The effect of triclosan on intergeneric horizontal transmission of plasmid-mediated tigecycline resistance gene tet(X4) from Citrobacter freundii isolated from grass carp gut.

The transmission of antibiotic resistance genes (ARGs) in pathogenic bacteria affects culture animal health, endangers food safety, and thus gravely threatens public health. However, information about the effect of disinfectants - triclosan (TCS) on ARGs dissemination of bacterial pathogens in aquatic animals is still limited. One Citrobacter freundii (C. freundii) strain harboring tet(X4)-resistant plasmid was isolated from farmed grass carp guts, and subsequently conjugative transfer frequency from C. freundii to Escherichia coli C600 (E. coli C600) was analyzed under different mating time, temperature, and ratio. The effect of different concentrations of TCS (0.02, 0.2, 2, 20, 200 and 2000 µg/L) on the conjugative transfer was detected. The optimum conditions for conjugative transfer were at 37 °C for 8h with mating ratio of 2:1 or 1:1 (C. freundii: E. coli C600). The conjugative transfer frequency was significantly promoted under TCS treatment and reached the maximum value under 2.00 µg/L TCS with 18.39 times that of the control group. Reactive oxygen species (ROS), superoxide dismutase (SOD) and catalase (CAT) activities, cell membrane permeability of C. freundii and E. coli C600 were obviously increased under TCS stress. Scanning electron microscope showed that the cell membrane surface of the conjugative strains was wrinkled and pitted, even broken at 2.00 µg/L TCS, while lysed or even ruptured at 200.00 µg/L TCS. In addition, TCS up-regulated expression levels of oxidative stress genes (katE, hemF, bcp, hemA, katG, ahpF, and ahpC) and cell membrane-related genes (fimC, bamE and ompA) of donor and recipient bacteria. Gene Ontology (GO) enrichment demonstrated significant changes in categories relevant to pilus, porin activity, transmembrane transporter activity, transferase activity, hydrolase activity, material transport and metabolism. Taken together, a tet(X4)-resistant plasmid could horizontal transmission among different pathogens, while TCS can promote the propagation of the resistant plasmid.

Hepcidin from Onychostoma macrolepis: Response to Aeromonas hydrophila infection by down-regulating expression of inflammatory factor genes and regulating iron homeostasis potentially.

Hepcidin, as an antimicrobial peptide, is associated with innate immunity and is considered a potential antibiotic substitute. In the present study, the hepcidin gene from the cavefish - Onychostoma macrolepis was identified and analyzed. The recombinant hepcidin protein (rOmhepc) was obtained by prokaryotic expression, evaluating the inhibitory effect of 5 pathogenic bacteria in vitro. Sixty O. macrolepis injected with 100 µL A. hydrophila (1.5 × 108 CFU/mL) were randomly divided into the therapeutic group and infection group, and therapeutic group was injected with 100 µL rOmhepc (100 µg/mL) at 6 and 18 h. The survival rates of O. macrolepis and bacterial load in liver were measured at 24 h. The liver tissues were collected at 0, 6, 12, and 24 h after A. hydrophila injection for investigating expression levels of immune-related, inflammatory factor genes and FPN1 gene. The results demonstrated that the hepcidin CDS contained 279 bp and encoded 93 aa. Hepcidin protein has a hydrophobic surface formed by multiple hydrophobic residues (CCGCCYC), and the theoretical pI was 7.53. Omhepc gene was expressed at varying levels in tested tissues, with the liver showing the highest expression, followed by the spleen. The expression of hepcidin gene following A. hydrophila infection was up-regulated and then down-regulated in liver, and the highest expression level was found at 12 h with a 10.93-fold. The rOmhepc remarkably inhibited the growth of A. hydrophila, Staphylococcus aureus, and Streptococcus agalactiae, with inhibition rates reaching 69.67 %, 42.97 %, and 65.74 % at 100 µg/mL. The mortality rates of O. macrolepis and bacterial load in liver were significantly decreased in the therapeutic group than that of infection group (p < 0.05). After the rOmhepc therapeutic, interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were significantly down-regulated with 14.4-fold and 106.07-fold at 24 h. Furthermore, the expression of immune-related genes (C3, TNF-α, IFN-γ) and Ferroportin gene (FPN1) significantly decreased (p < 0.05). The integrated analyses indicated that the rOmhepc could significantly inhibit the growth of A. hydrophila both in vitro and in vivo, attenuating the over-expression of inflammatory factor, FPN1 and immune-related genes.

Effects of 4-tert-butylpyrocatechol and tea polyphenol on growth, physiology and antioxidant responses in Microcystis aeruginosa.

Global warming has increased the frequency of Microcystis aeruginosa blooms, leading to the deterioration of water quality and loss of biodiversity. Therefore, developing effective strategies for controlling M. aeruginosa blooms has become an important research topic. Plant extracts, 4­tert-butylpyrocatechol (TBC) and tea polyphenol (TP) are commonly used for water purification and to increase fish immunity, which have great potential to inhibit cyanobacterial blooms. The inhibitory effects of TBC and TP on M. aeruginosa were investigated in terms of growth characteristics, cell membrane morphology, physiological, photosynthetic activities, and antioxidant enzymes activities. The results showed that TBC and TP inhibited the growth of M. aeruginosa by decreasing the chlorophyll fluorescence transients or increasing the antioxidant enzymes activities of M. aeruginosa. TBC damaged the cell morphology of M. aeruginosa, reduced extracellular polysaccharides and protein contents, and up-regulated the antioxidant activity-related gene (sod and gsh) expressions of M. aeruginosa. TP significantly decreased the photosynthetic pigment content, influenced the phycobiliprotein content, and strongly down-regulated the photosynthesis-related gene (psbA, psaB, and rbcL) relative expressions of M. aeruginosa. TBC caused significant oxidative stress, dysfunction of physiological metabolic processes, and damaged crucial biomacromolecules (e.g., lipids, proteins and polysaccharides), prompted the loss of cell integrity, ultimately leading to the death of M. aeruginosa. However, TP depressed photosynthetic activities and consequently inhibited the transfer of electrons, affected the electron transfer chain, decreased the photosynthetic efficiency, and eventually caused the death of M. aeruginosa cells. Our study showed the inhibitory effects and algicidal mechanisms of TBC and TP on M. aeruginosa, and provide a theoretical basis for restrain the overgrowth of M. aeruginosa.