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.

Integrated multifunctional nanoplatform for fluorescence detection and inactivation of Staphylococcus aureus.

Foodborne illness caused by Staphylococcus aureus (S. aureus) has posed a significant threat to human health. Herein, an integrated multifunctional nanoplatform was developed for fluorescence detection and inactivation of S. aureus based on cascade signal amplification coupled with single strand DNA-template copper nanoparticles (ssDNA-Cu NPs). Benefiting from reasonable design, one-step cascade signal amplification was achieved through strand displacement amplification combined with rolling circle amplification, followed by in-situ generation of copper nanoparticles. S. aureus detection could be performed through naked eye observation and microplate reader measurement of the red fluorescence signal. The multifunctional nanoplatform had satisfactory specificity and sensitivity, achieving 5.2 CFU mL-1 detection limit and successful detection of 7.3 CFU of S. aureus in spiked egg after < 5 h of enrichment. Moreover, ssDNA-Cu NPs could eliminate S. aureus to avoid secondary bacterial contamination without further treatment. Therefore, this multifunctional nanoplatform has potential application in food safety dtection.

Survival of Salmonella in Tea Under Different Storage Conditions and Brewing Methods.

Salmonella is one of the leading causes of bacterial gastroenteritis. High prevalence of Salmonella in environment is partially due to its ability to enter the "viable but non-culturable" (VBNC) state when they encounter unfavorable conditions. Dried teas are traditionally believed to have a low risk of causing salmonellosis. This study investigated the survival of Salmonella in four types of dried teas under different storage conditions and brewing methods. A method that coupled propidium monoazide (PMA) and quantitative PCR was optimized to quantify VBNC Salmonella cells to assess the risk of Salmonella contamination in teas after brewing. Each tea sample was inoculated with Salmonella at an 8 log CFU/ml concentration and stored at 4, 10, and 25°C. Under three storage conditions, the number of survived Salmonella was highest in teas stored at 4°C and lowest in teas stored at 25°C. After storage of 120 days, culturable Salmonella was detected from all samples ranging from 6-7 log CFU/g (4°C storage) to 3-4 log CFU/g (25°C storage). The effectiveness of brewing methods in inactivating Salmonella was assessed by brewing inoculated teas at room temperature, 55, 75, and 100°C for 10 min. Brewing teas at 75 and 100°C significantly (P < 0.05) reduced the number of viable Salmonella, but VBNC Salmonella formed when brewed at 75°C. Altogether, Salmonella can persist in dried teas for over 3 months at a temperature ranging from 4 to 25°C, and thermal treatment delivered during home brewing may not eradicate Salmonella in teas.

Production and Characterization of a Novel Low-Sugar Beverage from Red Jujube Fruits and Bamboo Shoots Fermented with Selected Lactiplantibacillus plantarum.

Red jujube fruits and bamboo shoots are rich in many nutrients and have the advantage of high yield in China. However, the storage of fresh fruits is difficult, and there are no fermented products using both as raw materials. In order to develop the two raw materials into novel products and improve their nutritional value, this study reports the production and characterization of a beverage via fermentation of red jujube fruits and bamboo shoots with Lactiplantibacillus plantarum. L. plantarum TUST-232 was selected as the starter from several different strains by comparing pH value and the number of viable cells, which reached 8.91 log CFU/mL in the beverage fermented for 14 h at 37 °C with 0.3% inoculation. After fermentation, the beverage showed improvement in the contents of several nutrients and antioxidant indices, with a decrease of 44.10% in sucrose content, along with increases of 11.09%, 12.30%, and 59.80% in total phenolic content, total antioxidant capacity, and superoxide anion scavenging ability, respectively. These results indicate that L. plantarum fermentation of red jujube fruits and bamboo shoots could be an effective way to develop a new beverage with high nutritional value, high antioxidant capacity, and high dietary fiber content. This research provided experimental support for the development of new fermentation products with the functions of improving health and body functions.

Protective effect and mechanism of Monascus-fermented red yeast rice against colitis caused by Salmonella enterica serotype Typhimurium ATCC 14028.

Red yeast rice (RYR), a traditional Chinese fermented food, has the effect of lowering blood lipid and cholesterol, but little information is available about whether RYR can inhibit pathogenic bacterial infection in vivo. The present study explored the effect of RYR on Salmonella enterica-induced intestinal inflammation and gut microbiota dysbiosis in mice as well as the underlying anti-inflammatory mechanism. Results showed that RYR can alleviate S. enterica infection in vivo and Monascus pigments are the main functional components. The analysis of microbiota, gene expression profile and serological immunology revealed that RYR can regulate the intestinal flora and increase the relative abundance of beneficial bacteria such as Lactobacillus and Akkermansia. Meanwhile, RYR is also found to regulate the expression of pro-inflammatory factors and tight junction-related genes to inhibit the NO and NF-κB-mediated inflammatory response and maintain the integrity of the intestinal barrier. This study provides a new dietary intervention strategy for the prevention of pathogenic bacterial infection.

Comparative Proteomic Analysis of Adhesion/Invasion Related Proteins in Cronobacter sakazakii Based on Data-Independent Acquisition Coupled With LC-MS/MS.

Cronobacter sakazakii is foodborne pathogen that causes serious illnesses such as necrotizing enterocolitis, meningitis and septicemia in infants. However, the virulence determinants and mechanisms of pathogenicity of these species remain unclear. In this study, multilocus sequence typing (MLST) was performed on 34 C. sakazakii strains and two strains with the same sequence type (ST) but distinct adhesion/invasion capabilities were selected for identification of differentially expressed proteins using data-independent acquisition (DIA) proteomic analysis. A total of 2,203 proteins were identified and quantified. Among these proteins, 210 exhibited differential expression patterns with abundance ratios ≥3 or ≤0.33 and P values ≤0.05. Among these 210 proteins, 67 were expressed higher, and 143 were expressed lower in C. sakazakii SAKA80220 (strongly adhesive/invasive strain) compared with C. sakazakii SAKA80221 (weakly adhesive/invasive strain). Based on a detailed analysis of the differentially expressed proteins, the highly expressed genes involved in flagellar assembly, lipopolysaccharide synthesis, LuxS/AI-2, energy metabolic pathways and iron-sulfur cluster may be associated with the adhesion/invasion capability of C. sakazakii. To verify the accuracy of the proteomic results, real-time qPCR was used to analyze the expression patterns of some genes at the transcriptional level, and consistent results were observed. This study, for the first time, used DIA proteomic to investigate potential adhesion/invasion related factors as a useful reference for further studies on the pathogenic mechanism of C. sakazakii.

Protective Effect of Recombinant Proteins of Cronobacter Sakazakii During Pregnancy on the Offspring.

Cronobacter sakazakii is a food-borne pathogen carried in milk powder that can cause severe bacteremia, enterocolitis, and meningitis in newborns, which can lead to death of newborns. Preventing infection by this pathogen is significant to the health of newborns. Since infants and young children are the main target group of C. sakazakii, it is considered that maternal immunity can enhance the protection of newborns. Previous studies showed that two proteins of C. sakazakii (GroEL and OmpX) exhibited high expression levels and elicited strong immune reactions, suggesting their potential as vaccine candidates. In this study, GroEL and OmpX were recombinantly expressed in Escherichia coli and purified as immunogens to immunize pregnant rats. Three days after birth, the progeny were challenged with C. sakazakii to determine the protective effect of maternal immunity on the offspring. The results showed that immunization during pregnancy decreased bacterial load in the brain and blood, reduced brain and intestine damage, and significantly increased specific antibody titers in the offspring. Immunization with the recombinant proteins significantly increased cytokine levels in the serum of the progeny. The group whose mothers were immunized with OmpX produced more IL-4, while the group whose mothers were immunized with GroEL produced more IFN-γ, indicating that the immunogens enhanced the Th2 and Th1 responses, respectively. However, although the immune response was induced by both proteins, only the offspring of the pregnant rats immunized with OmpX or OmpX/GroEL mixture showed delayed death, possibly because immunization with OmpX led to a stronger humoral immune response in the offspring, suggesting that OmpX was a better vaccine candidate than GroEL. This study first reported that exposure to C. sakazakii proteins during pregnancy could improve the offspring's ability to resist infection caused by this pathogen.

Functionalized AuMBA @Ag Nanoparticles as an Optical and SERS Dual Probe in a Lateral Flow Strip for the Quantitative Detection of Escherichia coli O157:H7.

A method combining surface-enhanced Raman scattering (SERS) with a lateral flow strip (LFS) was developed for the quantitative and sensitive analysis of Escherichia coli O157:H7. AuMBA @Ag nanoparticles were prepared as SERS probes, and 4-methylthiobenzoic acid (MBA) as a Raman reporter was inserted into the interior gap of the Au@Ag core-shell nanoparticles, which replaced the Au nanoparticles that serve as SERS nanotags in traditional LFS. Using this developed SERS-LFS, the presence of the target bacteria could be tested through the appearance of a red band on the test line. Furthermore, quantitative analysis of E. coli O157:H7 was achieved by measuring the specific Raman intensity of MBA on the test line. The sensitivity of this SERS-LFS biosensor is 5 × 104 CFU/mL of E. coli O157:H7, which is 10-fold higher than that of a naked eye-based colorimetric LFS. This quantitative detection of E. coli O157:H7 ( Y = 1993.86 X - 6812.17, R2 = 0.9947) was obtained with a wide linear range (5 × 104 to 5 × 108 ) due to the signal enhancement of the SERS nanotags. In addition, the SERS-LFS could differentiate E. coli O157:H7 from closely related bacterial species or nontarget contaminants, suggesting high specificity of this assay. The applicability of SERS-LFS to the analysis of E. coli O157:H7 in milk, chicken breast, and beef was also validated, indicating that the sensitivity was not disturbed by the food matrix. In summary, the SERS-LFS developed in this study could be a powerful tool for the quantitative and sensitive screening of E. coli O157:H7 in a food matrix. PRACTICAL APPLICATION: This study demonstrates that a surface-enhanced Raman scattering (SERS)-based lateral flow strip (LFS) could be used as a rapid and sensitive method for Escherichia coli O157:H7 detection. Furthermore, this SERS-based LFS could achieve quantitative detection of the target, eliminating the defect of the traditional colloidal gold LFS, which is not quantifiable.

Characterization of the Desiccation Tolerance of Cronobacter sakazakii Strains.

Strong desiccation tolerance is an outstanding feature of Cronobacter sakazakii and can enable the bacterium to survive in a dry food matrix (such as milk powder) for a long time. Therefore, contamination of food possessing low water activity with C. sakazakii can increase the risk of infection in human beings, particularly in neonates and infants. However, the mechanism underlying the desiccation tolerance property of C. sakazakii is largely unknown. In this study, the desiccation tolerance characteristics of 42 C. sakazakii strains were analyzed. Simultaneously, the sequence types and biofilm formation abilities of the strains were investigated, and their correlations with desiccation tolerance were analyzed. The results showed no significant correlation between desiccation tolerance and sequence type. However, there was a positive correlation between biofilm formation ability and desiccation tolerance. Raman spectroscopy was employed to investigate the biofilm formed by strains with distinct desiccation tolerance levels, and the results showed that the levels of polysaccharide, proteins and carotenoid might play important roles in the resistance to dry environments. In addition, 10 genes involved in osmoprotectant synthesis or transport were selected, and their differential expression in strains with diverse desiccation tolerance levels was compared to investigate whether these genes were responsible for cytoprotection in the dry environment. The results revealed a great difference in gene expression among strains with different desiccation tolerance levels, suggesting that these genes play a regulatory role in the resistance of C. sakazakii to dry environments. Our study provides a useful reference for follow-up studies investigating the mechanism of desiccation tolerance in C. sakazakii.

Investigation of Citrinin and Pigment Biosynthesis Mechanisms in Monascus purpureus by Transcriptomic Analysis.

Monascus purpureus YY-1 is widely used in food colorant production in China. Our previous study clearly illustrated the whole-genome data for YY-1 and provided useful insight into evolutionary research and industrial applications. However, the presence of citrinin, which has nephrotoxic, hepatotoxic, and carcinogenic activities, has attracted attention to the safety of Monascus products. In an effort to reduce the harmful effects of citrinin in Monascus-related products, a random mutant of M. purpureus YY-1 with low citrinin production (designated as "winter") was obtained in this study. To analyze the biosynthesis and regulation mechanisms of pigment and citrinin, a transcriptomic analysis of the M. purpureus YY-1 and winter strains was performed. Comparative transcriptomic analysis reveals pksCT, the essential gene for citrinin synthesis, showed a low expression level in M. purpureus YY-1 and winter, which suggested there might be isoenzymes in M. purpureus YY-1 that were responsible for the citrinin synthesis during evolution. In addition, changes in transcription factor expression may also influence the network regulating the citrinin synthesis pathway in M. purpureus. Moreover, the yields of pigments produced by the winter mutant were significantly increased. Repressing the central carbon metabolism and improving the acetyl-CoA pool can contribute to a high pigment yield, and enhanced NADPH regeneration can also lead to the metabolic flux of pigment production in M. purpureus. Investigations into the biosynthesis and regulation of citrinin and pigment production in M. purpureus will enhance our knowledge of the mechanisms behind the biosynthesis of fungal secondary metabolites.

Recombinase Polymerase Amplification Combined with Lateral Flow Strip for Listeria monocytogenes Detection in Food.

Listeria monocytogenes is an important food-borne pathogenic bacterium that causes human disease, resulting in economic losses worldwide. The current detection methods for L. monocytogenes are not well suited for direct field testing because they involve complicated, time-consuming operations. A simple, efficient method is vital for L. monocytogenes detection. In this study, we combined isothermal recombinase polymerase amplification (RPA) with a lateral flow (LF) strip to rapidly and reliably detect L. monocytogenes. In the presence of biotin- and digoxin-modified primers, RPA produced numerous digoxin- and biotin-attached duplex DNA products. These products were detected on an LF strip via dual immunoreactions (digoxin on the duplex DNA reacted with the anti-digoxin antibody on the gold nanoparticle (Au-NP) and the biotin on the duplex DNA captured by the streptavidin on the LF test zone). The accumulation of Au-NPs produced characteristic bands, enabling the visual detection of L. monocytogenes without instrumentation. This assay could be used to detect L. monocytogenes within 15 min, including DNA amplification with RPA for 10 min at 39 °C and visualization of the amplicons by LF strips for 5 min. Experiments confirmed a detection limit as low as 300 fg of DNA and 1.5 × 101 CFU in pure cultures. Furthermore, RPA-LF exhibited no cross-reactions with pathogens. Evaluation of the method with food samples indicated that the detection limit was substantially improved to 1.5 × 10° CFU for the original bacterial content in 25 g/mL samples after enrichment for 6 hr. RPA-LF can be used as a sensitive and rapid detection technique for L. monocytogenes. PRACTICAL APPLICATION: Recombinase polymerase amplification (RPA) can amplify target DNA at 37 to 42 °C without a thermal cycler. Lateral flow (LF) strips are portable, cheap and easy to operate. RPA combined with LF strips to detect Listeria monocytogenes can be widely used in remote areas.

SERS-Based Lateral Flow Strip Biosensor for Simultaneous Detection of Listeria monocytogenes and Salmonella enterica Serotype Enteritidis.

Rapid, sensitive, point-of-care detection of bacteria is extremely important in food safety. To address this requirement, we developed a new surface-enhanced Raman scattering (SERS)-based lateral flow (LF) strip biosensor combined with recombinase polymerase amplification (RPA) for simultaneous detection of Listeria monocytogenes and Salmonella enterica serotype Enteritidis. AuMBA@Ag core-shell nanoparticles were used in this SERS-LF. Highly sensitive quantitative detection is achieved by measuring the characteristic peak intensities of SERS tags. Under optimal conditions, the SERS intensities of MBA at 1077 cm-1 on test lines are used to measure S. Enteritidis (y = 1980.6x - 539.3, R2 = 0.9834) and L. monocytogenes (y = 1696.0x - 844, R2 = 0.9889), respectively. The limit of detection is 27 CFU/mL for S. Enteritidis and 19 CFU/mL for L. monocytogenes. Significantly, this SERS-LF has high specificity and applicability in the detection of L. monocytogenes and S. Enteritidis in food samples. Therefore, the SERS-LF is a feasible method for the rapid and quantitative detection of a broad range of bacterial pathogens in real food samples.

A Negative Regulator of Cellulose Biosynthesis, bcsR, Affects Biofilm Formation, and Adhesion/Invasion Ability of Cronobacter sakazakii.

Cronobacter sakazakii is an important foodborne pathogen that causes neonatal meningitis and sepsis, with high mortality in neonates. However, very little information is available regarding the pathogenesis of C. sakazakii at the genetic level. In our previous study, a cellulose biosynthesis-related gene (bcsR) was shown to be involved in C. sakazakii adhesion/invasion into epithelial cells. In this study, the detailed functions of this gene were investigated using a gene knockout technique. A bcsR knockout mutant (ΔbcsR) of C. sakazakii ATCC BAA-894 showed decreased adhesion/invasion (3.9-fold) in human epithelial cell line HCT-8. Biofilm formation by the mutant was reduced to 50% of that exhibited by the wild-type (WT) strain. Raman spectrometry was used to detect variations in biofilm components caused by bcsR knockout, and certain components, including carotenoids, fatty acids, and amides, were significantly reduced. However, another biofilm component, cellulose, was increased in ΔbcsR, suggesting that bcsR negatively affects cellulose biosynthesis. This result was also verified via RT-PCR, which demonstrated up-regulation of five crucial cellulose synthesis genes (bcsA, B, C, E, Q) in ΔbcsR. Furthermore, the expression of other virulence or biofilm-related genes, including flagellar assembly genes (fliA, C, D) and toxicity-related genes (ompA, ompX, hfq), was studied. The expression of fliC and ompA in the ΔbcsR mutant was found to be remarkably reduced compared with that in the wild-type and the others were also affected excepted ompX. In summary, bcsR is a negative regulator of cellulose biosynthesis but positively regulates biofilm formation and the adhesion/invasion ability of C. sakazakii.

Development of an isothermal amplification-based assay for the rapid visual detection of Salmonella bacteria.

The efficient and timely detection of pathogens is a major concern worldwide. The aim of this study was to establish a rapid detection method for Salmonella bacteria in food samples to facilitate timely treatment. Widely used detection methods currently include culture-based methods and PCR-based methods. The former are time consuming, requiring 2 to 3 d, whereas the latter have higher accuracy but are typically complicated, requiring expertise and expensive instruments. In this study, a sensitive and rapid approach for the visual and point-of-use detection of Salmonella bacteria based on recombinase polymerase amplification (RPA) and a lateral-flow (LF) nucleic acid strip was established. We designed a pair of primers according to the invA gene of Salmonella bacteria: one was modified with digoxin, and the other was modified with biotin. In the presence of the biotin- and digoxin-modified primers and target DNA, the RPA produced a substantial amount of duplex DNA attached to biotin and digoxin. The products were detected using LF strips through immunoreaction: anti-digoxin antibodies on the gold nanoparticles, digoxin on the duplex, streptavidin on the LF test line, and biotin on the duplex. The developed RPA-LF assay allowed detection of Salmonella genomic DNA in less than 20 min with simple water bath equipment or portable thermal equipment. In addition, the RPA-LF assay was highly sensitive, with a detection limit as low as 20 fg of target DNA or 1.05 × 101 cfu of bacteria in pure culture, and highly specific, exhibiting no cross-reaction with Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Shigella, Enterobacter aerogenes, or Campylobacter jejuni. Importantly, Salmonella could be detected in milk and chicken breast at concentrations as low as 1.05 × 100 cfu/mL or 1.05 × 100 cfu/g after enrichment for 2 h and in eggs at 1.05 × 100 cfu/g after enrichment for 4 h. Furthermore, RPA was more sensitive than PCR, which requires a thermal cycling device. In summary, this study describes a sensitive, simple, and point-of-use detection method for Salmonella bacteria.

Inhibitor-Assisted High-Pressure Inactivation of Bacteria in Skim Milk.

The combined inactivation effects of high hydrostatic pressure (HHP) and antimicrobial compounds (potassium sorbate and ε-polylysine [ε-PL]) on 4 different bacterial strains present in skim milk and the effect of these treatments on milk quality were investigated in this study. HHP treatment at 500 MPa for 5 min reduced the populations of Escherichia coli, Salmonella enterica Typhimurium, Listeria monocytogenes, and Staphylococcus aureus from 6.5 log colony-forming units (CFUs) or higher to less than 1 log CFU/mL. Compared to HHP alone, HHP with potassium or ε-PL resulted in significantly higher reductions in the bacterial counts. After 5 min of treatment with HHP (500 MPa) and ε-PL (2 mg/mL), no growth of E. coli, S. enterica Typhimurium, or L. monocytogenes in skim milk was observed during 15 d of refrigerated storage (4 ± 1 °C). Scanning electron microscopy analysis revealed that the synergistic treatments caused more serious damage to the bacterial cell walls. Quality assessments of the treated samples indicated that the combined treatments did not influence the color, the turbidity, the concentrations of -SH group of the proteins, or the in vitro digestion patterns of the milk. This study demonstrates that HHP with potassium or ε-PL may be useful in the processing of milk or milk-containing foods.

A rapid Salmonella detection method involving thermophilic helicase-dependent amplification and a lateral flow assay.

Salmonella is a major foodborne pathogen that is widespread in the environment and can cause serious human and animal disease. Since conventional culture methods to detect Salmonella are time-consuming and laborious, rapid and accurate techniques to detect this pathogen are critically important for food safety and diagnosing foodborne illness. In this study, we developed a rapid, simple and portable Salmonella detection strategy that combines thermophilic helicase-dependent amplification (tHDA) with a lateral flow assay to provide a detection result based on visual signals within 90 min. Performance analyses indicated that the method had detection limits for DNA and pure cultured bacteria of 73.4-80.7 fg and 35-40 CFU, respectively. Specificity analyses showed no cross reactions with Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Enterobacter aerogenes, Shigella and Campylobacter jejuni. The results for detection in real food samples showed that 1.3-1.9 CFU/g or 1.3-1.9 CFU/mL of Salmonella in contaminated chicken products and infant nutritional cereal could be detected after 2 h of enrichment. The same amount of Salmonella in contaminated milk could be detected after 4 h of enrichment. This tHDA-strip can be used for the rapid detection of Salmonella in food samples and is particularly suitable for use in areas with limited equipment.

Screening of genes involved in interactions with intestinal epithelial cells in Cronobacter sakazakii.

Cronobacter sakazakii possesses a significant ability to adhere to and invade epithelial cells in its host. However, the molecular mechanisms underlying this process are poorly understood. In the current study, the adhesive and invasive capabilities of 56 C. sakazakii strains against human epithelial cells were evaluated, and one of them was selected for construction of a mutant library using the Tn5 transposon. In a systematic analysis of the adhesive and invasive capabilities of 1084 mutants, 10 mutants that showed more than a 50 % reduction in adhesion or invasion were obtained. Tail-PCR was used to sequence the flanking regions of the inserted transposon and 8 different genes (in 10 different mutants) were identified that encoded an exonuclease subunit, a sugar transporter, a transcriptional regulator, two flagellar biosynthesis proteins, and three hypothetical proteins. Raman spectroscopy was used to analyze variations in the biochemical components of the mutants, and the results showed that there were fewer amide III proteins, protein -CH deformations, nucleic acids and tyrosines and more phenylalanine, carotenes, and fatty acids in the mutants than in the wild type strain. Real-time PCR was used to further confirm the involvement of the genes in the adhesive and invasive abilities of C. sakazakii, and the results indicated that the expression levels of the 8 identified genes were upregulated 1.2- to 11.2-fold. The results of this study provide us with insight into the mechanism by which C. sakazakii infects host cells at molecular level.

Development of an Immunoassay for Chloramphenicol Based on the Preparation of a Specific Single-Chain Variable Fragment Antibody.

Specific antibodies are essential for the immune detection of small molecule contaminants. In the present study, the heavy and light variable regions (V(H )and V(L)) of the immunoglobulin genes from a hybridoma secreting a chloramphenicol (CAP)-specific monoclonal antibody (mAb) were cloned and sequenced. In addition, the light and heavy chains obtained from the monoclonal antibody were separated using SDS-PAGE and analyzed using Orbitrap mass spectrometry. The results of DNA sequencing and mass spectrometry analysis were compared, and the V(H) and V(L) chains specific for CAP were determined and used to construct a single-chain variable fragment (scFv). This fragment was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein and used to develop a direct competitive ELISA. Compared with the parent mAb, scFv exhibits lower sensitivity but better food matrix resistance. This work highlights the application of engineered antibodies for CAP detection.

Transcriptome analysis of Cronobacter sakazakii ATCC BAA-894 after interaction with human intestinal epithelial cell line HCT-8.

Cronobacter spp. are opportunistic pathogens that are responsible for infections including severe meningitis, septicemia, and necrotizing enterocolitis in neonates and infants. To date, questions still remain regarding the mechanisms of pathogenicity and virulence determinants for each bacterial strain. In this study, we established an in vitro model for Cronobacter sakazakii ATCC BAA-894 infection of HCT-8 human colorectal epithelial cells. The transcriptome profile of C. sakazakii ATCC BAA-894 after interaction with HCT-8 cells was determined using high-throughput whole-transcriptome sequencing (RNA sequencing (RNA-seq)). Gene expression profiles indicated that 139 genes were upregulated and 72 genes were downregulated in the adherent C. sakazakii ATCC BAA-894 strain on HCT-8 cells compared to the cultured bacteria in the cell-free medium. Expressions of some flagella genes and virulence factors involved in adherence were upregulated. High osmolarity and osmotic stress-associated genes were highly upregulated, as well as genes responsible for the synthesis of lipopolysaccharides and outer membrane proteins, iron acquisition systems, and glycerol and glycerophospholipid metabolism. In sum, our study provides further insight into the mechanisms underlying C. sakazakii pathogenesis in the human gastrointestinal tract.

Comparative proteomic analysis of Cronobacter sakazakii isolates with different virulences.

Cronobacter is a genus of widespread, opportunistic, foodborne pathogens that can result in serious illnesses in at-risk infants because of their immature immunity and high dependence on powdered formula, which is one of the foods most often contaminated by this pathogen. However, limited information is available regarding the pathogenesis and the specific virulence factors of this species. In this study, the virulences of 42 Cronobacter sakazakii isolates were analyzed by infecting neonatal SD rats. A comparison of the typing patterns of the isolates enabled groups with close relationships but that exhibited distinct pathogenesis to be identified. Among these groups, 2 strains belonging to the same group but showing distinct virulences were selected, and 2-DE was applied to identify differentially expressed proteins, focusing on virulence-related proteins. A total of 111 protein spots were identified using matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS), and 89 were successfully identified. Further analysis suggested that at least 11 of these proteins may be involved in the pathogenesis of this pathogen. Real-time PCR was carried out to further confirm the differential expression pattern of the genes, and the results indicated that the mRNA expression levels were consistent with the protein expression levels. BIOLOGICAL SIGNIFICANCE: The virulence factors and pathogenesis of Cronobacter are largely unknown. In combination with animal toxicological experiments and subtyping results of C. sakazakii, comparative proteomics analysis was performed to comprehensively evaluate the differentially expressed proteins of two isolates that exhibited distinct virulence but were closely related. These procedures made it possible to identify the virulence-related of factors of Cronobacter. Among the 89 total identified proteins, at least 11 show virulence-related potential. This work provides comprehensive candidates for the further investigation of the pathogenesis of Cronobacter.