Molecular Resistance Mechanism And Targeted Blocking Of Cronobacter Sakazakii Based On The Regulation Of Quorum Sensing Receptor SdiA

Contamination of powdered infant formula（PIF）with Cronobacter sakazakii（C.sakazakii）poses serious food safety risks due to the bacterium’s unique stress resistance.Although sterilization of C.sakazakii has gained considerable attention in research,the current body of literature is often insufficient regarding the critical paths of contamination and the precise targeting mechanisms.As a result,the quorum sensing receptor SdiA of C.sakazakii was screened,the regulatory molecular mechanism of SdiA on environmental stress tolerance and biofilm formation were investigated,the natural targeted inhibitors against SdiA were screened through molecular docking,and combined stress conditions were cooperated to inactivate C.sakazakii.The main findings are as follows:（1）The survival of C.sakazakii wild-type（WT）,sdi A knockout strain（Δsdi A）,and sdi A complemented strain（sdi A⁺）under different stress conditions were investigated.The results showed that after a 60-min thermal treatment at 55℃,the survivors of WT and sdi A⁺in Tryptone Soya Broth（TSB）were high than those ofΔsdi A by 4.00 and 2.97 log,respectively（p<0.05）.This indicated that the absence of sdi A was detrimental to the survival of C.sakazakii during thermal treatment.Following a 6-hour exposure to a hyperosmotic TSB solution containing 12%Na Cl,the survival rates of WT,Δsdi A,and sdi A⁺exhibited reductions of92.11%,99.64%,and 97.18%,respectively.These results substantiated that both WT and sdi A⁺possess superior salt stress tolerance compared to theΔsdi A strain.In addition,the findings indicated that after 28 days of desiccation,the survivability of WT,Δsdi A and sdi A⁺in PIF was5.85,3.13 and 5.61 log,respectively,which demonstrated a significant reduction in the desiccation tolerance ofΔsdi A compared to WT and sdi A⁺（p<0.05）.After 3 hours of acid adaptation in simulated gastric fluid,the survival rate of WT and sdi A⁺improved,while the survival rate ofΔsdi A continued to decrease.Furthermore,after simulation of heating,evaporation,and drying conditions,remaining viable bacterial counts of WT and sdi A⁺were observed,however,the culturable bacterial population ofΔsdi A was undetectable.（2）The molecular mechanism underlying the regulation of desiccation tolerance in C.sakazakii by the SdiA receptor was investigated.Following the desiccation treatment,the WT cells exhibited cellular shrinkage but with a heightened presence of peritrichous flagella.In contrast,theΔsdi A cells demonstrated notable cellular damage.Compared to the desiccated WT sample（WT＿T）,1980 genes were significantly upregulated and 626 genes were significantly downregulated in the desiccatedΔsdi A（SdiA-MUT＿T）（p<0.05）.KEGG pathway enrichment analysis revealed that these differentially expressed genes were significantly enriched in pathways related to flagellar assembly,biofilm formation,two-component systems,and bacterial chemotaxis.Under desiccation conditions,compared withΔsdi A,the expression levels of several transcriptional regulatory factors such as sdi A,csr A,rpo N,and nha R were significantly upregulated in WT,and these regulatory factors might interact reciprocally,thereby regulating the expression of downstream genes.Additionally,the expression level of genes related to synthesis and transport of trehalose and flagellar assembly was greater in WT compared toΔsdi A,facilitating the accumulation of intracellular compatible solutes and flagellar synthesis under desiccation conditions.Furthermore,genes linked to ribosome and oxidative phosphorylation were expressed at higher levels in WT than inΔsdi A,indicating the energy supply and the provision of macromolecular proteins.（3）The molecular mechanism of the SdiA receptor regulating the biofilm formation of C.sakazakii was discussed.After the knockout of the sdi A gene,C.sakazakii cells exhibited a substantial increase in absorbance at 595 nm when combined with crystal violet,rising from0.59 to 1.23（p<0.05）,as evidenced by the formation of a thicker and denser biofilm.The swimming diameter of WT on semi-solid agar plates was measured as 2.60 cm,whereas theΔsdi A exhibited a reduced swimming diameter of 0.34 cm（p<0.05）.However,upon complementation of the sdi A gene,the swimming diameter increased to 2.17 cm.These findings suggested that the presence of sdi A contributed to the maintenance of motility in C.sakazakii.In addition,the self-aggregation ability,surface hydrophobicity,and outer membrane permeability ofΔsdi A were all markedly higher than those of WT and sdi A⁺（p<0.05）.Transcriptomic sequencing analysis revealed that genes related to flagella synthesis（flg,flh,and fli）were significantly downregulated after deletion of sdi A gene,whereas genes associated with capsule,cellulose,and polysaccharide synthesis（rcs,bcs A,etc.）were upregulated.That is to say,the bacterial motility and extracellular matrix synthesis might be regulated by SdiA to inhibit biofilm formation.It’s worth to notice that the biofilm formation of C.sakazakii could be promoted in low osmotic pressure（3%Na Cl）or mildly acidic（p H 5.0）conditions,but gradually decreased as the osmotic or acidic pressure increased.（4）Natural targeted inhibitors of the quorum sensing receptor SdiA were screened and combined stress conditions were cooperated to prevent C.sakazakii.A three-dimensional model of the SdiA receptor of C.sakazakii was developed,using Lib Dock and CDOCKER,eight targeted inhibitors were selected:gastrodin,phloretin,salidroside,naringenin,apigenin,vanillin,catechin,and baicalein.The findings indicated that salidroside and gastrodin had no inhibitory effect on the resistance of WT;apigenin,vanillin,and catechin exhibited slight inhibitory effects on the heat,osmotic,and desiccation resistance of WT（p>0.05）;whereas naringin and baicalein displayed significant inhibitory effects on the heat and osmotic resistance of the WT strain（p<0.05）.Importantly,it was found that phloretin exhibited significant inhibitory effects on the heat,osmotic,and desiccation resistance of WT at sub-inhibitory concentrations（p<0.05）,and at concentrations lower than 1.25 mg/m L,phloretin had no positive impact（p>0.05）on biofilm formation of WT.Additionally,it has been demonstrated that phloretin has the ability to bind to the SdiA protein and establish hydrogen bonds with ASP80,TRP 67,and TYR 71 within the hydrophobic cavity of the receptor.These interactions lead to a discernible increase in the UV absorption,α-helical content,and quenching of endogenous fluorescence of the SdiA protein.In whey protein inoculated with less than 5 log₁₀ CFU/m L of WT,phloretin at a concentration of 0.6 mg/m L was effective in eliminating WT when combined with simulation of evaporation and drying processes.When WT was inoculated in oligofructose at or below 5 log₁₀ CFU/m L,both 0.3 and 0.6 mg/m L concentrations of phloretin exhibited complete inactivation of all viable bacteria.The present study investigated the mechanisms involved in the resistance and biofilm formation of C.sakazakii mediated by the SdiA receptor.Through molecular docking,natural targeted inhibitors of the SdiA receptor were identified and the interactions between inhibitors and SdiA were elucidated.In particular,targeted inhibition of the SdiA receptor through the use of natural inhibitors was achieved in dry powder ingredients of PIF,and mild heat,osmotic,and drying stress were combined to ensure the safety of dry powder ingredients.The outcomes of this study provide valuable scientific evidence and insights for the development of innovative prevention and control strategies against C.sakazakii.