Revealing the mechanism of citral induced entry of Vibrio vulnificus into viable but not culturable (VBNC) state based on transcriptomics.

Citral has attracted much attention as a safe and effective plant-derived bacteriostatic agent. However, the ability of citral to induce the formation of VBNC state in Vibrio vulnificus has not been evaluated. In the present study, V. vulnificus was shown to be induced to form the VBNC state at 4.5 h and 3 h of citral treatment at 4MIC and 6MIC. Moreover, the citral-induced VBNC state of V. vulnificus maintained some respiratory chain activity and was able to recover well in both APW media, APW media supplemented with 5 % (v/v) Tween 80 and 2 mg/mL sodium pyruvate. Field emission and transmission electron microscopy showed that the external structure of the citral-induced VBNC V. vulnificus cells was shortened to short rods, with folded cell membrane, rough cell surface, and dense cytoplasm and loose nuclear material in the internal cell structure. In addition, the possible molecular mechanisms of citral-induced formation and recovery of V. vulnificus in the VBNC state were explored by transcriptomics. Transcriptome analyses revealed that 1118 genes were significantly altered upon entry into the VBNC state, and 1052 genes were changed after resuscitation. Most of the physiological activities related to energy production were inhibited in the citral-induced VBNC state of V. vulnificus; however, the bacteria retained its pathogenicity. The citral-induced resuscitation of V. vulnificus in the VBNC state selectively restored the activity of some genes related to bacterial growth and reproduction. Meanwhile, the expression levels of other genes may have been influenced by citral-induced resuscitation after the formation of the VBNC state. In conclusion, this study evaluated and analyzed the ability and possible mechanism of citral on the formation of VBNC state and the recovery of VBNC state of V. vulnificus, and made a comprehensive assessment for the safety of citral application in food production.

Antibacterial Mechanism of Shikonin Against Vibrio vulnificus and Its Healing Potential on Infected Mice with Full-Thickness Excised Skin.

Shikonin has anticancer, anti-inflammatory, and wound healing activities. Vibrio vulnificus is an important marine foodborne pathogen with a high fatality rate and rapid pathogenesis that can infect humans through ingestion and wounds. In this study, the antibacterial activity and possible antibacterial mechanism of shikonin against V. vulnificus were investigated. In addition, the ability of shikonin to control V. vulnificus infection in both pathways was assessed by artificially contaminated oysters and full-thickness excised skin-infected mice. Shikonin treatment can cause abnormal cell membrane function, as evidenced by hyperpolarization of the cell membrane, significant decreased intracellular ATP concentration (p < 0.05), significant increased intracellular reactive oxygen species and malondialdehyde content (p < 0.05), decreased cell membrane integrity, and changes in cell morphology. Shikonin at 40 and 80 µg/mL reduced bacterial numbers in shikonin-contaminated oysters by 3.58 and 2.18 log colony-forming unit (CFU)/mL. Shikonin can promote wound healing in mice infected with V. vulnificus by promoting the formation of granulation tissue, hair follicles, and sebaceous glands, promoting epithelial cell regeneration and epidermal growth factor production. These findings suggest that shikonin has a strong inactivation effect on V. vulnificus and can be used in food production and wound healing to effectively control V. vulnificus and reduce the number of diseases associated with it.

Inhibition of Cronobacter sakazakii by Litsea cubeba Essential Oil and the Antibacterial Mechanism.

Litsea cubeba essential oil (LC-EO) has anti-insecticidal, antioxidant, and anticancer proper-ties; however, its antimicrobial activity toward Cronobacter sakazakii has not yet been researched extensively. The objective of this study was to investigate the antimicrobial and antibiofilm effects of LC-EO toward C. sakazakii, along with the underlying mechanisms. The minimum inhibitory concentrations of LC-EO toward eight different C. sakazakii strains ranged from 1.5 to 4.0 µL/mL, and LC-EO exposure showed a longer lag phase and lower specific growth compared to untreated bacteria. LC-EO increased reactive oxygen species production, decreased the integrity of the cell membrane, caused cell membrane depolarization, and decreased the ATP concentration in the cell, showing that LC-EO caused cellular damage associated with membrane permeability. LC-EO induced morphological changes in the cells. LC-EO inhibited C. sakazakii in reconstituted infant milk formula at 50 °C, and showed effective inactivation of C. sakazakii biofilms on stainless steel surfaces. Confocal laser scanning and attenuated total reflection-Fourier-transform infrared spectrometry indicated that the biofilms were disrupted by LC-EO. These findings suggest a potential for applying LC-EO in the prevention and control of C. sakazakii in the dairy industry as a natural antimicrobial and antibiofilm agent.

Didecyldimethylammonium bromide: Application to control biofilms of Staphylococcus aureus and Pseudomonas aeruginosa alone and in combination with slightly acidic electrolyzed water.

This project explored the antibacterial mechanism of didecyldimethylammonium bromide (DDAB) toward Staphylococcus aureus and Pseudomonas aeruginosa, and its removal effect on biofilms. Furthermore, we explored the effect of treatment by DDAB combined with slightly acidic electrolyzed water (SAEW) on biofilms of S. aureus or P. aeruginosa. First, DDAB has bacteriostatic and biofilm removal effects. Second, The effect of DDAB combined with SAEW on biofilm is more obvious than that of the two alone. DDAB at a concentration of 16 MIC combined with SAEW (ACC 30 mg/L, ORP 875 mV, pH 6.30) completely cleared the biofilm. In addition, the results of Confocal laser scanning microscopy (CLSM), Field emission scanning electron microscopy (FESEM) and Attenuated total reflection-Fourier-transform infrared spectroscopy (ATR-FTIR) showed that DDAB combined with SAEW could disrupt the structure of biofilms, reduce polysaccharides, proteins and phospholipids in biofilms. This method has the potential to be used in food production chains to control S. aureus and P. aeruginosa and their biofilms, and it can be used in other industries.

Antibacterial Effect of Oregano Essential Oil against Vibrio vulnificus and Its Mechanism.

Oregano essential oil (OEO) is an effective natural antibacterial agent, but its antibacterial activity against Vibrio vulnificus has not been widely studied. The aim of this study was to investigate the inhibitory effect and germicidal activity of OEO on V. vulnificus and its possible inhibition mechanism. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of OEO against four V. vulnificus strains (ATCC 27562 and three isolates from seafoods) were from 0.06 to 0.15 µL/mL. Compared with untreated bacteria, OEO reduced the concentration of intracellular adenosine triphosphate (ATP), hyperpolarized the cell membrane, increased the level of reactive oxygen species (ROS), and increased the concentration of intracellular malondialdehyde (MDA), but there was no obvious DNA damage at the OEO test concentration. It was indicated that OEO inactivated V. vulnificus by generating ROS which caused lipid peroxidation of cell membranes, thereby reducing the permeability and integrity of cell membranes and causing morphological changes to cells, but there was no obvious damage to DNA. In addition, OEO could effectively kill V. vulnificus in oysters at 25 °C, and the number of bacteria decreased by 48.2% after 0.09% OEO treatment for 10 h. The good inhibitory effect and bactericidal activity of OEO showed in this study, and the economy and security of OEO make it possible to apply OEO to control V. vulnificus contamination in oysters and other seafoods.