Cross protection can undermine the effectiveness of control measures on foodborne pathogens,and therefore brings major implications for food safety.In this work,the capacity of Salmonella Enteritidis to mount ethanol ...Cross protection can undermine the effectiveness of control measures on foodborne pathogens,and therefore brings major implications for food safety.In this work,the capacity of Salmonella Enteritidis to mount ethanol tolerance following acid adaptation was characterized by analysis of cell viability and cell membrane property.It was observed that preadaptation to pH 4.5 significantly(P<0.05)increased the tolerance of log-phase cells to ethanol;in contrast,stationary-phase cells displayed reduced ethanol tolerance after acid adaptation.However,acid adaptation did not cause cell leakage and morphological change in both log-phase and stationary-phase S.Enteritidis.Fatty acid analysis further revealed that the amount of C_(14:0),C_(17:0 cyclo) and C_(19:0 cyclo) fatty acids was increased,while that of C_(16:1ω7c) and C_(18:1ω7c) fatty acids was decreased,respectively,in response to acid adaptation,regardless of bacterial growth phase.Notably,acid adaptation significantly(P<0.05)increased the proportion of C_(16:0) fatty acid in log-phase cells,but this effect did not occur in stationary-phase cells.Moreover,exogenous addition of C_(16:0) fatty acid to stationary-phase acid-adapted cultures was able to enhance bacterial ethanol tolerance.Taken together,C_(16:0) fatty acid is involved in the growth-phase-dependent protective effect of acid adaptation on ethanol tolerance in S.Enteritidis.展开更多
Sodium hypochlorite(NaClO)is a widely used disinfectant in food processing environments.However,its improper use can lead Salmonella to develop tolerance to other stresses,which threatens food safety and human health....Sodium hypochlorite(NaClO)is a widely used disinfectant in food processing environments.However,its improper use can lead Salmonella to develop tolerance to other stresses,which threatens food safety and human health.In this study,the survival ability assays showed that adaptation of S.Enteritidis to NaClO resulted in the development of heat tolerance at 55℃and 63℃.NaClO-adapted S.Enteritidis also showed a survival benefit in milk at 55℃and 63℃.NaClO-adapted S.Enteritidis exhibited a decrease in accumulation of reactive oxygen species(ROS)compared to non-adapted S.Enteritidis after exposure to 63℃.Moreover,compared to non-adapted S.Enteritidis,the membrane fatty acids C16:0 and cyclo-heptadecanoic acid Cyc17 were significantly increased in NaClO-adapted S.Enteritidis,whereas UFA to SFA ratio was observed to decrease from 0.52±0.04 to 0.41±0.04.These changes resulted in a decrease in membrane fluidity of NaClO-adapted S.Enteritidis.Furthermore,RT-qPCR analysis revealed a considerable increase in the expression levels of heat shock-related genes in NaClO-adapted S.Enteritidis with or without heat treatment,in comparison to non-adapted S.Enteritidis.It was noteworthy that upregulation was 7.30-fold for dnaK,11.62-fold for ibpA,and 3.92-fold for rpoH under heat treatment,suggesting that these genes played important roles in the development of heat tolerance.Taken together,adaptation of S.Enteritidis to NaClO resulted in the development of heat tolerance,which might increase the risk of cross-contamination and salmonellosis in the food industry.展开更多
Objectives:Salmonella spp.is a world-leading foodborne pathogen and its rapid detection is essential for ensuring food safety.Conventional methods require expensive instruments,considerable operational skills and cann...Objectives:Salmonella spp.is a world-leading foodborne pathogen and its rapid detection is essential for ensuring food safety.Conventional methods require expensive instruments,considerable operational skills and cannot provide fast mobile on-site systems to detect Salmonella in food.Materials and Methods:A visual method was established based on multiple recombinase polymerase amplification(RPA)coupled with lateral flow dipsticks(LFD)for the simultaneous detection of Salmonella spp.,Salmonella Enteritidis and Salmonella Typhimurium in vitro and food.Results:The optimal volume and temperature for the multiplex RPA-LFD method were determined to be 25μL and 38°C,respectively.The reaction process was completed within 25 min and the results were observed visually.The limits of detection(LODs)were 2.8×10^(2),5.9×10^(2),and 7.6×10^(2) CFU/mL for Salmonella spp.,S.Enteritidis and S.Typhimurium,respectively.Meanwhile,the results of the established method showed no cross-reactivity between the Salmonella cells and other common foodborne bacteria,which was highly specific for Salmonella.More importantly,the developed method exhibited good performance in artificially contaminated chicken samples with the LODs of 2.8×10^(3),5.9×10^(3),and 7.6×10^(3) CFU/mL for Salmonella spp.,S.Enteritidis,and S.Typhimurium,respectively.Finally,the application of the multiple RPA-LFD methods in retailed food samples displayed that this method was effective and practical for the detection of Salmonella spp.in food.Conclusion:The developed multiplex RPA-LFD method provides a new sensitive and rapid alternative for the specific detection of Salmonella spp.and its important serovars in food.展开更多
Stability assessme nt of observed tolerance phe no types is integral in un dersta nding stress adaptati on in food-borne pathogens.Therefore,the current work was carried out to determine whether ethanol adaptation ind...Stability assessme nt of observed tolerance phe no types is integral in un dersta nding stress adaptati on in food-borne pathogens.Therefore,the current work was carried out to determine whether ethanol adaptation induced by exposure to 5 percent ethanol for 60 min is a stable phenomenon in Salmonella enterica serovar Enteritidis.The capacity of Salmonella Enteritidis(S.Enteritidis)to maintain the acquired ethanol adaptation in the absenee of sublethal ethanol stress was investigated at 37℃,25℃ or 4℃ in Luria-Bertani broth and two types of meat juice.It was found that ethanol adaptation was completely reversed within 40 min at 37℃ or within 60 min at 25℃,but was stable at 4℃ for at least 48 h in the broth assay.Ethanol adaptation was retained in chicken juice during 60-min incubation at 25℃ or 48h incubation at 4℃.Moreover,exposure to pork juice stored at either 25℃ or 4℃ sigrdficantly(P<0.05)increased the ethanol toleranee of ethanol-adapted cells.Collectively,these fin dings suggest that ethanol adaptation stability in S.En teritidis under cold conditi ons and in meat juices should be take n into acco unt whe n con ducting a comprehensive risk analysis during food processing.展开更多
Benzalkonium chloride(BAC) is an effective disinfectant against Salmonella and widely used in the food industry.However,there are growing concerns about the risk of inducing Salmonella to produce crossresistance to an...Benzalkonium chloride(BAC) is an effective disinfectant against Salmonella and widely used in the food industry.However,there are growing concerns about the risk of inducing Salmonella to produce crossresistance to antibiotics resulting from the excessive use of BAC.In this study,50 foodborne Salmonella isolates were exposed to sub-inhibitory concentrations of BAC.It was demonstrated that 7 isolates showed direct resistance to BAC,and 35 isolates showed cross-resistance to at least one of 10 tested antibiotics.Among these 35 isolates,15 isolates exhibited an increase in minimum inhibitory concentration(MIC) for tetracycline,followed by 11 isolates for kanamycin and 9 isolates for ampicillin.Salmonella Enteritidis SJTUSM06 with the highest increase in MIC of tetracycline(from 4 to 32 μg/mL) was selected for further study.Tandem mass tag-labeled proteomics was used to determine key proteins involved in development of cross-resistance to tetracycline in BAC-adapted S.Enteritidis.It was identified that there were 146 differentially expressed proteins,including 95 up-regulated and 51 down-regulated proteins,most of which were involved in carbohydrate and lipid metabolism,ribosomes,transporters,virulence,motility,and stress response pathways.The efflux pump AcrB was significantly upregulated by 2.03-fold in BAC-adapted S.Enteritidis.It was also demonstrated that efflux pump inhibitor phenylalanine-arginine β-naphthylamide(PaβN) decreased the MIC of tetracycline in BAC-adapted S.Enteritidis from 32 to 8 μg/mL,indicating that active efflux pump played an important role in the development of resistance to tetracycline induced by BAC.Deletion of acrB resulted in a decrease in the resistance to tetracycline in BAC-adapted S.Enteritidis.Compared with the parent strain,MIC for tetracycline in ΔacrB mutant was 16 μg/mL with a 2-fold decrease.These results demonstrated that AcrB played a positive role in the development of crossresistance to tetracycline after adaptation of S.Enteritidis to BAC.展开更多
Salmonella Enteritidis presents a significant challenge to food safety due to its resilience against various foodrelated stresses.The cold shock protein A(CspA)has been extensively studied for its role in facilitating...Salmonella Enteritidis presents a significant challenge to food safety due to its resilience against various foodrelated stresses.The cold shock protein A(CspA)has been extensively studied for its role in facilitating the survival of S.Enteritidis at low temperatures.However,the broader biological functions of CspA in this pathogen remain inadequately characterized.This study aims to address this knowledge gap through a comprehensive approach involving mutagenesis,transcriptomic analysis,and phenotypic evaluation.RNA sequencing analysis identified 119 genes that were upregulated and 26 genes that were downregulated in theΔcspA deletion mutant compared to the wild-type strain.This observation was further substantiated through the RT-qPCR technique.Functional annotation of the differentially expressed genes indicated their involvement in various pathways,such as stress resistance(4 genes),flagellar assembly(6 genes),cellular metabolism(22 genes),ATP-bindingcassette(ABC)transporters(9 genes),regulators(7 genes),and the Type III secretion system(10 genes),thus highlighting the extensive influence of CspA on bacterial gene expression.Notably,the upregulation of genes associated with acid stress response(SEN_RS08115,10.74-fold;yhcN,3.11-fold)and osmotic stress response(osmX,1.69-fold;osmY,1.50-fold)significantly enhanced the resistance of S.Enteritidis to hydrochloric acid and sodium chloride,respectively.Furthermore,approximately 20%of genes related to flagellar assembly were repressed,which resulted in swimming defects and increased auto-aggregation,thereby promoting the formation of bacterial biofilms.Overall,these findings suggest that CspA plays a pivotal role in mediating stress resistance and biofilm formation in S.Enteritidis.展开更多
The utilization of cinnamon essential oil(CEO)as a natural antimicrobial agent for fresh-cut vegetables has encountered challenges owing to its low water solubility,insufficient stability,and high volatility.To overco...The utilization of cinnamon essential oil(CEO)as a natural antimicrobial agent for fresh-cut vegetables has encountered challenges owing to its low water solubility,insufficient stability,and high volatility.To overcome these limitations,CEO was incorporated into a microemulsion system consisting of CEO,ethanol,Tween-80,and deionized water in a ratio of 1:3:6:40,serving as a stock solution in the current work.The microemulsion underwent a series of physicochemical and antimicrobial characterizations,followed by application to fresh-cut cucumbers.The resultant oil-in-water microemulsion demonstrated a particle size of 10.26 nm and a spherical morphology,exhibiting uniform dispersion and considerable stability.The CEO microemulsion displayed a minimum inhibitory concentration of 0.05%and 0.025%,along with a minimum bactericidal concentration of 0.05%and 0.1%against Escherichia coli and Staphylococcus aureus,respectively.When applied to fresh-cut cucumbers at concentrations of 0.025%,0.05%,and 0.1%,the CEO microemulsion exerted a pronounced antimicrobial effect as indicated by a significant(P<0.05)reduction in total viable counts(0.29-0.92 log units)and total yeast and mould counts(0.26-2.20 log units)in comparison to the control group.Moreover,the micro-emulsion effectively mitigated the decline of vitamin C and soluble solid content while reducing the rate of discoloration during refrigerated storage of cucumbers.Sensory evaluations confirmed that acceptable characteristics in terms of color,aroma,taste,flavour,and appearance were also maintained by CEO microemulsions.Collectively,microemulsion technology provides a feasible way to load CEO for its application in the fresh-cut vegetable industry.展开更多
基金supported by the National Key Research and Development Program of China(2019YFE0119700)the National Natural Science Foundation of China(32001797)+1 种基金the Science and Technology Innovation Agricultural Project of Shanghai Science and Technology Commission(19391902100)the Natural Science Foundation of Shanghai(22ZR1429900).
文摘Cross protection can undermine the effectiveness of control measures on foodborne pathogens,and therefore brings major implications for food safety.In this work,the capacity of Salmonella Enteritidis to mount ethanol tolerance following acid adaptation was characterized by analysis of cell viability and cell membrane property.It was observed that preadaptation to pH 4.5 significantly(P<0.05)increased the tolerance of log-phase cells to ethanol;in contrast,stationary-phase cells displayed reduced ethanol tolerance after acid adaptation.However,acid adaptation did not cause cell leakage and morphological change in both log-phase and stationary-phase S.Enteritidis.Fatty acid analysis further revealed that the amount of C_(14:0),C_(17:0 cyclo) and C_(19:0 cyclo) fatty acids was increased,while that of C_(16:1ω7c) and C_(18:1ω7c) fatty acids was decreased,respectively,in response to acid adaptation,regardless of bacterial growth phase.Notably,acid adaptation significantly(P<0.05)increased the proportion of C_(16:0) fatty acid in log-phase cells,but this effect did not occur in stationary-phase cells.Moreover,exogenous addition of C_(16:0) fatty acid to stationary-phase acid-adapted cultures was able to enhance bacterial ethanol tolerance.Taken together,C_(16:0) fatty acid is involved in the growth-phase-dependent protective effect of acid adaptation on ethanol tolerance in S.Enteritidis.
基金supported by the National Natural Science Foundation of China(grant number 32172316).
文摘Sodium hypochlorite(NaClO)is a widely used disinfectant in food processing environments.However,its improper use can lead Salmonella to develop tolerance to other stresses,which threatens food safety and human health.In this study,the survival ability assays showed that adaptation of S.Enteritidis to NaClO resulted in the development of heat tolerance at 55℃and 63℃.NaClO-adapted S.Enteritidis also showed a survival benefit in milk at 55℃and 63℃.NaClO-adapted S.Enteritidis exhibited a decrease in accumulation of reactive oxygen species(ROS)compared to non-adapted S.Enteritidis after exposure to 63℃.Moreover,compared to non-adapted S.Enteritidis,the membrane fatty acids C16:0 and cyclo-heptadecanoic acid Cyc17 were significantly increased in NaClO-adapted S.Enteritidis,whereas UFA to SFA ratio was observed to decrease from 0.52±0.04 to 0.41±0.04.These changes resulted in a decrease in membrane fluidity of NaClO-adapted S.Enteritidis.Furthermore,RT-qPCR analysis revealed a considerable increase in the expression levels of heat shock-related genes in NaClO-adapted S.Enteritidis with or without heat treatment,in comparison to non-adapted S.Enteritidis.It was noteworthy that upregulation was 7.30-fold for dnaK,11.62-fold for ibpA,and 3.92-fold for rpoH under heat treatment,suggesting that these genes played important roles in the development of heat tolerance.Taken together,adaptation of S.Enteritidis to NaClO resulted in the development of heat tolerance,which might increase the risk of cross-contamination and salmonellosis in the food industry.
基金supported by the National Key R&D Program of China(No.2019YFE0119700)the National Natural Science Foundation of China(No.32172316).
文摘Objectives:Salmonella spp.is a world-leading foodborne pathogen and its rapid detection is essential for ensuring food safety.Conventional methods require expensive instruments,considerable operational skills and cannot provide fast mobile on-site systems to detect Salmonella in food.Materials and Methods:A visual method was established based on multiple recombinase polymerase amplification(RPA)coupled with lateral flow dipsticks(LFD)for the simultaneous detection of Salmonella spp.,Salmonella Enteritidis and Salmonella Typhimurium in vitro and food.Results:The optimal volume and temperature for the multiplex RPA-LFD method were determined to be 25μL and 38°C,respectively.The reaction process was completed within 25 min and the results were observed visually.The limits of detection(LODs)were 2.8×10^(2),5.9×10^(2),and 7.6×10^(2) CFU/mL for Salmonella spp.,S.Enteritidis and S.Typhimurium,respectively.Meanwhile,the results of the established method showed no cross-reactivity between the Salmonella cells and other common foodborne bacteria,which was highly specific for Salmonella.More importantly,the developed method exhibited good performance in artificially contaminated chicken samples with the LODs of 2.8×10^(3),5.9×10^(3),and 7.6×10^(3) CFU/mL for Salmonella spp.,S.Enteritidis,and S.Typhimurium,respectively.Finally,the application of the multiple RPA-LFD methods in retailed food samples displayed that this method was effective and practical for the detection of Salmonella spp.in food.Conclusion:The developed multiplex RPA-LFD method provides a new sensitive and rapid alternative for the specific detection of Salmonella spp.and its important serovars in food.
基金supported by the National Key Research and Development Program of China(No.2019YFE0119700)the National Natural Science Foundation of China(No.32001797)the China Postdoctoral Science Foundation(No.2019M661516).
文摘Stability assessme nt of observed tolerance phe no types is integral in un dersta nding stress adaptati on in food-borne pathogens.Therefore,the current work was carried out to determine whether ethanol adaptation induced by exposure to 5 percent ethanol for 60 min is a stable phenomenon in Salmonella enterica serovar Enteritidis.The capacity of Salmonella Enteritidis(S.Enteritidis)to maintain the acquired ethanol adaptation in the absenee of sublethal ethanol stress was investigated at 37℃,25℃ or 4℃ in Luria-Bertani broth and two types of meat juice.It was found that ethanol adaptation was completely reversed within 40 min at 37℃ or within 60 min at 25℃,but was stable at 4℃ for at least 48 h in the broth assay.Ethanol adaptation was retained in chicken juice during 60-min incubation at 25℃ or 48h incubation at 4℃.Moreover,exposure to pork juice stored at either 25℃ or 4℃ sigrdficantly(P<0.05)increased the ethanol toleranee of ethanol-adapted cells.Collectively,these fin dings suggest that ethanol adaptation stability in S.En teritidis under cold conditi ons and in meat juices should be take n into acco unt whe n con ducting a comprehensive risk analysis during food processing.
基金supported by the National Natural Science Foundation of China(32172316)。
文摘Benzalkonium chloride(BAC) is an effective disinfectant against Salmonella and widely used in the food industry.However,there are growing concerns about the risk of inducing Salmonella to produce crossresistance to antibiotics resulting from the excessive use of BAC.In this study,50 foodborne Salmonella isolates were exposed to sub-inhibitory concentrations of BAC.It was demonstrated that 7 isolates showed direct resistance to BAC,and 35 isolates showed cross-resistance to at least one of 10 tested antibiotics.Among these 35 isolates,15 isolates exhibited an increase in minimum inhibitory concentration(MIC) for tetracycline,followed by 11 isolates for kanamycin and 9 isolates for ampicillin.Salmonella Enteritidis SJTUSM06 with the highest increase in MIC of tetracycline(from 4 to 32 μg/mL) was selected for further study.Tandem mass tag-labeled proteomics was used to determine key proteins involved in development of cross-resistance to tetracycline in BAC-adapted S.Enteritidis.It was identified that there were 146 differentially expressed proteins,including 95 up-regulated and 51 down-regulated proteins,most of which were involved in carbohydrate and lipid metabolism,ribosomes,transporters,virulence,motility,and stress response pathways.The efflux pump AcrB was significantly upregulated by 2.03-fold in BAC-adapted S.Enteritidis.It was also demonstrated that efflux pump inhibitor phenylalanine-arginine β-naphthylamide(PaβN) decreased the MIC of tetracycline in BAC-adapted S.Enteritidis from 32 to 8 μg/mL,indicating that active efflux pump played an important role in the development of resistance to tetracycline induced by BAC.Deletion of acrB resulted in a decrease in the resistance to tetracycline in BAC-adapted S.Enteritidis.Compared with the parent strain,MIC for tetracycline in ΔacrB mutant was 16 μg/mL with a 2-fold decrease.These results demonstrated that AcrB played a positive role in the development of crossresistance to tetracycline after adaptation of S.Enteritidis to BAC.
基金supported by the Natural Science Foundation of Shanghai(No.22ZR1429900)the National Natural Science Foundation of China(No.32001797).
文摘Salmonella Enteritidis presents a significant challenge to food safety due to its resilience against various foodrelated stresses.The cold shock protein A(CspA)has been extensively studied for its role in facilitating the survival of S.Enteritidis at low temperatures.However,the broader biological functions of CspA in this pathogen remain inadequately characterized.This study aims to address this knowledge gap through a comprehensive approach involving mutagenesis,transcriptomic analysis,and phenotypic evaluation.RNA sequencing analysis identified 119 genes that were upregulated and 26 genes that were downregulated in theΔcspA deletion mutant compared to the wild-type strain.This observation was further substantiated through the RT-qPCR technique.Functional annotation of the differentially expressed genes indicated their involvement in various pathways,such as stress resistance(4 genes),flagellar assembly(6 genes),cellular metabolism(22 genes),ATP-bindingcassette(ABC)transporters(9 genes),regulators(7 genes),and the Type III secretion system(10 genes),thus highlighting the extensive influence of CspA on bacterial gene expression.Notably,the upregulation of genes associated with acid stress response(SEN_RS08115,10.74-fold;yhcN,3.11-fold)and osmotic stress response(osmX,1.69-fold;osmY,1.50-fold)significantly enhanced the resistance of S.Enteritidis to hydrochloric acid and sodium chloride,respectively.Furthermore,approximately 20%of genes related to flagellar assembly were repressed,which resulted in swimming defects and increased auto-aggregation,thereby promoting the formation of bacterial biofilms.Overall,these findings suggest that CspA plays a pivotal role in mediating stress resistance and biofilm formation in S.Enteritidis.
文摘The utilization of cinnamon essential oil(CEO)as a natural antimicrobial agent for fresh-cut vegetables has encountered challenges owing to its low water solubility,insufficient stability,and high volatility.To overcome these limitations,CEO was incorporated into a microemulsion system consisting of CEO,ethanol,Tween-80,and deionized water in a ratio of 1:3:6:40,serving as a stock solution in the current work.The microemulsion underwent a series of physicochemical and antimicrobial characterizations,followed by application to fresh-cut cucumbers.The resultant oil-in-water microemulsion demonstrated a particle size of 10.26 nm and a spherical morphology,exhibiting uniform dispersion and considerable stability.The CEO microemulsion displayed a minimum inhibitory concentration of 0.05%and 0.025%,along with a minimum bactericidal concentration of 0.05%and 0.1%against Escherichia coli and Staphylococcus aureus,respectively.When applied to fresh-cut cucumbers at concentrations of 0.025%,0.05%,and 0.1%,the CEO microemulsion exerted a pronounced antimicrobial effect as indicated by a significant(P<0.05)reduction in total viable counts(0.29-0.92 log units)and total yeast and mould counts(0.26-2.20 log units)in comparison to the control group.Moreover,the micro-emulsion effectively mitigated the decline of vitamin C and soluble solid content while reducing the rate of discoloration during refrigerated storage of cucumbers.Sensory evaluations confirmed that acceptable characteristics in terms of color,aroma,taste,flavour,and appearance were also maintained by CEO microemulsions.Collectively,microemulsion technology provides a feasible way to load CEO for its application in the fresh-cut vegetable industry.
