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.

Outer membrane defect and stronger biofilm formation caused by inactivation of a gene encoding for heptosyltransferase I in Cronobacter sakazakii ATCC BAA-894.

AIMS: To investigate the role of lipopolysaccharide (LPS) structure in the stability of outer membrane and the ability of biofilm formation in Cronobacter sakazakii. METHODS AND RESULTS: A C. sakazakii mutant strain LWW02 was constructed by inactivating the gene ESA_04107 encoding for heptosyltransferase I. LPS were purified from LWW02, and changes in their structure were confirmed by thin-layer chromatography and electrospray ionization mass spectrometry. Comparing with the wild-type strain BAA-894, slower growth, higher membrane permeability, higher surface hydrophobicity, stronger ability of autoaggregation and biofilm formation were observed for the mutant strain LWW02. CONCLUSIONS: The gene ESA_04107 encodes heptosyltransferase I in C. sakazakii ATCC BAA-894. The cleavage of LPS in C. sakazakii could cause its outer membrane defects and increase its ability to form biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The study is important for understanding the pathogenic mechanism and efficient control of C. sakazakii.

Environmental factors influencing the inactivation of Cronobacter sakazakii by high hydrostatic pressure.

The effect of High Hydrostatic Pressure (HHP) on the survival of Cronobacter sakazakii was investigated. Deviations from linearity were found on the survival curves and the Mafart equation accurately described the kinetics of inactivation. Comparisons between strains and treatments were made based on the time needed for a 5-log(10) reduction in viable count. The ability of C. sakazakii to tolerate high pressure was strain-dependent with a 26-fold difference in resistance among four strains tested. Pressure resistance was greatest in the stationary growth phase and at the highest growth temperatures tested (30 and 37 °C). Cells treated in neutral pH buffer were 5-fold more resistant than those treated at pH 4.0, and 8-fold more sensitive than those treated in buffer with sucrose added (a(w)=0.98). Pressure resistance data obtained in buffer at the appropriate pH adequately estimated the resistance of C. sakazakii in chicken and vegetables soups. In contrast, a significant protective effect against high pressure was conferred by rehydrated powdered milk. As expected, treatment efficacy improved as pressure increased. z values of 112, 136 and 156 MPa were obtained for pH 4.0, pH 7.0 and a(w)=0.98 buffers, respectively. Cells with sublethal injury to their outer and cytoplasmic membranes were detected after HHP under all the conditions tested. The lower resistance of C. sakazakii cells when treated in media of pH 4.0 seemed to be due to a decreased barostability of the bacterial envelopes. Conversely, the higher resistance displayed in media of reduced water activity may relate to a higher stability of bacterial envelopes.

Evaluation of a chromogenic medium supplemented with glucose for detecting Enterobacter sakazakii.

A commercial chromogenic agar medium (DFI) was supplemented with glucose (mDFI) to enhance the specificity of Enterobacter sakazakii (E. sakazakii) detection. Escherichia vulneris (E. vulneris), a putative false-positive strain on the DFI medium, produces alpha-glucosidase. The enzyme alpha- glucosidase hydrolyzes a substrate, 5-bromo-4-chloro-3- indolyl-alpha,D-glucopyranoside (XalphaGlc), producing green colonies. E. sakazakii strains produced green colonies on both DFI and mDFI agar, whereas E. vulneris produced green colonies on DFI agar but small white colonies on mDFI agar. E. sakazakii and E. vulneris were also readily differentiated by colony color when the mixed culture of the two strains was plated on mDFI agar and incubated for 24 h at 37 degrees C. The results indicate that the selectivity of the commercial chromogenic agar medium could be improved by a simple supplementation with glucose.