Amaranthus tricolor crude extract inhibits Cronobacter sakazakii isolated from powdered infant formula.

The purpose of this study was to elucidate the antibacterial activity and possible mechanism of action of Amaranthus tricolor crude extract (ATCE) against Cronobacter sakazakii isolated from powdered infant formula (PIF). The antibacterial activity of ATCE was assessed by measuring the diameter of inhibition zone (DIZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The possible mechanism of action of ATCE was revealed by analyzing the effects of ATCE on growth curves and changes in cell membrane potential, intracellular pH, content of bacterial protein and genomic DNA, and cell morphology. Finally, ATCE was applied to the disinfection of C. sakazakii in biofilm on stainless steel tube. The results showed that the DIZ, MIC, and MBC of ATCE against C. sakazakii strains were from 14.35 ± 0.67 to 14.84 ± 0.67 mm, 20 mg/mL, and 40 mg/mL, respectively. Treatment with ATCE ended the logarithmic growth phase of C. sakazakii, and led to depolarization of the cell membranes, reducing intracellular pH and bacterial protein and genomic DNA contents, and resulting in cytoplasmic leakage and deformation. In addition, ATCE effectively inactivated C. sakazakii in biofilm, reducing viable bacteria by approximately 6.5 log cfu/mL bacterial count after treatment with 1 MIC (1 MIC = 20 mg/mL) of ATCE for 20 min at 25°C. Our findings showed that ATCE inactivated C. sakazakii strains isolated from PIF and has potential as a natural disinfectant to reduce the contamination of PIF by C. sakazakii.

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.

Antimicrobial Activity of Ferulic Acid Against Cronobacter sakazakii and Possible Mechanism of Action.

Cronobacter sakazakii is an opportunistic pathogen transmitted by food that affects mainly newborns, infants, and immune-compromised adults. In this study, the antibacterial activity of ferulic acid was tested against C. sakazakii strains. Minimum inhibitory concentration of ferulic acid against C. sakazakii strains was determined using the agar dilution method. Changes in intracellular pH, membrane potential and intracellular ATP concentration were measured to elucidate the possible antibacterial mechanism. Moreover, SYTO 9 nucleic acid staining was used to assess the effect of ferulic acid on bacterial membrane integrity. Cell morphology changes were observed under a field emission scanning electron microscope. The minimum inhibitory concentrations of ferulic acid against C. sakazakii strains ranged from 2.5 to 5.0 mg/mL. Addition of ferulic acid exerted an immediate and sustained inhibition of C. sakazakii proliferation. Ferulic acid affected the membrane integrity of C. sakazakii, as evidenced by intracellular ATP concentration decrease. Moreover, reduction of intracellular pH and cell membrane hyperpolarization were detected in C. sakazakii after exposure to ferulic acid. Reduction of green fluorescence indicated the injury of cell membrane. Electronic microscopy confirmed that cell membrane of C. sakazakii was damaged by ferulic acid. Our results demonstrate that ferulic acid has moderate antimicrobial activity against C. sakazakii. It exerts its antimicrobial action partly through causing cell membrane dysfunction and changes in cellular morphology. Considering its antimicrobial properties, together with its well-known nutritional functions, ferulic acid has potential to be developed as a supplement in infant formula or other foods to control C. sakazakii.

Characterization of outer membrane vesicles from a neonatal meningitic strain of Cronobacter sakazakii.

Cronobacter sakazakii is associated with severe and often fatal cases of infant meningitis and necrotizing enterocolitis. The form of meningitis differs from that due to Neisseria meningitidis and Streptococcus spp., in that it is highly invasive and destructive towards human brain cells. However, there is relatively little understanding of the cytopathogenic interaction of C. sakazakii with host cells which results in stimulation of an inflammatory immune response. The production of Cronobacter outer membrane vesicles (OMV) and their potential pathogenic functions have not yet been elucidated. This study is the first to show that C. sakazakii produce OMV, which may play a role in the activation of cytopathogenic and host cell responses on human intestinal epithelial cells. Cronobacter sakazakii strain 767 was used which had been isolated from a fatal outbreak of neonatal meningitis and necrotizing enterocolitis. Cronobacter sakazakii OMV were internalized by Caco-2 cells, increased cell proliferation and stimulated the host's innate proinflammatory response without inducing overt toxicity. A total of 18 OMV-associated proteins were identified by mass spectrometry and their potential pathogenicity roles were evaluated. Collectively, these data indicate that C. sakazakii OMV could play a role in pathogenesis by delivering bacterial toxins into host epithelial cells, driving proliferative and proinflammatory responses.

Investigating the responses of Cronobacter sakazakii to garlic-drived organosulfur compounds: a systematic study of pathogenic-bacterium injury by use of high-throughput whole-transcriptome sequencing and confocal micro-raman spectroscopy.

We present the results of a study using high-throughput whole-transcriptome sequencing (RNA-seq) and vibrational spectroscopy to characterize and fingerprint pathogenic-bacterium injury under conditions of unfavorable stress. Two garlic-derived organosulfur compounds were found to be highly effective antimicrobial compounds against Cronobacter sakazakii, a leading pathogen associated with invasive infection of infants and causing meningitis, necrotizing entercolitis, and bacteremia. RNA-seq shows changes in gene expression patterns and transcriptomic response, while confocal micro-Raman spectroscopy characterizes macromolecular changes in the bacterial cell resulting from this chemical stress. RNA-seq analyses showed that the bacterial response to ajoene differed from the response to diallyl sulfide. Specifically, ajoene caused downregulation of motility-related genes, while diallyl sulfide treatment caused an increased expression of cell wall synthesis genes. Confocal micro-Raman spectroscopy revealed that the two compounds appear to have the same phase I antimicrobial mechanism of binding to thiol-containing proteins/enzymes in bacterial cells generating a disulfide stretching band but different phase II antimicrobial mechanisms, showing alterations in the secondary structures of proteins in two different ways. Diallyl sulfide primarily altered the α-helix and ß-sheet, as reflected in changes in amide I, while ajoene altered the structures containing phenylalanine and tyrosine. Bayesian probability analysis validated the ability of principal component analysis to differentiate treated and control C. sakazakii cells. Scanning electron microscopy confirmed cell injury, showing significant morphological variations in cells following treatments by these two compounds. Findings from this study aid in the development of effective intervention strategies to reduce the risk of C. sakazakii contamination in the food production environment and on food contact surfaces, reducing the risks to susceptible consumers.

Enterobacter sakazakii targets DC-SIGN to induce immunosuppressive responses in dendritic cells by modulating MAPKs.

Enterobacter sakazakii (ES) is an emerging pathogen that causes meningitis and necrotizing enterocolitis in infants. Dendritic cells (DCs) are professional phagocytic cells that play an essential role in host defense against invading pathogens; however, the interaction of ES with DCs is not known. In this study, we demonstrate that ES targets DC-specific ICAM nonintegrin (DC-SIGN) to survive in myeloid DCs for which outer membrane protein A (OmpA) expression in ES is critical, although it is not required for uptake. In addition, DC-SIGN expression was sufficient to cause a significant invasion by ES in HeLa cells and intestinal epithelial cells, which are normally not invaded by ES. OmpA(+) ES prevented the maturation of DCs by triggering the production of high levels of IL-10 and TGF-beta and by suppressing the activation of MAPKs. Pretreatment of DCs with Abs to IL-10 and TGF-beta or of bacteria with anti-OmpA Abs significantly enhanced the maturation markers on DCs. Furthermore, DCs pretreated with various inhibitors of MAPKs prohibited the increased production of proinflammatory cytokines stimulated by LPS or OmpA(-) ES. LPS pretreatment followed by OmpA(+) ES infection of DCs failed to induce maturation of DCs, indicating that OmpA(+) ES renders the cells in immunosuppressive state to external stimuli. Similarly, OmpA(+) ES-infected DCs failed to present Ag to T cells as indicated by the inability of T cells to proliferate in MLR. We conclude that ES interacts with DC-SIGN to subvert the host immune responses by disarming MAPK pathway in DCs.

The effect of temperature and length of heat shock treatment on the thermal tolerance and cell leakage of Cronobacter sakazakii BCRC 13988.

Enterobacter sakazakii is an emerging opportunistic pathogen associated with life-threatening illnesses in infants, with infant formula serving as the principal mode of transmission. In the present study, C. sakazakii (formely E. sakazakii) BCRC 13988 was subjected to various heat shock treatments (42-48 degrees C for 5-15 min). Its subsequent survival at 51 degrees C and the leakage of intracellular materials was investigated. It was found that 47 degrees C was the maximum growth temperature of the test organism. In addition, heat shock enhanced the thermal tolerance of C. sakazakii BCRC 13988. Within heat shock temperatures between 42 and 47 degrees C, the thermal tolerance enhancing effect increased as the length or temperature of the heat shock treatment was increased. However, increasing the heat shock temperature to 48 degrees C reduced the thermal tolerance enhancing effect. Among the various heat shocked cells examined, the 47 degrees C-15 min-heat shocked C. sakazakii exhibited the highest thermal tolerance. Moreover, electron micrograph analysis showed that heat shock treatment caused damage and disruption in C. sakazakii cells. There was a significant increase (P<0.05) in the leakage of nucleic acid and protein in the supernatant of the heat shocked cell suspension that increased as the temperature and duration of heat shock increased.

Inhibitory effect of caprylic acid and mild heat on Cronobacter spp. (Enterobacter sakazakii) in reconstituted infant formula and determination of injury by flow cytometry.

The combined effects of caprylic acid and mild heat were investigated to ascertain their impact on Cronobacter spp. (E. sakazakii) in reconstituted infant formula. Samples containing a mixture of 3 strains of Cronobacter spp. (10(7) to 10(8) CFU/ml) were prepared with various concentrations of caprylic acid (5, 10, 20, and 30 mM) and were then heated to 45, 50, and 55 degrees C. The inhibitory effect of the combined treatment resulted in a synergistic effect, in which Cronobacter spp. numbers were reduced much more rapidly with increased temperatures and concentrations of caprylic acid. When samples were treated with 30 mM caprylic acid, the time required to reduce Cronobacter spp. cell numbers to an approximate reduction of 7.8 log CFU/ml was 60 min at 45 degrees C, 20 min at 50 degrees C, and 10 min at 55 degrees C. In the validation assay using a low population of Cronobacter spp. (approximately 10(3) log CFU/ml), no recovery of injured cells was observed after samples were treated with 10 mM caprylic acid for 20 min at 55 degrees C, 20 mM caprylic acid for 10 min at 50 degrees C and 55 degrees C, and 30 mM caprylic acid for 10 min at 45 degrees C to 55 degrees C. To determine the bactericidal mechanism of caprylic acid, membrane integrity was examined by fluorescent staining followed by flow cytometry and confocal microscopy. Increased cellular inactivation was associated with increased propidium iodide staining, indicating damage to the cell membrane of Cronobacter spp.. Overall, these data indicate that the addition of this natural antimicrobial agent to infant formula may have potential use for controlling microbes prior to consumption at lower heating temperatures. The study also provides a complementary understanding of the mode of action of caprylic acid on Cronobacter spp.

Influences of milk components on biofilm formation of Cronobacter spp. (Enterobacter sakazakii).

AIM: To determine the critical component(s) of skim milk for biofilm formation of Cronobacter species. METHODS AND RESULTS: Biofilm forming ability of 72 Cronobacter strains in skim milk preparation was assayed by crystal violet staining. The results revealed that whey protein and casein are more important determinants of skim milk for biofilm formation than lactose, although there was a wide variation in biofilm forming ability. Biofilm structure and capsular material of six strains exhibiting different biofilm forming ability was investigated via electron microscopes. Scanning electron microscopy showed visually that while the strong biofilm formers (E27B, FSM 30 and 2.82) resulted in almost complete coagulation of skim milk, the weak biofilm formers (55, FSM 290 and 2.84) caused less coagulation. No capsule was clearly delineated in transmission electron micrographs of either strong or weak biofilm formers. CONCLUSION: These results indicate that, for biofilm formation of Cronobacter species in skim milk, nitrogen source is probably a more important determinant than carbohydrate, and that strong biofilm formers are responsible for substantial coagulation of skim milk. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information for better understanding of the underlying mechanisms by which Cronobacter species form biofilm in infant formula milk.