Effect And Molecular Mechanism Of LED Treatment On Pathogenicity Of Cronobacter Sakazakii

Cronobacter sakazakii as a Gram-negative,rod-shaped,non-sporeforming,and motile via peritrichous flagella bacterium could cause fatal neonatal meningitis,sepsis,necrotizing enterocolitis(NEC)with a high rate of mortality of 50～80%.In addition,C.sakazakii has a wide range of contamination,but powdered infant formula(PIF)is the main source of the outbreak.Therefore,it is imperative to find a safe and effective method for the control of C.sakazakii in PIF.Recently,some authors have proposed several decontamination methods effectively to reduce the level of C.sakazakii in PIF,including heat sterilization,simultaneous near-infrared radiant heating,UV radiation,and gamma radiation.However,these methods may cause a loss of sensitive nutrients in PIF and have limitations because of high capital costs for installation.In addition,in recent years,some researchers have focused on the use of plantderived active substances to inhibit the C.sakazakii in PIF.However,the use of plant-derived active substances alone also has lots of disadvantages such as high concentration,high cost,and strong smell,which limits its application in PIF.Therefore,it is of great significance to explore effective,safe,cheap methods,which do not affect the product quality at the same time,to control the C.sakazakii in PIF.Light-emitting diode(LED),as a new type of low temperature,efficient and environmentally friendly sterilization technology,has gradually attracted the attention.405 nm LED has been proved to have a wide range of bactericidal effect,and has a higher safety than ultraviolet radiation(UV)on human body.However,studies on the inhibitory effects and anti-infection molecular mechanism of 405 nm LED are rare,which is difficult to support its industrial applications.Therefore,in this study by building the PIF model and PBS model contaminated by C.sakazakii,on the basis of which studies the inhibition effects of 405 nm LED to C.sakazakii in PIF,effects of 405 nm LED on the environment tolerance of C.sakazakii in PIF,interactions of C.sakazakii with the host cells after LED illumination,and related expressions of resistance genes and virulence genes of C.sakazakii after LED illumination.The results were shown as following:1.Establishment of PIF model contaminated by C.sakazakii and determination of sublethal irradiation dose of 405 nm LED illuminationFirst,design and make the LED sterilization device,then build the PIF model contaminated by C.sakazakii,and determine the incubation temperatures of corresponding control groups,and then determine the inhibition effects of 405 nm LEDs in different power to C.sakazakii in PIF so as to ascertain the sublethal radiation dose of 405 nm LED illumination.The damage rates of C.sakazakii in PIF are also further determinated during the LED-illumination.The results showed that there was no significant difference in the inhibitory effect on C.sakazakii in PIF between 10 W and 20 W LED.After the treatment for 480 min of405 nm LED,the damage rates of bacteria reached the maximum,which was significantly different from that of the corresponding control groups and showed the dose dependence in degree.2.Influence of 405 nm LED on environmental tolerance of C.sakazakii in PIFA series of extreme environments faced by C.sakazakii were simulated in the production,processing,reconstitution,and gastrointestinal tract of host,such as drying,high temperature,gastric acid,bile salt,etc.,in this experiment and determined the effects of 405 nm LED on the tolerance to different adverse environments of C.sakazakii in PIF.The experimental results showed that the 405 nm LED significantly reduced the resistance of C.sakazakii to dry,heat,gastric acid,and bile salt.3.Influence of 405 nm LED on the interactions of C.sakazakii with host cellsConstructe the PBS model contaminated by C.sakazakii,then determine the incubation temperature of control group in PBS,based on which studies the inhibition effect of 405 nm LED on C.sakazakii,finally explore the effects of 405 nm LED on adhesion and intrusion ability of C.sakazakii to Caco-2 cells and proliferation and survival ability of it in macrophage RAW 264.7.The results showed that the adhesion ability of C.sakazakii to Caco-2 cells decreased but its invasion ability increased after 405-nm LED treatment.Additionally,405 nm LED can significantly promote the survival and proliferation ability of C.sakazakii in macrophage RAW 264.7.4.The molecular mechanism by which 405 nm LED regulates the pathogenic ability of C.sakazakiiThe effect of 405 nm LED on the cell membrane of C.sakazakii was observed by confocal laser microscopy,and the effects of 405 nm LED on the environmental tolerance genes and virulence genes of C.sakazakii were determined by RT-PCR.The results showed that the cell membrane integrity of C.sakazakii was damaged after 405 nm LED treatment.The genes(osm Y,dnak,grx B,hfq,fur,omp C,mfla,gro ES,rpos)related to environmental tolerance of C.sakazakii were significantly down-regulated.The genes(omp A,omp X,uvr Y)were related to adhesion and invasion,among them omp A was significantly up-regulated,while omp X and uvr Y were significantly down-regulated.The gene sod related to survival and proliferation was significantly up-regulated.