Cronobacter spp.has strong resistance to desiccation and high permeability in Enterobacteriaceae,and powdered infant formula(PIF)is one of the main contamination routes.In recent years,the contamination of Cronobacter...Cronobacter spp.has strong resistance to desiccation and high permeability in Enterobacteriaceae,and powdered infant formula(PIF)is one of the main contamination routes.In recent years,the contamination of Cronobacter spp.in PIF incidents occurs from time to time,causing infant serious diseases or death.In this investigation,matrix-assisted laser desorption/ionization time of flight mass spectrometry was used to identify the phenotypes of 35 Cronobacter strains isolated from PIF and its processing environment.Subsequently,the isolates were evaluated for drying and osmotic pressure tolerance.The results showed that the deactivation rate of the strains ranged from 9.01%to 77.57%,and the highest osmotic pressure condition the strains could tolerate was 6 g/100 mL Na Cl.In addition,there was a positive correlation between biofilm formation ability and desiccation resistance.Combined with transcriptomics,Cronobacter spp.could activate biofilm synthesis,produce more trehalose,accumulate betaine and electrolytes to stabilize intracellular structure under the two treatment conditions.A total of 31 and 43 genes were found related to desiccation and permeability resistance,respectively.And some genes(cysM,thuF,ycjO,etc.)were found to be associated with two tolerances for the first time.展开更多
Cronobacter sakazakii(C.sakazakii)is a foodborne opportunistic pathogen that can cause life-threatening invasive diseases,such as necrotizing enterocolitis,meningitis,and sepsis in infants.The potential risk of C.saka...Cronobacter sakazakii(C.sakazakii)is a foodborne opportunistic pathogen that can cause life-threatening invasive diseases,such as necrotizing enterocolitis,meningitis,and sepsis in infants.The potential risk of C.sakazakii contamination of powdered infant formula(PIF)is an issue that has attracted considerable attention from manufacturers,regulators,and consumers.C.sakazakii biofilms on the surfaces of equipment and in diverse food-production environments constitute a mode of cell growth that protects the pathogen from hostile environments,and are an important source of persistent contamination of food products.Bacterial biofilms are difficult to remove due to their resistant properties.Conventional cleaning and sterilizing procedures may be insufficient for biofilm control,and may lead to further biofilm development and dispersal.Consequently,novel control strategies are being developed,such as nanotechnology-based delivery systems,interspecies interactions,antimicrobial molecules of microbial origin,natural extracts,and phages.This review focuses on describing the mechanisms underlying the biofilm formation and behavior of C.sakazakii and discussing potential control strategies.展开更多
Infections by Cronobacter spp. are hazardous to infants since they can lead to neonatal meningitis, bacteremia, and necrotizing enterocolitis. Cronobacter spp. are frequently resistant to 13-1actam derivatives, macrol...Infections by Cronobacter spp. are hazardous to infants since they can lead to neonatal meningitis, bacteremia, and necrotizing enterocolitis. Cronobacter spp. are frequently resistant to 13-1actam derivatives, macrolides, and aminoglycosides. In addition, multi-resistant strains have also been detected. In China, the isolation rate of Cronobacter spp. from commercial powdered infant formula （PIF） or follow-up formula （FUF） is relatively high. Nevertheless, clinical cases of Cronobacter infection have been ignored to date. Here we describe two cases of Cronobacter infection detected at the Wuhan Women and Children Medical Care Center Hospital （Wuhan City, China）. We provide the genomic analysis of the isolates and the antibiotic-resistance profiles of the two strains. The Cronobacter strains identified in this study were not susceptible to third-generation cephalosporins, aminoglycoside, and/or trimethoprim-sulfamethoxazole. Whole genome sequencing revealed various genes known to encode antibiotic resistance. Future studies are needed to determine whether the genes predicted in this study are functional. As with Enterobocter spp., the antibiotic resistance of Cronobocter is a serious issue that requires more attention.展开更多
Objective To determine Cronobacter spp. contamination in infant and follow-up powdered formula in China. Methods All of 2282 samples were collected from the retail markets in China from January 2012 to December 2012, ...Objective To determine Cronobacter spp. contamination in infant and follow-up powdered formula in China. Methods All of 2282 samples were collected from the retail markets in China from January 2012 to December 2012, and analyzed for Cronobacter spp. by the Chinese National Food Safety Standard. Characterization of the isolates was analyzed by pulsed-field gel electrophoresis（PFGE） with XbaI and Spe I restriction enzymes. Results Cronobacter spp. strains were isolated from 25 samples, and the positive rates in infant powdered formulas and follow-up powdered formulas were 0.90%（10/1011） and 1.18%（15/1271）, respectively. Analysis of variable data regarding different purchasing store formats, seasonality, and production locations as well as comparison of infant versus follow-up formulas did not reveal statistically significant factors. During the sampling period, one of six surveillance zones did exhibit a statistically significant trend towards higher positive rate. PFGE characterization of Cronobacter spp. to elucidate genetic diversity revealed only three pairs of Cronobacter spp. out of 25 having the same PFGE patterns. Conclusion The current investigation indicated a lower positive rate of Cronobacter spp. in the powdered formula in China. This evidence suggested contamination originating from multiple different sources during the manufacturing process.展开更多
Cronobacter species are a group of Gram-negative opportunistic pathogens,which cause meningitis,sep-ticemia,and necrotizing enterocolitis in neonates and infants,with neurological sequelae in severe cases.Interest in ...Cronobacter species are a group of Gram-negative opportunistic pathogens,which cause meningitis,sep-ticemia,and necrotizing enterocolitis in neonates and infants,with neurological sequelae in severe cases.Interest in Cronobacter has increased significantly in recent years due to its high virulence in children.In this review,we summarize the current understanding of the prevalence of Cronobacter species in several important food types.We discuss the response mechanisms enabling persistence in adverse growth con-ditions,as well as its pathogenicity.We emphasize the food safety concerns caused by Cronobacter and subsequent control methods and clinical treatments.展开更多
We used a proteomic approach to identify IbpA in Cronobacter sakazokii （C. sakazaki）, which is related to heat tolerance in this strain. The abundance of IbpA in C. sakazakii strains strongly increased after heat sh...We used a proteomic approach to identify IbpA in Cronobacter sakazokii （C. sakazaki）, which is related to heat tolerance in this strain. The abundance of IbpA in C. sakazakii strains strongly increased after heat shock. C sakazakii CMCC 45402 ibpA deletion mutants were successfully constructed. The C. sakazakii CMCC 45402 AibpA and wild-type strains could not be distinguished based on colony morphology on LB agar plates or biochemical assays. The growth of the C. sakazakii CMCC 45402 AibpA mutant in heat shock conditions was indistinguishable from that of the isogenic wild-type, but showed greater heat resistance than E. coil O157：H7 strain CMCC 44828. This study suggests that the absence of a single ibpA gene has no obvious effect on the phenotype or heat resistance of the strain C. sakazakii CMCC 45402.展开更多
A total of 7 Cronobocter strains were isolated from 703 fecal samples collected in Jinan from June 13 to December 30, 2011, with the positive rate of Cronobacter spp. being 1.0% （95% confidence interval 0.6%-1.4%）. ...A total of 7 Cronobocter strains were isolated from 703 fecal samples collected in Jinan from June 13 to December 30, 2011, with the positive rate of Cronobacter spp. being 1.0% （95% confidence interval 0.6%-1.4%）. Three Cronobacter sakazakii stains were isolated from 157 fecal samples of healthy neonates （95% confidence interval 0.4%-5.5%）. This number was slightly higher than that isolated from 273 fecal samples of healthy adults, in which 1 strain of C. sakazakii and 1 strain of Cronobacter malonaticus were isolated, and that from 173 fecal samples of adults with acute diarrhea, in which 1 strain of C sakazakii and 1 strain of C. malonaticus were isolated, but the differences were not statistically significant （P〉O.05）. The Cronobocter isolates were all from different genetic sources. It should be noted that Cronobacter carriage may cause infection under certain conditions, especially in neonates.展开更多
[Objective] This study aimed to establish a Real-Time quantitative PCR method for the determination of transposon copy number in C. sakazakii. [ Method ] With single-copy housekeeping gene atpD as the reference gene, ...[Objective] This study aimed to establish a Real-Time quantitative PCR method for the determination of transposon copy number in C. sakazakii. [ Method ] With single-copy housekeeping gene atpD as the reference gene, recombinant plasmid containing both single-copy housekeeping gene atpD and EZ-TN5 transposon was constructed; based on the established standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon, copy number of atpD gene and EZ-TN5 transpason in three C. sakazakii mutants was detected and the ratio was calculated. [ Result] Correlation coefficients of the standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon were 0. 999 and 0.998, respectively ; the ratios of copy number of atpD gene and EZ-TN5 transposon in three C. sakazakii mutants were 0.98, 1.17 and 0.91, respectively, which indicates that EZ-TN5 transpeson in C. sakakii mutants is a single-copy. [ Conclusion] Real-time quantitative PCR method established in this study had high availability and could replace the Southern blot method to detect the copy num- ber of EZ-TN5 transposon in different bacteria.展开更多
Cronobacter sakazakii is an emerging pathogen that can cause diseases for several infant groups. These bacteria were contaminated in foods, clinical utensils, and environments. In Indonesia, C. sakazakii has been isol...Cronobacter sakazakii is an emerging pathogen that can cause diseases for several infant groups. These bacteria were contaminated in foods, clinical utensils, and environments. In Indonesia, C. sakazakii has been isolated from powdered infant formulas, weaning foods, and other dried foods such as cornstarch. The objective of this research is to trace survival of C. sakazakii during cornstarch production step using its mutant. Mutant was constructed by inserting Green Fluorescent Protein plasmid inside to the bacterial cell that appeared green fluorescent colonies under UV observation. The presence of C. sakazakii during processing was conducted by artificial contamination. This research consists of three steps, i.e. determination of the suitable enumeration method of C. sakazakii’s mutant, cornstarch production from yellow corn, and survival analysis of C. sakazakii during endosperm soaking and cornstarch drying. The suitable enumeration method was surface plating method on TSA-ampicillin medium combining with UV light application because of ineffectiveness of ampicillin inhibition for growth of yeasts and molds. The cornstarch produced in laboratory has the same properties with commercial cornstarch in parameters of moisture content, density, and starch granule structure. The yield of cornstarch final product was 48.90% (dry whole kernel-based). C. sakazakii cannot survive in 48 hours soaking process at 52?C and 24 hours drying process at 50?C that is applied during cornstarch production.展开更多
Cronobacter sakazakii is an opportunistic foodborne pathogen with remarkable resilience to environmental stressors,posing severe risks in powdered infant formula.Here,we identify the GlrKR two-component system(TCS)as ...Cronobacter sakazakii is an opportunistic foodborne pathogen with remarkable resilience to environmental stressors,posing severe risks in powdered infant formula.Here,we identify the GlrKR two-component system(TCS)as a master regulator that integrates environmental sensing with virulence control.Genetic deletion of either the sensor kinase(glrK)or the response regulator(glrR)markedly attenuates bacterial motility,epithelial cell adherence,and virulence in a neonatal rat model.GlrK is essential for acid and oxidative stress resistance,whereas both GlrK and GlrR suppress desiccation tolerance,revealing a regulatory trade-off between environmental persistence and host invasion.Quantitative proteomics and site-specific mutagenesis uncover enhanced deamidation of OmpA at N101 inΔglrR as a mechanistic basis for impaired adherence.Additionally,GlrKR modulates sensitivity to p-coumaric acid,a plant-derived antimicrobial,via transcriptional control of the efflux pump TolC.These findings establish GlrKR as a central node linking environmental adaptation,antimicrobial defense,and host-pathogen interaction in C.sakazakii,offering a promising target for next-generation food safety interventions.展开更多
Enterobacteriaceae species have caused foodborne illnesses through consumption of a variety of foods,including infant foods.A total of 915 samples representing 4 types of samples,were collected from 7 powdered infant ...Enterobacteriaceae species have caused foodborne illnesses through consumption of a variety of foods,including infant foods.A total of 915 samples representing 4 types of samples,were collected from 7 powdered infant formula factories in Heilongjiang Province,China.All samples were processed for enumeration of Enterobac-teriaceae and isolation of Cronobacter sakazakii,which were detected using the ISO enrichment procedure and a chromogenic medium.Multi-locus sequence typing(MLST),and pulsed field gel electrophoresis(PFGE)were applied to characterize the C.sakazakii isolates to trace back the original source of contamination.Samples were considered Enterobacteriaceae positive at 10 CFU/g in powdered samples,1 CFU/mL in water samples,and 10 CFU/100 cm^(2) or larger surface area.Enterobacteriaceae positive rate were 0/35 water samples associated with production,11/56 raw materials,147/761 environmental samples and 3/63 finished products respectively.C.sakazakii strains were isolated from 7.0%of environmental samples followed by 1.6%of the finished products.A total of 20 C.sakazakii isolates were recovered in Factory 4,1 strain derived from a final product and 19 strains recovered from the environment representing 4 MLST sequence types and 10 PFGE pulse types.Most environ-mental sites positive for C.sakazakii had higher counts of Enterobacteriaceae,except for 11 sites where Enterobacteriaceae did not reach minimal levels for reporting a positive sample.The present study,provides insight to the correlation between the prevalence of Enterobacteriaceae and contamination of C.sakazakii in powdered infant formula factories;there is no one-to-one relation between number of Enterobacteriaceae and C.sakazakii.Identifying environmental sites positive for C.sakazakii and/or Enterobacteriaceae will aid in development of appropriate corrective measures to improve environmental hygiene and enhance safety of powdered infant formula.展开更多
Cronobacter sakazakii is a foodborne pathogen,and the use of effective and non-hazardous bacteriostatic agents to manage C.sakazakii infection has garnered attention.Portulaca oleracea L.is a food plant with several p...Cronobacter sakazakii is a foodborne pathogen,and the use of effective and non-hazardous bacteriostatic agents to manage C.sakazakii infection has garnered attention.Portulaca oleracea L.is a food plant with several pharmaceutical properties.However,the products of kombucha-fermented P.oleracea(FP)have been scarcely studied with respect to their metabolite profiles and for their effects against food-borne pathogens.Therefore,in this study,the chemical and microbiological composition and inhibitory effects of FP against C.sakazakii were evaluated.The activities and molecular mechanisms of FP against C.sakazakii were investigated for the first time using transcriptomic and physiological experiments.Komagataeibacter was the most abundant bacterium in fermentation broth(>79%),and chlorogenic and acetic acids were the most abundant phenolics and organic acids in fermentation products.Transcriptomic analysis showed that FP induced significant changes in C.sakazakii as demonstrated by the differential expression of 506 genes.The genes that exhibited differential expression were primarily involved in ATP-related energy metabolism,cell membrane structure,protein biosynthesis,amino acid transport,and metabolism.These roles suggest multifaceted potential effects including cell membrane damage,protein synthesis limitation,metabolism disorder,reduced virulence factor expression,and reduced cell mobility.FP addition significantly inhibited C.sakazakii growth in PIF.These results provide insights into the anti-C.sakazakii mechanism of FP,which is valuable for its development and application in food safety.展开更多
Cronobacter spp.are classified as class A pathogens in powdered infant formula(PIF),and the capsular poly-saccharide is an important hazard-forming factor of Cronobacter.Currently,there are few studies about the capsu...Cronobacter spp.are classified as class A pathogens in powdered infant formula(PIF),and the capsular poly-saccharide is an important hazard-forming factor of Cronobacter.Currently,there are few studies about the capsular polysaccharide on tolerance of Cronobacter to environmental stress.Herein,we investigated the function of variable region genes of Cronobacter K1 antigen in stress response.The results demonstrated that key genes of K1 antigen could enhance acid,salt,and oxidative tolerance,and decrease desiccation tolerance.In addition,deletion of ESA_03354,ESA_03355,and ESA_03356 significantly reduced the biofilm capacity,cellular auto-aggregation capacity,and cell surface hydrophobicity of Cronobacter,while enhanced its motility.Thus,ESA_03354,ESA_03355,and ESA_03356 genes might involve in the stress response through biofilm formation when Cronobacter cell confronted with gastric acid,bile salt,and oxidative stress,whereas ESA_03357 preferred to produce yellow pigment to resist adverse stress.And ESA_03355 and ESA_03356 might mediate stress response by the polymerization of teichoic acid.Therefore,the key K1 antigen genes displayed different properties under various stresses,which provided important information for targeted control of Cronobacter.展开更多
Cronobacter sakazakii is a facultative anaerobic gram-negative pathogen that has attracted widespread attention for causing necrotizing enterocolitis in infants(premature and low-weight newborns)by contaminating powde...Cronobacter sakazakii is a facultative anaerobic gram-negative pathogen that has attracted widespread attention for causing necrotizing enterocolitis in infants(premature and low-weight newborns)by contaminating powdered infant formula.Several studies have found that the antibiotic tolerance of Cronobacter sakazakii isolated from food sources is increasing,thereby markedly increasing the difficulty of clinical treatment of Cronobacter sakazakii infection.At present,most studies are focused on clarifying the pathogenicity of Cronobacter sakazakii,but there is still a gap in the study of necrotizing enterocolitis in neonatal rats caused by Cronobacter sakazakii with different antibiotic tolerance.To fill this gap,we analyzed and compared the ability of Cronobacter sakazakii induced by different classes of antibiotics to induce necrotizing enterocolitis in neonatal rats by mediating TLR4-NLRP3-IL-1β pathway.In the results,Cronobacter sakazakii acquired tolerance to aminoglycosides,quinolones and cephalosporins,and caused more inflammatory damage in neonatal rats,improved the ability of mediating TLR4-NLRP3-IL-1β pathway to induce necrotizing enterocolitis.Compared with the XS001 group,the NEC scores of neonatal rats in XS001-Amp,XS001-Ami,XS001-Ofl and XS001-Ceo groups were increased by 8.3%,58.3%,50.0% and 58.3%.The acquisition of penicillin and tetracycline tolerance had little effect on the ability of Cronobacter sakazakii to induce necrotizing enterocolitis in neonatal rats.The ability of Cronobacter sakazakii to induce necrotizing enterocolitis in neonatal rats was significantly different due to the type and tolerance of antibiotics.展开更多
Cronobacter spp.(formerly Enterobacter sakazakii) are special foodborne pathogens. Cronobacter infection can cause necrotizing enterocolitis, sepsis and meningitis in all age groups, especially neonates and infants, w...Cronobacter spp.(formerly Enterobacter sakazakii) are special foodborne pathogens. Cronobacter infection can cause necrotizing enterocolitis, sepsis and meningitis in all age groups, especially neonates and infants, with a high fatality of up to 80%, although the infection is rare. Outbreaks of Cronobacter infection are epidemiologically proven to be associated with contaminated powdered infant formula(PIF). Cronobacter spp.can resist dry environments and survive for a long period in food with low water activity. Therefore, Cronobacter spp.have become serious pathogens of neonates and infants, as well as in the dairy industry. In this review, we present the taxonomy, pathogenesis, resistance, detection and control of Cronobacter spp.展开更多
Objectives:Cronobacter sakazakii,formerly Enterobacter sakazakii,is an emerging ubiquitous and opportunistic foodborne pathogen with a high mortality rate.It has been implicated in cases of meningitis,septicaemia,and ...Objectives:Cronobacter sakazakii,formerly Enterobacter sakazakii,is an emerging ubiquitous and opportunistic foodborne pathogen with a high mortality rate.It has been implicated in cases of meningitis,septicaemia,and necrotizing enterocolitis among infants worldwide in association with powdered infant formula(PIF).This study was an insilico designed peptide base kit framework,using immunoinformatic techniques for quick detection of C.sakazakii in PIF.Materials and Methods:In the present study,a peptide-based kit was designed with a bioinformatic technique to rapidly identify C.sakazakii in PIF using fhE,secY,and bcsC,which are genes responsible for its bioflm formation,as target genes.The antigenicity,membrane topology,and the presence of signal peptides of the target genes were analysed using VaxiJen,DeepTMHMM,and SignalP servers.To provide stability and fexibility to the multiple-epitope construct,the linear B cells and helper T cells(IL-4(interleukin 4)and IL-10(interleukin 10)inducing epitopes)were linked with a GSGSG linker followed by the addition of protein disulphide bonds.To ascertain specifcity,the multi-epitope construct was molecularly docked against genes from sources other than PIF,like alfalfa,and the environment,with PIF being the highest:–328.48.Finally,the codons were modifed using the pET28a(+)vector,and the resultant multi-epitope construct was successfully cloned in silico.Results:The fnal construct had a length of 486 bp,an instability index of 23.26,a theoretical pI of 9.34,a molecular weight of 16.5 kDa,and a Z-score of–3.41.Conclusions:The multi-epitope peptide construct could be a conceptual framework for creating a C.sakazakii peptide-based detection kit,which has the potential to provide fast and effcient detection.However,there is a need for additional validation through the in vitro and in vivo techniques.展开更多
Cronobacter sakazakii may encounter subinhibitory concentrations of ethanol stress over its lifecycle.Bacterial tolerance to homologous or heterologous stress agents may be altered as a result of ethanol adaptive resp...Cronobacter sakazakii may encounter subinhibitory concentrations of ethanol stress over its lifecycle.Bacterial tolerance to homologous or heterologous stress agents may be altered as a result of ethanol adaptive responses.Therefore,the tolerance of ethanol-exposed and control cells to subsequent lethal stresses was evaluated in the current work.It was discovered that sublethal ethanol exposure increased the susceptibility to lethal ethanol stress in C.sakazakii as determined by the Weibull model.Furthermore,sublethal ethanol concentration exposure in C.sakazakii did not lead to any cross-tolerance against other stressors such as benzalkonium chloride(120 mg/L),heat(55°C),cold(4°C),simulated gastric fuid(pH 3.0),osmotic stress(sorbitol,0.75 g/mL),and desiccation stress.Analysis of zeta potential,scanning electron microscope,and surface-enhanced Raman spectroscopy spectra revealed that cellular injury and changes in cellular chemical composition may contribute to the reduced resistance of C.sakazakii after ethanol exposure.Furthermore,sublethal ethanol exposure resulted in an elevated proportion of unsaturated fatty acids(USFA),while reducing the proportion of saturated fatty acids(SFA)and the ratio of SFA to USFA.The developed inactivation models can serve as a valuable source of data to support quantitative microbial risk assessment.Moreover,a better understanding of the response of C.sakazakii to sublethal ethanol exposure may provide valuable insights into the prevention and control of C.sakazakii.展开更多
Cronobacter sakazakii(C.sakazakii),a food-borne pathogen,can cause severe intestinal diseases,such as intestinal damage and necrotizing enterocolitis,and even death in neonates and infants through its adhesion to inte...Cronobacter sakazakii(C.sakazakii),a food-borne pathogen,can cause severe intestinal diseases,such as intestinal damage and necrotizing enterocolitis,and even death in neonates and infants through its adhesion to intestinal epithelium cells by protein-protein interaction and translocation across the intestinal barrier.Nevertheless,the mechanisms of adhesion,translocation,and pathogenesis remain unclear.This study aimed to screen and validate the outer membrane proteins of C.sakazakii capable of adhering to the surface of intestinal cells through biotinylated protein adhesion and LC-MS/MS identification.A total of 319 proteins were identified.Gene Ontology and KEGG analysis revealed significant enrichment of ligand proteins in metabolism-related processes and catalytic activity.The identified 37 outer membrane proteins and 41 proteins of unknown subcellular localization or function mainly enriched in five protein-protein interaction clusters.Ten of these outer membrane/unknown proteins were selected to test their roles on C.sakazakii adhesion and invasion of intestinal cells by silencing the encoding gene expression and nine of the ten proteins,Protein_26/30/34/38/42/98/104/194/282,were shown to be critical in this process.This study contributes to the understanding of proteins involved in the host adhesion process by C.sakazakii as well as the mechanisms for its translocation across the intestinal barrier.展开更多
基金supported by the Joint Funds of the National Natural Science Foundation of China(U21A20272)。
文摘Cronobacter spp.has strong resistance to desiccation and high permeability in Enterobacteriaceae,and powdered infant formula(PIF)is one of the main contamination routes.In recent years,the contamination of Cronobacter spp.in PIF incidents occurs from time to time,causing infant serious diseases or death.In this investigation,matrix-assisted laser desorption/ionization time of flight mass spectrometry was used to identify the phenotypes of 35 Cronobacter strains isolated from PIF and its processing environment.Subsequently,the isolates were evaluated for drying and osmotic pressure tolerance.The results showed that the deactivation rate of the strains ranged from 9.01%to 77.57%,and the highest osmotic pressure condition the strains could tolerate was 6 g/100 mL Na Cl.In addition,there was a positive correlation between biofilm formation ability and desiccation resistance.Combined with transcriptomics,Cronobacter spp.could activate biofilm synthesis,produce more trehalose,accumulate betaine and electrolytes to stabilize intracellular structure under the two treatment conditions.A total of 31 and 43 genes were found related to desiccation and permeability resistance,respectively.And some genes(cysM,thuF,ycjO,etc.)were found to be associated with two tolerances for the first time.
基金financial support of National Key Research and Development Program of China(2017YFC1601200)the Science and Technology Planning Project of Guangdong Province(2017A070702018)+1 种基金the Science and Technology Planning Project of Guangzhou(201604020003)Guangdong Academy of Sciences Special Project of Science and Technology Development(2017GDASCX-0201).
文摘Cronobacter sakazakii(C.sakazakii)is a foodborne opportunistic pathogen that can cause life-threatening invasive diseases,such as necrotizing enterocolitis,meningitis,and sepsis in infants.The potential risk of C.sakazakii contamination of powdered infant formula(PIF)is an issue that has attracted considerable attention from manufacturers,regulators,and consumers.C.sakazakii biofilms on the surfaces of equipment and in diverse food-production environments constitute a mode of cell growth that protects the pathogen from hostile environments,and are an important source of persistent contamination of food products.Bacterial biofilms are difficult to remove due to their resistant properties.Conventional cleaning and sterilizing procedures may be insufficient for biofilm control,and may lead to further biofilm development and dispersal.Consequently,novel control strategies are being developed,such as nanotechnology-based delivery systems,interspecies interactions,antimicrobial molecules of microbial origin,natural extracts,and phages.This review focuses on describing the mechanisms underlying the biofilm formation and behavior of C.sakazakii and discussing potential control strategies.
基金supported by the National Natural Science Foundation of China(No.81501799)
文摘Infections by Cronobacter spp. are hazardous to infants since they can lead to neonatal meningitis, bacteremia, and necrotizing enterocolitis. Cronobacter spp. are frequently resistant to 13-1actam derivatives, macrolides, and aminoglycosides. In addition, multi-resistant strains have also been detected. In China, the isolation rate of Cronobacter spp. from commercial powdered infant formula （PIF） or follow-up formula （FUF） is relatively high. Nevertheless, clinical cases of Cronobacter infection have been ignored to date. Here we describe two cases of Cronobacter infection detected at the Wuhan Women and Children Medical Care Center Hospital （Wuhan City, China）. We provide the genomic analysis of the isolates and the antibiotic-resistance profiles of the two strains. The Cronobacter strains identified in this study were not susceptible to third-generation cephalosporins, aminoglycoside, and/or trimethoprim-sulfamethoxazole. Whole genome sequencing revealed various genes known to encode antibiotic resistance. Future studies are needed to determine whether the genes predicted in this study are functional. As with Enterobocter spp., the antibiotic resistance of Cronobocter is a serious issue that requires more attention.
基金supported by National High-tech R&D Program of China(863 Program)(2012AA101603)
文摘Objective To determine Cronobacter spp. contamination in infant and follow-up powdered formula in China. Methods All of 2282 samples were collected from the retail markets in China from January 2012 to December 2012, and analyzed for Cronobacter spp. by the Chinese National Food Safety Standard. Characterization of the isolates was analyzed by pulsed-field gel electrophoresis（PFGE） with XbaI and Spe I restriction enzymes. Results Cronobacter spp. strains were isolated from 25 samples, and the positive rates in infant powdered formulas and follow-up powdered formulas were 0.90%（10/1011） and 1.18%（15/1271）, respectively. Analysis of variable data regarding different purchasing store formats, seasonality, and production locations as well as comparison of infant versus follow-up formulas did not reveal statistically significant factors. During the sampling period, one of six surveillance zones did exhibit a statistically significant trend towards higher positive rate. PFGE characterization of Cronobacter spp. to elucidate genetic diversity revealed only three pairs of Cronobacter spp. out of 25 having the same PFGE patterns. Conclusion The current investigation indicated a lower positive rate of Cronobacter spp. in the powdered formula in China. This evidence suggested contamination originating from multiple different sources during the manufacturing process.
基金the financial support of National Key Research and Development program, China (2017YFC1601202 and 2017YFC1601200)China Postdoctoral Science Foundation (2021M690854)+2 种基金National Natural Science Foundation of China (31671951 and 31972175)Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01S174)Fundamental Research Funds for the Central Universities, China (JZ2020HGQA0151)
文摘Cronobacter species are a group of Gram-negative opportunistic pathogens,which cause meningitis,sep-ticemia,and necrotizing enterocolitis in neonates and infants,with neurological sequelae in severe cases.Interest in Cronobacter has increased significantly in recent years due to its high virulence in children.In this review,we summarize the current understanding of the prevalence of Cronobacter species in several important food types.We discuss the response mechanisms enabling persistence in adverse growth con-ditions,as well as its pathogenicity.We emphasize the food safety concerns caused by Cronobacter and subsequent control methods and clinical treatments.
基金funded by National Science and Technology Major Project of the Ministry of Science and Technology of China(2013ZX09304101)
文摘We used a proteomic approach to identify IbpA in Cronobacter sakazokii （C. sakazaki）, which is related to heat tolerance in this strain. The abundance of IbpA in C. sakazakii strains strongly increased after heat shock. C sakazakii CMCC 45402 ibpA deletion mutants were successfully constructed. The C. sakazakii CMCC 45402 AibpA and wild-type strains could not be distinguished based on colony morphology on LB agar plates or biochemical assays. The growth of the C. sakazakii CMCC 45402 AibpA mutant in heat shock conditions was indistinguishable from that of the isogenic wild-type, but showed greater heat resistance than E. coil O157：H7 strain CMCC 44828. This study suggests that the absence of a single ibpA gene has no obvious effect on the phenotype or heat resistance of the strain C. sakazakii CMCC 45402.
文摘A total of 7 Cronobocter strains were isolated from 703 fecal samples collected in Jinan from June 13 to December 30, 2011, with the positive rate of Cronobacter spp. being 1.0% （95% confidence interval 0.6%-1.4%）. Three Cronobacter sakazakii stains were isolated from 157 fecal samples of healthy neonates （95% confidence interval 0.4%-5.5%）. This number was slightly higher than that isolated from 273 fecal samples of healthy adults, in which 1 strain of C. sakazakii and 1 strain of Cronobacter malonaticus were isolated, and that from 173 fecal samples of adults with acute diarrhea, in which 1 strain of C sakazakii and 1 strain of C. malonaticus were isolated, but the differences were not statistically significant （P〉O.05）. The Cronobocter isolates were all from different genetic sources. It should be noted that Cronobacter carriage may cause infection under certain conditions, especially in neonates.
基金Supported by Key Project of Tianjin Municipal Education Commission(2010ZD01)
文摘[Objective] This study aimed to establish a Real-Time quantitative PCR method for the determination of transposon copy number in C. sakazakii. [ Method ] With single-copy housekeeping gene atpD as the reference gene, recombinant plasmid containing both single-copy housekeeping gene atpD and EZ-TN5 transposon was constructed; based on the established standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon, copy number of atpD gene and EZ-TN5 transpason in three C. sakazakii mutants was detected and the ratio was calculated. [ Result] Correlation coefficients of the standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon were 0. 999 and 0.998, respectively ; the ratios of copy number of atpD gene and EZ-TN5 transposon in three C. sakazakii mutants were 0.98, 1.17 and 0.91, respectively, which indicates that EZ-TN5 transpeson in C. sakakii mutants is a single-copy. [ Conclusion] Real-time quantitative PCR method established in this study had high availability and could replace the Southern blot method to detect the copy num- ber of EZ-TN5 transposon in different bacteria.
文摘Cronobacter sakazakii is an emerging pathogen that can cause diseases for several infant groups. These bacteria were contaminated in foods, clinical utensils, and environments. In Indonesia, C. sakazakii has been isolated from powdered infant formulas, weaning foods, and other dried foods such as cornstarch. The objective of this research is to trace survival of C. sakazakii during cornstarch production step using its mutant. Mutant was constructed by inserting Green Fluorescent Protein plasmid inside to the bacterial cell that appeared green fluorescent colonies under UV observation. The presence of C. sakazakii during processing was conducted by artificial contamination. This research consists of three steps, i.e. determination of the suitable enumeration method of C. sakazakii’s mutant, cornstarch production from yellow corn, and survival analysis of C. sakazakii during endosperm soaking and cornstarch drying. The suitable enumeration method was surface plating method on TSA-ampicillin medium combining with UV light application because of ineffectiveness of ampicillin inhibition for growth of yeasts and molds. The cornstarch produced in laboratory has the same properties with commercial cornstarch in parameters of moisture content, density, and starch granule structure. The yield of cornstarch final product was 48.90% (dry whole kernel-based). C. sakazakii cannot survive in 48 hours soaking process at 52?C and 24 hours drying process at 50?C that is applied during cornstarch production.
基金supported by Tianjin Key Medical Discipline Con-struction(TJYXZDXK-3-004A-3).
文摘Cronobacter sakazakii is an opportunistic foodborne pathogen with remarkable resilience to environmental stressors,posing severe risks in powdered infant formula.Here,we identify the GlrKR two-component system(TCS)as a master regulator that integrates environmental sensing with virulence control.Genetic deletion of either the sensor kinase(glrK)or the response regulator(glrR)markedly attenuates bacterial motility,epithelial cell adherence,and virulence in a neonatal rat model.GlrK is essential for acid and oxidative stress resistance,whereas both GlrK and GlrR suppress desiccation tolerance,revealing a regulatory trade-off between environmental persistence and host invasion.Quantitative proteomics and site-specific mutagenesis uncover enhanced deamidation of OmpA at N101 inΔglrR as a mechanistic basis for impaired adherence.Additionally,GlrKR modulates sensitivity to p-coumaric acid,a plant-derived antimicrobial,via transcriptional control of the efflux pump TolC.These findings establish GlrKR as a central node linking environmental adaptation,antimicrobial defense,and host-pathogen interaction in C.sakazakii,offering a promising target for next-generation food safety interventions.
基金supported by the Natural Science Foundation Project of Heilongjiang Province,China(Joint Guidance Project)Project title:“Prevalence and exposure risk assessment of pathogenic strains from infant formula processing environments(LH2023C110)".
文摘Enterobacteriaceae species have caused foodborne illnesses through consumption of a variety of foods,including infant foods.A total of 915 samples representing 4 types of samples,were collected from 7 powdered infant formula factories in Heilongjiang Province,China.All samples were processed for enumeration of Enterobac-teriaceae and isolation of Cronobacter sakazakii,which were detected using the ISO enrichment procedure and a chromogenic medium.Multi-locus sequence typing(MLST),and pulsed field gel electrophoresis(PFGE)were applied to characterize the C.sakazakii isolates to trace back the original source of contamination.Samples were considered Enterobacteriaceae positive at 10 CFU/g in powdered samples,1 CFU/mL in water samples,and 10 CFU/100 cm^(2) or larger surface area.Enterobacteriaceae positive rate were 0/35 water samples associated with production,11/56 raw materials,147/761 environmental samples and 3/63 finished products respectively.C.sakazakii strains were isolated from 7.0%of environmental samples followed by 1.6%of the finished products.A total of 20 C.sakazakii isolates were recovered in Factory 4,1 strain derived from a final product and 19 strains recovered from the environment representing 4 MLST sequence types and 10 PFGE pulse types.Most environ-mental sites positive for C.sakazakii had higher counts of Enterobacteriaceae,except for 11 sites where Enterobacteriaceae did not reach minimal levels for reporting a positive sample.The present study,provides insight to the correlation between the prevalence of Enterobacteriaceae and contamination of C.sakazakii in powdered infant formula factories;there is no one-to-one relation between number of Enterobacteriaceae and C.sakazakii.Identifying environmental sites positive for C.sakazakii and/or Enterobacteriaceae will aid in development of appropriate corrective measures to improve environmental hygiene and enhance safety of powdered infant formula.
基金financially supported by Shaanxi science and technology plan projects of China(2022KXJ-010,2022ZDLNY04-09)Xi’an city science and technology plan projects of China(22NYGG0012,23KGDW0021-2022)the Fundamental Research Funds for the Central Universities in China(GK202306005).
文摘Cronobacter sakazakii is a foodborne pathogen,and the use of effective and non-hazardous bacteriostatic agents to manage C.sakazakii infection has garnered attention.Portulaca oleracea L.is a food plant with several pharmaceutical properties.However,the products of kombucha-fermented P.oleracea(FP)have been scarcely studied with respect to their metabolite profiles and for their effects against food-borne pathogens.Therefore,in this study,the chemical and microbiological composition and inhibitory effects of FP against C.sakazakii were evaluated.The activities and molecular mechanisms of FP against C.sakazakii were investigated for the first time using transcriptomic and physiological experiments.Komagataeibacter was the most abundant bacterium in fermentation broth(>79%),and chlorogenic and acetic acids were the most abundant phenolics and organic acids in fermentation products.Transcriptomic analysis showed that FP induced significant changes in C.sakazakii as demonstrated by the differential expression of 506 genes.The genes that exhibited differential expression were primarily involved in ATP-related energy metabolism,cell membrane structure,protein biosynthesis,amino acid transport,and metabolism.These roles suggest multifaceted potential effects including cell membrane damage,protein synthesis limitation,metabolism disorder,reduced virulence factor expression,and reduced cell mobility.FP addition significantly inhibited C.sakazakii growth in PIF.These results provide insights into the anti-C.sakazakii mechanism of FP,which is valuable for its development and application in food safety.
基金National Natural Science Foundation of China(Grant No.32102104,Grant No.32230084)Guangdong S&T Program(2022B1111040002)+2 种基金the National Key Research and Development Program(2022YFF1100703)Provincial Key Laboratory of Microbial Safety and Health(2020B121201009-PKLMSH002-2021)Anhui Provincial Natural Science Foundation(2208085J11).
文摘Cronobacter spp.are classified as class A pathogens in powdered infant formula(PIF),and the capsular poly-saccharide is an important hazard-forming factor of Cronobacter.Currently,there are few studies about the capsular polysaccharide on tolerance of Cronobacter to environmental stress.Herein,we investigated the function of variable region genes of Cronobacter K1 antigen in stress response.The results demonstrated that key genes of K1 antigen could enhance acid,salt,and oxidative tolerance,and decrease desiccation tolerance.In addition,deletion of ESA_03354,ESA_03355,and ESA_03356 significantly reduced the biofilm capacity,cellular auto-aggregation capacity,and cell surface hydrophobicity of Cronobacter,while enhanced its motility.Thus,ESA_03354,ESA_03355,and ESA_03356 genes might involve in the stress response through biofilm formation when Cronobacter cell confronted with gastric acid,bile salt,and oxidative stress,whereas ESA_03357 preferred to produce yellow pigment to resist adverse stress.And ESA_03355 and ESA_03356 might mediate stress response by the polymerization of teichoic acid.Therefore,the key K1 antigen genes displayed different properties under various stresses,which provided important information for targeted control of Cronobacter.
基金support by the Joint Funds of the National Natural Science Foundation of China(No.U21A20272).
文摘Cronobacter sakazakii is a facultative anaerobic gram-negative pathogen that has attracted widespread attention for causing necrotizing enterocolitis in infants(premature and low-weight newborns)by contaminating powdered infant formula.Several studies have found that the antibiotic tolerance of Cronobacter sakazakii isolated from food sources is increasing,thereby markedly increasing the difficulty of clinical treatment of Cronobacter sakazakii infection.At present,most studies are focused on clarifying the pathogenicity of Cronobacter sakazakii,but there is still a gap in the study of necrotizing enterocolitis in neonatal rats caused by Cronobacter sakazakii with different antibiotic tolerance.To fill this gap,we analyzed and compared the ability of Cronobacter sakazakii induced by different classes of antibiotics to induce necrotizing enterocolitis in neonatal rats by mediating TLR4-NLRP3-IL-1β pathway.In the results,Cronobacter sakazakii acquired tolerance to aminoglycosides,quinolones and cephalosporins,and caused more inflammatory damage in neonatal rats,improved the ability of mediating TLR4-NLRP3-IL-1β pathway to induce necrotizing enterocolitis.Compared with the XS001 group,the NEC scores of neonatal rats in XS001-Amp,XS001-Ami,XS001-Ofl and XS001-Ceo groups were increased by 8.3%,58.3%,50.0% and 58.3%.The acquisition of penicillin and tetracycline tolerance had little effect on the ability of Cronobacter sakazakii to induce necrotizing enterocolitis in neonatal rats.The ability of Cronobacter sakazakii to induce necrotizing enterocolitis in neonatal rats was significantly different due to the type and tolerance of antibiotics.
基金supported by the China Postdoctoral Science Foundation(2017M611478)Fundamental Research Funds for the Central Universities
文摘Cronobacter spp.(formerly Enterobacter sakazakii) are special foodborne pathogens. Cronobacter infection can cause necrotizing enterocolitis, sepsis and meningitis in all age groups, especially neonates and infants, with a high fatality of up to 80%, although the infection is rare. Outbreaks of Cronobacter infection are epidemiologically proven to be associated with contaminated powdered infant formula(PIF). Cronobacter spp.can resist dry environments and survive for a long period in food with low water activity. Therefore, Cronobacter spp.have become serious pathogens of neonates and infants, as well as in the dairy industry. In this review, we present the taxonomy, pathogenesis, resistance, detection and control of Cronobacter spp.
文摘Objectives:Cronobacter sakazakii,formerly Enterobacter sakazakii,is an emerging ubiquitous and opportunistic foodborne pathogen with a high mortality rate.It has been implicated in cases of meningitis,septicaemia,and necrotizing enterocolitis among infants worldwide in association with powdered infant formula(PIF).This study was an insilico designed peptide base kit framework,using immunoinformatic techniques for quick detection of C.sakazakii in PIF.Materials and Methods:In the present study,a peptide-based kit was designed with a bioinformatic technique to rapidly identify C.sakazakii in PIF using fhE,secY,and bcsC,which are genes responsible for its bioflm formation,as target genes.The antigenicity,membrane topology,and the presence of signal peptides of the target genes were analysed using VaxiJen,DeepTMHMM,and SignalP servers.To provide stability and fexibility to the multiple-epitope construct,the linear B cells and helper T cells(IL-4(interleukin 4)and IL-10(interleukin 10)inducing epitopes)were linked with a GSGSG linker followed by the addition of protein disulphide bonds.To ascertain specifcity,the multi-epitope construct was molecularly docked against genes from sources other than PIF,like alfalfa,and the environment,with PIF being the highest:–328.48.Finally,the codons were modifed using the pET28a(+)vector,and the resultant multi-epitope construct was successfully cloned in silico.Results:The fnal construct had a length of 486 bp,an instability index of 23.26,a theoretical pI of 9.34,a molecular weight of 16.5 kDa,and a Z-score of–3.41.Conclusions:The multi-epitope peptide construct could be a conceptual framework for creating a C.sakazakii peptide-based detection kit,which has the potential to provide fast and effcient detection.However,there is a need for additional validation through the in vitro and in vivo techniques.
基金the Shanghai Project of Food Safety Risk Assessment(RA-2022-10)the Shanghai Agriculture Applied Technology Development Program(2022-02-08-00-12-F01144),China.
文摘Cronobacter sakazakii may encounter subinhibitory concentrations of ethanol stress over its lifecycle.Bacterial tolerance to homologous or heterologous stress agents may be altered as a result of ethanol adaptive responses.Therefore,the tolerance of ethanol-exposed and control cells to subsequent lethal stresses was evaluated in the current work.It was discovered that sublethal ethanol exposure increased the susceptibility to lethal ethanol stress in C.sakazakii as determined by the Weibull model.Furthermore,sublethal ethanol concentration exposure in C.sakazakii did not lead to any cross-tolerance against other stressors such as benzalkonium chloride(120 mg/L),heat(55°C),cold(4°C),simulated gastric fuid(pH 3.0),osmotic stress(sorbitol,0.75 g/mL),and desiccation stress.Analysis of zeta potential,scanning electron microscope,and surface-enhanced Raman spectroscopy spectra revealed that cellular injury and changes in cellular chemical composition may contribute to the reduced resistance of C.sakazakii after ethanol exposure.Furthermore,sublethal ethanol exposure resulted in an elevated proportion of unsaturated fatty acids(USFA),while reducing the proportion of saturated fatty acids(SFA)and the ratio of SFA to USFA.The developed inactivation models can serve as a valuable source of data to support quantitative microbial risk assessment.Moreover,a better understanding of the response of C.sakazakii to sublethal ethanol exposure may provide valuable insights into the prevention and control of C.sakazakii.
基金supported by National Key R&D Program of China(2023YFF1103900).
文摘Cronobacter sakazakii(C.sakazakii),a food-borne pathogen,can cause severe intestinal diseases,such as intestinal damage and necrotizing enterocolitis,and even death in neonates and infants through its adhesion to intestinal epithelium cells by protein-protein interaction and translocation across the intestinal barrier.Nevertheless,the mechanisms of adhesion,translocation,and pathogenesis remain unclear.This study aimed to screen and validate the outer membrane proteins of C.sakazakii capable of adhering to the surface of intestinal cells through biotinylated protein adhesion and LC-MS/MS identification.A total of 319 proteins were identified.Gene Ontology and KEGG analysis revealed significant enrichment of ligand proteins in metabolism-related processes and catalytic activity.The identified 37 outer membrane proteins and 41 proteins of unknown subcellular localization or function mainly enriched in five protein-protein interaction clusters.Ten of these outer membrane/unknown proteins were selected to test their roles on C.sakazakii adhesion and invasion of intestinal cells by silencing the encoding gene expression and nine of the ten proteins,Protein_26/30/34/38/42/98/104/194/282,were shown to be critical in this process.This study contributes to the understanding of proteins involved in the host adhesion process by C.sakazakii as well as the mechanisms for its translocation across the intestinal barrier.
