| Citrobacter freundii（C.freundii）is a normal flora of the animal gut,widely dispersed in the natural environment such as soil and water with the excretion of the animal,acquiring numerous drug resistance in the environment and carrying several drug resistance genes.C.freundii is a conditionally pathogenic bacterium that assaults the body when the animal’s resistance is diminished,migrating from the intestine to other internal organs and showing itself as diarrhea,fever,and other symptoms.Plant essential oils（PEO）are volatile chemicals collected from different parts of the plant and contain a variety of active components such as phenol,aldehyde,and alcohol.The active compounds in PEO have various physiological effects and have a role in suppressing the development of inflammation,the growth of pathogenic microorganisms,and the body’s oxidative stress.In this experiment,the inhibition effect of PEO on C.freundii was investigated both in vitro and in vivo,with the following main results:1、A total of 11 PEO,namely Oregano essential oil,Cinnamon essential oil,Litsea cubeba essential oil,Clove essential oil,Rosemary Essential Oil,Essential oil of Artemisia argyi,Eucalyptus Essential Oil,Natural garlic essential oil,star anise essential oil,perilla essential oil and Murraya paniculata essential oil were tested for their antibacterial activity against C.freundii by Minimal inhibition concentration（MIC）test,Oregano essential oil and Cinnamon essential oil were selected for their good antibacterial activity against C.freundii at MIC of 0.78125 mg/m L.2、The results of the fractional inhibitory concentration（FIC）test revealed that the Oregano essential oil and Cinnamon essential oil showed a synergistic inhibition against C.freundii with a FICI of 0.5,which was used as a Combine essential oil（CEO）.The results of the time-inhibition curve,Malondialdehyde（MDA）and Reactive oxygen species（ROS）content,Adenosine triphosphate（ATP）content,and Adenosine triphosphatase enzyme（ATPase）activity showed that,the CEO causes oxidative stress and increases lipidation in C.freundii,as evidenced by a sharp increase in intracellular MDA and ROS;the metabolism,respiration and energy transfer of C.freundii is reduced and interrupted,as evidenced by a decrease in ATP content and ATPase activity;the growth of C.freundii is inhibited,as evidenced by a decrease in absorbance at OD600.3、Intracellular nucleic acid and protein content,virulence gene expression,extracellular nucleic acid and protein leakage,relative conductivity,Alkaline phosphatase（AKP/ALP）content,and scanning electron microscopy showed that after the action of the CEO on C.freundii,the content of intracellular nucleic acid and protein decreased,the expression level of bacterial motility,adhesion,and colonization-related genes declined,the extracellular nucleic acid and protein content increased after the cell membrane permeability increased,the integrity of the cell wall was disrupted,and the bacteria crumpled and showed a vacuolated appearance.4、A mice infection model was established by intraperitoneal injection of C.freundii to investigate the pathogenicity of C.freundii in mice.The mice showed clinical signs of chilling,eyelid swelling,and eye congestion and then died of convulsions,5×109 CFU/m L concentrations of C.freundii caused nearly half of the mice to die and 1×1010 CFU/m L concentrations of C.freundii caused nearly all of the mice to die.The 5×109 CFU/m L concentrations of C.freundii was selected for the prophylactic experiment,and the results showed that CEO has a good preventative effect against C.freundii infection in the mice.It strengthens the healthy barrier of the mice intestine by increasing the villi height,crypt depth,villi crypt ratio,and the expression of tight junction protein,preventing the transfer of C.freundii from the intestine to major organs such as liver and spleen of the mice,reduces the colonization of C.freundii in the liver and spleen,reduces the inflammatory response induced by C.freundii colonization in mice,thereby increasing the survival rate of C.freundii-infected mice.5、A bullfrog infection model was established by intramuscular injection of C.freundii to investigate the pathogenicity of C.freundii in bullfrogs.The bullfrogs died after showing clinical signs of skin ulceration,and both legs’redness,and a dose of 5×109 CFU/m L of C.freundii caused nearly half of the bullfrogs to die.The dose of 5×109 CFU/m L was selected for a prophylactic experiment,the results show that the addition of CEO to bullfrog diets strengthens the intestinal barrier and reduces the burden on the main organs of bullfrogs,thus increasing the survival rate of C.freundii-infected bullfrogs and CEO can be used as a safe and effective feed additive to prevent the occurrence of bacterial diseases in bullfrog farming. |
