EnvZ/OmpR Controls Protein Expression and Modifications in Cronobacter sakazakii for Virulence and Environmental Resilience.

Cronobacter sakazakii is a notorious foodborne opportunistic pathogen, particularly affecting vulnerable populations such as premature infants, and poses significant public health challenges. This study aimed to elucidate the role of the envZ/ompR genes in environmental tolerance, pathogenicity, and protein regulation of C. sakazakii. An envZ/ompR knockout mutant was constructed and assessed for its impact on bacterial growth, virulence, environmental tolerance, and protein regulation. Results demonstrate that deletion of envZ/ompR genes leads to reduced growth rate and attenuated virulence in animal models. Additionally, the knockout strain exhibited compromised environmental tolerance, particularly in desiccation and oxidative stress conditions, along with impaired adhesion and invasion abilities in epithelial cells. Proteomic analysis revealed significant alterations in protein expression and phosphorylation patterns, highlighting potential compensatory mechanisms triggered by gene deletion. Furthermore, investigation into protein deamidation and glucose metabolism uncovered a link between envZ/ompR deletion and energy metabolism dysregulation. Interestingly, the downregulation of MalK and GrxC proteins was identified as contributing factors to altered desiccation tolerance and disrupted redox homeostasis, respectively, providing mechanistic insights into the phenotypic changes observed. Overall, this study enhances understanding of the multifaceted roles of envZ/ompR in C. sakazakii physiology and pathogenesis, shedding light on potential targets for therapeutic intervention and food safety strategies.

Use of proteomics to elucidate characteristics of Cronobacter sakazakii under mild heat stress.

Cronobacter sakazakii is an opportunistic pathogen known for causing severe diseases. Mild heat treatment is commonly used in food processing, however, the pathogenic characteristics and underlying mechanisms of Cronobacter sakazakii strains remain poorly understood. In this study, we found that mild heat stress (MHS) at 52 °C can induce several deleterious effects in Cronobacter sakazakii, including damage to the cell wall, genomic DNA breakage, and misfolding of cytoplasmic proteins. These conditions lead to a decreased survival ability under acid, desiccation, and osmotic stress; a reduction in biofilm formation; and diminished motility. Notably, surviving C. sakazakii cells retain their pathogenicity, causing significant intestinal damage in newborn mice. This damage is characterized by epithelial sloughing and disruption of the intestinal structure. Tandem mass tag (TMT)-based proteomics identified 736 proteins with differential abundance across C. sakazakii strains subjected to mild heat stress, highlighting adaptations in biofilm formation, motility, and stress tolerance. Key regulatory changes were observed in phospholipid metabolism and protein synthesis, which underpin this complex stress response. This data illustrates a sophisticated balance between environmental adaptability and pathogenic potential. The metabolic and pathogenic responses of C. sakazakii to mild heat stress are closely linked to its phospholipid metabolism and the production of secretory proteins, both crucial for its virulence and reliant on membrane transport. This complex interplay emphasizes the need to understand these mechanisms to develop effective control strategies.

Effect of Bacterial Endophytes Isolated from Tropical Fruits against Listeria monocytogenes and Cronobacter sakazakii in Model Food Products.

Listeria monocytogenes and Cronobacter sakazakii are two important foodborne bacterial pathogens. Bacterial endophytes, which reside in plant cells, can produce antimicrobial compounds to protect the host organism or inhibit pathogens. This study investigated the bacterial community of tropical fruits for their potential to inactivate L. monocytogenes or C. sakazakii in cantaloupe and liquid infant formula, respectively. Tropical fruits including papayas, dragon fruits, and sugar apples, were sourced from several countries. Candidate bacterial endophytes were recovered from these tropical fruits using blood agar and Reasoner's 2A (R2A) agar and tested for potential inhibition against L. monocytogenes and C. sakazakii. A total of 196 bacterial endophytes were recovered from papayas, dragon fruits, and sugar apples. Among these bacterial endophytes, 33 (16.8%) and 13 (6.6%) of them demonstrated an inhibition zone against L. monocytogenes and C. sakazakii, respectively. The inhibitory strains were identified using 16S rRNA sequencing as Bacillus spp., Enterobacter spp., Klebsiella spp., Microbacterium spp., Pantoea spp., and Pseudomonas spp. A cocktail of Pantoea spp. and Enterobacter spp. was used in challenge studies with cantaloupe and significantly reduced the number of L. monocytogenes by approximately 2.5 log10 CFU/g. In addition, P. stewartii demonstrated antagonistic activity against C. sakazakii in liquid infant formula, i.e., it significantly decreased the number of C. sakazakii by at least 1 log10 CFU/mL. Thus, the use of bacterial endophytes recovered from fruits and vegetables could be a promising area of research. Their use as potential biocontrol agents to control bacterial pathogens in ready-to-eat foods warrants further investigation.

Microbiological survey and genomic analysis of Cronobacter sakazakii strains isolated from US households and retail foods.

Cronobacter species are opportunistic pathogens that are capable of causing morbidity and mortality, particularly in infants. Although the transmission dynamics involved in Cronobacter infections remain largely unknown, contaminated powdered infant formula (PIF) has been linked to 30% of Cronobacter sakazakii cases involving invasive illness in infants. As several lines of evidence have implicated the domestic environment in PIF contamination, we undertook a microbiological survey of homes (N = 263) across the US. Cronobacter spp. and C. sakazakii were isolated from 36.1% and 24.7% of US homes, respectively, with higher recovery rates observed for floor and kitchen surfaces. Multi-locus sequence typing indicated that the dominant strain was C. sakazakii ST4, the sequence type most commonly associated with neonatal meningitis. For comparison purposes, retail foods (N = 4,009) were also surveyed, with the highest contamination frequencies (10.1%-26.3%) seen for nut products, seeds, and grains/baked goods/flours. The sequence type profile of isolates recovered from homes mirrored that of isolates recovered from retail foods, with increased representation of ST1, ST4, ST13, ST17, and ST40. Analysis of 386 whole genomic sequences revealed significant diversity. Redundancies were only observed for isolates recovered from within the same domicile, and there were no identical matches with sequences archived at the NCBI pathogen database. Genes coding for putative virulence and antibiotic resistance factors did not segregate with clinically significant sequence types. Collectively, these findings support the possibility that contamination events occurring within the home should not be overlooked as a contributor to community-onset Cronobacter infections. IMPORTANCE: Cronobacter sakazakii is an opportunistic pathogen that can cause significant morbidity and mortality in neonates. Its transmission dynamics are poorly understood, though powered infant formula (PIF) is thought to be the major transmission vehicle. How the PIF becomes contaminated remains unknown. Our survey shows that roughly 1/4 of US homes are contaminated with Cronobacter sakazakii, particularly in the kitchen setting. Our analyses suggest that the domestic environment may contribute to contamination of PIF and provides insights into mitigating the risk of transmission.

Assessment of the Sensitivity of Foodborne Cronobacter spp. Strains and Other Enterobacteria to Temperature Stresses and Chlorine-Containing Biocides.

We examined the effects of elevated temperatures and biocides on survivability of food isolates of Cronobacter spp. (C. sakazakii) and concomitant enterobacteriaceae obtained in microbiological control of infant nutrition products. Increased resistance of certain strains of Cronobacter, Enterobacter cloacae, and Pantoea spp. to thermal processing was revealed. Salmonella, Pantoea, and Cronobacter bacteria were least sensitive to antimicrobial action of chlorine-containing agents. The above properties varied in the strains of the same species. Specifically, only two of three examined isolates of Cronobacter spp. demonstrated lower sensitivity to heat in comparison with the enterobacterial test-cultures of other species.

Genome Mining of Novel Targets and Construction of Ladder-shaped melting temperature isothermal amplification Assays for the Identification of Cronobacter sakazakii and Cronobacter malonaticus.

Cronobacter species are potential pathogens that can contaminate powdered infant formula. C. sakazakii and C. malonaticus are the most common species of Cronobacter associated with infections. This study mined new molecular targets for the detection of C. sakazakii and C. malonaticus by using comparative genome approaches. Specific target genes mngB and ompR were obtained and used to detect C. sakazakii and C. malonaticus, respectively. A novel detection method, termed ladder-shape melting temperature isothermal amplification (LMTIA), was developed and evaluated. The detection limit for pure C. sakazakii DNA was 1 pg per reaction and 1 fg per reaction for C. malonaticus. The C. sakazakii, C. malonaticus, and the reference stains were all correctly identified. The amplicons can be successfully visualized and identified by naked eyes when hydroxy naphthol blue dye (HNB dye) was used in the reaction. Therefore, the LMTIA assays developed in this study showed potential application for microorganism identification and detection.

Emergent methods for inactivation of Cronobacter sakazakii in foods: A systematic review and meta-analysis.

Cronobacter sakazakii is a potentially pathogenic bacterium that is resistant to osmotic stress and low aw, and capable of persisting in a desiccated state in powdered infant milks. It is widespread in the environment and present in various products. Despite the low incidence of cases, its high mortality rates of 40 to 80 % amongst neonates make it a microorganism of public health interest. This current study performed a comparative assessment between current reduction methods applied for C. sakazakii in various food matrices, indicating tendencies and relevant parameters for process optimization. A systematic review and meta-analysis were conducted, qualitatively identifying the main methods of inactivation and control, and quantitatively evaluating the effect of treatment factors on the reduction response. Hierarchical clustering dendrograms led to conclusions on the efficiency of each treatment. Review of recent research trend identified a focus on the potential use of alternative treatments, with most studies related to non-thermal methods and dairy products. Using random-effects meta-analysis, a summary effect-size of 4-log was estimated; however, thermal methods and treatments on dairy matrices displayed wider dispersions - of τ2 = 8.1, compared with τ2 = 4.5 for vegetal matrices and τ2 = 4.0 for biofilms. Meta-analytical models indicated that factors such as chemical concentration, energy applied, and treatment time had a more significant impact on reduction than the increase in temperature. Non-thermal treatments, synergically associated with heat, and treatments on dairy matrices were found to be the most efficient.

The role of DsbA and PepP genes in the environmental tolerance and virulence factors of Cronobacter sakazakii.

Cronobacter sakazakii, an opportunity foodborne pathogen, could contaminate a broad range of food materials and cause life-threatening symptoms in infants. The bacterial envelope structure contribute to bacterial environment tolerance, biofilm formation and virulence in various in Gram-negative bacteria. DsbA and PepP are two important genes related to the biogenesis and stability of bacterial envelope. In this study, the DsbA and PepP were deleted in C. sakazakii to evaluate their contribution to stress tolerance and virulence of the pathogen. The bacterial environment resistance assays showed DsbA and PepP are essential in controlling C. sakazakii resistance to heat and desiccation in different mediums, as well as acid, osmotic, oxidation and bile salt stresses. DsbA and PepP also played an important role in regulating biofilm formation and motility. Furthermore, DsbA and PepP deletion weaken C. sakazakii adhesion and invasion in Caco-2, intracellular survival and replication in RAW 264.7. qRT-PCR results showed that DsbA and PepP of C. sakazakii played roles in regulating the expression of several genes associated with environment stress tolerance, biofilm formation, bacterial motility and cellular invasion. These findings indicate that DsbA and PepP played an important regulatory role in the environment resisitance, biofilm formation and virulence of C. sakazakii, which enrich understanding of genetic determinants of adaptability and virulence of the pathogen.

Machine-Learning-Assisted Aggregation-Induced Emissive Nanosilicon-Based Sensor Array for Point-of-Care Identification of Multiple Foodborne Pathogens.

How timely identification and determination of pathogen species in pathogen-contaminated foods are responsible for rapid and accurate treatments for food safety accidents. Herein, we synthesize four aggregation-induced emissive nanosilicons with different surface potentials and hydrophobicities by encapsulating four tetraphenylethylene derivatives differing in functional groups. The prepared nanosilicons are utilized as receptors to develop a nanosensor array according to their distinctive interactions with pathogens for the rapid and simultaneous discrimination of pathogens. By coupling with machine-learning algorithms, the proposed nanosensor array achieves high performance in identifying eight pathogens within 1 h with high overall accuracy (93.75-100%). Meanwhile, Cronobacter sakazakii and Listeria monocytogenes are taken as model bacteria for the quantitative evaluation of the developed nanosensor array, which can successfully distinguish the concentration of C. sakazakii and L. monocytogenes at more than 103 and 102 CFU mL-1, respectively, and their mixed samples at 105 CFU mL-1 through the artificial neural network. Moreover, eight pathogens at 1 × 104 CFU mL-1 in milk can be successfully identified by the developed nanosensor array, indicating its feasibility in monitoring food hazards.

Global and regional prevalence of Cronobacter sakazakii in powdered milk and flour.

Cronobacter sakazakii (Cz) infections linked with powdered milk/flour (PMF) are on the increase in recent times. The current study aimed at assessing worldwide and regional prevalence of Cz in PMF. Cz-PMF-directed data were conscientiously mined in four mega-databases via topic-field driven PRISMA protocol without any restriction. Bivariate analysis of datasets was conducted and then fitted to random-intercept logistic mixed-effects regressions with leave-one-study-out-cross-validation (LOSOCV). Small-study effects were assayed via Egger's regression tests. Contributing factors to Cz contamination/detection in PMF were determined using 1000-permutation-bootstrapped meta-regressions. A total of 3761 records were found out of which 68 studies were included. Sample-size showed considerable correlation with Cz positivity (r = 0.75, p = 2.5e-17), Milkprod2020 (r = 0.33, p = 1.820e-03), and SuDI (r = - 0.30, p = 4.11e-03). The global prevalence of Cz in PMF was 8.39% (95%CI 6.06-11.51, PI: 0.46-64.35) with LOSOCV value of 7.66% (6.39-9.15; PI: 3.10-17.70). Cz prevalence in PMF varies significantly (p < 0.05) with detection methods, DNA extraction method, across continents, WHO regions, and world bank regions. Nation, detection method, world bank region, WHO region, and sample size explained 53.88%, 19.62%, 19.03%, 15.63%, and 9.22% of the true differences in the Cz prevalence in PMF, respectively. In conclusion, the results indicated that national will power in the monitoring and surveillance of Cz in PMF matched with adequate sample size and appropriate detection methods will go a long way in preventing Cz contamination and infections.

Integrating Recombinase Polymerase Amplification and Photosensitization Colorimetric Detection in One Tube for Fast Screening of C. sakazakii in Formula Milk Powder.

Cronobacter sakazakii (C. sakazakii) is a widely existing opportunistic pathogen and thus threatens people with low immunity, especially infants. To prevent the outbreak, a rapid and accurate on-site testing method is required. The current standard culture-based method is time-consuming (3-4 days), while the nucleic acid amplification (PCR)-based detection is mostly carried out in central laboratories. Herein, isothermal recombinase polymerase amplification (RPA) coupled with a photosensitization colorimetric assay (PCA) was adopted for the on-site detection of C. sakazakii in powdered infant formulas (PIFs). The lowest visual detection concentration of C. sakazakii is 800 cfu/mL and 2 cfu/g after 8 h bacteria pre-enrichment. Furthermore, to avoid typical cap opening-resulted aerosol pollution, the PCA reagents were lyophilized onto the cap of the RPA tube (containing lyophilized RPA reagents). After amplification, the tube was subjected to simple shaking to mix the PCA reagents with the amplification products for light-driven color development. Such a one-tube assay offered a lowest concentration of 1000 copies of genomic DNA of C. sakazakii within 1 h. After 8 h of bacterial enrichment, the lowest detecting concentration could be pushed down to 5 cfu/g bacteria in PIF. To facilitate on-site monitoring, a portable, battery-powered PCA device was designed to mount the typical RPA 8-tube strip, and a color analysis cellphone APP was further employed for facile readout.

Survival and Expression of rpoS and grxB of Cronobacter sakazakii in Powdered Infant Formula Under Simulated Gastric Conditions of Newborns.

Cronobacter sakazakii can cause severe illnesses in infants, predominantly in preterm newborns, with consumption of contaminated powdered infant formula (PIF) being the major vehicle of infection. Using a dynamic human gastrointestinal simulator called the SHIME, this study examined the effects of gastric acidity and gastric digestion time of newborns on the survival and expression of stress genes of C. sakazakii. Individual strains, inoculated at 7 log CFU/mL into reconstituted PIF, were exposed to gastric pH values of 4.00, 5.00 and 6.00 for 4 h with gradual acidification. The survival results showed that C. sakazakii grew in the stomach portion of the SHIME during a 4-h exposure to pH 4.00, 5.00 and 6.00 by 0.96-1.05, 1.02-1.28 and 1.11-1.73 log CFU/mL, respectively. The expression of two stress genes, rpoS and grxB, throughout gastric digestion was evaluated using reverse transcription qPCR. The upregulation of rpoS and grxB during the 4-h exposure to simulated gastric fluid at pH 4.00 showed that C. sakazakii strains may be experiencing the most stress in the pH 4.00 treatment. The gene expression results also suggest that C. sakazakii strains appeared to develop an acid adaptation response during the 4-h exposure that may facilitate their survival. Altogether, this study highlights that a combination of low gastric acidity, long digestion time in the presence of reconstituted PIF, created a favorable environment for the adaptation and survival of C. sakazakii in the simulation of a newborn's stomach. This study gives directions for future research to further advance our understanding of the behavior of C. sakazakii in the GI tract of newborns.

Maltodextrin-binding protein as a key factor in Cronobacter sakazakii survival under desiccation stress.

Cronobacter sakazakii (C. sakazakii) is a notorious pathogen responsible for infections in infants and newborns, often transmitted through contaminated infant formula. Despite the use of traditional pasteurization methods, which can reduce microbial contamination, there remains a significant risk of pathogenic C. sakazakii surviving due to its exceptional stress tolerance. In our study, we employed a comparative proteomic approach by comparing wild-type strains with gene knockout strains to identify the essential genes crucial for the successful survival of C. sakazakii during desiccation. Our investigation revealed the significance of envZ-ompR, recA, and flhD gene cassettes in contributing to desiccation tolerance in C. sakazakii. Furthermore, through our comparative proteomic profiling, we identified the maltodextrin-binding protein encoded by ESA_03421 as a potential factor influencing dry tolerance. This protein is regulated by EnvZ-OmpR, RecA, and FlhD. Notably, the knockout of ESA_03421 resulted in a 150% greater reduction in Log CFU compared to the wild-type C. sakazakii. Overall, our findings offer valuable insights into the mechanisms underlying C. sakazakii desiccation tolerance and provide potential targets for the development of new antimicrobial strategies aimed at reducing the risk of infections in infants and newborns.

Rosa roxburghii Tratt Pomace Crude Extract Inactivates Cronobacter sakazakii Isolated from Powdered Infant Formula.

Cronobacter sakazakii is an important foodborne pathogen in powder infant formula (PIF). The objective of this study was to evaluate the inactivation effect of Rosa roxburghii Tratt pomace crude extract (RRPCE) on C. sakazakii isolated from PIF and to reveal the mechanism of action. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were used to evaluate the inhibitory activity of RRPCE against C. sakazakii. The inhibitory mechanism was revealed from the perspective of effects of RRPCE on intracellular adenosine 5'-triphosphate (ATP), reactive oxygen species (ROS), membrane potential, protein and nucleic acid leakage, and cell morphology of C. sakazakii. The inactivation effects of RRPCE on C. sakazakii in biofilms on stainless steel, tinplate, glass, silica gel, polyethylene terephthalate, and polystyrene to evaluate its potential as a natural disinfectant. The results showed that the MIC and MBC of RRPCE against C. sakazakii were 7.5 and 15 mg/mL, respectively. After treatments with RRPCE, intracellular ATP content decreased significantly while intracellular ROS level increased significantly (p < 0.05). The cell membrane depolarization, large leakage of proteins and nucleic acids, and severely damaged cell morphology also occurred in C. sakazakii treated with RRPCE. In addition, a 20-minute treatment with 2 MIC (15 mg/mL) of RRPCE could inactivate all C. sakazakii (from 6.10 to 6.40 CFU/mL) in biofilms on all six contact surfaces. Our findings suggest that RRPCE is ideal for the inactivation of C. sakazakii and has the potential to be used as a natural disinfectant for the inactivation of PIF packaging materials and containers.

Effect of glutathione-transport-related gene gsiD on desiccation tolerance of Cronobacter sakazakii and its related regulatory mechanism.

The outstanding desiccation tolerance of Cronobacter sakazakii (C. sakazakii) enables long-term persistence in food products with low-water activity to increase the infection risk, especially in low-birth-weight, immuno-compromised neonates, and infants less than 4 weeks of age. In our previous study, the disruption of glutathione transport-related gene gsiD by transposon was found to significantly increase its inactivation rate under drying stress challenges. However, the mechanism underlying the association between glutathione transport and desiccation tolerance of C. sakazakii remains to be clarified. In this study, the mechanism underlying their association was investigated in detail by constructing the gsiD gene deletion mutant. gsiD gene deletion was found to cause the dysfunction of the glutathione transport system GsiABCD and the limitation of glutathione import. The resulting decrease in intracellular glutathione caused the decreased potassium ions uptake and increased potassium ions efflux, inhibited the proline synthesis process, limited extracellular glutathione utilization, increased oxidant stress, reduced biofilm formation, and increased outer membrane permeability, which may be the main reasons for the significant reduction of the desiccation tolerance of C. sakazakii.IMPORTANCEContributing to its superior environmental adaptability, Cronobacter sakazakii can survive under many abiotic stress conditions. The outstanding desiccation tolerance makes this species persist in low-water activity foods, which increases harm to humans. For decades, many studies have focused on the desiccation tolerance of C. sakazakii, but the existing research is still insufficient. Our study found that gsiD gene deletion inhibited glutathione uptake and further decreased intracellular glutathione content, causing a decrease in desiccation tolerance and biofilm formation and an increase in outer membrane permeability. Moreover, the expression level of relative genes verified that gsiD gene deletion made the mutant not conducive to surviving in dry conditions due to restricting potassium ions uptake and efflux, inhibiting the conversion of glutamate to compatible solute proline, and increasing the oxidative stress of C. sakazakii. The above results enrich our knowledge of the desiccation tolerance mechanism of C. sakazakii.

Electrotransformation of Foodborne Pathogen Cronobacter sakazakii by a Simple Method.

Cronobacter sakazakii is an opportunistic foodborne pathogen that mainly infects infants and immunocompromised people, with a high mortality rate. However, the efficient transformation method of this bacterium has not been systematically reported. In this study, we developed a fast and efficient transformation method for C. sakazakii by cold sucrose treatment. Compared with CaCl2 or glycerol treatment, the transformation efficiency of this method is significantly high when bacteria were cultured overnight at 42°C before cold sucrose treatment. Furthermore, applying this method, we successfully knocked out the pppA gene by direct electroporation. Collectively, our study provides a simple, time-saving, and efficient method for competent cell preparation of C. sakazakii, which is conducive to the further research of C. sakazakii.

Cronobacter sakazakii infection implicates multifaceted neuro-immune regulatory pathways of Caenorhabditis elegans.

The neural pathways of Caenorhabditis elegans play a crucial role in regulating host immunity and inflammation during pathogenic infections. To understand the major neuro-immune signaling pathways, this study aimed to identify the key regulatory proteins in the host C. elegans during C. sakazakii infection. We used high-throughput label-free quantitative proteomics and identified 69 differentially expressed proteins. KEGG analysis revealed that C. sakazakii elicited host immune signaling cascades primarily including mTOR signaling, axon regeneration, metabolic pathways (let-363 and acox-1.4), calcium signaling (mlck-1), and longevity regulating pathways (ddl-2), respectively. The abrogation in functional loss of mTOR-associated players deciphered that C. sakazakii infection negatively regulated the lifespan of mutant worms (akt-1, let-363 and dlk-1), including physiological aberrations, such as reduced pharyngeal pumping and egg production. Additionally, the candidate pathway proteins were validated by transcriptional profiling of their corresponding genes. Furthermore, immunoblotting showed the downregulation of mTORC2/SGK-1 during the later hours of pathogen exposure. Overall, our findings profoundly provide an understanding of the specificity of proteome imbalance in affecting neuro-immune regulations during C. sakazakii infection.

Group selective aptamers: Broad-spectrum recognition of target groups in Cronobacter species and implementation of electrochemical biosensors as receptors.

Aptamers are a versatile class of receptors with a high affinity and selectivity for specific targets. Although their ability to recognize individual targets has been extensively studied, some scenarios require the development of receptors capable of identifying all target groups. This study investigated the use of aptamers to achieve the broad-spectrum recognition of groups instead of individual targets. Aptamers were screened for selectively distinct groups of Cronobacter species associated with foodborne diseases. Seven Cronobacter spp. were divided into Group A (C. sakazakii, C. malonaticus, C. turicensis, and C. muytjensii) and Group B (C. dublinensis, C. condimenti, and C. universalis). Aptamers with exclusive selectivity for each group were identified, allowing binding to the species within their designated group while excluding those from the other group. The screened aptamers demonstrated reliable affinity and specificity with dissociation constants ranging from 1.3 to 399.7 nM for Group A and 4.0-24.5 nM for Group B. These aptamers have also been successfully employed as receptors in an electrochemical biosensor platform, enabling the selective detection of each group based on the corresponding aptamer (limit of detection was 7.8 and 3.2 CFU for Group A and Group B, respectively). The electrochemical sensor effectively detected the extent of infection in each group in powdered infant formula samples. This study highlights the successful screening and application of group-selective aptamers as sensing receptors, emphasizing their potential for diverse applications in different fields such as food safety, environmental monitoring, and clinical diagnostics, where the selective biosensing of target groups is crucial.

Inactivation of Carotenogenic-Biosynthesizing Genes Altered Lipids Composition and Intensity in Cronobacter sakazakii.

Cronobacter sakazakii, an opportunistic milk-borne pathogen responsible for severe neonatal meningitis and bacteremia, can synthesize yellow pigment (various carotenoids) benefiting for bacterial survival, while little literature was available about the influence of various carotenoids on bacterial resistance to a series of stresses and the characteristics of cell membrane, obstructing the development of novel bactericidal strategies overcoming the strong tolerance of C. sakazakii. Thus in this study, for the first time, five carotenogenic genes of C. sakazakii BAA-894 were inactivated, respectively, to construct a series of mutants producing various carotenoids and their effects on the cell membrane properties, and resistances to food- and host-related stresses, were investigated systematically. Furthermore, to explore its possible mode of action, comparative lipidomics analysis was performed to reveal the change of lipids that were mainly located at cell membranes. The results showed that five mutants (ΔcrtB, ΔcrtI, ΔcrtY, ΔcrtZ, and ΔcrtX) displayed negligible change in growth rate but higher permeability of the outer membrane and lower fluidity of cell membrane compared to the wild type. Besides, these mutants exhibited poorer ability of biofilm formation and lower resistances to acid, oxidative, osmotic, and desiccation stresses, indicating that different carotenoid composition significantly affected environmental tolerance of C. sakazakii. To discover the possible causes, lipidomics analysis of C. sakazakii was conducted and more than 500 lipid species belonging to 27 classes had been identified at first. Compared to that of BAA-894, the composition and relative intensity of lipid species in five mutants varied significantly, especially the monounsaturated and biunsaturated phosphatidylethanolamine. The evidence presented in this study demonstrated that the varied composition of carotenoids in C. sakazakii significantly altered the lipid profile and intensity, which maybe a crucial means to influencing the characteristics of cell membranes and resistance to environmental stresses.

Effects and molecular mechanism of sugar transporter ESA_RS15745 on desiccation resistance, motility, and biofilm formation of Cronobacter sakazakii.

Cronobacter sakazakii, an important Gram-negative foodborne pathogen, can cause neonatal meningitis and sepsis with high rates of infection and death. Gene ESA_RS15745 encodes a sugar transporter protein, which is not only essential for osmotic pressure maintenance during bacterial growth and reproduction but also associated with their desiccation tolerance, motility, and biofilm formation. Here, a mutant strain of ESA_RS15745 (ΔESA_RS15745) and the complementation strain (cpESA_RS15745) were constructed using a suicide vector knockout and gene complementation. ΔESA_RS15745 was found to have a decrease in its ability to transport maltose and trehalose and resist desiccation, whereas an increase in the ability of motility and biofilm formation, implying that ESA_RS15745 may positively regulate sugar transport and desiccation tolerance and negatively regulate motility and biofilm formation. To further investigate the molecular mechanisms underlying the function of related genes, RNA-seq was performed to explore the differentially expressed genes in the mutants. RNA-seq results showed the upregulation of 114 genes (mainly including those regulating chemotaxis and flagellar motility) and the downregulation of 22 genes (mainly including those regulating sugar transport). qRT-PCR analysis supported the RNA-seq results and showed that ESA_RS15745 may influence the dehydration tolerance though decreasing the intracellular trehalose content and negatively regulate the motility though the chemotactic signaling pathway. In addition, the biofilm formation of C. sakazakii should also be speculated to negatively regulate by ESA_RS15745 by consuming the extracellular carbohydrates concentration and then downregulating the intracellular cyclic diguanosine monophosphate. This study offers a reference for comprehending the molecular mechanism of gene ESA_RS15745 in C. sakazakii.