Aquaculture industry needs new options to prevent and control bacterial infections in its different areas, and perhaps many treatments were explored, the use of phages is still and attractive option to substitute or complement other therapies [47]. One of the main advantage of phages is their quantity and diversity found in nature, where is a limitless source of new phages [48]. Considering this, the isolation of new phages is necessary to access a new phage strains with the potential for phage therapy and provide new genomic information inside them. Here, wWe isolated a new lytic phage vB_Vc_SrVc2 against Vibrio parahaemolyticus AHPND and described its potential for phage therapy in aquaculture.
According to Fernandez et al, (2019) phage particles consist only of acid nucleic with a proteinaceous envelope, making them a more complex and labile compounds than other antimicrobials, so a previous analysis of their stability to different environmental conditions (marine water, fresh water, digestive tract, soil, etc.) is necessary for phage selection [48]. Also, the isolation of phages from same sites where we found the pathogenic bacteria, it is a good option to increase the proportion of adequate phages; because a high probability to found specific phages well adapted to site conditions is expected, considering a posterior re-introduction in similar sites during phage therapy application [48, 49]. Based on this point, wWe isolated vB_Vc_SrVc2 phage from samples of white shrimp ponds previously reported with AHPND problems, who which was stable at different values of temperature and salinity as expected, with at least a 40 % survival of phage virions. Also, the similar phage survival results were obtained at different UV radiation and chloroform values.
vB_Vc_SrVc2 phage was able to recognize and replicate using three vibrio species, with EOP values from 0.04 and 0.5, however a reduced host availability limited a deeper characterization. Also, vB_Vc_SrVc2 has a lower EOP with CIBGEN-001 and CIBGEN-004 host compared to EOP values of phage vB_Vc_SrVc9 using the same host. The smaller number of genetic differences between the two phages, narrows down the possible genes involved in this. However, further work is; however a new test is needed to determine the exact mechanism that causes the difference in EOP if phages differences are due to recognitions sites or other characteristics. Recognition of strains or even species inside a genera is a desirable result because bacteriophages could use similar receptors on membrane surface of related bacteria, like Harveyi clade [50, 51]; but this characteristic is not common in all phages [17]. Also, phage vB_Vc_SrVc2 was able to inhibit bacterial growth during 12 h with a unique dose of phage (MOI 0.1 for each strain). All these characteristic, showed that vB_Vc_SrVc2 phage is a good candidate for phage therapy against Vibrio infections and also could be used with other phages (phage cocktail) [52, 53] or other agents like probiotics [54, 55] and even antibiotics [56, 57].
vB_Vc_SrVc2 is incorporated into the recently created genus Maculvirus with a genomic organization and GC content similar to other vibriophages previously reported in GenBank (March 2021). The variation between the genomes of vB_Vc_SrVc2, VP93 and OWB is primarily concentrated in regions containing protein-coding genes that include transcriptional regulators, bacterial surface binding receptors and proteins with unknown function, while the main differences between vB_Vc_SrVc2 and vB_Vc_SrVc9 are in nucleotide metabolism, such as amino acid substitutions in the primase in vB_Vc_SrVc2, however, these changes were not found in the active or metal-binding site of the protein [58] and appear unlikely to have marked effects on function, suggesting alternative events, like competitive interaction between phages [59], host-specific changes [60] or genomic variations or small intraspecies genomic variations [61] that may favor slightly different infection profiles between vB_Vc_SrVc2 and vB_Vc_SrVc9 despite their high genomic identity. Further evaluation would be necessary to determine whether minor changes in the genome were solely responsible for the shift in infectivity range.
Interestingly, although several host recognition proteins in vB_Vc_SrVc2 maintain a percentage of identity above 90% compared to phages of the same genus, major differences in the amino acid sequence of the tail fiber protein between vB_Vc_SrVc2, VP93 and OWB may indicate distinct cell-surface targets for each of these phages since the changes observed between vB_Vc_SrVc2 and VP93 are conserved with little change, while greater divergence exists between vB_Vc_SrVc2 and OWB. These changes in host recognition patterns have been previously described in genetically identical phages infecting Vibrio anguillarum, suggesting that non-genomic mechanisms affect the functional properties of phages, expanding diversity beyond genomic variations [60] However, it has also been documented, that small intraspecies genomic variations can have phenotypic consequences due to minor changes in the genome, which can cause a significant change in the range of infectivity [61], for example, small point mutations can help in the evasion of host restriction-modification systems [62] or specific nucleotide variations, over generations, can change the phage's host range [63].
It is important to note, that the phages most closely related to SrVc2 like vB_VpaS_OWB isolated from the Atlantic Ocean water [64], Vibriophage VP93 isolated from the Pacific Ocean of Chile [65], and vB_VpaP_KF1 (KF1) & B_VpaP_KF2 (KF2) isolated from Western and Southern coastal areas of Korea [66]; are also capable to infect Vibrio parahaemolyticus strains. Previous studies on protein expression of KF1 and KF2 phages confirmed the role of two homologous proteins found in vB_Vc_SrVc2, both ORF 8 coding for a peptidase and ORF 41 coding for a glycosyl hydrolase, demonstrated to be lytic proteins, being the peptidase, the one that showed the highest lytic activity [67]. Genomic information on MGD1, DE17, and FE11 phages are available in GenBank but there are no references on the biology or host of these phages yet. To date, vB_VcaS_HC (Accession number: MK559459.1) comprises the second reference (besides vB_Vc_SrVc9) to a vibriophage capable of infecting Vibrio campbellii [68] however vB_VcaS_HC contains a potential lysogeny genepotential lysogeny related gene (RecA) in its genome. BLASTnp analysis showed no significant similarity between vB_Vc_SrVc2 and vB_VcaS_HC.
On the other hand, using genes encoding for individual proteins (major capsid protein and DNA polymerase) we concluded that phage vB_Vc_SrVc2 belonged to the Autographiviridae family. These genes are widely used to assess viral diversity [69], because containas they several conserved regions [70] that allow a better resolution for phage relationships. Additionally, the high identity with other vibriophages also classified within this family as well as the systematic classification resulting from using VICTOR Victor's server, were indicatativeors of phage vB_Vc_SrVc2 taxonomic position. In addition, the phylogenetic classification obtained from the DNA polymerase allowed a much better differentiation among members of the Maculvirus genus, even with the high genetic identity presented by phage vB_Vc_SrVc2 and phage vB_Vc_SrVc9. The aforementioned could indicate a useful phylogenetic marker for this specific genus. Although it is recommended that it be used with additional phylogenetic markers, known for their ubiquity among phages and high level of sequence conservation such as the terminase large subunit (TerL) to avoid false phylogenetic signals [71].
Phage therapy represents an opportunity against the spread of multi-resistant bacterial pathogens. Phage vB_Vc_SrVc2 offers an alternative against infections caused by Vibrio campbellii due to its genetic composition characterized for therapeutic applications [72]. The absence of genes that promote lysogeny or multi-resistance, the use of lytic lifestyle, and antibacterial efficacy evaluated by experimental techniques make phage vB_Vc_SrVc2 a suitable candidate for therapeutic use in the aquaculture industry.