| Cronobacter sakazakii is an opportunistic pathogen transmitted by food,which has been implicated in severe forms of neonatal infections such as bacteraemia,necrotizing enterocolitis and neonatal meningitis with reported case fatality rates of 50-80%.It is very meaningful to search for an effective method that controls C.sakazakii.Recently,some authors have proposed several decontamination methods in an effort to reduce levels of C.sakazakii in powdered infant formula,including simultaneous near-infrared radiant heating and UV radiation and gamma radiation.However,these methods may cause a loss of sensitive nutrients from the formula and have limitations because of high capital costs for installation.Chemical preservatives have also been commonly used to prevent microbial contamination in various foods,but their application in food is not favored by consumers due to their synthetic nature and possible negative effect on human health.There is currently an increased trend in food industry to use natural antimicrobials,which gain increased consumer acceptance.Recently,anti-virulence strategy is considered to be a promising avenue to combat bacterial infection,which was thought to impose less selective pressure on bacteria to reduce the chance of selecting resistant strains.However,the research of plant-derived compounds on antimicrobial mechamism and anti-infection activity were scare.Therefore,in my study,based on the evaluation of the effective antimicrobial against C.sakazakii,citral was selected as the antimicrobial component investigated in this study.The antimicrobial activity,inhibitory activity of virulence and the protective effect of intestinal inflammation of citral were clarified.In addition,the inactivation of nondesiccated and desiccated C.sakazakii in reconstituted infant formula by combination of citral and mild heat was investigated,the context and results were shown as following: 1.Evaluation of antibacterial activities of plant-derived compounds against Cronobacter sakazakiiThe antibacterial activity of fifty plant-derived compounds was evaluated to screen the effective antimicrobials.The growth inhibition zone of plant-derived compounds against C.sakazakii was first determined via Oxford cup method.Moreover,the MICs of compouds which caused inhibition zone were determined using agar dilution method.Finally,the MICs of plant-derived compounds against four strains from American Type Culture Collection and five food isolates strains were assessed.It was shown that 40 compounds had obvious growth inhibition zone,and the diameters of 7 compounds(carvacrol,thymoquinone,thymol,cinnamaldehyde,citral,protocatechuic acid and protocatechualdehyde)were more than 13 mm.In addition,the MICs of eighteen compounds against C.sakazakii ATCC 29544 were less than or equal to 5 mg/mL.The most effective plant-derived compounds were thymol and carvacrol,and their MICs ranged from 0.1 to 0.2 mg/mL.Thymoquinone,cinnamaldehyde,citral and protocatechualdehyde demonstrated a stronger effect with the MICs ranged from 0.3 to 1.25 mg/m L.Nine compouds(ferulic acid,chlorogenic acid,syringic acid,lipoid acid,protocatechuic acid,epicatechin,caffeic acid,paeonol and cichoric acid)have moderate antimicrobial activity against C.sakazakii with the MICs ranged from 2.5 to 5 mg/m L.These findings suggest that some plant-derived compounds exhibit antimicrobial effect against C.sakazakii strains and could be potentially used to control C.sakazakii in the process of food processing,circulation and storage.2.Antimicrobial activity of citral against Cronobacter sakazakii and possible mechanism of actionThe antimicrobial mechanism of citral against C.sakazakii strains was investigated.The minimum inhibitory concentration(MIC)of citral against C.sakazakii was determined via agar dilution method,then Gompertz models were used to quantitate the effect of citral on microbial growth kinetics.Changes in intracellular pH(pHin),membrane potential,intracellular ATP concentration and membrane integrity were measured to elucidate the possible antimicrobial mechanism.Moreover,cell morphology and integrity changes were observed under field emission scanning electron microscope and confocal laser scanning microscope.The results showed that the MICs of citral against C.sakazakii strains ranged from 0.27 to 0.54 mg/mL,the addition of citral resulted in a lower specific growth rate and a lower maximum optical density of C.sakazakii.Citral at MIC and 2MIC caused a decrease in pHin of C.sakazakii from 6.06 to 5.01 and 4.84,and caused intracellular ATP concentrations reduced from 0.416 ?mol/L to 0.078 and 0.056 ?mol/L,both tested concentrations of citral caused 56% and 85% reduction in cell integrity,respectively.In addition,cells treated with citral showed hyperpolarized cell membranes.Scanning electron microscopy analysis further confirmed that C.sakazakii cell membranes were damaged by citral.In summary,citral exerts its inhibitory effect through inducing excessive permeability of the cell membrane associated with generalized membrane-disrupting effects.3.Inactivation of nondesiccated and desiccated Cronobacter sakazakii in reconstituted infant formula by combination of citral and mild heatThe combined effect of citral and mild heat on nondesiccated and desiccated C.sakazakii in reconstituted infant formula was evaluated.Various concentrations of citral(0,0.3%,0.6%,and 0.9%)combined with different temperatures(25°C,45°C,50°C,and 55°C)were applied to nondesiccated and desiccated cocktails of three C.sakazakii strains(approximately 6.0 log CFU/m L)in reconstituted infant formula,and the bacterial populations were assayed periodically.Our results showed that the combined treatments had marked antimicrobial effects on C.sakazakii,compared with the control.Desiccated cells were more susceptible to citral than nondesiccated cells in reconstituted infant formula.These findings suggest a potential application of citral in combination with mild heat in controlling C.sakazakii during preparation of reconstituted infant formula.4.Citral attenuate virulence in Cronobacter sakazakii and possible mechanism of actionThe aim of this study was to determine whether citral could suppress putative virulence factors of C.sakazakii that contribute to infection.Swimming and swarming were evaluated in LB containing different agar concentrations,and transmission electron microscopy was used to examine the morphology of flagella;The effect of citral on the adhesion and invasion of C.sakazakii was investigated by using Caco-2 cells and the effect on the intracellular survival and replication was determined by using RAW 264.7 cells;Changes in endotoxin production were also analyzed.In addition,expression of different virulence genes were determined by reverse-transcriptase(RT)PCR.The results showed that sub-inhibitory concentrations of citral significantly decreased motility and endotoxin production.Citral substantially reduced the adhesion and invasion of C.sakazakii to Caco-2 cells and decreased bacterial survival and replication within the RAW 264.7 macrophage cells.Citral also repressed the expression of fifteen genes involved in the virulence.These findings suggest that citral has potential to be developed as an alternative or supplemental agent to mitigate the infections caused by C.sakazakii.5.The effect of citral on biofilm formation and quorum sensing in Cronobacter sakazakiiThe effects of surface type(stainless steel,glass,silicone),growth media(typtic soy broth,rconstituted infant formula),and growth temperature(12°C,25°C,37°C)on biofilm formation of C.sakazakii.The effect of citral on biofilm formation was determined by crystal violet test and FESEM.The effect on the QS inhibitory activity was measured by quantifying the violacein production with the indicator strain.The results showed that the higher populations attached and formed biofilms at 25°C and 37°C than at 12°C,the biofilm formation ability of C.sakazakii in TSB is stronger than its ability in reconstituted infant formula,and biofilm formed on three kinds of surface without specific differences;The efficiency of disinfectants was dependent on the growth temperature,the surface type and the disinfectant products.A significant decrease in biofilm formation in the presence of citral at 12°C and 25°C was demonstarted.In addition,it was shown that citral inhibited violacein pigment production of AHL quorum sensing indicator strain,Chromobacterium violaceum.Our results also revealed that citral significantly down-regulated the expression of the genes(bcsA and bcsG)which encode the cellulose biosynthesis operon,and the expression of luxR which encodes a transcriptional regulator associated with AHL type QS system was also down-regulated.These findings suggest that citral has potential application for controlling C.sakazakii biolilms and quorun sensing in food industry.6.Citral protects against Cronobacter sakazakii-induced intestinal inflammationCaco-2 cell infected with C.sakazakii was used as a model to detect the release of NO,LDH,TNF-? and the enterocyte apoptosis in vitro.We also tested in vivo whether citral could protect Kunming newborn mice from developing intestinal inflammation induced by exposure to C.sakazakii.It was found that concentrations of citral(?10 ?g/mL)has no effect on cell proliferation and survival rate.Citral decreased the concentrations of inflammatory cytokines TNF-? and could significantly decrease the levels of nitric oxide from Caco-2 cells infected with the C.sakazakii.The TUNEL dyeing results showed that citral can significantly delay the Caco-2 cells apoptosis.In addition,it inhibited Caspase-3 activity in Caco-2 cells infected with C.sakazkii.Pretreatment with citral prevented infection and maintained ileal structure in Kunming newborn mice.Moreover,citral downregulated several genes related to apoptosis,cytokine production,and various signaling pathways.This findings suggests that pretreatment with citral can protect Caco-2 cells and newborn mice infected with C.sakazakii from inflammation.Citral has the potential to prevent and treat intestinal inflammation infected with C.sakazakii. |
PDF Full Text Request