Prophages carrying Zot toxins on different Vibrio genomes: A comprehensive assessment using multilayer networks.

Vibrios, a group of bacteria that are among the most abundant in marine environments, include several species such as Vibrio cholerae and Vibrio parahaemolyticus, which can be pathogenic to humans. Some species of Vibrio contain prophages within their genomes. These prophages can carry genes that code for toxins, such as the zonula occludens toxin (Zot), which contribute to bacterial virulence. Understanding the association between different Vibrio species, prophages and Zot genes can provide insights into their ecological interactions. In this study, we evaluated 4619 Vibrio genomes from 127 species to detect the presence of prophages carrying the Zot toxin. We found 2030 potential prophages with zot-like genes in 43 Vibrio species, showing a non-random association within a primarily modular interaction network. Some prophages, such as CTX or Vf33, were associated with specific species. In contrast, prophages phiVCY and VfO3K6 were found in 28 and 20 Vibrio species, respectively. We also identified six clusters of Zot-like sequences in prophages, with the ZOT2 cluster being the most frequent, present in 34 Vibrio species. This analysis helps to understand the distribution patterns of zot-containing prophages across Vibrio genomes and the potential routes of Zot-like toxin dissemination.

Valp1, a Newly Identified Temperate Phage Facilitating Coexistence of Lysogenic and Non-Lysogenic Populations of Vibrio anguillarum.

Vibrio anguillarum is a pathogen for several fish and shellfish species. Its ecology is influenced by diverse factors, including bacteriophages. Here, we identify and characterize a new temperate bacteriophage (Valp1) of V. anguillarum. Valp1 is a myovirus with a 60 nm head and a 90 nm contractile tail. Its double-stranded DNA genome of 42,988 bp contains 68 genes, including a protelomerase gene, typical of telomeric phages. Valp1 inhibits the growth of the virulent strain of V. anguillarum PF4, while the derived lysogenic strain P1.1 presents a slight reduction in its growth but is not affected by the presence of Valp1. Both strains present similar virulence in a larval zebrafish (Danio rerio) model, and only slight differences have been observed in their biochemical profile. Co-culture assays reveal that PF4 and P1.1 can coexist for 10 h in the presence of naturally induced Valp1, with the proportion of PF4 ranging between 28% and 1.6%. By the end of the assay, the phage reached a concentration of ~108 PFU/mL, and all the non-lysogenic PF4 strains were resistant to Valp1. This equilibrium was maintained even after five successive subcultures, suggesting the existence of a coexistence mechanism between the lysogenic and non-lysogenic populations of V. anguillarum in conjunction with the phage Valp1.

ECOPHAGE: Combating Antimicrobial Resistance Using Bacteriophages for Eco-Sustainable Agriculture and Food Systems.

The focus of this meeting was to discuss the suitability of using bacteriophages as alternative antimicrobials in the agrifood sector. Following a One Health approach, the workshop explored the possibilities of implementing phage application strategies in the agriculture, animal husbandry, aquaculture, and food production sectors. Therefore, the meeting had gathered phage researchers, representatives of the agrifood industry, and policymakers to debate the advantages and potential shortcomings of using bacteriophages as alternatives to traditional antimicrobials and chemical pesticides. Industry delegates showed the latest objectives and demands from consumers. Representatives of regulatory agencies (European Medicines Agency (EMA) and Spanish Agency of Medicines and Health Products (AEMPS)) presented an update of new regulatory aspects that will impact and support the approval and implementation of phage application strategies across the different sectors.

Correction: Villalobos-González et al. Photoprotection Is Achieved by Photorespiration and Modification of the Leaf Incident Light, and Their Extent Is Modulated by the Stomatal Sensitivity to Water Deficit in Grapevines. Plants 2022, 11, 1050.

In the original publication [...].

Long-Term Interactions of Salmonella Enteritidis With a Lytic Phage for 21 Days in High Nutrients Media.

Salmonella spp. is a relevant foodborne pathogen with worldwide distribution. To mitigate Salmonella infections, bacteriophages represent an alternative to antimicrobials and chemicals in food animals and food in general. Bacteriophages (phages) are viruses that infect bacteria, which interact constantly with their host. Importantly, the study of these interactions is crucial for the use of phages as a mitigation strategy. In this study, experimental coevolution of Salmonella Enteritidis (S. Enteritidis) and a lytic phage was conducted in tryptic soy broth for 21 days. Transfer to fresh media was conducted daily and every 24 hours, 2 mL of the sample was collected to quantify Salmonella OD600 and phage titter. Additionally, time-shift experiments were conducted on 20 colonies selected on days 1, 12, and 21 to evaluate the evolution of resistance to past (day 1), present (day 12), and future (day 21) phage populations. The behavior of the dynamics was modeled and simulated with mathematical mass-action models. Bacteria and phage from days 1 and 21 were sequenced to determine the emergence of mutations. We found that S. Enteritidis grew for 21 days in the presence and absence of the phage and developed resistance to the phage from day 1. Also, the phage was also able to survive in the media for 21 days, however, the phage titer decreased in approx. 3 logs PFU/mL. The stability of the lytic phage population was consistent with the leaky resistance model. The time-shift experiments showed resistance to phages from day 1 of at least 85% to the past, present, and future phages. Sequencing of S. Enteritidis showed mutations in genes involved in lipopolysaccharide biosynthesis genes rfbP and rfbN at day 21. The phage showed mutations in the tail phage proteins responsible for recognizing the cell surface receptors. These results suggest that interactions between bacteria and phage in a rich resource media generate a rapid resistance to the infective phage but a fraction of the population remains susceptible. Interactions between Salmonella and lytic phages are an important component for the rational use of phages to control this important foodborne pathogen.

Photoprotection Is Achieved by Photorespiration and Modification of the Leaf Incident Light, and Their Extent Is Modulated by the Stomatal Sensitivity to Water Deficit in Grapevines.

Absorbed energy in excess of that used by photosynthesis induces photoinhibition, which is common in water deficit conditions, resulting in reductions in stomatal conductance. In grapevines, controlled water deficit is a common field practice, but little is known about the impact of a given water shortage on the energy transduction processes at the leaf level in relation to contrasting stomatal sensitivities to drought. Here, we assessed the effect of a nearly similar water deficit condition on four grapevine varieties: Cabernet Sauvignon (CS) and Sauvignon Blanc (SB), which are stomatal sensitive, and Chardonnay (CH) and Carménère (CM), which are less stomatal sensitive, grown in 20 L pots outdoors. Plants were maintained to nearly 94% of field capacity (WW) and 83% field capacity (WD). We have assessed plant water status, photosynthesis (AN), photorespiration, AN vs. PAR, ACi curves, photochemical (qP) and non-photochemical (qN) fluorescence quenching vs. PAR, the photoprotective effectiveness of NPQ (qPd) and light interception by leaves. Photorespiration is important under WD, but to a different extent between varieties. This is related to stomatal sensitivity, maintaining a safe proportion of PSII reaction centres in an open state. Additionally, the capacity for carboxylation is affected by WD, but to a greater extent in more sensitive varieties. As for qN, in WD it saturates at 750 µmol PAR m-2s-1, irrespective of the variety, which coincides with PAR, from which qN photoprotective effectiveness declines, and qP is reduced to risky thresholds. Additionally, that same PAR intensity is intercepted by WD leaves from highly stomatal-sensitive varieties, likely due to a modification of the leaf angle in those plants. Pigments associated with qN, as well as chlorophylls, do not seem to be a relevant physiological target for acclimation.

Comparison of Haptic and Biometric Properties, Bacterial Load, and Student Perception of Fixative Solutions: Formaldehyde Versus Chilean Conservative Fixative Solution with and without Formaldehyde in Pig Kidneys.

One of the most widely used solutions to fix and preserve organic tissues is formaldehyde, despite reservations regarding its toxicity and the fact that formaldehyde-embalmed bodies lose their original characteristics. Anatomy laboratories have been replacing formaldehyde with solutions that retain the characteristics of fresh tissue. For this purpose, alternative solutions with a very low concentration of formaldehyde or without any formaldehyde have been analyzed. The objective of this study was to compare biometry, coloration, haptic properties, and bacterial load on animal specimens (pig kidneys) embalmed with formaldehyde, and with Chilean Conservative Fixative Solution with and without formaldehyde (formaldehyde chCFS and formaldehyde-free chCFS). Also, the perception of health and biological science students toward specimens treated with different solutions was assessed. The results indicated that there were no significant differences in specimens' retraction, or bacterial load. Students showed a preference for organs embalmed in formaldehyde chCFS and formaldehyde-free chCFS; indicating that with these treatments they could better visualize structures and that the prosections had greater flexibility and the colors were more similar to those of fresh tissue. Additionally, students recommended the material embalmed in formaldehyde chCFS and formaldehyde-free chCFS for anatomy learning. In contrast, students indicated that formaldehyde-fixation negatively affected their practical experience. In conclusion, embalming with formaldehyde chCFS or formaldehyde-free chCFS provides an advantageous practical experience over the use of formaldehyde and may be an alternative to replace the use of formaldehyde in anatomy laboratories.

Analysis of the Zonula occludens Toxin Found in the Genome of the Chilean Non-toxigenic Vibrio parahaemolyticus Strain PMC53.7.

Vibrio parahaemolyticus non-toxigenic strains are responsible for about 10% of acute gastroenteritis associated with this species, suggesting they harbor unique virulence factors. Zonula occludens toxin (Zot), firstly described in Vibrio cholerae, is a secreted toxin that increases intestinal permeability. Recently, we identified Zot-encoding genes in the genomes of highly cytotoxic Chilean V. parahaemolyticus strains, including the non-toxigenic clinical strain PMC53.7. To gain insights into a possible role of Zot in V. parahaemolyticus, we analyzed whether it could be responsible for cytotoxicity. However, we observed a barely positive correlation between Caco-2 cell membrane damage and Zot mRNA expression during PMC53.7 infection and non-cytotoxicity induction in response to purified PMC53.7-Zot. Unusually, we observed a particular actin disturbance on cells infected with PMC53.7. Based on this observation, we decided to compare the sequence of PMC53.7-Zot with Zot of human pathogenic species such as V. cholerae, Campylobacter concisus, Neisseria meningitidis, and other V. parahaemolyticus strains, using computational tools. The PMC53.7-Zot was compared with other toxins and identified as an endotoxin with conserved motifs in the N-terminus and a variable C-terminal region and without FCIGRL peptide. Notably, the C-terminal diversity among Zots meant that not all of them could be identified as toxins. Structurally, PMC53.7-Zot was modeled as a transmembrane protein. Our results suggested that it has partial 3D structure similarity with V. cholerae-Zot. Probably, the PMC53.7-Zot would affect the actin cytoskeletal, but, in the absence of FCIGRL, the mechanisms of actions must be elucidated.

Characterization of Bacteriophages against Pseudomonas Syringae pv. Actinidiae with Potential Use as Natural Antimicrobials in Kiwifruit Plants.

Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of a bacterial canker in kiwifruit plants and has caused economic losses worldwide. Currently, the primary strategies to control this pathogen include the use of copper-based compounds and even antibiotics. However, the emergence of isolates of Psa that are resistant to these agrochemicals has raised the need for new alternatives to control this pathogen. Bacteriophages have been proposed as an alternative to control bacterial infections in agriculture, including Psa. Here, we show the isolation and characterization of 13 phages with the potential to control Psa infections in kiwifruit plants. The phages were characterized according to their host range and restriction fragment length polymorphism (RFLP) pattern. Four phages were selected according to their lytic effect on the bacteria and their tolerance to different environmental conditions of pH (4-7), temperature (4-37 °C), and solar radiation exposure (30 and 60 min). The selected phages (CHF1, CHF7, CHF19, and CHF21) were sequenced, revealing a high identity with the podophage of Psa phiPSA2. In vitro assays with kiwifruit leaf samples demonstrated that the mixture of phages reduced the Psa bacterial load within three hours post-application and was able to reduce the damage index in 50% of cases. Similarly, assays with kiwifruit plants maintained in greenhouse conditions showed that these phages were able to reduce the Psa bacterial load in more than 50% of cases and produced a significant decrease in the damage index of treated plants after 30 days. Finally, none of the selected phages were able to infect the other bacteria present in the natural microbiota of kiwifruit plants. These results show that bacteriophages are an attractive alternative to control Psa infections in kiwifruit plants.

Diversification of Vibrio anguillarum Driven by the Bacteriophage CHOED.

Bacteriophages are an important factor in bacterial evolution. Some reports suggest that lytic bacteriophages can select for resistant mutant strains with reduced virulence. The present study explores the role of the CHOED bacteriophage in the diversification and virulence of its host Vibrio anguillarum. Nine phage-resistant strains were analyzed for their phenotype and different virulence factors, showing alterations in their fitness, motility, biofilm formation, lipopolysaccharide profiles and/or protease activity. Seven of the nine phage-resistant strains showed virulence reduction in a Sparus aurata larvae model. However, this is not generalized since two of the resistant strains show equal virulence compared with the parental strain. The genomic analysis of representative resistant strains displayed that the majority of the mutations are specific for each isolate, affecting genes related to lipopolysaccharide biosynthesis, quorum sensing, motility, toxin and membrane transport. The observed mutations were coherent with the phenotypic and virulence differences observed. These results suggest that the CHOED phage acts as a selective pressure on V. anguillarum, allowing proliferation of resistant strains with different genotypes, phenotypes and degrees of virulence, contributing to bacterial diversification.

Bacteriophage Production Models: An Overview.

The use of bacteriophages has been proposed as an alternative method to control pathogenic bacteria. During recent years several reports have been published about the successful use of bacteriophages in different fields such as food safety, agriculture, aquaculture, and even human health. Several companies are now commercializing bacteriophages or bacteriophage-based products for therapeutic purposes. However, this technology is still in development and there are challenges to overcome before bacteriophages can be widely used to control pathogenic bacteria. One big hurdle is the development of efficient methods for bacteriophage production. To date, several models for bacteriophage production have been reported, some of them evaluated experimentally. This mini-review offers an overview of different models and methods for bacteriophage production, contrasting their principal differences.

Conservation of Small Regulatory RNAs in Vibrio parahaemolyticus: Possible role of RNA-OUT Encoded by the Pathogenicity Island (VPaI-7) of Pandemic Strains.

Small regulatory RNAs (sRNAs) are molecules that play an important role in the regulation of gene expression. sRNAs in bacteria can affect important processes, such as metabolism and virulence. Previous studies showed a significant role of sRNAs in the Vibrio species, but knowledge about Vibrio parahaemolyticus is limited. Here, we examined the conservation of sRNAs between V. parahaemolyticus and other human Vibrio species, in addition to investigating the conservation between V. parahaemolyticus strains differing in pandemic origin. Our results showed that only 7% of sRNAs were conserved between V. parahaemolyticus and other species, but 88% of sRNAs were highly conserved within species. Nonetheless, two sRNAs coding to RNA-OUT, a component of the Tn10/IS10 system, were exclusively present in pandemic strains. Subsequent analysis showed that both RNA-OUT were located in pathogenicity island-7 and would interact with transposase VPA1379, according to the model of pairing of IS10-encoded antisense RNAs. According to the location of RNA-OUT/VPA1379, we also investigated if they were expressed during infection. We observed that the transcriptional level of VPA1379 was significantly increased, while RNA-OUT was decreased at three hours post-infection. We suggest that IS10 transcription increases in pandemic strains during infection, probably to favor IS10 transposition and improve their fitness when they are facing adverse conditions.

Genetic and Phenotypic Characterization of Indole-Producing Isolates of Pseudomonas syringae pv. actinidiae Obtained From Chilean Kiwifruit Orchards.

In recent years, Chilean kiwifruit production has been affected by the phytopathogen Pseudomonas syringae pv. actinidiae (Psa), which has caused losses to the industry. In this study, we report the genotypic and phenotypic characterization of 18 Psa isolates obtained from Chilean kiwifruits orchards between 2012 and 2016 from different geographic origins. Genetic analysis by multilocus sequence analysis (MLSA) using four housekeeping genes (gyrB, rpoD, gltA, and gapA) and the identification of type III effector genes suggest that the Chilean Psa isolates belong to the Psa Biovar 3 cluster. All of the isolates were highly homogenous in regard to their phenotypic characteristics. None of the isolates were able to form biofilms over solid plastic surfaces. However, all of the isolates formed cellular aggregates in the air-liquid interface. All of the isolates, except for Psa 889, demonstrated swimming motility, while only isolate Psa 510 demonstrated swarming motility. The biochemical profiles of the isolates revealed differences in 22% of the tests in at least one Psa isolate when analyzed with the BIOLOG system. Interestingly, all of the isolates were able to produce indole using a tryptophan-dependent pathway. PCR analysis revealed the presence of the genes aldA/aldB and iaaL/matE, which are associated with the production of indole-3-acetic acid (IAA) and indole-3-acetyl-3-L-lysine (IAA-Lys), respectively, in P. syringae. In addition, IAA was detected in the cell free supernatant of a representative Chilean Psa strain. This work represents the most extensive analysis in terms of the time and geographic origin of Chilean Psa isolates. To our knowledge, this is the first report of Psa being able to produce IAA. Further studies are needed to determine the potential role of IAA in the virulence of Psa during kiwifruit infections and whether this feature is observed in other Psa biovars.

Exploring the Genomic Traits of Non-toxigenic Vibrio parahaemolyticus Strains Isolated in Southern Chile.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis worldwide. As reported in other countries, after the rise and fall of the pandemic strain in Chile, other post-pandemic strains have been associated with clinical cases, including strains lacking the major toxins TDH and TRH. Since the presence or absence of tdh and trh genes has been used for diagnostic purposes and as a proxy of the virulence of V. parahaemolyticus isolates, the understanding of virulence in V. parahaemolyticus strains lacking toxins is essential to detect these strains present in water and marine products to avoid possible food-borne infection. In this study, we characterized the genome of four environmental and two clinical non-toxigenic strains (tdh-, trh-, and T3SS2-). Using whole-genome sequencing, phylogenetic, and comparative genome analysis, we identified the core and pan-genome of V. parahaemolyticus of strains of southern Chile. The phylogenetic tree based on the core genome showed low genetic diversity but the analysis of the pan-genome revealed that all strains harbored genomic islands carrying diverse virulence and fitness factors or prophage-like elements that encode toxins like Zot and RTX. Interestingly, the three strains carrying Zot-like toxin have a different sequence, although the alignment showed some conserved areas with the zot sequence found in V. cholerae. In addition, we identified an unexpected diversity in the genetic architecture of the T3SS1 gene cluster and the presence of the T3SS2 gene cluster in a non-pandemic environmental strain. Our study sheds light on the diversity of V. parahaemolyticus strains from the southern Pacific which increases our current knowledge regarding the global diversity of this organism.

In situ antimicrobial behavior of materials with copper-based additives in a hospital environment.

Copper and its alloys are effective antimicrobial surface materials in the laboratory and in clinical trials. Copper has been used in the healthcare setting to reduce environmental contamination, and thus prevent healthcare-associated infections, complementing traditional protocols. The addition of copper nanoparticles to polymer/plastic matrices can also produce antimicrobial materials, as confirmed under laboratory conditions. However, there is a lack of studies validating the antimicrobial effects of these nanocomposite materials in clinical trials. To satisfy this issue, plastic waiting room chairs with embedded metal copper nanoparticles, and metal hospital IV pools coated with an organic paint with nanostructured zeolite/copper particles were produced and tested in a hospital environment. These prototypes were sampled once weekly for 10 weeks and the viable microorganisms were analysed and compared with the copper-free materials. In the waiting rooms, chairs with copper reduced by around 73% the total viable microorganisms present, showing activity regardless of the microorganism tested. Although there were only low levels of microorganisms in the IV pools installed in operating rooms because of rigorous hygiene protocols, samples with copper presented lower total viable microorganisms than unfilled materials. Some results did not have statistical significance because of the low load of microorganisms; however, during at least three weeks the IV pools with copper had reduced levels of microorganisms by a statistically significant 50%. These findings show for the first time the feasibility of utilizing the antimicrobial property of copper by adding nanosized fillers to other materials in a hospital environment.

Isolation and characterization of a novel bacteriophage infecting Vibrio alginolyticus.

Vibrio alginolyticus is a common marine bacterium implicated in disease outbreaks in marine farmed fish and invertebrates. Due to the inappropriate use of antibiotics in aquaculture, alternative therapies have been proposed. One of the most promising options is the use of lytic bacteriophages to control pathogenic bacteria. This work describes the isolation and characterization of a lytic phage (VEN) against a V. alginolyticus strain (V2) isolated from a disease outbreak in common dentex (Dentex dentex) cultured at the Hellenic Centre for Marine Research (HCMR) in Crete, Greece. The bacteriophage is morphologically similar to phages from Podoviridae family and remained stable for 1 year at 4 °C and over 1 h when kept at 50 °C. VEN was able to lyse the host bacteria at several multiplicity of infection (MOI) (0.1-100) in liquid cultures. However, it was unable to infect other V. alginolyticus strains. Its genome consists of 44,603 bp with a GC content of 43.5%, while sequence analysis revealed the presence of 54 potential ORFs with a T7-like genomic organization. Almost 65% of the predicted ORFs presented homology with proteins of the vibriophages Vc1 and phi-A318 infecting Vibrio cyclitrophicus and Vibrio alginolyticus, respectively. Phylogenetic analysis applying the amino acid sequence of the large terminase subunit confirmed the close relationship of these phages. Furthermore, the comparison of the RNA polymerase of these phages revealed that the motifs A, B and C related to the catalytic activity and the recognition loop related to promotor identification were also conserved. VEN has an obligate lytic life cycle demonstrated by experimental data and genomic analysis. These results suggest that VEN may provide a good candidate to control recurrent diseases caused by V. alginolyticus at HCMR.

Encapsulation of specific Salmonella Enteritidis phage f3αSE on alginate-spheres as a method for protection and dosification

Background: Bacteriophages have been proposed as an alternative to control pathogenic bacteria resistant to antibiotics. However, they are not extensively used due to different factors such as vulnerability under environmental conditions and the lack of efficient administration methods. A potential solution is the encapsulation of bacteriophages in hydrogel polymers to increase their viability and as a controlled release method. This work describes the use of alginate-Ca+2 matrixes as mechanisms for protection and dosification of the phage f3αSE which has been successfully used to prevent infections produced by Salmonella Enteritidis. Results: The viability of the pure phage is reduced in near 100% after 1-h incubation at pH 2 or 3. However, the encapsulated phage remains active in 80, 6% at pH 3, while no differences were observed at pH 2, 4 or 7. Exposition of f3αSE to different T° showed that the viability of this phage decreased with increased T° to near 15% at 60°C, while the encapsulated phage remains with 50% viability at same temperature. Finally, the encapsulation of phages showed to extend their presence for 100 h in the medium compared to non-encapsulated phages in a water flow system, which simulate automatic birdbath used in poultry industry, maintaining the phage concentration between 102 and 104 PFU/mL during 250 h. Conclusions: Encapsulation in alginate-Ca+2 spheres can be a good alternative to extend viability of phages and can be used as a phage method dosification method in water flow systems.

Bacteriophages in the control of pathogenic vibrios

Vibrios are common inhabitants of marine and estuarine environments. Some of them can be pathogenic to humans and/or marine animals using a broad repertory of virulence factors. Lately, several reports have indicated that the incidence of Vibrio infections in humans is rising and also in animals constitute a continuing threat for aquaculture. Moreover, the continuous use of antibiotics has been accompanied by an emergence of antibiotic resistance in Vibrio species, implying a necessity for efficient treatments. One promising alternative that emerges is the use of lytic bacteriophages; however, there are some drawbacks that should be overcome to make phage therapy a widely accepted method. In this work, we discuss about the major pathogenic Vibrio species and the progress, benefits and disadvantages that have been detected during the experimental use of bacteriophages to their control.

Isolation and Characterization of Two Lytic Bacteriophages, φSt2 and φGrn1; Phage Therapy Application for Biological Control of Vibrio alginolyticus in Aquaculture Live Feeds.

Bacterial infections are a serious problem in aquaculture since they can result in massive mortalities in farmed fish and invertebrates. Vibriosis is one of the most common diseases in marine aquaculture hatcheries and its causative agents are bacteria of the genus Vibrio mostly entering larval rearing water through live feeds, such as Artemia and rotifers. The pathogenic Vibrio alginolyticus strain V1, isolated during a vibriosis outbreak in cultured seabream, Sparus aurata, was used as host to isolate and characterize the two novel bacteriophages φSt2 and φGrn1 for phage therapy application. In vitro cell lysis experiments were performed against the bacterial host V. alginolyticus strain V1 but also against 12 presumptive Vibrio strains originating from live prey Artemia salina cultures indicating the strong lytic efficacy of the 2 phages. In vivo administration of the phage cocktail, φSt2 and φGrn1, at MOI = 100 directly on live prey A. salina cultures, led to a 93% decrease of presumptive Vibrio population after 4 h of treatment. Current study suggests that administration of φSt2 and φGrn1 to live preys could selectively reduce Vibrio load in fish hatcheries. Innovative and environmental friendly solutions against bacterial diseases are more than necessary and phage therapy is one of them.