Short communication: Roles of outer membrane protein W on survival, cellular morphology, and biofilm formation of Cronobacter sakazakii in response to oxidative stress.

Cronobacter species are a group of opportunistic food-borne pathogens that cause rare but severe infections in neonates. Tolerance to environmental stress in Cronobacter is known; however, factors involved in oxidative stress are undefined. In this study, Cronobacter sakazakii survival, cellular morphology, and biofilm formation in response to oxidative stress were evaluated between the wild type (WT) and an outer membrane protein W (OmpW) mutant. The survival rates of ΔOmpW strain after treatment with 1.0 and 1.5 mM hydrogen peroxide were significantly reduced compared with those of WT. Morphological changes, including cell membrane damage and cell fragmentation, in ΔOmpW were more predominant than those in WT. By crystal violet staining, we also observed increased biomass in ΔOmpW biofilms as compared with WT following treatment with 0.5 and 1.0 mM H2O2. Biofilms using scanning electron microscopy and confocal laser scanning microscopy further confirmed the structural changes of biofilms between WT and ΔOmpW in response to oxidative stress. The current findings show that OmpW contributed to survival of planktonic cells under oxidative stress and the deletion of OmpW facilitated the biofilm formation in C. sakazakii to adapt to oxidative stress.

Roles of outer membrane protein W (OmpW) on survival, morphology, and biofilm formation under NaCl stresses in Cronobacter sakazakii.

Cronobacter sakazakii is an important foodborne pathogen associated with rare but severe infections through consumption of powdered infant formula. Tolerance to osmotic stress in Cronobacter has been described. However, the detailed factors involved in tolerance to osmotic stress in C. sakazakii are poorly understood. In this study, roles of outer membrane protein W (OmpW) on survival rates, morphologic changes of cells, and biofilm formation in C. sakazakii under different NaCl concentrations between wild type (WT) and OmpW mutant (ΔOmpW) were determined. The survival rates of ΔOmpW in Luria-Bertani medium with 3.5% or 5.5% NaCl were reduced significantly, and morphological injury of ΔOmpW was significantly increased compared with survival and morphology of WT. Compared with biofilm formation of the WT strain, biofilms in ΔOmpW were significantly increased in Luria-Bertani with 3.5% or 5.5% NaCl using crystal violet staining assay after 48 and 72 h of incubation. Detection of biofilms using confocal laser scanning microscopy and scanning electron microscopy further confirmed the changes of biofilm formation under different NaCl stresses. This study demonstrates that OmpW contributes to survival of cells in planktonic mode under NaCl stresses, and biofilm formation is increased in ΔOmpW in response to NaCl stress.

Short communication: Roles of outer membrane protein W (OmpW) on survival and biofilm formation of Cronobacter sakazakii under neomycin sulfate stress.

Cronobacter sakazakii is associated with severe infections including sepsis, neonatal meningitis, and necrotizing enterocolitis. Antibiotic resistance in Cronobacter species has been documented in recent years, but the genes involved in resistance in Cronobacter strains are poorly understood. In this study, we determined the role of outer membrane protein W (OmpW) on survival rates, morphologic changes, and biofilm formation between wild type (WT) and an OmpW mutant strain (ΔOmpW) under neomycin sulfate stress. Results indicated that the survival rates of ΔOmpW were significantly reduced after half minimum inhibitory concentration (½ MIC) treatment compared with the WT strain. Filamentation of C. sakazakii cells was observed after ½ MIC treatment in WT and ΔOmpW, and morphologic injury, including cell disruption and leakage of cells, was more predominant in ΔOmpW. Under ½ MIC stress, the biofilms of WT and ΔOmpW were significantly decreased, but decreasing rates of biofilm formation in mutant strain were more predominant compared with WT strain. This is the first report to determine the role of OmpW on survival, morphological changes, and biofilm formation in C. sakazakii under neomycin sulfate stress. The findings indicated that OmpW contributed to survival and reduction of morphological injury under neomycin sulfate stress. In addition, enhancing biofilm formation in ΔOmpW may be an alternative advantage for adaptation to neomycin sulfate stress.

Potential factors involved in virulence of Cronobacter sakazakii isolates by comparative transcriptome analysis.

Cronobacter species are important foodborne pathogens causing severe infections in neonates through consumption of contaminated powdered infant formula. However, the virulence-associated factors in Cronobacter are largely unknown. In this study, the transcriptome analysis between highly virulent Cronobacter sakazakii G362 and attenuated L3101 strains was used to reveal the potential factors involved in virulence. The total transcripts were grouped into 20 clusters of orthologous group categories and summarized in 3 gene ontology categories (biological process, cellular component, and molecular function). In addition, the differentially expressed genes (DEG) between these isolates were analyzed using Volcano plots and gene ontology enrichment. The predominant DEG were flagella-associated genes such as flhD, motA, flgM, flgB, and fliC. Furthermore, the expression abundance of outer membrane protein or lipoprotein genes (ompW, slyB, blc, tolC, and lolA), potential virulence-related factors (hlyIII and hha), and regulation factors (sdiA, cheY, Bss, fliZ) was also significantly different between G362 and L3101. Interestingly, 3 hypothetical protein genes (ESA_01022, ESA_01609, and ESA_00609) were found to be expressed only in G362. Our findings provide valuable transcriptomic information about potential virulence factor genes, which will be needed in future molecular biology studies designed to understand the pathogenic mechanism of Cronobacter.

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.

Short communication: Effects of high-pressure processing on the inactivity of Cronobacter sakazakii in whole milk and skim milk samples.

Powdered infant formula is considered as the main transmission vehicle for Cronobacter sakazakii infections including meningitis, septicemia, and necrotizing enterocolitis. The effects of high-pressure processing treatment on inactivation of C. sakazakii ranging from 100 to 400 MPa for 3.0, 5.0, and 7.0 min in whole milk and skim milk were studied. Significant differences in inactivation of C. sakazakii were observed in milk samples under different pressures for 3 to 7 min compared with untreated samples, and C. sakazakii was not detected after 400 MPa for 3 min. The lethality rates of C. sakazakii cells in whole and skim milk with an initial level of 10(4) cfu/mL after 100 and 200 MPa treatments were not significantly different, but relatively higher lethality rates were found in whole milk after 300 MPa treatment than in skim milk. Finally, the scanning electron micrographs indicated that cellular envelope and intracellular damage of C. sakazakii cells were apparent after 300 and 400 MPa for 5.0 min compared with the untreated cells, and a progressive increase of injured cells with increased pressure treatment was found. It was concluded that C. sakazakii was sensitive to high-pressure processing treatment and that high-pressure processing treatment with 400 MPa for 3.0 min can be used to control C. sakazakii contamination in milk samples.

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 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.