Aeromonas hydrophila is a ubiquitous Gram-negative opportunistic pathogen in the freshwater environment and the most common cause of bacterial septicemia in aquaculture. In this study, we investigated the impact of carvacrol, a natural monoterpenoid found in herbs, on the virulence of A. hydrophila in vitro and the antibacterial effect in combination with antibiotics. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of carvacrol against A. hydrophila NJ-35 were 125 µg/mL and 250 µg/mL, respectively. Carvacrol could inhibit the virulence factors (biofilm, protease, exopolysaccharide, and hemolysin) of A. hydrophila, and the antibiofilm potential of carvacrol was further verified by microscopic detection. Transcriptional analyses showed that the gene expression of flaB, ompA, aha, ahp, ela, act, aerA, AhyR, and hly were marked as downregulated. The checkerboard assay results showed that carvacrol did not have an antagonistic effect in combination with antibiotics (florfenicol, enrofloxacin, thiamphenicol, or doxycycline hydrochloride) commonly used in aquaculture but possessed an additive-synergistic effect with neomycin sulfate. In vivo studies demonstrated that carvacrol protected grass carp (Ctenopharyngodon idella) from A. hydrophila infection. Our results indicated that carvacrol possessed significant anti-bacterial and anti-virulence effects on A. hydrophila.

External segmentation and internal proglottization are important evolutionary characters of the Eucestoda. The monozoic caryophyllideans are considered the earliest diverging eucestodes based on partial mitochondrial genes and nuclear rDNA sequences, yet, there are currently no complete mitogenomes available. We have therefore sequenced the complete mitogenomes of three caryophyllideans, as well as the polyzoic Schyzocotyle acheilognathi, explored the phylogenetic relationships of eucestodes and compared the gene arrangements between unsegmented and segmented cestodes. The circular mitogenome of Atractolytocestus huronensis was 15,130 bp, the longest sequence of all the available cestodes, 14,620 bp for Khawia sinensis, 14,011 bp for Breviscolex orientalis and 14,046 bp for Schyzocotyle acheilognathi. The A-T content of the three caryophyllideans was found to be lower than any other published mitogenome. Highly repetitive regions were detected among the non-coding regions (NCRs) of the four cestode species. The evolutionary relationship determined between the five orders (Caryophyllidea, Diphyllobothriidea, Bothriocephalidea, Proteocephalidea and Cyclophyllidea) is consistent with that expected from morphology and the large fragments of mtDNA when reconstructed using all 36 genes. Examination of the 54 mitogenomes from these five orders, revealed a unique arrangement for each order except for the Cyclophyllidea which had two types that were identical to that of the Diphyllobothriidea and the Proteocephalidea. When comparing gene order between the unsegmented and segmented cestodes, the segmented cestodes were found to have the lower similarities due to a long distance transposition event. All rearrangement events between the four arrangement categories took place at the junction of rrnS-tRNA Arg (P1) where NCRs are common. Highly repetitive regions are detected among NCRs of the four cestode species. A long distance transposition event is inferred between the unsegmented and segmented cestodes. Gene arrangements of Taeniidae and the rest of the families in the Cyclophyllidea are found be identical to those of the sister order Proteocephalidea and the relatively basal order Diphyllobothriidea, respectively.

Stress is an important contributing factor in the outbreak of infectious fish diseases. To comprehensively understand the impact of catecholamine stress hormone norepinephrine (NE) on the pathogenicity of Aeromonas hydrophila, we assessed variations in bacterial growth, virulence‐related genes expression and virulence factors activity after NE addition in serum‐SAPI medium. Further, we assessed the effects of NE on A. hydrophila virulence in vivo by challenging fish with pathogenic strain AH196 and following with or without NE injection. The NE‐associated stimulation of A. hydrophila strain growth was not linear‐dose‐dependent, and only 100 μM, or higher concentrations, could stimulate growth. Real‐time PCR analyses revealed that NE notably changed 13 out of the 16 virulence‐associated genes (e.g. ompW, ahp, aha, ela, ahyR, ompA, and fur) expression, which were all significantly upregulated in A. hydrophila AH196 (p < 0.01). NE could enhance the protease activity, but not affect the lipase activity, hemolysis, and motility. Further, the mortality of crucian carp challenged with A. hydrophila AH196 was significantly higher in the group treated with NE (p < 0.01). Collectively, our results showed that NE enhanced the growth and virulence of pathogenic bacterium A. hydrophila.

Aeromonas hydrophila is a Gram-negative bacterium that widely exists in various aquatic environments and causes septicemia in fish and humans. Resveratrol, a natural polyterpenoid product, has potential chemo-preventive and antibacterial properties. In this study, we investigated the effect of resveratrol on A. hydrophila biofilm formation and motility. The results demonstrated that resveratrol, at sub-MIC levels, can significantly inhibit the biofilm formation of A. hydrophila, and the biofilm was decreased with increasing concentrations. The motility assay showed that resveratrol could diminish the swimming and swarming motility of A. hydrophila. Transcriptome analyses (RNA-seq) showed that A. hydrophila treated with 50 and 100 μg/mL resveratrol, respectively, presented 230 and 308 differentially expressed genes (DEGs), including 90 or 130 upregulated genes and 130 or 178 downregulated genes. Among them, genes related to flagellar, type IV pilus and chemotaxis were significantly repressed. In addition, mRNA of virulence factors OmpA, extracellular proteases, lipases and T6SS were dramatically suppressed. Further analysis revealed that the major DEGs involved in flagellar assembly and bacterial chemotaxis pathways could be regulated by cyclic-di-guanosine monophosphate (c-di-GMP)- and LysR-Type transcriptional regulator (LTTR)-dependent quorum sensing (QS) systems. Overall, our results indicate that resveratrol can inhibit A. hydrophila biofilm formation by disturbing motility and QS systems, and can be used as a promising candidate drug against motile Aeromonad septicemia.

Bacterial quorum sensing (QS) plays a crucial role in chemical communication between bacteria involving autoinducers and receptors and controls the production of virulence factors in bacteria. Therefore, reducing the concentration of signaling molecules in QS is an effective strategy for mitigating the virulence of pathogenic bacteria. In this study, we demonstrated that carvacrol at 15.625 μg/mL (1/4 MIC), a natural compound found in plants, exhibits potent inhibitory activity against QS in Chromobacterium violaceum, as evidenced by a significant reduction (62.46%) in violacein production. Based on its impressive performance, carvacrol was employed as a natural QS inhibitor to suppress the pathogenicity of Aeromonas hydrophila NJ-35. This study revealed a significant reduction (36.01%) in the concentration of N-acyl-homoserine lactones (AHLs), a QS signal molecular secreted by A. hydrophila NJ-35, after 1/4 MIC carvacrol treatment. Moreover, carvacrol was found to down-regulate the expression of ahyR/I, two key genes in the QS system, which further inhibited the QS system of A. hydrophila NJ-35. Finally, based on the above results and molecular docking, we proposed that carvacrol alleviate the pathogenicity of A. hydrophila NJ-35 through QS inhibition. These results suggest that carvacrol could serve as a potential strategy for reducing the virulence of pathogenic bacteria and minimizing the reliance on antibiotics in aquaculture.