ATP Leakage Research Articles

Synergistic inactivation effect and mechanism of ultrasound combined with Zanthoxylum schinifolium essential oil nanoemulsions against Escherichia coli O157:H7 and its application on fresh-cut cucumber

Fresh-cut produce is often contaminated with Escherichia coli O157, posing a significant health risk. This study investigated the synergistic inactivation effect and underlying mechanism of ultrasound (US) combined with Zanthoxylum schinifolium essential oil nanoemulsion (ZEO-NE) against E. coli O157:H7, and explored its potential application in fresh-cut cucumbers. The results demonstrated that combined treatment of US and ZEO-NE significantly reduced the viable counts of E. coli O157:H7 compared to treatment with US or ZEO-NE alone. In particular, US (20 kHz, 345 W/cm2) + ZEO-NE (1.0 mg/mL) treatment for 12 min achieved an 8.91 log colony-forming units (CFU)/mL reduction in viable counts of E. coli O157:H7. Moreover, biofilm formation assay revealed that US+EO-NE treatment significantly reduced mobility, altered surface properties, and inhibited biofilm formation of E. coli O157:H7 cells compared to single treatments. The synergistic inactivation mechanism of US+ZEO-NE treatment against E. coli O157:H7 was revealed to involve increased cell membrane permeability, resulting in leakage of nucleic acids, proteins, and LDH, AKP, and ATP, disruption of cell membrane integrity, and alterations in membrane potential. Additionally, the US+ZEO-NE treatment induced reactive oxygen species accumulation and disrupted cell morphology, ultimately leading to E. coli O157:H7 cell death. Furthermore, the US+EO-NE treatment significantly reduced the E. coli O15:H7 counts in fresh-cut cucumbers compared to single treatments, without adversely affecting the quality and sensory properties of the produce during storage at 4 °C for 12 days. Overall, these findings suggest that US+EO-NE is a promising technique for bacterial inactivation, and holds potential for improving microbial safety in fresh-cut produce.

A systematic evaluation of quenching, extraction and analysis procedures for metabolomics study of the mechanism of QYSLD intervention in A549 cells.

The preparation of cellular metabolomics samples and how to achieve comprehensive coverage of different polar metabolites in cell samples in the analysis pose a challenge for cellular metabolomics. In this study, we optimized a metabolomics protocol based on ultra-high-performance liquid chromatography high-resolution mass spectrometry (UPLC/HRMS) for theextraction and detection of metabolites in A549 cells and exploration of the intervention effect of Qi-Yu-San-Long decoction (QYSLD) on A549 cells. The results indicate that the lowest level of ATP leakage was observed when A549 cells were quenched under liquid nitrogen. MeOH/chloroform/H2O (1:2:1) extraction yielded more chromatographic peaks and excellent reproducibility, and the relative extraction efficiency of most target metabolites was also high. And we optimized thechromatographic separation conditions in both HILIC and RPLC modes, enabling comprehensive detection and analysis of metabolites with varying polarities. Then, we applied the optimized method to UPLC-Q-TOF/MS-based metabolomics of A549 cells to study the mechanism of QYSLD intervention in non-small cell lung cancer (NSCLC). The CCK-8, EdU staining, and cell cycle assay showed that QYSLD inhibited the proliferation of A549 cells by interfering with the cell cycle and blocking them in theG1 phase. A total of 36 differential metabolites associated with the antitumor effects of QYSLD on NSCLC were identified, mainly involving nicotinate and nicotinamide metabolism, sphingolipid metabolism, and glycerophospholipid metabolism. And western blotting confirmed that thechange in 1-methylnicotinamide levels after QYSLD intervention was associated with theinhibition of nicotinamide N-methyltransferase expression in A549 cells.

Exploring the Membrane-Active Interactions of Antimicrobial Long-Chain Fatty Acids Using a Supported Lipid Bilayer Model for Gram-Positive Bacterial Membranes.

The dynamic nature of bacterial lipid membranes significantly impacts the efficacy of antimicrobial therapies. However, traditional assay methods often fall short in replicating the complexity of these membranes, necessitating innovative approaches. Herein, we successfully fabricated model bacterially supported lipid bilayers (SLBs) that closely mimic the characteristics of Gram-positive bacteria using the solvent-assisted lipid bilayer (SALB) technique. By employing a quartz crystal microbalance with dissipation and fluorescence microscopy, we investigated the interactions between these bacterial mimetic membranes and long-chain unsaturated fatty acids. Specifically, linolenic acid (LNA) and linoleic acid (LLA) demonstrated interaction behaviors correlated with the critical micelle concentration (CMC) on Gram-positive membranes, resulting in membrane remodeling and removal at concentrations above their respective CMC values. In contrast, oleic acid (OA), while showing similar membrane remodeling patterns to LNA and LLA, exhibited membrane insertion and CMC-independent activity on the Gram-positive membranes. Particularly, LNA and LLA demonstrated bactericidal effects and promoted membrane permeability and ATP leakage in the bacterial membranes. OA, characterized by a CMC-independent activity profile, exhibited potent bactericidal effects due to its robust penetration into the SLBs, also enhancing membrane permeability and ATP leakage. These findings shed light on the intricate molecular mechanisms governing the interactions between long-chain unsaturated fatty acids and bacterial membranes. Importantly, this study underscores the potential of using biologically relevant model bacterial membrane systems to develop innovative strategies for combating bacterial infections and designing effective therapeutic agents.

Biological Activity of Hybrid Vaterite-Pectin Microparticles Towards Bacteria E. coli and Human Neutrophils.

Interaction of microbiota with hybrid vaterite-pectin microparticles as an attractive multifunctional vehicle for mucosal delivery should not provoke inflammation. Our purpose was to study the reaction of bacteria E. coli strain Mg1655 and isolate SharL from a patient with Crohn disease on the cultivation with hybrid microparticles and vaterite, and the subsequent activation of neutrophils. Vaterite-pectin microparticles enhanced leakage of ATP from bacteria. For E. coli Mg1655, the concentration of DNA decreased, while intracellular ATP increased. For E. coli SharL, the intracellular ATP decreased with simultaneous growth of DNA. Bacteria and microparticles together did not enhance activation of neutrophils in comparison with the particles per se in the medium without serum and in comparison with bacteria in the medium supplemented with serum; microparticles did not reduce functional activity of neutrophils.

Self-assembly of sophorolipid and eugenol into stable nanoemulsions for synergetic antibacterial properties through alerting membrane integrity

Exploring the natural, safe, and effective antimicrobial is one of the preferable ways to control foodborne bacteria. In this work, novel oil-in-water nanoemulsions were formulated with sophorolipids and eugenol without any co-surfactant using a self-assembling strategy. These nanoemulsions showed high stability with sizes less than 200 nm when exposure to low concentrations of salt ions, various pH values (5.0, 7.0, 10.0), storage temperature and time. The synergistic antibacterial effects against both Gram-negative Escherichia coli and Gram-positive Bacillus cereus were determined with a minimum inhibitory concentration (MIC) value of 0.5 mg/mL and 0.125 mg/mL, respectively. Further microscopy (SEM, TEM, LCSM) examination and ATP/Na+-K+-ATPase assay results showed that the morphological changes, intensive cell membrane permeability, leakage of ATP, and decreased Na+-K+-ATPase contributed to the antibacterial effects. Moreover, the bonding mechanism between nanoemulsions and cell membranes were further evaluated by FTIR and ITC using a DPPC vesicle model, which demonstrated that the nanoemulsions adsorbed on the surface of bilayer, interacted with the hydrophobic chains of DPPC membrane mainly through the hydrophobic interaction, and altered the structural integrity of the lipid bilayer. These results not only provide a facile green strategy for fabricating stable nanoemulsions, but also highlight a new perspective for stabilizing essential oils for their widely application in food industry.

Combination treatment of slightly acidic electrolyzed water with electron beam irradiation to mono- and mixed biofilms of Vibrio parahaemolyticus and Pseudomonas fluorescens

Effective antimicrobial measures are needed to control cross-contamination and rapid spoilage of food caused by Vibrio parahaemolyticus (V. parahaemolyticus) and Pseudomonas fluorescens (P. fluorescens). This study evaluated the combined effect of electron beam irradiation (EBI) combined with slightly acidic electrolytic water (SAEW) treatment on suspended bacteria and single and dual-species biofilms of V. parahaemolyticus and P. fluorescens. Colony counting results showed that EBI combined with SAEW treatment effectively removed biofilm cells. Confocal laser scanning microscopy and ATP leakage analysis showed that EBI combined with SAEW treatment damaged the membrane integrity of biofilm cells and caused ATP leakage. Field emission scanning electron microscopy (SEM) observations showed that EBI with or without SAEW significantly disrupted the three-dimensional reticular structure of mono/co-cultured biofilm (extracellular polymeric substances) EPS, producing reticular perforation damage. Combined treatment with 60 mg/L SAEW and 0.5 kGy (kGy) EBI caused serious damage to the three-dimensional structure of the biofilms. The detection results of biofilm protein and DNA showed that EBI combined with SAEW could effectively destroy the extracellular polymeric matrix of biofilm, but the primary structure of the protein had no significant effect. These results suggest that SAEW(60 mg/L) combined with EBI(0.5 kGy) is effective in reducing microbial and biofilm attachment during the large yellow croaker storage processing.

Ligilactobacillus salivarius 7247 Strain: Probiotic Properties and Anti-Salmonella Effect with Prebiotics.

The Ligilactobacillus salivarius 7247 (LS7247) strain, originally isolated from a healthy woman's intestines and reproductive system, has been studied for its probiotic potential, particularly against Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) as well as its potential use in synbiotics. LS7247 showed high tolerance to gastric and intestinal stress and effectively adhered to human and animal enterocyte monolayers, essential for realizing its probiotic properties. LS7247 showed high anti-Salmonella activity. Additionally, the cell-free culture supernatant (CFS) of LS7247 exhibited anti-Salmonella activity, with a partial reduction upon neutralization with NaOH (p < 0.05), suggesting the presence of anti-Salmonella factors such as lactic acid (LA) and bacteriocins. LS7247 produced a high concentration of LA, reaching 124.0 ± 2.5 mM after 48 h of cultivation. Unique gene clusters in the genome of LS7247 contribute to the production of Enterolysin A and metalloendopeptidase. Notably, LS7247 carries a plasmid with a gene cluster identical to human intestinal strain L. salivarius UCC118, responsible for class IIb bacteriocin synthesis, and a gene cluster identical to porcine strain L. salivarius P1ACE3, responsible for nisin S synthesis. Co-cultivation of LS7247 with SE and ST pathogens reduced their viability by 1.0-1.5 log, attributed to cell wall damage and ATP leakage caused by the CFS. For the first time, the CFS of LS7247 has been shown to inhibit adhesion of SE and ST to human and animal enterocytes (p < 0.01). The combination of Actigen prebiotic and the CFS of LS7247 demonstrated a significant combined effect in inhibiting the adhesion of SE and ST to human and animal enterocytes (p < 0.001). These findings highlight the potential of using the LS7247 as a preventive strategy and employing probiotics and synbiotics to combat the prevalence of salmonellosis in animals and humans caused by multidrug resistant (MDR) strains of SE and ST pathogens.

Exploring the potential of 1,8-cineole from cardamom oil against food-borne pathogens: Antibacterial mechanisms and its application in meat preservation

Food-borne pathogenic bacteria are a major public health concern globally. Traditional control methods using antibiotics have limitations, leading to the exploration of alternative strategies. Essential oils such as cardamom possess antimicrobial properties and have shown efficacy against food-borne pathogenic bacteria. The utilization of essential oils and their bioactive constituents in food preservation is a viable strategy to prolong the shelf-life of food products while ensuring their quality and safety. To the best of our knowledge, there are no studies that have utilized 1,8-cineole (the main active constituent of cardamom essential oil) as a preservative in meat, so this study might be the first to utilize 1,8-cineole as an antibacterial agent in meat preservation. The application of 1,8-cineole had a significant suppressive impact on the growth rate of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonella Typhimurium in meat samples stored for 7 days at 4 °C. Additionally, the surface color of the meat samples was not negatively impacted by the application of 1,8-cineole. The minimum inhibitory concentration was 12.5–25 mg/ml, and the minimum bactericidal concentration was 25–50.0 mg/ml. The bacterial cell membrane may be the target of cardamom, causing leakage of intracellular proteins, ATP, and DNA. The obtained data in this study may pave a new avenue for using 1,8-cineole as a new perspective for dealing with this problem of food-borne pathogens and food preservation, such as meat.

Antibacterial activity and mechanisms of α-terpineol against foodborne pathogenic bacteria.

This study aimed to evaluate the antibacterial activities of α-terpineol against common foodborne pathogenic bacteria by agar well diffusion, broth microdilution, and colony counting assay. Propulsive research was conducted to reveal the antibacterial mechanisms, including morphology, infrared spectroscopy, membrane fluidity, membrane permeability, proton motive force, and oxidative phosphorylation. Results indicated that the antibacterial activity of α-terpineol decreased in the following order: Escherichia coli O157:H7, Salmonella typhimurium, Listeria monocytogenes, and Staphylococcus aureus. With an initial cell count of 8 log CFU/mL, α-terpineol at 0.8% (v/v) reduced E. coli O157:H7 and S. aureus by approximately 5.6 and 3.9 log CFU/mL within 1 h, respectively. Remarkable destruction in cell envelopes and intracellular organizations was observed. The hydroxyl of α-terpineol might form glycosidic bonds with carbohydrates and hydrogen bonds with PO2- and COO- via infrared spectroscopy analysis. Generalized polarization of Laurdan revealed that the polar head groups of phospholipids transformed into close packed. The anisotropy variations of trimethyl amino-diphenylhexatriene (TMA-DPH) and DPH suggested membrane fluidity decreased. The N-phenyl-1-naphthylamine intake assay indicated that α-terpineol impairedthe cell wall. Propidium iodide staining was indicative of damaged plasma membranes. Electron transport in the cytoplasmic membrane was impaired, inducing reactive oxygen species accumulation. Both membrane electrical potential and membrane pH gradient collapsed. The disruption of proton motive force and the leakage of ATP resulted in a deficit of intracellular ATP. Our research revealed the interaction between the hydroxyl group of α-terpineol and bacteria affects membrane function contributing to the bacteria's death. KEY POINTS: • α-Terpineol hydroxy formed glycosidic bonds and hydrogen bonds with bacteria • α-Terpineol increased the membrane gelation and reduced the membrane fluidity • Proton motive force and oxidative phosphorylation were impaired.

Fabrication of Co-Assembly from Berberine and Tannic Acid for Multidrug-Resistant Bacteria Infection Treatment.

Long-term antibiotic use induces drug resistance in bacteria. This has given rise to the challenge of refractory infections, which have become a global health threat. Berberine (BBR) and tannic acid (TA) from plants exhibit promising antibacterial activities and may overcome antibiotic resistance. However, poor solubility and/or low penetration capability have limited their application. Carrier-free co-assembled nanocomposites composed entirely of BBR and TA exhibit improved or new properties and produce improved efficacy. Herein, we demonstrated that an ordered nanostructure could be spontaneously co-assembled by the solvent evaporation method using the two natural products. These co-assembled berberine-tannic acid nanoparticles (BBR-TA NPs) exhibited the best antibacterial effect compared with the corresponding physical mixture, pristine BBR, and some first-line antibiotics (benzylpenicillin potassium-BP and ciprofloxacin-Cip) against Staphylococcus aureus (S. aureus) and multidrug-resistant Staphylococcus aureus (MRSA). Even if the concentration of BBR-TA NPs was as low as 15.63 μg/mL, the antibacterial rate against S. aureus and MRSA was more than 80%. In addition to the synergistic effect of the two compounds, the antibacterial mechanism underlying the nanostructures was that they strongly adhered to the surface of the bacterial cell wall, thereby inducing cell membrane damage and intracellular ATP leakage. Furthermore, the in vivo wound healing effect of BBR-TA NPs was verified using an MRSA wound infection mouse model. The BBR-TA NPs achieved the best efficacy compared with BP and Cip. Moreover, cytotoxic and histopathological evaluations of mice revealed that the nanodrug had good biological safety. This facile and green co-assembly strategy for preparing nanoparticles provides a feasible reference for the clinical treatment of bacterial infection.

Cold atmospheric pressure plasma-antibiotic synergy in Pseudomonas aeruginosa biofilms is mediated via oxidative stress response

Cold atmospheric-pressure plasma (CAP) has emerged as a potential alternative or adjuvant to conventional antibiotics for the treatment of bacterial infections, including those caused by antibiotic-resistant pathogens. The potential of sub-lethal CAP exposures to synergise conventional antimicrobials for the eradication of Pseudomonas aeruginosa biofilms is investigated in this study. The efficacy of antimicrobials following or in the absence of sub-lethal CAP pre-treatment in P. aeruginosa biofilms was assessed. CAP pre-treatment resulted in an increase in both planktonic and biofilm antimicrobial sensitivity for all three strains tested (PAO1, PA14, and PA10548), with both minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs) of individual antimicrobials, being significantly reduced following CAP pre-treatment of the biofilm (512-fold reduction with ciprofloxacin/gentamicin; and a 256-fold reduction with tobramycin). At all concentrations of antimicrobial used, the combination of sub-lethal CAP exposure and antimicrobials was effective at increasing time-to-peak metabolism, as measured by isothermal microcalorimetry, again indicating enhanced susceptibility. CAP is known to damage bacterial cell membranes and DNA by causing oxidative stress through the in situ generation of reactive oxygen and nitrogen species (RONS). While the exact mechanism is not clear, oxidative stress on outer membrane proteins is thought to damage/perturb cell membranes, confirmed by ATP and LDH leakage, allowing antimicrobials to penetrate the bacterial cell more effectively, thus increasing bacterial susceptibility. Transcriptomic analysis, reveals that cold-plasma mediated oxidative stress caused upregulation of P. aeruginosa superoxide dismutase, cbb3 oxidases, catalases, and peroxidases, and upregulation in denitrification genes, suggesting that P. aeruginosa uses these enzymes to degrade RONS and mitigate the effects of cold plasma mediated oxidative stress. CAP treatment also led to an increased production of the signalling molecule ppGpp in P. aeruginosa, indicative of a stringent response being established. Although we did not directly measure persister cell formation, this stringent response may potentially be associated with the formation of persister cells in biofilm cultures. The production of ppGpp and polyphosphate may be associated with protein synthesis inhibition and increase efflux pump activity, factors which can result in antimicrobial tolerance. The transcriptomic analysis also showed that by 6 h post-treatment, there was downregulation in ribosome modulation factor, which is involved in the formation of persister cells, suggesting that the cells had begun to resuscitate/recover. In addition, CAP treatment at 4 h post-exposure caused downregulation of the virulence factors pyoverdine and pyocyanin; by 6 h post-exposure, virulence factor production was increasing. Transcriptomic analysis provides valuable insights into the mechanisms by which P. aeruginosa biofilms exhibits enhanced susceptibility to antimicrobials. Overall, these findings suggest, for the first time, that short CAP sub-lethal pre-treatment can be an effective strategy for enhancing the susceptibility of P. aeruginosa biofilms to antimicrobials and provides important mechanistic insights into cold plasma-antimicrobial synergy. Transcriptomic analysis of the response to, and recovery from, sub-lethal cold plasma exposures in P. aeruginosa biofilms improves our current understanding of cold plasma biofilm interactions.

Limosilactobacillus fermentum 3872 That Produces Class III Bacteriocin Forms Co-Aggregates with the Antibiotic-Resistant Staphylococcus aureus Strains and Induces Their Lethal Damage

LF3872 was isolated from the milk of a healthy lactating and breastfeeding woman. Earlier, the genome of LF3872 was sequenced, and a gene encoding unique bacteriocin was discovered. We have shown here that the LF3872 strain produces a novel thermolabile class III bacteriolysin (BLF3872), exhibiting antimicrobial activity against antibiotic-resistant Staphylococcus aureus strains. Sequence analysis revealed the two-domain structural (lysozyme-like domain and peptidase M23 domain) organization of BLF3872. At least 25% residues of this protein are expected to be intrinsically disordered. Furthermore, BLF3872 is predicted to have a very high liquid-liquid phase separation. According to the electron microscopy data, the bacterial cells of LF3872 strain form co-aggregates with the S. aureus 8325-4 bacterial cells. LF3872 produced bacteriolysin BLF3872 that lyses the cells of the S. aureus 8325-4 mastitis-inducing strain. The sensitivity of the antibiotic-resistant S. aureus collection strains and freshly isolated antibiotic-resistant strains was tested using samples from women with lactation mastitis; the human nasopharynx and oral cavity; the oropharynx of pigs; and the cows with a diagnosis of clinical mastitis sensitive to the lytic action of the LF3872 strain producing BLF3872. The co-cultivation of LF3872 strain with various antibiotic-resistant S. aureus strains for 24 h reduced the level of living cells of these pathogens by six log. The LF3872 strain was found to be able to co-aggregate with all studied S. aureus strains. The cell-free culture supernatant of LF3872 (CSLF3872) induced S. aureus cell damage and ATP leakage. The effectiveness of the bacteriolytic action of LF3872 strain did not depend on the origin of the S. aureus strains. The results reported here are important for the creation of new effective drugs against antibiotic-resistant strains of S. aureus circulating in humans and animals.

Oxidative Stress-Induced Overactivation of Frog Eggs Triggers Calcium-Dependent Non-Apoptotic Cell Death.

Excessive activation of frog eggs (overactivation) is a pathological process that renders eggs unfertilizable. Its physiological inducers are unknown. Previously, oxidative stress was shown to cause time- and dose-dependent overactivation of Xenopus laevis frog eggs. Here, we demonstrate that the oxidative stress-induced egg overactivation is a calcium-dependent phenomenon which can be attenuated in the presence of the selective calcium chelator BAPTA. Degradation of cyclin B2, which is known to be initiated by calcium transient in fertilized or parthenogenetically activated eggs, can also be observed in the overactivated eggs. Decline in mitochondrial membrane potential, ATP depletion and termination of protein synthesis manifest in the eggs within one hour of triggering overactivation. These intracellular events occur in the absence of caspase activation. Furthermore, plasma membrane integrity is compromised in the overactivated eggs, as evidenced by ATP leakage and egg swelling. In sum, our data demonstrate that oxidative stress-induced overactivation of frog eggs causes fast and dramatic disruption of cellular homeostasis, resulting in robust and expedited cell death by a calcium-dependent non-apoptotic mechanism.

Limosilactobacillus fermentum Strain 3872: Antibacterial and Immunoregulatory Properties and Synergy with Prebiotics against Socially Significant Antibiotic-Resistant Infections of Animals and Humans.

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. The high-quality genome sequencing of LF3872 was performed, and a gene encoding a unique bacteriocin was discovered. It was established that the bacteriocin produced by LF3872 (BLF3872) belongs to the family of cell-wall-degrading proteins that cause cell lysis. The antibacterial properties of LF3872 were studied using test cultures of antibiotic-resistant Gram-positive and Gram-negative pathogens. Gram-positive pathogens (Staphylococcus aureus strain 8325-4 and S. aureus strain IIE CI-SA 1246) were highly sensitive to the bacteriolytic action of LF3872. Gram-negative pathogens (Escherichia coli, Salmonella strains, and Campylobacter jejuni strains) were more resistant to the bacteriolytic action of LF3872 compared to Gram-positive pathogens. LF3872 is a strong co-aggregator of Gram-negative pathogens. The cell-free culture supernatant of LF3872 (CSLF3872) induced cell damage in the Gram-positive and Gram-negative test cultures and ATP leakage. In the in vitro experiments, it was found that LF3872 and Actigen prebiotic (Alltech Inc., Nicholasville, KY, USA) exhibited synergistic anti-adhesive activity against Gram-negative pathogens. LF3872 has immunoregulatory properties: it inhibited the lipopolysaccharide-induced production of proinflammatory cytokines IL-8, IL-1β, and TNF-α in a monolayer of Caco-2 cells; inhibited the production of IL-12 and stimulated the production of IL-10 in immature human dendritic cells; and stimulated the production of TGF-β, IFN-γ, and IgA in the immunocompetent cells of intestinal Peyer’s patches (PPs) in mice. These results indicate the possibility of creating a synbiotic based on LF3872 and a prebiotic derived from Saccharomyces cerevisiae cell wall components. Such innovative drugs and biologically active additives are necessary for the implementation of a strategy to reduce the spread of antibiotic-resistant strains of socially significant animal and human infections.

Phillygenin Inhibits Helicobacter pylori by Preventing Biofilm Formation and Inducing ATP Leakage.

With the widespread use and abuse of antibiotics, Helicobacter pylori (H. pylori) has become seriously drug resistant. The development of new antibiotics is an important way to solve H. pylori's drug resistance. Screening antibacterial ingredients from natural products is a convenient way to develop new antibiotics. Phillygenin, an effective antibacterial component, was selected from the natural product, forsythia, in this study. Its minimal inhibitory concentration (MIC) for 18 H. pylori strains was 16–32 μg/ml. The minimum bactericidal concentration (MBC) of H. pylori G27 was 128 μg/ml; the higher the drug concentration and the longer the time, the better the sterilization effect. It was non-toxic to gastric epithelial cell (GES)-1 and BGC823 cells at the concentration of 100 μg/ml. It presented a better antibacterial effect on H. pylori in an acidic environment, and after 24 days of induction on H. pylori with 1/4 MIC of phillygenin, no change was found in the MIC of H. pylori. In the mechanism of action, phillygenin could cause ATP leakage and inhibit the biofilm formation; the latter was associated with the regulation of spoT and Hp1174 genes. In addition, phillygenin could regulate the genes of Nhac, caggamma, MATE, MdoB, flagellinA, and lptB, leading to the weakening of H. pylori's acid resistance and virulence, the diminishing of H. pylori's capacity for drug efflux, H. pylori's DNA methylation, the initiation of human immune response, and the ATP leakage of H. pylori, thus accelerating the death of H. pylori. In conclusion, phillygenin was a main ingredient inhibiting H. pylori in Forsythia suspensa, with a good antibacterial activity, high safety, strong specificity, better antibacterial effect under acidic conditions, and low risk of resistance development by H. pylori. Its mechanism of action was mainly associated with inhibiting the biofilm formation and resulting in ATP leakage. In addition, phillygenin was shown to be able to reduce the acid resistance and virulence of H. pylori.

Ofloxacin‐loaded HMPB NPs for Klebsiella pneumoniae eradication in the surgical wound with the combination of PTT

Klebsiella pneumoniae (K. pneumoniae) is a common bacterium whose drug-resistant can cause surgical failures and incurable infections in hospital patients. Thus, how to reverse or delay the resistance induction has become a great challenge for development antiresistant drug. Recently, the combination of nanomaterial-loaded antibiotics with photothermal therapy showed the efficient antibacteria ability under a low dosage of antibiotics. In this study, a nanocomposite of HMPB NPs with inherent photothermal therapy capability was used to eradicate K. pneumoniae after loading with Ofloxacin, an antibiotic against K. pneumoniae in vitro and in vivo. The nanocomplexes named as Ofloxacin@HMPB@HA NPs showed a higher effect against K. pneumoniae by destroying cell integrity and inducing ATP leakage with the assistance of laser irradiation, compared with sole Ofloxacin@HMPB@HA NPs or laser irradiation. Surgical wound infection assay further demonstrated the efficient killing K. pneumoniae and promoting the formation of new tissues, as well, which was reflected by the rapid healing of surgical wound. In summary, these results indicate the great potential of this combinational tactic based on Ofloxacin@HMPB@HA NPs for preventing the failure caused by K. pneumoniae infection.

Contribution of ultrasound in combination with chlorogenic acid against Salmonella enteritidis under biofilm and planktonic condition

The antibacterial and antibiofilm mechanisms of ultrasound combined with chlorogenic acid treatment for Salmonella enteritidis under biofilm and planktonic condition were investigated. S. enteritidis under biofilm and planktonic cells were treated with ultrasound, chlorogenic acid, and their combination for 5, 10, 20, 30, and 60 min. Results showed that the combined treatments exhibited synergistic effects that inactivated the S. enteritidis biofilm and planktonic cells. The nucleic acids and ATP leakage and CLSM imagines showed that the combining chlorogenic acid and ultrasound treatment significantly increased the permeability of the S. enteritidis cell membrane. SEM indicated that the combining chlorogenic acid and ultrasound treatment quickly destroyed the integrity of the S. enteritidis cell membrane, and the activity of respiratory chain dehydrogenase sharply decreased. Additionally, the amounts of polysaccharides in the S. enteritidis biofilms significantly decreased after the combined treatments. Hence, the combining chlorogenic acid and ultrasound treatment have potential applications in food preservation.

Discovery of Mycobacterium tuberculosis Rv3364c-Derived Small Molecules as Potential Therapeutic Agents to Target SNX9 for Sepsis.

The serine protease inhibitor Rv3364c of Mycobacterium tuberculosis (MTB) is highly expressed in cells during MTB exposure. In this study, we showed that the 12WLVSKF17 motif of Rv3364c interacts with the BAR domain of SNX9 and inhibits endosome trafficking to interact with p47phox, thereby suppressing TLR4 inflammatory signaling in macrophages. Derived from the structure of this Rv3364c peptide motif, 2,4-diamino-6-(4-tert-butylphenyl)-1,3,5-trazine, DATPT as a 12WLVSKF17 peptide-mimetic small molecule has been identified. DATPT can block the SNX9-p47phox interaction in the endosome and suppress reactive oxygen species and inflammatory cytokine production; it demonstrated significant therapeutic effects in a mouse model of cecal ligation and puncture-induced sepsis. DATPT has considerably improved potency, with an IC50 500-fold (in vitro) or 2000-fold (in vivo) lower than that of the 12WLVSKF17 peptide. Furthermore, DATPT shows potent antibacterial activities by reduction in ATP production and leakage of intracellular ATP out of bacteria. These results provide evidence for peptide-derived small molecule DATPT with anti-inflammatory and antibacterial functions for the treatment of sepsis.

Antibacterial mechanism of apple phloretin on physiological and morphological properties of Listeria monocytogenes

Green bactericides are excellent strategies for reducing food spoilage. Phloretin was regarded as efficient, broad-spectrum and safe preservative, which showed enormous potential in food industry. Inhibitory mechanism of phloretin on Listeria monocytogenes was systematically elucidated through integrity of cell membrane, intracellular protein content, energy level, and nucleic acid morphological character. SEM and TEM results showed minimal injury of cell membrane, which was further proved by a leakage of potassium ions, ATP and little protein and nucleic acid. There were drastic reduction of intracellular or total protein and ATP compared to cell without phloretin treatment. However, the specific activity of hexokinase and isocitrate dehydrogenase were unaffected after phloretin treatment. From these results, we concluded that phloretin’s bacteriostatic mechanism is decreasing the intracellular protein content and energy level resulting from DNA aggregation. This finding was prospective to fully illustrate the phloretin’s mechanism and develop it into a natural preservative in food industry.

Use of Acetic Acid to Partially Replace Lactic Acid for Decontamination against Escherichia coli O157:H7 in Fresh Produce and Mechanism of Action.

Escherichia coli O157:H7 is frequently detected in ready-to-eat produce and causes serious food-borne diseases. The decontamination efficacy of lactic acid (LA) is clearly established. In this study, LA was mixed with acetic acid (AA) to reduce costs while achieving consistent or better inhibitory effects. Time-kill curves and inoculation experiments using fresh-cut spinach and arugula indicated that 0.8%LA+0.2%AA shows similar antibacterial effects to those of 1%LA. To determine whether 1%LA and 0.8%LA+0.2%AA exert antibacterial effects by similar mechanisms, proteomics analysis was used. The proteins related to macromolecule localization, cellular localization, and protein unfolding were uniquely altered after the treatment with 1%LA, and the proteins related to taxis, response to stress, catabolic process, and the regulation of molecular function were uniquely altered after the treatment with 0.8%LA+0.2%AA. Based on these findings, combined with the results of a network clustering analysis, we speculate that cell membrane damage is greater in response to LA than to 0.8%LA+0.2%AA. This prediction was supported by cell membrane permeability experiments (analyses of protein, nucleotide, ATP, and alkaline phosphatase leakage), which showed that LA causes greater membrane damage than 0.8%LA+0.2%AA. These results provide a theoretical basis for the application of an acid mixture to replace LA for produce decontamination.