Nihed Ajmi

Morphological and skeletal deformities compromise growth, swimming, feeding, welfare, and market value in farmed fish, but externally visible abnormalities may not reveal the underlying tissue lesions. This study characterized naturally occurring deformities in farmed rainbow trout (Oncorhynchus mykiss) using integrated macroscopic, radiographic, and histopathological evaluation. A total of 54 morphologically abnormal fish and one clinically normal fish were examined. Deformities were grouped by predominant external region, whole-body radiographs were evaluated for skeletal and structural abnormalities, and affected tissues were processed for routine histopathology. Caudal peduncle or tail-region deformities were most frequent (37.0%), followed by trunk/body deformities (25.9%), head-region deformities (24.1%), and specific or combined deformity patterns (13.0%). Vertebral compression was the dominant radiographic lesion (74.1%), followed by swim bladder anomalies (48.1%), contour irregularities (31.5%), combined deformity patterns (29.6%), and peduncle thickening (25.9%). Histopathology revealed ocular lesions compatible with cataract and microphthalmia, vertebral asymmetry and compression, cartilage and bone disorganization, intervertebral space loss, fibrosis, and skeletal muscle degeneration and atrophy. These findings show that visible deformities often reflect complex skeletal and soft-tissue pathology. Integrating radiography with histopathology provides a more informative framework for deformity assessment and welfare-oriented management in rainbow trout production.

Flavobacterium psychrophilum , the causative agent of bacterial cold‐water disease (BCWD) and rainbow trout fry syndrome (RTFS), remains one of the most important bacterial pathogens affecting salmonid aquaculture. In this study, 24 F. psychrophilum isolates recovered during a 12‐month surveillance programme from two rainbow trout farms in Türkiye were characterized using phenotypic, serological, antigenic, phylogenomic and experimental infection approaches. Phylogenomic analyses showed that most isolates belonged to CC‐ST10, whereas four isolates were assigned to the CC‐ST90/ST108 lineage, representing the first report of this lineage in Türkiye. Comparative genomic analysis revealed extensive conservation of virulence‐associated loci, with only FP0006 and FP0506 showing variable distribution among isolates. Notably, FP0506 was absent exclusively from ST108/CC‐ST90 isolates. Experimental immersion challenge assays demonstrated that all selected isolates were able to induce mortality in rainbow trout under the conditions tested. However, isolate‐dependent differences in survival were not statistically significant in the standard immersion model and became more evident following H ₂ O ₂ pre‐treatment, with the FP1 group showing the lowest survival. These findings suggest that in vivo pathogenicity may be influenced by host barrier status and cannot be fully predicted by phylogenomic lineage, serotype or in vitro virulence‐associated phenotypes alone. Phenotypic serotyping identified Th as the predominant serotype, although discrepancies were observed between phenotypic and genome‐based serotype classifications. Whole‐cell protein profiling and Western blot analyses revealed both antigenic heterogeneity and conserved immunoreactive structures across phylogenetically distinct isolates. Antimicrobial susceptibility testing showed a high proportion of non‐wild‐type phenotypes for oxytetracycline, florfenicol and enrofloxacin; however, no acquired antimicrobial resistance genes were detected by genome‐based screening. Collectively, these findings demonstrate that Turkish F. psychrophilum populations are phylogenomically diverse but retain largely conserved virulence‐associated loci and immunoreactive antigenic structures. This study provides the first evidence of the CC‐ST90/ST108 lineage in Türkiye and highlights the value of integrating genomic, serological, phenotypic, antigenic and experimental infection data for understanding the population structure, serotype heterogeneity and pathogenic potential of F. psychrophilum in rainbow trout aquaculture.

Outbreaks of vibriosis caused by Vibrio anguillarum are a persistent constraint on rainbow trout ( Oncorhynchus mykiss ) aquaculture. However, information on the population structure of field strains in Turkey has been lacking. Here, we report the first systematic serotypic, proteomic, and genomic characterization of 23 V. anguillarum isolates collected over 10 years from rainbow trout farms located in six major aquaculture regions of Turkey. Serological analyses based on microagglutination, supported by ELISA characterization of hyperimmune sera, identified a clear predominance of serotype O1, whereas isolate V12 exhibited a non‐agglutinating, atypical O‐antigen profile. Protein profiling (SDS‐PAGE) and immunoblotting showed largely conserved whole‐cell protein patterns among the isolates, but distinct immunogenic bands at 14, 18, and 40 kDa were detected in isolates V18 and V21. Long‐read whole‐genome sequencing revealed that most Turkish isolates grouped within the global O1 clade, while V12, V25, and V28 isolates occupied more distant branches. Comparative genomics demonstrated a conserved core virulence gene set (RTX toxins, siderophore and iron‐uptake systems, motility and adhesion factors, Type VI secretion system), with strain‐dependent variation in accessory loci such as anguibactin and T6SS‐I. Experimental infections of rainbow trout demonstrated significant differences in virulence among isolates ( p < 0.05), with the V18 isolate showing high, the V15 intermediate, and the V12 low‐mortality rates. By elucidating the relationship among the serotype, immunogenic protein profiles, virulence gene repertoires, and in vivo pathogenicity, this study provides a comprehensive overview of the antigenic and genomic diversity of Vibrio anguillarum isolates from Turkey. Notably, the identification of V18 and V21 as promising candidate strains for further vaccine evaluation, characterized by high virulence and unique immunogenic features, provides a scientific foundation for the development of serotype‐specific vaccination strategies to mitigate vibriosis‐associated losses in aquaculture.

Edwardsiella tarda is a facultatively anaerobic, Gram-negative bacterium that is widely distributed in aquatic environments. With the increasing interactions between aquatic animals and humans, E. tarda has become an important pathogen with significant implications for global public health. In this study, we characterized strain ET-1, isolated from an aquarium housing Pangasius pangasius, to evaluate its ecological presence, antimicrobial resistance, and pathogenic potential using a combination of phenotypic and genome-based approaches. Biochemical characteristics were determined using the API 20E system. Antimicrobial susceptibility was evaluated using MIC testing to determine resistance to clinically relevant antibiotics. Whole-genome sequencing using Oxford Nanopore Technologies revealed a genome size of 3.76 Mb with a GC content of 57.12%. Genome-based identification using digital DNA–DNA hybridization (dDDH, 86.6%) confirmed the classification as E. tarda. Several virulence-associated genes (flgG – flagellar basal body-rod protein; etfC – electron transfer flavoprotein subunit C) and antimicrobial resistance determinants (qnrS1 – quinolone resistance protein S1; efflux pump genes such as kpnH and kpnF) were identified. Pathogenic potential analysis via PathogenFinder2 indicated a high likelihood of human pathogenicity. Additionally, 16S rRNA-based profiling revealed the widespread environmental and host-associated presence of ET-1-like sequences. These results underscore the zoonotic and environmental significance of E. tarda ET-1 and support its relevance for public health surveillance within the One Health framework.

Aquarium fish are increasingly being recognized as reservoirs of zoonotic pathogens, with Aeromonas species posing a notable risk because of their environmental resilience and opportunistic pathogenicity. This study presents the most comprehensive genome-based investigation to date of Aeromonas diversity in aquarium fish, aiming to characterize their taxonomic distribution, population structure, and genomic features. A total of 64 Aeromonas isolates were collected from various aquarium fish species. Wholegenome sequencing was conducted on all isolates to facilitate comparative genomic analyses. Key approaches included multilocus sequence typing (MLST), pairwise Digital DNA-DNA hybridization (dDDH), and the construction of a phylogenomic tree for species-level classification. Furthermore, a population structure analysis was performed to explore genomic diversity and evolutionary trends among the isolates. The results identified 14 distinct Aeromonas species, with A. veronii, A. caviae, and A. hydrophila being the most common. Importantly, several isolates exhibited taxonomic ambiguity, indicating the possible presence of new species or subspecies lineages. Furthermore, antimicrobial resistance gene profiles and virulence factor distributions varied significantly across clades, indicating genomic plasticity. This study highlights the underappreciated genomic complexity of Aeromonas populations in aquarium environments and raises concerns about the public health implications of pathogen reservoirs in ornamental fish.

Co-infections are increasingly recognized as drivers of disease in ornamental fish, yet their genomic underpinnings and zoonotic implications remain underexplored compared to farmed species. Leveraging a One Health perspective, we investigated an acute mortality event in koi carp and characterized a co-infection by opportunistic aquatic bacteria that are also implicated in human disease. We isolated Aeromonas veronii and Shewanella sp. from a moribund koi using culture, biochemical assays, and MALDI-TOF MS, then generated draft genomes and performed orthology (OrthoVenn3), pathway annotation (KEGG BlastKOALA/Mapper), secondary-metabolite mining (antiSMASH), and virulence/resistome screening (VFDB/CARD), complemented by antimicrobial susceptibility testing. Clinically, affected fish showed dropsy/ascites, scale loss, abnormal buoyancy, and reduced activity. Phylogenomics positioned A. veronii Koi-2 within the A. veronii complex near species thresholds (ANI ~96.1%; dDDH ~70.2%), while Shewanella sp. Koi-1 formed a distinct lineage below accepted cut-offs relative to S. seohaensis (ANI ~95.9%; dDDH ~67.6%). The virulome comprised 194 loci in A. veronii Koi-2 and 152 in Shewanella sp. Koi-1 is enriched for adhesion, secretion, iron uptake, and immune-evasion functions. Genotype–phenotype agreement was high for multidrug resistance: Shewanella sp. encoded OXA-436 and rsmA, matching β-lactam resistance and reduced fluoroquinolone/phenicol susceptibility, whereas A. veronii carried tet(A), OXA-1157, cphA3, sul1, and aadA3 consistent with tetracycline, β-lactam, sulfonamide, and aminoglycoside resistance profiles. In conclusion, genome-resolved diagnostics confirmed a mixed Aeromonas–Shewanella co-infection with broad virulence potential and convergent resistance mechanisms, supporting the routine use of genomics to distinguish single- versus mixed-agent disease and to guide dual-coverage, mechanism-aware therapy in ornamental fish medicine while informing zoonotic risk mitigation.

The strain A-9^T, isolated from Oncorhynchus mykiss (rainbow trout) in a Turkish aquaculture facility, was characterized through integrated phenotypic, phylogenetic, and genomic analyses. Whole-genome sequencing revealed a 5.21 Mb circular chromosome (GC content: 58.16%) and three plasmids encoding proteins for mobilization and toxin–antitoxin systems. Multilocus phylogenetic analysis (MLPA) using seven housekeeping genes supported the distinct lineage of A-9^T. Digital DNA–DNA hybridization (77.6–78.6%) and average nucleotide identity values (96.59–97.58%) confirmed taxonomic divergence from all currently recognized A. salmonicida subspecies. Comparative proteomic and pangenomic analyses identified 328 strain-specific genes, including virulence factors, secretion system components (Type II and Type VI), and efflux-related proteins. Although genes encoding Type III secretion systems and biofilm formation were absent, A-9^T harbored a broad virulence gene repertoire and resistance determinants, including OXA-956, cphA5, and FOX-20, supporting a multidrug-resistant phenotype. Based on its genomic, phenotypic, and functional distinctiveness, we propose the novel taxon Aeromonas salmonicida subsp. oncorhynchi subsp. nov. (type strain A-9^T = LMG 33538^T = DSM 117494^T), expanding the taxonomic landscape of the A. salmonicida complex and offering insights into fish-associated bacterial evolution.

Piscine lactococcosis poses a significant threat to a wide range of cultured and wild fish populations worldwide, typically presenting as acute haemorrhagic septicemia with high morbidity and mortality. Although Lactococcus garvieae was historically considered the sole causative agent of piscine lactococcosis, recent studies have identified L. petauri and L. formosensis as additional, highly pathogenic species. In this study, we developed a novel TaqMan‐based multiplex qPCR assay for the simultaneous detection and differentiation of L. garvieae , L. petauri and L. formosensis , following a pangenome analysis of the publicly available genomes of these bacterial species. The assay demonstrated high sensitivity and specificity across 156 bacterial isolates obtained from various cultured fish species and geographical locations between 2008 and 2024, as well as against a panel of non‐target bacteria. It also successfully detected target pathogens in 146 field tissue samples, including tissues preserved in 70% ethanol, formalin‐fixed paraffin‐embedded (FFPE) tissues and tissues fixed on FTA cards. Compared to classical bacteriology, the multiplex qPCR assay yielded higher detection rates and enabled precise identification of the causative species of piscine lactococcosis. Overall, the multiplex qPCR assay developed in this study provides a reliable, rapid, highly sensitive and species‐specific molecular approach for diagnosing piscine lactococcosis, contributing to better surveillance and management of the disease in aquaculture.

The Gram-negative genus Aeromonas contains diverse bacterial species that are prevalent in aquatic environments. This present study describes three novel Aeromonas strains: A. ichthyocola sp. nov. A-5^T and A. mytilicola subsp. aquatica subsp. nov. A-8^T isolated from rainbow trout (Oncorhynchus mykiss), and A. mytilicola sp. nov. A-7^T isolated from mussels (Mytilus galloprovincialis), respectively. Genomic analyses revealed that strains A-5^T and A-7^T shared the highest 16S rRNA gene sequence similarity with A. rivipollensis P2G1^T (99.7% and 99.8%, respectively), while strain A-8^T exhibited 99.7% identity with A. media RM^T. Together with morphological, physiological, and biochemical data, genome-based analyses provided additional evidence for species differentiation. Digital DNA–DNA hybridization (dDDH; 56.8−65.9%) and average nucleotide identity (ANI; 94.2–95.7%) values fell below the species delineation thresholds, confirming that these isolates represent distinct taxa. Pathogenicity assays using greater wax moth (Galleria mellonella) larvae demonstrated strain-specific virulence profiles. Further genomic analyses identified biosynthetic gene clusters for nonribosomal peptides (NRPs) and ribosomally synthesized and post-translationally modified peptides (RiPPs), which often have roles in secondary metabolite production. Ecological analyses, based on genomic comparisons and metagenomic database searches, revealed the adaptability of the strains to diverse habitats, including freshwater, wastewater, and activated sludge. Based on the genetic and phenotypic data, the novel taxa Aeromonas ichthyocola sp. nov. A-5ᵀ (LMG 33534ᵀ = DSM 117488ᵀ), Aeromonas mytilicola sp. nov. A-7ᵀ (LMG 33536ᵀ = DSM 117490ᵀ), and Aeromonas mytilicola subsp. aquatica subsp. nov. A-8ᵀ (LMG 33537ᵀ = DSM 117493ᵀ) are proposed.

This study explores the biotechnological potential of Planococcus strain S3-L1, isolated from Horseshoe Island in Antarctica during the 6th Turkish Antarctic Expedition in 2022. Marine bacteria inhabiting polar environments play essential roles in ecosystem dynamics and biogeochemical cycles, largely due to their unique adaptations. The Planococcus genus, noted for its halotolerance and ability to thrive in cold environments, has garnered interest for its capability to produce bioactive metabolites, support bioremediation, and facilitate various industrial processes. In this research, Planococcus sp. S3-L1 was isolated and underwent 16S rRNA and wholegenome analysis, revealing a close genetic affinity with Planococcus kocurii and Planococcus faecalis. Genomic analysis identified genes responsible for carotenoid pigment production and aromatic compound degradation, underscoring its potential in environmental remediation and low-temperature pigment production. Furthermore, quorum-quenching enzymes suggest that S3-L1 could serve as an antimicrobial agent. These findings show that Planococcus sp. S3-L1 is a promising candidate for biotechnological applications, particularly within polar marine environments, where its cold adaptation and metabolic diversity offer potential benefits in aquaculture, bioremediation, and the synthesis of commercially valuable compounds.