Antimicrobial Effect and the Mechanism of Diallyl Trisulfide against Campylobacter jejuni.
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ABSTRACT: Campylobacter jejuni is an important foodborne pathogen causing campylobacteriosis. It can infect humans through the consumption of contaminated chicken products or via the direct handling of animals. Diallyl trisulfide (DATS) is a trisulfide compound from garlic extracts that has a potential antimicrobial effect on foodborne pathogens. This study investigated the antimicrobial activity of DATS on C. jejuni by evaluating the minimal inhibitory concentrations (MICs) of C. jejuni 81-168, and fourteen C. jejuni isolates from chicken carcasses. Thirteen of 14 C. jejuni isolates and 81-176 had MICs ≤ 32 μg/mL, while one isolate had MIC of 64 μg/mL. Scanning electron microscopy (SEM) analysis showed the disruption and shrink of C. jejuni bacterial cell membrane after the DATS treatment. A time-killing analysis further showed that DATS had a dose-dependent in vitro antimicrobial effect on C. jejuni during the 24 h treatment period. In addition, DATS also showed an antimicrobial effect in chicken through the decrease of C. jejuni colony count by 1.5 log CFU/g (cloacal sample) during the seven-day DATS treatment period. The transcriptional analysis of C. jejuni with 16 μg/mL (0.5× MIC) showed 210 differentially expression genes (DEGs), which were mainly related to the metabolism, bacterial membrane transporter system and the secretion system. Fourteen ABC transporter-related genes responsible for bacterial cell homeostasis and oxidative stress were downregulated, indicating that DATS could decrease the bacterial ability to against environmental stress. We further constructed five ABC transporter deletion mutants according to the RNA-seq analysis, and all five mutants proved less tolerant to the DATS treatment compared to the wild type by MIC test. This study elucidated the antimicrobial activity of DATS on C. jejuni and suggested that DATS could be used as a potential antimicrobial compound in the feed and food industry.
Project description:ObjectivesBacterial biofilms pose significant food safety risks because of their attachment to fomites and food surfaces, including fresh produce surfaces. The purpose of this study was to systematically investigate the activity of selected antimicrobials on Campylobacter jejuni biofilms.MethodsC. jejuni biofilms and planktonic cells were treated with ciprofloxacin, erythromycin and diallyl sulphide and examined using infrared and Raman spectroscopies coupled with imaging analysis.ResultsDiallyl sulphide eliminated planktonic cells and sessile cells in biofilms at a concentration that was at least 100-fold less than used for either ciprofloxacin or erythromycin on the basis of molarity. Distinct cell lysis was observed in diallyl sulphide-treated planktonic cells using immunoblot analysis and was confirmed by a rapid decrease in cellular ATP. Two phases of C. jejuni biofilm recalcitrance modes against ciprofloxacin and erythromycin were validated using vibrational spectroscopies: (i) an initial hindered adsorption into biofilm extracellular polymeric substance (EPS) and delivery of antibiotics to sessile cells within biofilms; and (ii) a different interaction between sessile cells in a biofilm compared with their planktonic counterparts. Diallyl sulphide destroyed the EPS structure of the C. jejuni biofilm, after which the sessile cells were killed in a similar manner as planktonic cells. Spectroscopic models can predict the survival of sessile cells within biofilms.ConclusionsDiallyl sulphide elicits strong antimicrobial activity against planktonic and sessile C. jejuni and may have applications for reducing the prevalence of this microbe in foods, biofilm reduction and, potentially, as an alternative chemotherapeutic agent for multidrug-resistant bacterial strains.
Project description:The aim of this study is to investigate the antimicrobial synergistic effect against Campylobacter jejuni, a leading foodborne pathogen that causes human gastroenteritis, by cinnamon oil, encapsulated curcumin, and zinc oxide nanoparticles (ZnO NPs). We compared three approaches to study the antimicrobial interactions, including the time-killing method, the fractional inhibitory concentration index (FICI) method, and a mathematical concentration-effect model. Isobologram analysis was performed to evaluate the synergy in different combinations, and a median-effect equation was applied to identify the combinations of synergistic effects at median, bacteriostatic, and bactericidal reduction levels. The time-killing method overestimated the synergistic interaction between antimicrobials, while the FICI method failed to detect an existing synergistic phenomenon. This lack of accuracy and sensitivity was mainly due to combining antimicrobials without a deep understanding of their concentration-effect relationships. Our results showed that each antimicrobial had a unique concentration-effect curve. Specifically, encapsulated curcumin showed a sharp sigmoidal curve unlike cinnamon oil and ZnO NPs. A mathematical model was applied to study the interaction between antimicrobials with a different shape of concentration-effect curve. We observed an additive effect of cinnamon oil/ZnO NPs and synergistic interactions of other binary combinations (cinnamon oil/encapsulated curcumin and ZnO NPs/encapsulated curcumin). The tertiary combination of cinnamon oil/ZnO NPs/encapsulated curcumin at IC25 (additive line <1-log CFU/mL) presented the greatest synergistic effect by reducing the bacterial population over 8-log CFU/mL. This mathematical model provided an alternative strategy to develop a new antimicrobial strategy.
Project description:BackgroundDiallyl trisulfide (DATS) is a garlic-derived organosulfur compound. As it has been shown to have anti-viral activity, we hypothesized that it may alleviate infections caused by H9N2 avian influenza virus (AIV), which is prevalent in poultry with pandemic potential.MethodsHuman lung A549 epithelial cells were treated with three different concentrations of DATS 24 h before (pre-treatment) or one hour after (post-treatment) H9N2 AIV infection. Culture supernatants were collected 24 h and 48 h post-infection and analyzed for viral titers and levels of inflammatory and anti-viral immune responses. For in vivo experiments, BABL/c mice were administered daily by intraperitoneal injection with DATS (30 mg/kg) for 2 weeks starting 1 day after H9N2 AIV infection. Clinical signs, lung pathology, and inflammatory and anti-viral immune responses were assessed 2, 4, and 6 days after infection.ResultsBoth pre-treatment and post-treatment of A549 cells with DATS resulted in reduced viral loads, increased expression of anti-viral genes (RIG-I, IRF-3, and interferon-β), and decreased expression of inflammatory cytokines (TNF-α and IL-6). These effects were also observed in H9N2 AIV-infected mice treated with DATS. Such treatment also reduced lung edema and inflammation in mice.ConclusionsResults suggest that DATS has anti-viral activity against H9N2 AIV and may be used as an alternative treatment for influenza virus infection.
Project description:Diallyl trisulfide (DATS) is an attractive anti-cancer phytochemical with in vitro and in vivo growth inhibitory effects against different solid tumors including breast cancer. We have shown previously that an immortalized mammary epithelial cell line (MCF-10A) is resistant to growth inhibition by DATS. In this study, we performed RNA-seq analysis using a breast cancer cell line (SK-BR-3) and MCF-10A cells to gain insights into cancer selective effects of DATS. The Gene Ontology analysis revealed upregulation of genes associated with actin cytoskeleton but downregulation of mitochondria-related genes in the SK-BR-3 human breast cancer cell line but not in the non-oncogenic MCF-10A cell line upon treatment with DATS. Quantitative real-time reverse transcription polymerase chain reaction confirmed DATS-mediated upregulation of several actin cytoskeleton-related genes in the SK-BR-3 cell line. The DATS treatment dose-dependently disrupted actin cytoskeleton in the SK-BR-3 cell line, whereas the MCF-10A cell line was more resistant to this effect. The DATS treatment caused a marked increase in phosphorylation of dynamin-1-like (DRP1) protein in the SK-BR-3 cell line. However, the DATS-mediated apoptosis was not affected by genetic deletion of DRP1 protein. The Reactome pathway analysis showed downregulation of genes associated with citric acid cycle in the SK-BR-3 cell line but not in the MCF-10A cells. However, expression of aconitase 2 or dihydrolipoamide S-succinyltransferase was not affected by DATS treatment. In conclusion, this study reveals that actin cytoskeleton is a novel target of DATS in the SK-BR-3 cell line, which may explain its inhibitory effect on breast cancer cell migration.
Project description:The multiterritory perforator flap is one of the widest flap patterns used to repair tissue defects. However, flap necrosis of the distal part is still a challenging issue for plastic surgeons. Diallyl trisulfide (DATS) is an efficient ingredient extracted from garlic, exerting many important effects on different diseases. Our experiment aims to reveal whether DATS has a beneficial effect on the survival of perforator flaps and to explore its mechanism of action. The results showed that DATS enhanced angiogenesis and autophagy and reduced cell apoptosis and oxidative stress, thereby improving the survival rate of skin flaps. After co-administration with autophagy inhibitor 3-methyladenine (3MA), perforator flap survival was further improved. Mechanistically, we showed that PI3K/Akt and AMPK-HIF-1α signaling pathways in flap were activated under DATS treatment. All in all, DATS promoted the survival of multiterritory perforator flaps via the synergistic regulation of PI3K/Akt and AMPK-HIF-1α signaling pathways, and inhibition of DATS-induced autophagy further improves flap survival.
Project description:The carbohydrate 2, 4-diacetamido-2, 4, 6-trideoxy-alpha-D-glucopyranose (BacAc(2)) is found in a variety of eubacterial pathogens. In Campylobacter jejuni, PglD acetylates the C4 amino group on UDP-2-acetamido-4-amino-2, 4, 6-trideoxy-alpha-D-glucopyranose (UDP-4-amino-sugar) to form UDP-BacAc(2). Sequence analysis predicts PglD to be a member of the left-handed beta helix family of enzymes. However, poor sequence homology between PglD and left-handed beta helix enzymes with existing structural data precludes unambiguous identification of the active site. The co-crystal structures of PglD in the presence of citrate, acetyl coenzyme A, or the UDP-4-amino-sugar were solved. The biological assembly is a trimer with one active site formed between two protomers. Residues lining the active site were identified, and results from functional assays on alanine mutants suggest His-125 is critical for catalysis, whereas His-15 and His-134 are involved in substrate binding. These results are discussed in the context of implications for proteins homologous to PglD in other pathogens.
Project description:Using laboratory challenge experiments, we examined whether Campylobacter-specific maternal antibody (MAB) plays a protective role in young chickens, which are usually free of Campylobacter under natural production conditions. Kinetics of C. jejuni colonization were compared by infecting 3-day-old broiler chicks, which were naturally positive for Campylobacter-specific MAB, and 21-day-old broilers, which were negative for Campylobacter-specific MAB. The onset of colonization occurred much sooner in birds challenged at the age of 21 days than it did in the birds inoculated at 3 days of age, which suggested a possible involvement of specific MAB in the delay of colonization. To further examine this possibility, specific-pathogen-free layer chickens were raised under laboratory conditions with or without Campylobacter infection, and their 3-day-old progenies with (MAB(+)) or without (MAB(-)) Campylobacter-specific MAB were orally challenged with C. jejuni. Significant decreases in the percentage of colonized chickens were observed in the MAB(+) group during the first week compared with the MAB(-) group. These results indicate that Campylobacter-specific MAB plays a partial role in protecting young chickens against colonization by C. jejuni. Presence of MAB in young chickens did not seem to affect the development of systemic immune response following infection with C. jejuni. However, active immune responses to Campylobacter occurred earlier and more strongly in birds infected at 21 days of age than those infected at 3 days of age. Clearance of Campylobacter infection was also observed in chickens infected at 21 days of age. Taken together, these findings (i) indicate that anti-Campylobacter MAB contributes to the lack of Campylobacter infection in young broiler chickens in natural environments and (ii) provide further evidence supporting the feasibility of development of immunization-based approaches for control of Campylobacter infection in poultry.
Project description:Campylobacter (C.) spp. from poultry is the main source of foodborne human campylobacteriosis, but diseased pets and cattle shedding Campylobacter spp. may contribute sporadically as a source of human infection. As fluoroquinolones are one of the drugs of choice for the treatment of severe human campylobacteriosis, the resistance rates of C. jejuni and C. coli from poultry against antibiotics, including fluoroquinolones, are monitored within the European program on antimicrobial resistance (AMR) in livestock. However, much less is published on the AMR rates of C.jejuni and C. coli from pets and cattle. Therefore, C. jejuni and C. coli isolated from diseased animals were tested phenotypically for AMR, and associated AMR genes or mutations were identified by whole genome sequencing. High rates of resistance to (fluoro)quinolones (41%) and tetracyclines (61.1%) were found in C. jejuni (n = 29/66). (Fluoro)quinolone resistance was associated with the known point mutation in the quinolone resistance-determining region (QRDR) of gyrA, and tetracycline resistance was mostly caused by the tet(O) gene. These high rates of resistance, especially to critically important antibiotics in C. jejuni and C. coli, are worrisome not only in veterinary medicine. Efforts to preserve the efficacy of important antimicrobial treatment options in human and veterinary medicine have to be strengthened in the future.
Project description:Diallyl sulfide, diallyl disulfide, and daillyl trisulfide (DATS) are major volatile components of garlic oil. In this study, we assessed their relative potency in inducing antioxidant enzyme expression. Among the three organosulfur compounds, DATS was found to be most potent in inducing heme oxygenase-1 (HO-1) andNad(p)hquinone oxidoreductase-1 (NQO1) in human gastric epithelial (AGS) cells. Furthermore, DATS administration by gavage increased the expression of HO-1 and NQO1 in C57BL/6 mouse stomach. Treatment with DATS increased the accumulation of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus of cultured AGS cells and in mouse stomach in vivo. The DATS-induced expression of HO-1 and NQO1 was abrogated in the cells transiently transfected with Nrf2-siRNA or in the embryonic fibroblasts from Nrf2-null mice, indicating that Nrf2 is a key mediator of the cytoprotective effects of DATS. Pretreatment of AGS cells with N-acetylcysteine or dithiothreitol attenuated DATS-induced nuclear localization of Nrf2 and the expression of HO-1 and NQO1. Cysteine-151, -273 and -288 of Kelch-like ECH-associated protein-1 (Keap1), a cytosolic repressor of Nrf2, have been considered to act as a redox sensor and play a role in Nrf2 activation. To determine whether DATS could inactivate Keap1 through thiol modification, we established cell lines constitutively expressing wild type-Keap1 or three different mutant constructs in which cysteine-151, -273, or -288 of Keap1 was replaced with serine by retroviral gene transfer. DATS failed to activate Nrf2, and to induce expression of HO-1 and NQO1 only in Keap1-C288S mutant cells. LC-ESI-MS/MS analysis of recombinant Keap1 treated with DATS revealed that the peptide fragment containing Cys288 gained a molecular mass of 72.1 Da equivalent to the molecular weight of mono-allyl mono-sulfide. Taken together, these findings suggest that DATS may directly interact with the Cys288 residue of Keap1, which partly accounts for its ability to induce Nrf2 activation and upregulate defensive gene expression.