60AhydR15PP, 22PfluR64PP, 67PfluR64PP, 71PfluR64PP) have a potential to be used in phage therapy due to confirmed lytic lifestyle and absence of virulence or resistance genes. At the same time, we observed (Table 3.) that most of the bacteriophages identified exhibited relatively narrow specificity to bacterial isolates. Selected Aeromonas phages revealed activity against only 8–35% of the whole examined Aeromonas collection but at the same time shows specificity towards A. hydrophila, A. salmonicida and A. sobria. This characteristic is contrary to previously described broad host range Aeromonas phages that act against only one Aeromonas species [13]. Narrow specificity of selected phages might create an obstacle in the development of effective phage treatment for Aeromonas sp. and Pseudomonas sp. infections. One potential way to overcome this challenge would be the creation of a multicomponent phage cocktail, consisting of completely characterized lytic bacteriophages. Using a mixture of bacteriophages to expand the coverage of treatment for heterogenous bacterial populations in bacteriophage therapy has shown high efficacy for many years. However, in most, if not all cases such bacteriophage mixtures do not consist of fully characterized bacteriophages [48, 49]. Selecting multiple components, that fulfil certain criteria, such as full genomic information, a lack of genes that create safety concerns, negligable risk of lysogenic lifestyle, and reproducible stability in the production environment, creates a significant challenge.

Bacteriophages isolated in this study have also significantly increased the knowledge about Aeromonas and Pseudomonas phages. In currently available genomes in the NCBI database among 19 described Aeromonas phages, 16 belong to Myoviridae (84% of whole population), only 2 to Podoviridae (10% of whole population) and 1 to Siphoviridae (6% of whole population). Myoviridae family members are most likely to be abundant in natural environments [13, 39, 50,51,52] and when compared to public databases, it is represented on a similar level in this study (71%). A comparable situation can be observed for Podoviridae phage population which is represented by 14% of Aeromonas phages in this study. However among the described collection there was one unclassified phage proving to be unique comparing in the public database. In the case of Pseudomonas phages, 41 genomes from the database belong to Myoviridae (28% of whole population), 46 belong to Podoviridae (32% of whole population), 44 belong to Siphoviridae (31% of whole population), 4 belong to Cystoviridae (3% of whole population), 2 belong to Leviviridae (1% of whole population), 2 belong to Inoviridae (1% of whole population) and 5 are unclassified (3% of whole population). When compared to the publicly available genomes, in the collection described in this study, only the Podoviridae family is represented and no other phage families were observed in the analyzed set of Pseudomonas phages. The summary of these findings is presented in Table 5.

Table 5 Taxonomy comparison of Aeromonas and Pseudomonas phage genomes from the database and from this study
Full size table

Conclusions

To conclude, 11 new bacteriophages were isolated and described on genomic level. Of these, only six have potential for phage therapy in aquaculture due to their proven lytic nature and their lack of antibiotic resistance and virulence genes. Four of them belong to the Podoviridae, while two to the Myoviridae family. The composition of these phages could be used as a therapeutic cocktail giving the cover of 41% of the Aeromonas and 44% of Pseudomonas pathogenic environmental isolates. Other phages described in this study should be excluded from any therapeutic cocktail composition due to identification of genes responsible for lysogenesis in their genomes.