Study On The Biofilm Formation And Quorum Sensing Mechanism Of Cronobacter Sakazakii

Cronobacter sakazakii(C.sakazakii)is a kind of emerging pathogenic bacteria,adults with compromised immune systems,especially infants are susceptible to C.sakazakii,and causing infections such as necrotizing enterocolitis and meningitis.It is from a wide range of sources which can be found in different kinds of food and production environment,putting people at the risk of infection.Quorum sensing(QS)is an intercellular communication process that relies on cell density.It promotes special gene expression and controls density-dependent behavior by producing signal molecules and spreading into the environment.The biofilms synthesized by bacteria serve as a defensive barrier for bacteria,protecting bacteria against all kinds of hostile environment,such as water or nutrition deficiency,the existence of fungicides or other antibacterial agents,leading to serious public health hazards and economic losses.Therefore,the aim of this study is to establish a method to detect N-acyl-homoserine lactones produced by four different C.sakazakii strains.In addition,investigating how environmental factors affected four C.sakazakii strains’ biofilm forming and the best conditions for biofilm fomation were determined.Discussing the effects of quorum sensing signal molecules on C.sakazakii’s biofilm formation.Finally,through detecting the production of C.sakazakii’s signal molecules at different times,establishing mathematical models by using IPMP software and the growth mechanism was studied.The contents and results of this subject were as follows:1.Two biosensing bacteria(C.violaceum CV026 and A.tumefaciens NTL4(pZLR4))were applied to detect N-acyl-homoserine lactones produced by four different C.sakazakii strains,the methods including parallel streak method and reporter plate drilling method.The results showed that the concentration of signal molecules produced by C.sakazakii was low,the short chain AHLs did not reach the detection limit of C.violaceum CV026.A.tumefaciens NTL4(pZLR4)was positive for the detection of N-acyl-homoserine lactones produced by four different C.sakazakii strains.2.UPLC-MS/MS physical and chemical quantitative detection methods were established to detect six different AHLs signal molecules(C6-HSL,C7-HSL,3-oxo-C8-HSL,C10-HSL,C12-HSL,C14-HSL)produced by four different C.sakazakii strains.The results showed that the species and content of the signal molecules from different C.sakazakii strains were specific to the strains.The number of AHLs signal molecules synthesized by C.sakazakii CICC21550 was the most,all of them were detected except C10-HSL,and the content of C14-HSL was the highest;Only a small number of C7-HSL signal molecules was detected in C.sakazakii CICC21562;Three kinds of AHLs(C6-HSL,C7-HSL,3-oxo-C8-HSL)were detected in C.sakazakii CICC21563 and C.sakazakii CICC21544.3.The effects of various environmental factors(initial pH value,culture time and incubation temperature)on biofilm formation of different C.sakazakii strains were investigated by microplate method,and the role of C14-HSL signal molecules in the development of C.sakazakii CICC21550 biofilm were was also studied.The results showed that environmental factors had significant effects on biofilm formation,the optimum environmental conditions for forming the maximum amount of biofilm is:pH=7,incubation temperature 30℃,culture time 36h for C.sakazakii CICC21550;pH=5,incubation temperature 30℃,culture time 36h for C.sakazakii CICC21563;pH=7,incubation temperature 42℃,culture time 24h for C.sakazakii CICC21544;pH=5,culture time 48h for C.sakazakii CICC21562,and the biofilm formation of this strain is insensitive to the temperature factor.In addition,quorum sensing signal molecules have effect on the biofilm development of C.sakazakii CICC21550.In heavy concentrations(2μM),the C14-HSL signal molecule significantly increased the biofilm formation of the C.sakazakii CICC21550.4.A mathematical model was established by IPMP program to explore the rule of C14-HSL signal molecule produced during the growth process of C.sakazakii.Meanwhile,the growth mechanism of C.sakazakii was discussed from microcosmic angle combined with the macroscopic model of bacterial growth.The results showed that C14-HSL signal molecules were detected in the early stage of logarithmic growth of C.sakazakii CICC21550,and it reached the maximum value in the stable period,and then decreased gradually,showing apparent density dependence.The IPMP program is not only suitable for the model to fit the growth of bacterial,but also suitable for the model to fit quorum sensing signal molecules of bacteria.The one-level model of Baranyi model,Huang model and Gompertz model gave good fit to the experimental data of C14-HSL signal molecule produced during the growth process of C.sakazakii CICC21550.The curve of each fitting model had obvious lag period,and the lag period is obviously longer than that of bacterial growth,it showed that the start of the bacterial quorum sensing system should be based on the adaptation of the bacteria to the environment.