Chemotaxis and Shorter O-Antigen Chain Length Contribute to the Strong Desiccation Tolerance of a Food-Isolated Cronobacter sakazakii Strain

Cronobacter sakazakii is an opportunistic pathogen causing a lethality rate as high as 80% in infants. Desiccation tolerance ensures its survival in powdered infant formula (PIF) and contributes to the increased exposure to neonates, resulting in neonatal meningitis, septicemia, and necrotizing enterocolitis. This study showed that a food-isolated C. sakazakii G4023 strain exhibited a stronger desiccation tolerance than C. sakazakii ATCC 29544 strain. Considering the proven pathogenicity of G4023, it could be a big threat to infants. Transcriptome and proteome were performed to provide new insights into the desiccation adaptation mechanisms of G4023. Integrated analyses of these omics suggested that 331 genes were found regulated at both transcriptional and protein levels (≥2.0- and ≥1.5-fold, respectively). Deletion of chemotaxis system encoded genes cheA and cheW resulted in decreased tolerance in both short- and long-term desiccation. Reduced O-antigen chain length contributed to the biofilm formation and desiccation tolerance in the short term rather than the long term. In addition, biosynthesis of flagella, arginine and its transport system, and Fe/S cluster were also observed regulated in desiccated G4023. A better understanding of desiccation adaptation mechanisms of G4023 could in turn guide the operations during production and preservation of PIF or other food to reduce survival odds of G4023 and lower its exposure to get to infants.

Biomedical Applications Of Novel Green Biomaterial Synthesized From Endophytic Bacteria Cronobacter Sakazakii

Endophytic bacteria from the flower of Nyctanthes arbor-tristis was isolated and identified as Cronobacter sakazakii. The supernatant of the biomass of this endophyte was utilized for biomaterial synthesis which was confirmed by a change in colour and characterized by using UV-Visible spectrophotometer, FTIR, FESEM, EDAX, Zeta potential and DLS. The antibacterial efficacy of the biomaterial against Gram positive and Gram negative bacteria was determined via agar well diffusion, MIC, MBC and biofilm formation assays. The antioxidant and anti-arthritic assays were also performed. The result reveals antibacterial effect of biomaterial against the multi-drug-resistant E. coli, S. aureus and K. pneumonia strains. This report is the first to reveal the isolation and identification of endophytic bacteria from the flower of Nyctanthes arbor-tristis, alongside its biomaterial synthesis, characterization and biomedical applications. Our result implies that the synthesized biomaterial can be utilized as an effective antimicrobial, antioxidant and anti-arthritic agent and can be incorporated in pharmaceutical formulation for drug development.

Why Does Cronobacter sakazakii Survive for a Long Time in Dry Environments? Contribution of the Glass Transition of Dried Bacterial Cells

The mechanical glass transition temperature ( T g ) of dried Cronobacter sakazakii cells varied depending on differences in drying methods and water activity (a w ) levels. Because the T g of the dried bacterial cells varied depending on the drying method and a w , the T g will play an important role as an operational factor in the optimization of dry food processing for controlling microbial contamination in the future.

Efficacy of slightly acidic electrolyzed water on inactivation of Cronobacter sakazakii and biofilm cell

The disinfection efficacy and mechanism of slightly acid electrolyzed water (SAEW) on Cronobacter sakazakii were investigated. SAEW solutions in three concentrations were carried on C. sakazakii which decreased in a range of 23%-55% in 2 minutes. The propidium iodide (PI) uptake and electronic microscopy (SEM) images indicated that SAEW treatment damaged cell integrity and changed membrane permeability with leaking nucleic acid (109.7%), intercellular protein (692.3%) and K + (53.6%). It was accompanied with lower ability of biofilm formation. SAEW treatment reduced the activity of SOD and CAT from 100.73 U/mgprot and 114.18 U/mgprot to 50.03 U/mgprot and 50.13 U/mgprot, respectively. It lowered down the gene expression of response regulator (katG, rpoS, phoP, glpK,dacC and CSK29544_RS05515 ) which made C. sakazakii failed to repair osmotic stress-induced damage and inhibited their biofilm formation. These findings provide an understanding of associations between bacterial genotype and phenotype induced by SAEW.

Outer Membrane Protein F Is Involved in Biofilm Formation, Virulence and Antibiotic Resistance in Cronobacter sakazakii

In some Gram-negative bacteria, ompF encodes outer membrane protein F (OmpF), which is a cation-selective porin and is responsible for the passive transport of small molecules across the outer membrane. However, there are few reports about the functions of this gene in Cronobacter sakazakii. To investigate the role of ompF in detail, an ompF disruption strain (ΔompF) and a complementation strain (cpompF) were successfully obtained. We find that OmpF can affect the ability of biofilm formation in C. sakazakii. In addition, the variations in biofilm composition of C. sakazakii were examined using Raman spectroscopy analyses caused by knocking out ompF, and the result indicated that the levels of certain biofilm components, including lipopolysaccharide (LPS), were significantly decreased in the mutant (ΔompF). Then, SDS-PAGE was used to further analyze the LPS content, and the result showed that the LPS levels were significantly reduced in the absence of ompF. Therefore, we conclude that OmpF affects biofilm formation in C. sakazakii by reducing the amount of LPS. Furthermore, the ΔompF mutant showed decreased (2.7-fold) adhesion to and invasion of HCT-8 cells. In an antibiotic susceptibility analysis, the ΔompF mutant showed significantly smaller inhibition zones than the WT, indicating that OmpF had a positive effect on the influx of antibiotics into the cells. In summary, ompF plays a positive regulatory role in the biofilm formation and adhesion/invasion, which is achieved by regulating the amount of LPS, but is a negative regulator of antibiotic resistance in C. sakazakii.

Alterations in the transcriptional landscape allows differential desiccation tolerance in different strains of Cronobacter sakazakii

Cronobacter sakazakii is a typical example of a xerotolerant bacterium. It is epidemiologically linked to low moisture foods like powdered infant formula (PIF) and is associated with high fatality rates among neonates. We characterized the xerotolerance in a clinically isolated strain, C. sakazakii ATCC™29544 T , and compared the desiccation tolerance with an environmental strain, C. sakazakii SP291, whose desiccation tolerance was previously characterized. We found that, although the clinical strain was desiccation-tolerant, the level of tolerance was compromised when compared to the environmental strain. RNA-seq based deep transcriptomic characterization identified a unique transcriptional profile in the clinical strain compared to what was already known for the environmental strain. As RNA-seq was also carried out in different TSB growth conditions, genes that were expressed specifically under desiccated conditions were identified and denoted as desiccation responsive genes (DRGs). Interestingly, these DRGs included transcriptomic factors like fnr , ramA, and genes associated with inositol metabolism, a phenotype as yet unreported in C. sakazakii . Further, the clinical strain did not express the proP gene, which was previously reported to be very important for desiccation survival and persistence. Interestingly, analysis of the plasmid genes showed that the iron metabolism in desiccated C. sakazakii ATCC™29544 T cells specifically involved the siderophore cronobactin encoded by the iucABCD genes. Confirmatory studies using qRT-PCR determined that, though the secondary desiccation response genes were upregulated in C. sakazakii ATCC™29544 T , the level of up-regulation was lower compared to that in C. sakazakii SP291. All these factors could collectively contribute to the compromised desiccation tolerance in the clinical strain. IMPORTANCE Cronobacter sakazakii has in past led to outbreaks, particularly associated with food that are low in moisture content. This species has adapted to survive in low water conditions and can survive in such environments for long periods. These characteristics have enabled the pathogen to contaminate powder infant formula, a food matrix with which the pathogen has been epidemiologically associated. Even though clinically adapted strains can also be isolated, there is no information on how the clinical strains adapt to low moisture environments. Our research assessed the adaptation of a clinically isolated strain to low moisture survival on sterile stainless steel coupons and compared the survival to a highly desiccation-tolerant environmental strain. We found that, even though the clinical strain is desiccation-tolerant, the rate of tolerance was compromised compared to the environmental strain. A deeper investigation using RNA-seq identified that the clinical strain used pathways different from that of the environmental strain to adapt to low moisture conditions. This shows that the adaptation to desiccation conditions, at least for C. sakazakii , is strain-specific and that different strains have used different evolutionary strategies for adaptation.