Identification and characterization of Pseudomonas aeruginosa in the intestines of large Scophthalmus maximus

Objective Pseudomonas aeruginosa is an opportunistic pathogen that has been extensively studied in freshwater aquaculture animals, but rarely investigated in marine aquaculture species. In this study, we isolated P. aeruginosa from the intestines of Scophthalmus maximus and conducted a comprehensive investigation into its drug susceptibility and pathogenicity, aiming to provide theoretical basis and practical guidance for the prevention and treatment of P. aeruginosa infection in S. maximus culture.

Methods The intestinal bacteria of S. maximus were identified through bacterial isolation and purification, morphological observation, physiological and biochemical tests, 16S rRNA gene sequencing and phylogenetic tree construction. The dominant strain was selected for artificial regression infection test, virulence gene detection and antimicrobial susceptibility test. Furthermore, the changes in the activities of antioxidant enzymes and immune-related enzymes in infected turbot were analyzed.

Results A dominant bacterial strain (Jda) was isolated from subhealthy S. maximus, and identified as P. aeruginosa by Gram staining, physiological and biochemical identification, 16S rRNA sequence alignment and phylogenetic analysis. The artificial regression infection test showed that the cumulative mortality rates of S. maximus body mass about (30±1) g reached 30% and 65% at 15 days post-infection with medium (3×10⁹ CFU/mL) and high (3×10¹⁰ CFU/mL) concentrations of P. aeruginosa, respectively, with a median lethal concentration (LD₅₀) of 1.0×10¹⁰ CFU/mL. Histopathological sections revealed that, compared with the control group, the intestinal villi structure of infected S. maximus was damaged and incomplete, and the hepatocytes exhibited obvious pathological changes including irregular morphology, numerous vacuoles, and pyknosis or migration of cell nuclei. Polymerase chain reaction (PCR) detection identified 16 virulence genes in the P. aeruginosa strain, including toxA, lasA, lasB, exoS, exoY, phzM and phzS, confirming the pathogenicity of this strain to S. maximus. Determination of enzyme activities in S. maximus liver showed that catalase (CAT) activity in the low (3×10⁸ CFU/mL) and high concentration infection groups was significantly lower than that in the control group (P<0.05); superoxide dismutase (SOD) activity in the low concentration group was significantly higher than that in the control group (P<0.05), while that in the medium and high concentration groups was significantly lower (P<0.05); acid phosphatase (ACP) activity in the medium concentration group was significantly lower than that in the control group (P<0.05); and there was no significant difference in alkaline phosphatase (AKP) activity between the three experimental groups and the control group (P>0.05). Antimicrobial susceptibility test indicated that the strain was highly sensitive to ciprofloxacin and enrofloxacin, and moderately sensitive to neomycin, doxycycline, florfenicol and trimethoprim sulfamethoxazole.

Conclusion This study confirmed the pathogenicity of P. aeruginosa to S. maximus. Although the S. maximus activated antioxidant and immune defense mechanisms to varying degrees, it was unable to resist the body damage caused by high-concentration P. aeruginosa infection. On the premise of ensuring the safety of aquatic drug use, ciprofloxacin and enrofloxacin can be used as effective drugs for the treatment of P. aeruginosa infection in S. maximus. This study provides technical support for the prevention and control of P. aeruginosa in S. maximus aquaculture.