Introduction
Nile tilapia (Oreochromis niloticus) is a cornerstone of global aquaculture, prized for fast growth and adaptability. In Ethiopia, expanding aquaculture brings attention to bacterial pathogens that threaten productivity and public health. Among these, Pseudomonas spp. are ubiquitous Gram-negative bacteria capable of causing disease in fish and posing zoonotic risks. This article summarizes a study that integrated phenotypic, molecular (PCR-based) detection, and antibiogram analyses of Pseudomonas isolates from Nile tilapia raised in Ethiopian aquaculture ponds.
Study aims and design
The study had three main objectives: (1) to isolate and identify Pseudomonas spp. associated with Nile tilapia in three Ethiopian stations, (2) to characterize isolates molecularly using 16S rDNA–targeted PCR, and (3) to determine antibiotic susceptibility patterns to a panel of veterinary and human-relevant antimicrobials. A cross-sectional design was implemented from December 2022 to November 2023 across three sites: Sebeta (NFALRC), Hawassa CARE, and Batu Fisheries Research Center. A total of 240 fish were sampled (80 per site) to capture diversity in tissue involvement and regional prevalence.
Clinical and postmortem observations
Infected tilapia exhibited clinical signs such as body depigmentation, corneal opacity, fin erosion, exophthalmia, scale detachment, abdominal distention, ulcers, and hemorrhages. Postmortem findings included splenomegaly, congested gills and kidneys, distended gall bladders, liver color changes, and ascitic fluids. These signs are consistent with systemic bacterial infection and align with prior reports on pseudomonal disease in fish.
Isolation, biochemical identification, and MALDI-TOF confirmation
From tissue homogenates, 81 presumptive Pseudomonas isolates were recovered based on colony morphology and Gram-negative, rod-shaped characteristics. Biochemical tests (oxidase and catalase positive, citrate utilization, glucose fermentation with gas and acid production, motility) aligned with Pseudomonas spp. A subset was confirmed to species level using MALDI-TOF mass spectrometry, corroborating biochemical results and enhancing diagnostic confidence.
Phenotypic traits and virulence indicators
Hemolysis assays on 5% sheep blood revealed a predominance of beta-hemolytic activity among isolates (approximately 85% of Pseudomonas aeruginosa, P. putida, and P. fluorescens groups), indicating robust virulence potential. A smaller fraction demonstrated alpha-hemolysis. Phenotypic profiling also noted green-blue pigmentation for many Pseudomonas colonies and variable results on API20E testing, underscoring the diversity within fish-associated Pseudomonas spp.
Molecular detection: 16S rDNA PCR and species distribution
Conventional PCR targeted a conserved 16S rDNA region with primers designed to detect Pseudomonas spp. A total of 75 samples were PCR-positive for Pseudomonas DNA, with species distribution showing: 8 P. aeruginosa, 28 P. putida, and 39 P. fluorescens. This molecular confirmation adds precision beyond phenotypic methods and highlights the relative predominance of P. fluorescens in these Ethiopian ponds.
Organ distribution and tissue tropism
Organ-specific prevalence varied by species. P. putida was most common in muscle tissue, while P. fluorescens dominated renal samples; P. aeruginosa showed a tendency toward the spleen. These patterns reflect pathogen-host-environment interactions and may influence sampling strategies for surveillance and diagnosis in tilapia farming.
Antibiogram analysis and antimicrobial resistance
Antibiotic susceptibility testing followed the Kirby-Bauer method across a panel of ten antibiotics. Key findings include high resistance to amoxicillin-clavulanate and ampicillin, with complete resistance to some beta-lactams such as amoxicillin and penicillin in all isolates. Conversely, isolates showed relatively strong sensitivity to ceftriaxone, ciprofloxacin, streptomycin, and tetracycline. Gentamicin also demonstrated high effectiveness against the isolates. The Multi-Antibiotic Resistance Index (MARI) calculated at 0.018 suggests a low overall resistance burden in this dataset, though stewardship remains essential to prevent escalation.
Implications for aquaculture and public health
The detection of Pseudomonas spp. in Nile tilapia across multiple Ethiopian sites underscores the need for robust biosecurity and routine microbial surveillance in ponds. The combination of phenotypic, MALDI-TOF, and PCR approaches provides a strong framework for rapid diagnosis. The observed antibiotic resistance profile emphasizes prudent use of antimicrobials in fish farming to curb AMR risks to animal and human health. Given the zoonotic potential of Pseudomonas spp., ensuring proper cooking, handling, and wastewater management is critical for protecting consumers and ecosystems.
Conclusions
This study demonstrates a multifaceted approach to characterizing Pseudomonas infections in Nile tilapia within Ethiopian aquaculture. By integrating phenotypic traits, molecular detection, and antibiogram data, researchers and farmers can better monitor disease, tailor treatment strategies, and implement preventive measures to sustain tilapia production and public health.
