Yingwang Ye

The Cronobacter sp. in milk and dairy products: Detection and typing

Cronobacter is a group of important foodborne pathogens that have been implicated in meningitis, sepsis and necrotising enterocolitis, especially in neonates through consumption of contaminated powdered infant formula (PIF). The detection of PIF contamination is of great concern to the infant formula industry. In this article, phenotypic and genotypic methods for detecting and typing Cronobacter are discussed in relation to minimising and controlling the risk of Cronobacter sp. contamination. To reliably detect and type Cronobacter strains, the use of conventional microbiological methods in combination with molecular assays is recommended.

Identification of potential virulence factors of Cronobacter sakazakii isolates by comparative proteomic analysis.

Cronobacter is a group of important foodborne pathogens associated with neonatal meningitis, septicemia, and necrotizing enterocolitis. Among Cronobacter species, Cronobacter sakazakii is the most common species in terms of isolation frequency. However, the molecular basis involved in virulence differences among C. sakazakii isolates is still unknown. In this study, based on the determination of virulence differences of C. sakazakii G362 (virulent isolate) and L3101 (attenuated isolate) through intraperitoneal injection, histopathologic analysis (small intestine, kidney, and liver) further confirmed virulence differences. Thereafter, the potential virulence factors were determined using two-dimensional electrophoresis (2-DE) coupled with MALDI/TOP/TOF mass spectrometry. Among a total of 36 protein spots showing differential expression (fold change>1.2), we identified 31 different proteins, of which the expression abundance of 22 was increased in G362. These up-regulated proteins in G362 mainly contained DNA starvation/stationary phase protection protein Dps, OmpA, LuxS, ATP-dependent Clp protease ClpC, and ABC transporter substrate-binding proteins, which might be involved in virulence of C. sakazakii. This is the first report to determine the potential virulence factors of C. sakazakii isolates at the proteomic levels.

The driving force of prophages and CRISPR-Cas system in the evolution of Cronobacter sakazakii

Cronobacter sakazakii is an important foodborne pathogens causing rare but life-threatening diseases in neonates and infants. CRISPR-Cas system is a new prokaryotic defense system that provides adaptive immunity against phages, latter play an vital role on the evolution and pathogenicity of host bacteria. In this study, we found that genome sizes of C. sakazakii strains had a significant positive correlation with total genome sizes of prophages. Prophages contributed to 16.57% of the genetic diversity (pan genome) of C. sakazakii, some of which maybe the potential virulence factors. Subtype I-E CRISPR-Cas system and five types of CRISPR arrays were found in the conserved site of C. sakazakii strains. CRISPR1 and CRISPR2 loci with high variable spacers were active and showed potential protection against phage attacks. The number of spacers from two active CRISPR loci in clinical strains was significant less than that of foodborne strains, it maybe a reason why clinical strains were found to have more prophages than foodborne strains. The frequently gain/loss of prophages and spacers in CRISPR loci is likely to drive the quick evolution of C. sakazakii. Our study provides a new insight into the co-evolution of phages and C. sakazakii.

The Membrane Proteins Involved in Virulence of Cronobacter sakazakii Virulent G362 and Attenuated L3101 Isolates

Cronobacter sakazakii is an opportunistic foodborne pathogen and the virulence differences were previously documented. However, information about membranous proteins involved in virulence differences was not available. In this study, virulent characterization such as biofilm formation and flagella motility between virulent C. sakazakii isolate G362 and attenuated L3101 were determined. Then, two-dimensional gel electrophoresis (2-DE) technology was used to preliminarily reveal differential expression of membranous proteins between G362 and L3101. On the mass spectrometry (MS) analysis and MASCOT research results, fourteen proteins with differential expression were successfully identified. At the threshold of twofold changes, five out of eight membranous proteins were up-regulated in G362. Using RT-PCR, the expression abundance of the protein (enzV, ompX, lptE, pstB, and OsmY) genes at mRNA levels was consistent with the results by 2-DE method. The findings presented here provided novel information and valuable knowledge for revealing pathogenic mechanism of C. sakazakii.

Detection and prevalence of pathogenic Yersinia enterocolitica in refrigerated and frozen dairy products by duplex PCR and dot hybridization targeting the virF and ail genes

Pathogenic Yersinia enterocolitica is involved in yersiniosis through expression of chromosome-borne or plasmid-borne virulence factors. Yersinia enterocolitica is a cold-tolerant pathogen frequently isolated from refrigerated or frozen foods. However, little attention has been focused on the prevalence of pathogenic Y. enterocolitica in refrigerated or frozen dairy samples in China. In this study, we developed a new duplex PCR targeting the plasmid-borne virF gene and chromosome-borne ail gene for detection of pathogenic Y. enterocolitica isolates. We established a detection limit for the duplex PCR of 6.5 × 10(2)cfu/mL in artificially contaminated dairy samples. In addition, the duplex PCR could detect directly 4.5 to 5.7 cfu of Y. enterocolitica in 5 mL of brain heart infusion broth after 6 h of enrichment at 28 °C. A newly developed dot hybridization assay further confirmed specificity of the duplex PCR for detection of virulent Y. enterocolitica. Furthermore, 13 Y. enterocolitica and 5 pathogenic strains, from 88 commercial frozen or refrigerated dairy products, were detected successfully by the China National Standard method (GB/T4789.8-2008) and the duplex PCR, respectively. Finally, biotypes and serotypes of pathogenic Y. enterocolitica strains were further characterized. The duplex PCR developed here is reliable for large-scale screening, routine monitoring, and risk assessment of pathogenic Y. enterocolitica in refrigerated or frozen dairy products.

Isolation and phenotypic characterization of Cronobacter from dried edible macrofungi samples.

UNLABELLED Cronobacter is a group of food-borne pathogens including 10 species associated with severe infections in infant by consumption of contaminated powdered infant formula. The information about the prevalence of Cronobacter in dried edible macrofungi samples is also not available. In combination with PCR targeting gluB gene, the traditional ISO method was modified for determining the prevalence of Cronobacter in dried edible macrofungi samples. In addition, the antibiotics susceptibility test, biofilm formation, osmotic, and desiccation resistance of Cronobacter strains were also tested. Results indicated that 18 dried edible macrofungi samples (n = 60) were found to be positive for Cronobacter. All isolated Cronobacter strains were resistant to vancomycin and penicillin G, and sensitive to chloramphenicol, norfloxacin, streptomycin, tetracycline, and cephazolin. The abilities to form biofilm and survive when exposed to osmotic and dry stresses were different. This study contributes to a valid method for detection of Cronobacter and phenotypic characterization of Cronobacter, promoting the necessary measures for control and precaution of Cronobacter in dried edible macrofungi samples. PRACTICAL APPLICATION In this study, the traditional ISO method was modified for detecting successfully Cronobacter in dried edible macrofungi samples. Severe contamination rate of Cronobacter (18/60, 30%) in dried edible macrofungi promotes risk analysis and assessment about Cronobacter. In addition, phenotypic characterization of Cronobacter is also helpful for understanding about control and precaution of Cronobacter in dried edible macrofungi samples.

Detection of Viable Cronobacter spp. (Enterobacter sakazakii) by One-Step RT-PCR in Dry Aquatic Product

UNLABELLED Cronobacter are opportunistic food-borne pathogens associated with meningitis, sepsis, and necrotizing enterocolitis. Little attempt has focused on detection of viable cell of Cronobacter spp. in dry aquatic products, which were frequently used for raw materials of infant foods due to high nutrition. In this paper, one-step reverse transcription polymerase chain reaction (RT-PCR) was developed for detection of viable Cronobacter spp. in dry aquatic products. Specificity test indicated that clearly expected amplicon in size 469 bp was amplified from RNA of Cronobacter, but not from RNA of negative controls and DNA of Cronobacter strains. The sensitivity was 10(4) CFU/mL of Cronobacter strain in artificially fish meal samples and 10(1) CFU/mL of Cronobacter after 10-h enrichment. In a total of 81 dry aquatic products, 9.8%, 8.6%, and 9.8% of samples were found to be positive for Cronobacter by one-step RT-PCR, U.S. Food and Drug Administration method, and Druggan-Forsythe-Iversen medium, respectively. The results clearly indicated that one-step RT-PCR could avoid the interference of residual DNA of Cronobacter in food samples and be used to specifically detect viable Cronobacter spp. for large-scale monitoring of food samples. PRACTICAL APPLICATION The use of rapid and specific detection of food borne pathogens in food samples was most of importance for control and precaution of food borne diseases. In this study, one-step RT-PCR was developed for detection of Cronobacter spp. in aquatic products. A comparison of different methods for detection of Cronobacter indicated that the newly developed method could be widely used to specifically detect Cronobacter spp. in food samples.

Short communication: Effects of vacuum freeze-drying on inactivation of Cronobacter sakazakii ATCC29544 in liquid media with different initial inoculum levels

Vacuum freeze-drying is an important food-processing technology for valid retention of nutrients and bioactive compounds. Cronobacter sakazakii has been reported to be associated with severe infections in neonates through consumption of contaminated powdered infant formula. In this study, effects of vacuum freeze-drying treatment for 12, 24, and 36 h on inactivation of C. sakazakii with different initial inoculum levels in sterile water, tryptic soy broth (TSB), skim milk, and whole milk were determined. Results indicated that the lethality rate of C. sakazakii in each sample increased with the extension of vacuum freeze-drying time. With initial inoculum levels of 102 and 103 cfu/mL, the survival of C. sakazakii in different liquid media was significantly affected by vacuum freeze-drying for 12, 24, and 36 h. In addition, the lethality rates of C. sakazakii in whole milk, skim milk, and TSB was significantly reduced compared with those in sterile water. Furthermore, whole milk showed the strongest protective role for C. sakazakii cells, followed by skim milk and TSB medium. Using the scanning electron microscope, the intracellular damage and obvious distortion of C. sakazakii cells were observed after vacuum freeze-drying for 24 and 36 h compared with the untreated sample, and the injured cells increased with the extension of vacuum-drying time. We concluded that inactivation of vacuum freeze-drying on C. sakazakii cells is related to the food matrix, and a combination with other methods for inactivating C. sakazakii is required for ensuring microbial safety of powdered infant formula.

Genes involved in tolerance to osmotic stress by random mutagenesis in Cronobacter malonaticus

Cronobacter malonaticus is one of the opportunistic food-borne pathogens in powdered infant formula and has unusual abilities to survive under environmental stresses such as osmotic conditions. However, the genes involved in osmotic stress have received little attention in C. malonaticus. Here, genes involved in osmotic stress were determined in C. malonaticus using a transposon mutagenesis approach. According to the growth of mutants (n = 215) under 5.0% NaCl concentration, the survival of 5 mutants under osmotic stress was significantly decreased compared with that of the wild type strain. Five mutating sites, including potassium efflux protein KefA, inner membrane protein YqjF, peptidylprolyl isomerase, Cys-tRNA(Pro)/Cys-tRNA(Cys) deacylase, and oligogalacturonate lyase were successfully identified. In addition, the biofilm formation of 5 mutants was determined using crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy, and the biofilms of 5 mutants significantly decreased within 72 h compared with that of wild type strain. This is the first report to determine the genes involved in osmotic tolerance in C. malonaticus. The findings provided valuable information for deep understanding of the mechanism of survival of C. malonaticus under osmotic stress, and a possible relationship between biofilm formation and tolerance to osmotic stress was also demonstrated in C. malonaticus.

The Glutaredoxin Gene, grxB, Affects Acid Tolerance, Surface Hydrophobicity, Auto-Aggregation, and Biofilm Formation in Cronobacter sakazakii

Cronobacter species are foodborne pathogens that can cause neonatal meningitis, necrotizing enterocolitis, and sepsis; they have unusual abilities to survive in environmental stresses such as acid stress. However, the factors involved in acid stress responses and biofilm formation in Cronobacter species are poorly understood. In this study, we investigated the role of grxB on cellular morphology, acid tolerance, surface hydrophobicity, auto-aggregation (AAg), motility, and biofilm formation in Cronobacter sakazakii. The deletion of grxB decreased resistance to acid stresses, and notably led to weaker surface hydrophobicity, AAg, and biofilm formation under normal and acid stress conditions, compared with those of the wild type strain; however, motility was unaffected. Therefore, grxB appears to contribute to the survival of C. sakazakii in acid stresses and biofilm formation. This is the first report to provide valuable evidence for the role of grxB in acid stress responses and biofilm formation in C. sakazakii.