This study aimed to investigate the molecular basis of high-level tetracycline resistance to tetracycline antibiotics in Aeromonas hydrophila isolated from fish farming. Comparative genomic analysis of the tetracycline-sensitive strain NJ-35 and the tetracycline-resistant strain AH823 revealed that the tetracycline repressor gene tetR in AH823 had undergone base mutations, resulting in premature translational termination. The tetR gene in NJ-35 was inhibited using a plasmid-based antisense RNA strategy, and the knockdown efficiency was confirmed by RT-qPCR. The resulting tetR antisense RNA-expressing strain, AHtetR-as, exhibited significantly increased resistance to tetracycline antibiotics (minocycline, tetracycline, and doxycycline), but did not affect biofilm formation or hemolysis. Moreover, tetR knockdown in NJ-35 was associated with increased efflux activity and reduced intracellular doxycycline accumulation. Transcriptomic analysis revealed that genes encoding the 30S ribosomal subunit proteins showed a differential expression pattern, with rpsO upregulated and rpsD and rpsP downregulated. These findings suggest that tetR contributes to tetracycline resistance in A. hydrophila and is associated with broad transcriptional changes related to cellular transport and ribosomal function.

In the spring of 2023, a disease outbreak occurred in Lake Taihu in China, which caused a large number of deaths of H. nobilis. In order to investigate the cause of morbidity and mortality of the H. nobilis, the diseased fish were collected for histopathological and etiological studies. Histopathological observation revealed that substantial inflammatory cell infiltration was observed around skin lesion in diseased fish, extensive degeneration and necrosis were observed in the hepatic parenchymal cells, the spleen exhibited congestion, and the kidney showed congestion. A bacterial strain, C1, isolated from diseased H. nobilis was identified as Iodobacter fluviatilis through 16S rRNA gene sequencing and biochemical phenotypic characterization. Experimental infection of the fish via intramuscular injection induced a localized abscess in a subset of fish. Antimicrobial susceptibility testing revealed that the isolate was susceptible to aminoglycosides, tetracyclines, quinolones and amphenicols, but resistant to sulfonamides commonly used in aquaculture. Here, we describe an association between I. fluviatilis and skin lesions in H. nobilis. Furthermore, we report the biochemical characteristics and drug resistance profile of the isolated bacteria. These findings also facilitate further investigations into the role of I. fluviatilis associated with skin diseases of H. nobilis and other freshwater fish.