Claudio D. Miranda

Background: Bacteriophages and phage-derived lytic enzymes are increasingly considered to be targeted antimicrobial tools in aquaculture; however, their compatibility with non-target microbial communities under hatchery-relevant conditions remains insufficiently characterized. Objectives This study evaluates the impact of a lytic phage (CH20) and a phage-derived lysin (LysVp1), applied under previously validated conditions for rapid Vibrio control, on the microbiota associated with seawater, rotifers, and zebrafish larvae challenged with Vibrio alginolyticus GV09. Methods: Treatments were independently applied to each biological matrix using short exposure times representative of hatchery practices, intentionally capturing the critical window during which microbial transfer from live feed to larvae occurs. Microbial communities were analyzed using 16S rRNA gene sequencing, with DNA- and RNA-derived datasets evaluated separately. Results: Alpha diversity indices were compared using appropriate statistical tests, while beta diversity was assessed using Aitchison distance, PERMANOVA, and dispersion analyses, and differential abundance was evaluated using ANCOM-BC2. Alpha diversity metrics showed no significant differences among treatments across all matrices, indicating the preservation of microbial richness and diversity. Beta diversity patterns differed according to the nucleic acid source, with RNA-based analyses revealing treatment-associated shifts in rotifer and water microbiota that were not consistently detected at the DNA level. In zebrafish larvae, neither phage nor lysin treatment significantly altered overall community structure, although dispersion effects reflected limitations related to sample size. Conclusions: Overall, these results indicate that phage CH20 and lysin LysVp1 exert minimal impact on alpha diversity and limited, context-dependent effects on microbial community structure, supporting their microbiota-safe potential for aquaculture applications.

Objective: The main aim of the study was to evaluate the role of wild fishes inhabiting in three anthropogenic-impacted Bays in Chile as reservoirs of antimicrobial resistance genes (ARGs). Methods: A total of 245 antimicrobial-resistant isolates were isolated from fish captured in the Coquimbo (142 isolates), Concepción (44 isolates), and Puerto Montt (59 isolates) Bays, and were identified by 16S rRNA gene sequence analysis, Antimicrobial-resistant isolates were tested for susceptibility to 12 antimicrobials by an agar disk diffusion method, and the carriage of genes encoding for resistance to main antimicrobial classes, such as β-lactams, amphenicols, tetracyclines, and sulfonamides by PCR (Polymerase Chain Reaction). Results: A predominance of the Pseudomonas (37.04%), Vibrio (14.40%), and Shewanella (13.99%) genera. Antimicrobial-resistant isolates were tested for susceptibility to 12 antimicrobials by an agar disk diffusion method, showing highest resistance to streptomycin (82.4%), amoxicillin (67.4%), and furazolidone (63.3%), and lowest resistance to ciprofloxacin (3.7%), meropenem (22.5%), and oxytetracycline (29.8%) and exhibiting a high occurrence of the multi-drug resistance phenotype (76.9%). Furthermore, an important number of isolates recovered from sampled fish species carried plasmids (53.5%), floR gene (36.7%), and tet genes (19.2%), whereas the detection of sul genes and class 1-integron was rare. As an overall result, 10.6% of isolates carried at least one bla gene, encoding an extended-spectrum-β-lactamase, with a high predominance of the bla_CTX-M1 gene (23 isolates), whereas 14 out of 245 isolates (5.7%) were positive for the carriage of carbapenemases encoding genes, which both groups exhibited the β-lactam resistance phenotype. Conclusions: The wide distribution of ARG-carrying bacteria in wild fishes from all sampled Bays provides evidence that wild fish are important reservoirs and drivers of spread of ARGs in the marine environment, prompting the need of a continuous surveillance of these genes in wild fishes inhabiting anthropic impacted coastal marine environments in Chile.

Bacteriophages and phage-derived lytic enzymes are emerging as promising alternatives to antibiotics in aquaculture; however, their effects on non-target microbial communities remain insufficiently characterized. This study evaluated the impact of a lytic phage (CH20) and a phage-derived lysin (LysVp1) on the microbiota associated with seawater, rotifers, and zebrafish larvae challenged with Vibrio alginolyticus GV09. Parallel treatments with phage CH20, lysin LysVp1, or no treatment were independently applied to each biological matrix. Microbial commu-nities were analyzed using 16S rRNA gene sequencing, with DNA- and RNA-derived datasets evaluated separately. Alpha diversity indices were compared using appropriate statistical tests, while beta diversity was assessed using Aitchison distance, PERMANOVA, and dispersion anal-yses, and differential abundance was evaluated using ANCOM-BC2. Alpha diversity metrics (Chao1, Shannon, and Simpson indices) showed no significant differences among treatments across all matrices, indicating preservation of microbial richness and diversity. Beta diversity pat-terns differed according to the nucleic acid source, with RNA-based analyses revealing treat-ment-associated shifts in rotifer and water microbiota that were not consistently detected at the DNA level. In zebrafish larvae, neither phage nor lysin treatment significantly altered overall community structure, although dispersion effects reflected limitations related to sample size. Overall, these results indicate that phage CH20 and lysin LysVp1 exert minimal impact on alpha diversity and limited, context-dependent effects on microbial community structure, supporting their microbiota-safe potential for aquaculture applications.

Studies on preventing antimicrobial resistance in aquaculture emphasise the need to responsibly and prudently use antimicrobials, selecting those most effective in controlling and/or reducing mortalities caused by vibriosis. In this study, the distribution of the antimicrobial susceptibility of 28 Vibrio anguillarum isolates from Turkish fish farms was determined using the broth microdilution minimum inhibitory concentration (MIC) test according to the Clinical and Laboratory Standards Institute (CLSI) guideline. The epidemiological cut‐off (CO_WT) values of the V. anguillarum isolates of florfenicol (FLO), tetracycline (TET), doxycycline (DOX), oxolinic acid (OXO), enrofloxacin (ENR) and trimethoprim/sulfamethoxazole (SXT) were calculated using the normalised resistance interpretation (NRI) and ECOFFinder methods. Isolates were categorised as belonging to the fully susceptible wild‐type (WT) or non‐wild‐type (NWT) populations. Calculated CO_WT values (in μg mL⁻¹) were ≤ 1.0 for FLO and DOX, ≤ 0.5 for TET, ≤ 0.016 for ENR, ≤ 0.032 for OXO, and ≤ 4.0 for SXT using the NRI analysis. Percentages of V. anguillarum isolates categorised as belonging to the NWT population were small for the antimicrobials FLO (10.7%) and SXT (0%), whereas they were higher for the antimicrobials OXO (39.3%) and ENR (39.3%). This is the first study to estimate the local CO_WT values for antibiotics used in the control of V. anguillarum isolates recovered from farmed fish in Turkey.

In an aquaponic system, fish and plants are cultivated together in a symbiotic environment where they mutually benefit, using significantly less water than traditional farming methods. The main aim of this study was to investigate the occurrence of antimicrobial resistance in two aquaponic systems implemented in two Chilean high schools using rainbow trout and lettuce cultures. When water samples (fish tank, biofilter, and plant raft) were analyzed over a three-month period, no resistance to oxytetracycline was detected, whereas the occurrence of resistance to florfenicol was rather small, ranging from 0.01% to 3.1% of bacterial culturable counts. Eighteen isolates were recovered from various sources as representatives of the florfenicol-resistant population, and all of them belonged to the Pseudomonas genus, showing a multi-drug-resistance phenotype and exhibiting simultaneous resistance to 7–13 antimicrobials. All isolates exhibited resistance to amoxicillin, chloramphenicol, florfenicol, and furazolidone and susceptibility to meropenem, oxytetracycline, oxolinic acid, flumequine, ciprofloxacin, and enrofloxacin. Five and two isolates carried the amphenicol-resistance-encoding genes floR and cmlA, respectively, whereas no carriage of integrons or the fexA, fexB, pexA, optrA, and cfr genes encoding for florfenicol resistance was detected. Eleven isolates carried plasmids, but only two of them were able to transfer their plasmid content by conjugation. The knowledge of the microbiome associated with aquaponic systems is still scarce, and their role as potential reservoirs of antimicrobial-resistant bacteria and related genes of these systems must be elucidated.

Vibrio species are naturally found in estuarine and marine ecosystems, but are also recognized as significant human enteropathogens, often linked to seafood-related illnesses. In aquaculture settings, Vibrio poses a substantial risk of infectious diseases, resulting in considerable stock losses and prompting the use of antimicrobials. However, this practice contributes to the proliferation of antimicrobial-resistant (AMR) bacteria and resistance genes. Our investigation aimed to explore the potential of biological agents such as bacteriophage CH20 and endolysin LysVPp1 in reducing Vibrio bacterial loads in both rotifer and fish larvae. LysVPp1’s lytic activity was assessed by measuring absorbance reduction against various pathogenic Vibrio strains. Phage CH20 exhibited a limited host range, affecting only Vibrio alginolyticus GV09, a highly pathogenic strain. Both CH20 and LysVPp1 were evaluated for their effectiveness in reducing Vibrio load in rotifers or fish larvae through short-setting bioassays. Our results demonstrated the significant lytic effect of endolysin LysVPp1 on strains of Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio splendidus. Furthermore, we have showcased the feasibility of reducing the load of pathogenic Vibrio in live feed and fish larvae by using a non-antibiotic-based approach, such as lytic phage and endolysin LysVPp1, thus contributing to the progress of a sustainable aquaculture from a One Health perspective.

Antimicrobial-resistant bacteria (ARB) from the intestinal contents of wild fish may have a relevant ecological significance and could be used as indicators of antimicrobial-resistance dissemination in natural bacterial populations in water bodies impacted by urban contamination. Thus, the occurrence of ARB in the intestinal contents of pelagic and demersal wild fishes captured in anthropogenic-impacted Coquimbo Bay in Chile was studied. Culturable counts of total and antimicrobial-resistant bacteria were determined by a spread plate method using Trypticase soy agar and R2A media, both alone and supplemented with the antimicrobials amoxicillin, streptomycin, florfenicol, oxytetracycline and ciprofloxacin, respectively. Heterotrophic plate counts of pelagic and demersal fishes ranged from 1.72 × 10⁶ CFU g⁻¹ to 3.62 × 10⁹ CFU g⁻¹, showing variable proportions of antimicrobial resistance. Representative antimicrobial-resistant isolates were identified by 16S rRNA gene sequencing, and isolates (74) from pelagic fishes mainly belonged to Pseudomonas (50.0%) and Shewanella (17.6%) genera, whereas isolates (68) from demersal fishes mainly belonged to Vibrio (33.8%) and Pseudomonas (26.5%) genera. Antimicrobial-resistant isolates were tested for susceptibility to 12 antimicrobials by an agar disk diffusion method, showing highest resistance to streptomycin (85.2%) and amoxicillin (64.8%), and lowest resistance to oxytetracycline (23.2%) and ciprofloxacin (0.7%). Only furazolidone and trimethoprim/sulfamethoxazole were statistically different (p < 0.05) in comparisons between isolates from pelagic and demersal wild fishes. Furthermore, an important number of these isolates carried plasmids (53.5%) and produced Extended-Spectrum-β-lactamases (ESBL) (16.9%), whereas the detection of Metallo–β–Lactamases and class 1-integron was rare. This study provides evidence that wild fish are important reservoirs and spreading-vehicles of ARB, carrying plasmids and producing ESBLs in Chilean marine environments.

Flavobacterial infection associated with diseased fish is caused by multiple bacterial species within the family Flavobacteriaceae. In the present study, the Chilean isolate FP99, from the gills of a diseased, farmed rainbow trout (Oncorhynchus mykiss), was characterized using phenotypic and genomic analyses. Additionally assessed was pathogenic activity. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that isolate FP99 belonged to the genus Epilithonimonas, an average nucleotide identity value of 100% was detected with the Chilean isolate identified as Epilithonimonas sp. FP211‐J200. In silico genome analysis, mechanisms for toxins production, and superantigens, adhesion, or other genes associated with virulence were not detected. However, genes encoding proteins for antibiotic resistance were found, including the chrA gene and the nucleotide sequence that encodes for multiple antibiotic resistance MarC proteins. Furthermore, the bla_ESP‐1 gene (87.85% aminoacidic sequence identity), encoding an extended‐spectrum subclass B3 metallo‐β‐lactamase and conferring carbapenem‐hydrolysing activity, and the tet(X) gene, which encodes a monooxygenase that catalyses the degradation of tetracycline‐class antimicrobials were carried by this isolate. Phenotyping analyses also supported assignment as E. ginsengisoli. Challenge trials against healthy rainbow trout resulted in no observed pathogenic effect. Our findings identify for the first time the species E. ginsengisoli as associated with fish farming, suggesting that this isolate may be a component of the microbiota of the freshwater system. Notwithstanding, poor environmental conditions and any stressors associated with aquaculture situations or lesions caused by other pathogenic bacteria, such as F. psychrophilum, could favour the entry of E. ginsengisoli into rainbow trout.

Intestinal microbiota has been involved in several processes that benefit the host, such as digestion, nutrient metabolism, resistance to pathogens colonization and immune function. In this study, we investigated the diversity, composition and functional prediction of microbiota of reared Seriolella violacea (palm ruff) in the same cohort sampled at different times (7-, 8- and 9-month-old). Microbial community structure analyses, using 16S rRNA amplicon sequencing, revealed that the intestinal microbiota was dominated by the phyla Firmicutes, Proteobacteria, Fusobacteria and Tenericutes. At the genus level, Candidatus Arthromitus was the most abundant in all sampled timepoints, representing in average 78% of the bacterial community (ranging from 18 to 98%), corresponding to segmented filamentous bacteria, which are interesting because they have been associated with the maturation of immune responses in the gut and protecting the host from bacterial infections. The comparisons of the intestinal microbiota among the three groups showed differences in abundance of bacterial taxa and also in alpha diversity indexes (Shannon and Simpson), as well as beta diversity metrics (weighted and unweighted UniFrac). Potential functions of the intestinal microbiota of palm ruff were retrieved using Philipin and Tax4fun and these analyses revealed high levels of genes for sugar metabolism. To our knowledge, this study represents the first description of the intestinal microbiota of S. violacea.

Two Vibrio strains (VPAP36 and VPAP40) were isolated from moribund-settled larvae of the Chilean scallop Argopecten purpuratus during vibriosis outbreaks that occurred in two commercial scallop larvae hatcheries located in the Inglesa and Tongoy bays in Northern Chile. The strains were identified as Vibrio chagasii using phenotypic characterization and whole genome sequence analysis. Both strains exhibited the phenotypic properties associated with virulence, gelatin hydrolysis and β-hemolysis, whereas only VPAP36 produced phospholipase and only VPAP40 produced caseinase. The whole genome analysis showed that the strains harbored genes encoding for the virulence factors, the EPS type II secretion system, and Quorum Sensing (auto-inductor 1 and auto-inductor 2), whereas genes encoding a metalloproteinase and a capsular polysaccharide were detected only in the VPAP40 genome. When challenge bioassays using healthy 11-day-old scallop larvae were performed, the V. chagasii VPAP36 and VPAP40 strains exhibited significant (p < 0.05) differences in their larval lethal activity, producing, after 48 h, larval mortalities of 65.51 ± 4.40% and 28.56 ± 5.35%, respectively. Otherwise, the cell-free extracellular products of the VPAP36 and VPAP40 strains produced larval mortalities of 20.86 ± 2.40% and 18.37 ± 2.40%, respectively, after 48 h of exposure. This study reports for the first time the isolation of V. chagasii from the massive larval mortalities of the farmed scallop (Argopecten purpuratus) in Chile, and demonstrates the pathogenic activity of V. chagasii towards the Chilean scallop, the second most important species for Chilean mariculture.