Xingchen Zhao

Antimicrobial, antioxidant, and antitumor activity of epsilon-poly-L-lysine and citral, alone or in combination

Background Food safety is an important worldwide public health concern, and microbial contamination in foods not only leads to food deterioration and shelf life reduction but also results in economic losses and disease. Objective The main aim of the present study was to evaluate the effect of epsilon-poly-L-lysine (ε-PL) and citral combination against Escherichia coli O157:H7 (E. coli O157:H7) strains. The preliminary antioxidant and antitumor activities were also studied. Design Synergism is a positive interaction created when two compounds combine and exert an inhibitory effect that is greater than the sum of their individual effects. The synergistic antimicrobial effect of ε-PL and citral was studied using the checkerboard method against E. coli O157:H7. The minimal inhibitory concentration, time-kill, and scanning electron microscope assays were used to determine the antimicrobial activity of ε-PL and citral alone or in combination; 2,2-diphenyl-1-picrylhydrazyl-scavenging assay and western blotting were used in antioxidant activity assays; cell viability assay was carried out to finish preliminary antitumor test. Results Minimal inhibitory concentrations of ε-PL and citral resisted to the five E. coli O157:H7 strains were 2–4 µg/mL and 0.5–1 µg/mL, and the fractional inhibitory concentration indices were 0.25–0.375. The results of time-kill assay revealed that a stronger bactericidal effect in a laboratory medium might be exerted in the combination against E. coli O157:H7 than that in a food model. The compounds alone or in combination exhibited a potential 2,2-diphenyl-1-picrylhydrazyl radical–scavenging activity, and the expression of superoxide dismutase 1 and glutathione peroxidase 1 protein increased. The preliminary antitumor activity effect of the combination was better than ε-PL or citral alone. Conclusions These findings indicated that the combination of ε-PL and citral could not only be used as a promising naturally sourced food preservative but also be used in the pharmaceutical industry.

The synergy of berberine chloride and totarol against Staphylococcus aureus grown in planktonic and biofilm cultures.

Staphylococcus aureus (S. aureus) is commonly associated with hospital-acquired infections and is known to form biofilms. Bacteria inside biofilms display an increased resistance to chemotherapeutics and host immune defences. Efficient antibiotics or combination therapy are urgently needed to treat patients with biofilm-associated MRSA infections. The objective of the current study was to evaluate the in vitro antimicrobial activities of totarol alone or in combination with berberine chloride (BBR) against S. aureus grown in planktonic and biofilm cultures. The synergistic antimicrobial effects between BBR and totarol were observed in all tested strains grown in biofilms using a chequerboard microdilution method, with the fractional inhibitory concentration index values ranging from 0.125 to 0.375. No antagonistic activity was observed in any of the strains tested in suspension or biofilm cultures. The synergistic activity against S. aureus biofilms was also corroborated by confocal laser scanning microscopy and adhesion assays. Moreover, the present study demonstrated that combination BBR and totarol treatment effectively decreased the formation of S. aureus biofilms by affecting extracellular genomic DNA release and polysaccharide intercellular adhesin expression. Subsequently, real-time reverse transcriptase PCR analysis revealed that the combination of BBR and totarol effectively inhibited the transcription of the biofilm-related genes sarA, cidA and icaA. These results suggest that the combination of totarol and BBR is momentous for the further development of a therapy protocol against S. aureus biofilms.

Inhibitory effect of totarol on exotoxin proteins hemolysin and enterotoxins secreted by Staphylococcus aureus

Staphylococcus aureus (S. aureus) causes a wide variety of infections, which are of major concern worldwide. S. aureus produces multiple virulence factors, resulting in food infection and poisoning. These virulence factors include hyaluronidases, proteases, coagulases, lipases, deoxyribonucleases and enterotoxins. Among the extracellular proteins produced by S. aureus that contribute to pathogenicity, the exotoxins α-hemolysin, staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) are thought to be of major significance. Totarol, a plant extract, has been revealed to inhibit the proliferation of several pathogens effectively. However, there are no reports on the effects of totarol on the production of α-hemolysin, SEA or SEB secreted by S. aureus. The aim of this study was to evaluate the effects of totarol on these three exotoxins. Hemolysis assay, western blotting and real-time reverse transcriptase-PCR assay were performed to identify the influence of graded subinhibitory concentrations of totarol on the production of α-hemolysin and the two major enterotoxins, SEA and SEB, by S. aureus in a dose-dependent manner. Moreover, an enzyme linked immunosorbent assay showed that the TNF-α production of RAW264.7 cells stimulated by S. aureus supernatants was inhibited by subinhibitory concentrations of totarol. Form the data, we propose that totarol could potentially be used as a promising natural compound in the food and pharmaceutical industries.

Oleuropein protects L-02 cells against H2O2-induced oxidative stress by increasing SOD1, GPx1 and CAT expression

Oleuropein (OL), a natural phenolic compound, comprises the major constituent of Olea europea leaves and unprocessed olives, and OL is considered to be the principal components that confer the characteristic taste and stability of olive oil. Oxidative damage induced by H2O2 treatment can irreversibly damage the L-02 cells, resulting in cell death and apoptosis. Whether the effects of oxidative stress could be attenuated in cultured human L-02 cells by incubation with OL is still unknown. In this research, the function of OL in protecting human L-02 cells against H2O2 induced cell death and cell apoptosis was investigated, and the mechanism by which OL underlies the effect was also examed. L-02 cells were exposed to 100μM H2O2 with or without OL pretreatment at different concentrations. Cell viabilities were monitored by WST-1 assay. ALT, AST and LDH production in culture medium were also determined. ROS levels were detected by L-012 chemiluminescence, and OL increased SOD1, CAT and GPx1 expression in a concentration-dependent manner. Further studies showed that OL also inhibited H2O2-induced P38 and JNK phosphorylation but enhanced ERK1/2 phosphorylation in a dose-dependent manner. These findings suggested that OL as a potent antioxidant agent and a natural compound found in several plants, may be exploited as a potentially useful method for hepatopathy prevention.

Effect of nisin and perilla oil combination against Listeria monocytogenes and Staphylococcus aureus in milk

In the present study, in vitro interaction of nisin and perilla oil (PO) against 20 food-borne isolates of L. monocytogenes and S. aureus were assessed using a checkerboard microdilution method. Synergism was observed in tested strains with the fractional inhibitory concentration indexs (FICIs) ranges from 0.125–0.25 and 0.19–0.375, respectively. Scanning electron microscopy was carried out to investigate the effect of nisin and PO on the integrity of cell wall and membrane of L. monocytogenes and S. aureus. The results showed that nisin and PO were more effective in damaging cell wall and membrane in combination.

In vitro activity of isoimperatorin, alone and in combination, against Mycobacterium tuberculosis

Previous studies have shown that isoimperatorin (IO), a furanocoumarin isolated from several medicinal plants, has antimycobacterial activity against Mycobacterium tuberculosis strain H37Rv (ATCC 27294). This study demonstrated that IO has antimycobacterial activity against 2 drug‐sensitive and 6 drug‐resistant isolates, with minimum inhibitory concentrations (MICs) of 50–100 μg ml−1 and 100–200 μg ml−1, respectively. IO exhibited synergistic antimycobacterial effects with rifampin (RMP), isoniazid (INH) and ethambutol (EMB) against 6 drug‐resistant strains, with fractional inhibitory concentration index (FICI) values of 0·133–0·472, 0·123–0·475 and 0·124–0·25, respectively. The IO/RMP, IO/INH and IO/EMB combination treatments had synergistic effects or no interaction in the 2 drug‐sensitive strains and the standard strain ATCC 27294. The synergism of combined drugs against drug‐resistant strains was better than drug‐sensitive strains. No antagonism was observed in with the aforementioned combinations against all strains tested. IO exhibited relatively low cytotoxicity to Vero cells. Our results indicate that IO may serve as promising a template for future antimycobacterial drug development.

In vitro antimicrobial activity of honokiol against Staphylococcus aureus in biofilm mode.

Abstract Staphylococcus aureus (S. aureus) can attach to food, host tissues and the surfaces of medical implants and form a biofilm, which makes it difficult to eliminate. The purpose of this study was to evaluate the effect of honokiol on biofilm-grown S. aureus. In this report, honokiol showed effective antibacterial activity against S. aureus in biofilms. S. aureus isolates are capable of producing distinct types of biofilms mediated by polysaccharide intercellular adhesion (PIA) or extracellular DNA (eDNA). The biofilms’ susceptibility to honokiol was evaluated using confocal laser scanning microscopy (CLSM) analysis. The transcript levels of the biofilm-related genes, the expression of PIA, and the amount of eDNA of biofilm-grown S. aureus exposed to honokiol were also investigated. The results of this study show that honokiol can detach existing biofilms, kill bacteria in biofilms, and simultaneously inhibit the transcript levels of sarA, cidA and icaA, eDNA release, and the expression of PIA.

Phenotype and RNA-seq-Based transcriptome profiling of Staphylococcus aureus biofilms in response to tea tree oil.

Staphylococcus aureus (S. aureus) is a Gram-positive bacterium that causes a wide range of diseases, including food poisoning. Tea tree oil (TTO), an essential oil distilled from Melaleuca alternifolia, is well-known for its antibacterial activities. TTO effectively inhibited all 19 tested strains of S. aureus biofilm and planktonic cells. Phenotype analyses of S. aureus biofilm cells exposed to TTO were performed by biofilm adhesion assays, eDNA detection and PIA release. RNA sequencing (RNA-seq) was used in our study to elucidate the mechanism of TTO as a potential antibacterial agent to evaluate differentially expressed genes (DEGs) and the functional network in S. aureus ATCC 29213 biofilms. TTO significantly changed (greater than a 2- or less than a 2-fold change) the expression of 304 genes in S. aureus contained in biofilms. The levels of genes related to the glycine, serine and threonine metabolism pathway, purine metabolism pathway, pyrimidine metabolism pathway and amino acid biosynthesis pathway were dramatically changed in the biofilm exposed to TTO. Furthermore, the expression changes identified by RNA-seq analysis were verified by real-time RT-PCR. To the best of our knowledge, this research is the first study to report the phenotype and expression profiles of S. aureus in biofilms exposed to TTO.

Inhibition effect of tea tree oil on Listeria monocytogenes growth and exotoxin proteins listeriolysin O and p60 secretion

Listeria monocytogenes (L. monocytogenes) is a Gram‐positive bacterium that causes infections in humans. In this study, the effects of tea tree oil (TTO) at subinhibitory concentrations on L. monocytogenes growth and two important exotoxin proteins secreted by L. monocytogenes were researched. Treatment with half of minimal inhibitory concentration of TTO demonstrated very little or no reduction in numbers of viable ATCC 19115 cells. Listeriolysin O (LLO) and p60, were investigated. A listeriolysin assay was used to investigate the hemolytic activities of L. monocytogenes exposed to TTO, and the secretion of LLO and p60 was detected by immunoblot analysis. Additionally, real‐time RT‐PCR was used to analyse the influence of TTO on the transcription of LLO and p60 encoded genes hly and iap respectively. According to our experimental results, we propose that TTO could be used as a promising natural compound against L. monocytogenes and its virulence factors.

Effect of honokiol on exotoxin proteins listeriolysin O and p60 secreted by Listeria monocytogenes.

Listeria monocytogenes is considered one of the most important foodborne pathogens. The virulence-related proteins listeriolysin O (LLO) and p60 are critical factors involved in Listeria pathogenesis. In the present study, we investigated the effect of honokiol on LLO and p60 secreted from L. monocytogenes. A listeriolysin assay was used to investigate the haemolytic activities of L. monocytogenes exposed to honokiol, and the secretion of LLO and p60 was detected by immunoblot analysis. Additionally, the influence of honokiol on the transcription of LLO and p60 genes (hly and iap, respectively) was analysed by real-time reverse transcription PCR. TNF-α release assays were performed to elucidate the biological relevance of changes in LLO and p60 secretion induced by honokiol. According to the data, honokiol showed good anti-L. monocytogenes activity, with MICs of 8-16 μg ml(-1), and the secretion of LLO and p60 was decreased by honokiol. In addition, the transcription of hly and iap was inhibited by honokiol. Our results indicate that TNF-α production by RAW264.7 cells stimulated with L. monocytogenes supernatants was inhibited by honokiol. Based on these data, we propose that honokiol could be used as a promising natural compound against L. monocytogenes and its virulence factors.