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A combination method of real‐time PCR and high‐resolution melting (HRM) analysis for the rapid detection and specific classification of six pathogenic Cronobacter species based on gene cgcA was developed. HRM profiles with distinct Tm (melting temperature) peaks were consistently obtained with each species, represented by a single peak ranging from 86.02 to 86.80 °C. The detection limit ranged from 0.1 to 1 pg per reaction. Desiccated Cronobacter sakazakii and Cronobacter muytjensii of 2 cfu/25 g were successfully detected after 4‐week storage at room temperature. The newly developed method provided a molecular tool for direct detection and simultaneous identification of pathogenic Cronobacter species in powdered infant formulae. [ABSTRACT FROM AUTHOR]

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Cronobacter is a group of opportunistic pathogen responsible for necrotizing enterocolitis, meningitis and septicaemia especially to infant and neonate, with high lethality ranging in 40%–80%. Some of them are able to survive in infant milk formula and still possesses capability of pathogenicity and virulence, biofilm formation, and high resistance to elevated osmotic pressure, low pH, heat, oxidation and desiccation. The genus Cronobacter consists of a diverse group of Gram-negative bacteria and comprises seven species: C. sakazakii , C. turicensis , C. malonaticus , C. universails , C. muytjensii , C. condiment and C. dublinensis with the three subspecies dublinensis , lausannensis and lactatidi . Sub-lethal treatments on Cronobacter strains including heat shock, acid shock, ethanol shock and desiccation would enhance its tolerance to adverse environments. Here, we discuss the stress resistance, rapid detection and disinfection methods of Cronobacter spp. in recent literature. [ABSTRACT FROM AUTHOR]

Cronobacter sakazakii is a foodborne pathogen throughout the world and survives extremely desiccation stress. However, the molecular basis involved in desiccation resistance of C. sakazakii is still unknown. In this study, the potential desiccation resistance factors of C. sakazakii ATCC 29544 were determined using iTRAQ-based quantitative proteomic analysis. A total of 2775 proteins were identified by iTRAQ, of which 233 showed a different protein expression between control group and desiccation stress group. Among these 233 proteins identified as desiccation resistance proteins, there were 146 proteins downregulated and 87 proteins upregulated. According to the comprehensive proteome coverage analysis, C. sakazakii increased its resistance to desiccation by reducing the gene involved with unnecessary survival functions such as those used for virulence, adhesion, invasion and flagella assembly, while increasing gene expression of genes used in withstanding osmotic stress such as those genes involved in trehalose and betaine uptake. However, the mechanism involved in amino acid metabolism in an osmotic stress response, including the producing of γ-aminobutyric acid in C. sakazakii is still uncertain. This is the first report to determine the potential desiccation resistant factors of C. sakazakii at the proteomic levels. [ABSTRACT FROM AUTHOR]

[Display omitted] • The Δ sdiA formed more biofilms with extracellular matrix compared to the wild type. • The motility of C. sakazakii was reduced in Δ sdiA compared to the wild type. • C. sakazakii wild type and Δ sdiA formed stronger biofilm in TSB 3% NaCl. • C. sakazakii wild type and Δ sdiA did not grown in TSB 12% NaCl. This study characterizes the impact of sdiA on biofilm formation under normal or osmotic stress conditions in Cronobacter sakazakii by constructing a sdiA deletion mutant (Δ sdiA). Here, the downregulation of flagellar assembly-related genes and upregulation of capsular, cellulose and lipopolysaccharide biosynthesis-associated genes in Δ sdiA were observed when compared to the wild type strain (WT) through transcriptomic analysis. Meanwhile, reduced ability of motility, enhanced cell surface hydrophobicity and stronger biofilms with extracellular matrix were observed in WT with deletion of sdiA. Both WT and Δ sdiA formed more biofilm in low osmotic stress medium, while in hyperosmolarity conditions, formation of biofilm was dramatically reduced. Our findings supported that sdiA might suppress biofilm formation of C. sakazakii by regulating biosynthesis of flagellar and extracellular polymeric substances. This study investigates the role of sdiA on biofilm formation in C. sakazakii , and provides the basis for the inhibition of C. sakazakii in food industry and infant-feeding. [ABSTRACT FROM AUTHOR]

The combined effects of four substances, carvacrol, citral, thymol and caprylic acid, on the growth of Cronobacter sakazakii CICC 21544 were evaluated. Among the six combinations, only the combination of citral and carvacrol demonstrated a synergistic effect. This combination had a stronger growth inhibition effect than treatment with citral or carvacrol alone, which led to more portions of cells in the propidium iodide (PI) fluorescent region and caused more severe cell damage. Citral and carvacrol also showed promising and potent antibiofilm activity against C. sakazakii , which was evaluated using crystal violet assay, field emission scanning electron microscopy (FESEM) and qRT-PCR. Mild heat treatment combined with citral and carvacrol at a concentration of 2 or 3 MIC completely eradicated C. sakazakii in reconstituted infant formula. Moreover, the combination at 2 MIC had no significant effect on the color and aroma scores of reconstituted infant formulae. The combination of citral and carvacrol could potentially be developed as an alternative or supplemental agent to reduce infections caused by C. sakazakii in reconstituted infant formula. • Combination of citral and carvacrol demonstrated a synergistic effect. • Citral and carvacrol showed antibiofilm activity. • Mild heat combined with citral and carvacrol could eradicate C. sakazakii in RIF. [ABSTRACT FROM AUTHOR]

Response mechanism of Cronobacter sakazakii under stress of combination of citral and carvacrol was investigated by transcriptome sequencing. Twenty-five significantly differentially expressed genes were identified to be closely related to the ability of C. sakazakii tolerating the stress after analysis, mainly involving glycerol metabolism; citrate metabolism; formate metabolism; ribosome function and transmembrane transport system. Combination of carvacrol and citral could cause dissipation of proton motive force (PMF) and the psp regulon is an important switch in the response of C. sakazakii to this condition. To maintain PMF, psp regulated cellular processes to reduce the consumption of cellular energy for cell membrane repair and proton consumption. In T1 group (low concentration group), C. sakazakii remained aerobic metabolism and expression of glycerol metabolism related genes were upregulated for cell membrane repair and proton consumption. In T2 group (high concentration group), bacteria convert from aerobic metabolism to anaerobic metabolism to reduce energy consumption and expression of genes in formate metabolism were upregulated to consume excessive intracellular protons; citrate lyase-related genes were upregulated to decompose citrate, the intermediate of the tricarboxylic acid cycle, to generate acetyl CoA for production of cell membrane phospholipids. • Citral and carvacrol caused dissipation of proton motive force of C. sakazakii. • Psp regulon regulated cellular processes to maintain PMF. • Energy preservation, membrane repair and proton consuming helped to maintain PMF. [ABSTRACT FROM AUTHOR]