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Objective To study the inhibitory effect of Huidu Yinhua powder from the Orthodox Manual of External Medicine on methicillin-resistant Staphylococcus aureus(MRSA),virulence factor α-hemolysin(Hla)activity,and biofilm formation,and to explore the optimal ratios of Huidu Yinhua powder and provide experimental support for its use.Methods The inhibitory effects of Huidu Yinhua powder and the herbs in the formula on USA300 were analyzed by the minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC),and disk diffusion assay(K-B method).Hemolysis,neutralization,oligomerization,and Western blot assays were used to verify in which form the drug inhibits the activity of virulence factor α-hemolysin(Hla).A biofilm assay was performed to evaluate the inhibitory effect of Huidu Yinhua powder on biofilm.Orthogonal experiments were performed to explore the optimal ratio of Huidu Yinhua powder.Results Huidu Yinhua powder inhibited the MRSA strain with a MIC90 of 64 mg/mL and an MBC of 256 mg/mL with antibacterial circle diameter of(7.50±0.50)mm.Huidu Yinhua powder inhibited Hla activity by inhibiting Hla secretion.The minimum effective concentration(MEC)was 16 mg/mL,and the MEC of biofilm was 8 mg/mL.In Huidu Yinhua powder,honeysuckle and astragalus only affected the hemolytic activity of MRSA and biofilm formation without inhibiting bacterial growth.The hemolytic activity and biofilm of MEC were both 32 mg/mL.Glycyrrhiza had a strong bacterial inhibitory capacity with a MIC90 of 8 mg/mL and biofilm MEC of 1 mg/mL without showing inhibitory hemolytic activity at subinhibitory concentrations.The orthogonal experiment showed that,at a ratio of honeysuckle,astragalus,and glycyrrhiza in Huidu Yinhua powder of 1∶2∶4,the MIC90 was 16 mg/mL,MEC of hemolytic activity was 8 mg/mL and that of biofilm was 4 mg/mL,both of which were the lowest among the nine groups.Conclusions Huidu Yinhua powder affects the hemolytic activity and biofilm formation of MRSA at subinhibitory concentrations with the optimal ratio of honeysuckle,astragalus,and glycyrrhiza being 1∶2∶4.
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Objective:To study whether Tanreqing injection （TRQ） can alleviate the body injury in the process of infection by inhibiting the production and release of <italic>α</italic>-hemolysin of <italic>Staphylococcus aureus</italic> under sub-minimal inhibitory concentration， and to provide experimental basis for better guidance of clinical medication. Method:The effects of TRQ on the minimum inhibitory concentration （MIC） and bacterial growth of <italic>S.aureus</italic> were determined firstly by microplate method and time-growth curve. The different sub-minimal inhibitory concentrations of TRQ were co-cultured with bacteria or bacterial supernatants， and then co-incubated with defibrillated rabbit blood to detect the inhibitory and neutralizing effects of TRQ on <italic>S.aureus</italic> <italic>α</italic>-hemolysin. Cell counting kit-8 （CCK-8） cell viability assay was used to detect the protective effect of TRQ on <italic>S. aureus</italic>-mediated damage to human alveolar epithelial cells （A549）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the effect of sub-minimal inhibitory concentration of TRQ on the mRNA expression of <italic>S.aureus</italic> <italic>α</italic>-hemolysin regulatory genes hla and agrA. Result:The MIC of TRQ to <italic>S.aureus </italic>was 1/8 of the stock solution， and the sub-minimal inhibitory concentration （1/64MIC-1/16MIC） TRQ used in this study did not affect the growth of bacteria. 1/64MIC-1/16 MIC TRQ had the effect of inhibiting and neutralizing the hemolytic activity of <italic>α</italic>-hemolysin， with a protective effect on <italic>S.aureus</italic> supernatant-mediated A549 cell damage， and its inhibitory effect on <italic>α</italic>-hemolysin was closely related to the inhibition of hla and agrA mRNA expression. Conclusion:The sub-minimal inhibitory concentration TRQ can inhibit and neutralize the hemolytic activity of <italic>α</italic>-hemolysin of <italic>S.aureus</italic>， with a protective effect on A549 cell damage mediated by <italic>S.aureus</italic> infection， and its mechanism of inhibiting <italic>α</italic>-hemolysin is closely related to the interference with agr regulatory system.
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Single nanopore current pulse method is a new, rapid and simple detection method, which is promising for single-molecule DNA sequencing and bio-sensing. Due to the short duration and the low current amplitude of the pulses caused by molecular translocation under normal conditions, pulse detection system with fast response and high sensitivity is required. In this work, based on a lab-established pulse detection system, the effect of protamine in the regulation of single-stranded (ssDNA) current pulses with α-hemolysin (α-HL) single nanopore interface was investigated. Experimental results showed that the pulses positive charged protamine and negative ssDNA probes were both well observed with the established system, and both the pulse amplitude and duration of ssDNA were increased as a result of interaction with protamine. This study provides a way to improve the resolving power of current pulses based on molecular interactions.
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Due to the difference in spatial configuration and charge of the bases in a DNA molecule, characteristic translocation current pulses through a single nanopore could be obtained. This could become the basis of DNA sequencing method. However, due to the fast translocation speed (sub-micro seconds) and the small current change (about pA), it is still a challenge to obtain the accurate molecular substructure with present electronic techniques. In this work, in order to control the translocation behavior of ssDNA, two kinds of ionic liquids with high viscosity and conductivity were introduced to establish a viscosity gradient with the α-hemolysin single nanopore interface and the acidity of the solution was optimized. The trans chamber contained pure BmimPF6 and the cis chamber contained 1 mol/ L BmimCl and 10 mmol/ L Tris-HCl ( pH 5. 5 ). Preliminary experiment results under this electrolyte configuration showed that poly ( dC) 15 , poly ( dC) 15 , poly(dC) 30 and poly(dC) 50 exhibited obvious long duration pulses with high current suppression ratio. The blocking depth reached more than 95％ of long blocking events. The duration time of long blocking events prolonged to tens or hundreds of milliseconds. Meanwhile, the peak-peak of baseline noise was reduced by about 30％ .
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Objective To investigate the inhibitory effects of aloin on the growth of Staphylococcus aureus and its virulence factors α-hemolysin in vitro.Methods Broth dilution was used to measure the minimum inhibitory concentration (MIC) of water-soluble aloin on S.aureus.Agar drilling method was used to observe the size of inhibition zone of aloin for S.aureus.Plasma coagulase test was used to detect the changes of S.aureus coagulase and absorbance was measured to detect the changes of hemolytic activity when S.aureus was exposed to aloin.Real time PCR was used to detect the effects of aloin on the expressions of hla and agrA mRNA.Results The soluble aloin inhibited the growth of S.aureus in a dose-dependent manner.The inhibition zone diameter of a standard strain of S.aureus (ATCC 25923) was 21.5 mm with MIC of 12.5 mg/mL and 17 mm for the clinical isolate SA1.5 with MIC of 15 mg/mL.After treated with soluble aloin,the coagulase titers of ATCC 25923 were 16,4 and 2 for 1/2 MIC,1 MIC and 2 MIC respectively compared with titer 32 of the control group without soluble aloin.The expression of α-hemolysin of S.aureus ATCC 25923 was down-regulated by soluble aloin and the hemolytic activity of S.aureus ATCC 25923 with 1/2 MIC,1 MIC and 2 MIC groups were (77.4 ±3.41) ％,(42.2 ± 2.4) ％ and (38.7 ± 2.4) ％ respectively.The expression levels of hla were 0.020 3 (0.019 6,0.028 8),0.011 6(0.010 6,0.013 1) and 0.033 7(0.020 2,0.042 9) respectively in the 1/2 MIC,1 MIC and 2 MIC group respectively,and there were significant differences among the three groups (H =16.807,P ＜ 0.05).The expression levels of agrA was 0.074 6 (0.066 2,0.098 2),0.020 8 (0.012 2,0.032 6) and 0.021 3 (0.010 2,0.029 6) in the 1/2 MIC,1 MIC and 2 MIC group respectively,and there were significant differences among the three groups (H =16.320,P ＜ 0.05).Conclusion Aloin may inhibit the growth of S.aureus and could effectively inhibit the expression of α-hemolysin.
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Staphylococcus aureus(S.aureus) secretes a variety of pathogenic toxins and one of the most prominent toxins is α-hemolysin,which is considered as the main virulence factor in skin necrosis and severe infections caused by S.aureus infection. α-hemolysin is so named because of its ability to dissolve red blood cells. However,it has a wide range of effects on various cells and may cause different pathogenic re-sponses in different host cells. This review summarizes recent studies on the structure of α-hemolysin and its interaction with different cells in order to better understand the role of α-hemolysin in the process of S.aureus infection,which may provide a reference for developing new approaches to the prophylaxis and treatment of S.aureus infection.
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Methicillin-resistant staphylococcus aureus (MRSA) remains a prominent human pathogen.Traditionally, MRSA infections occurred exclusively in hospitals and were limited to immunocompromised patients or individuals with predisposing risk factors. However, recently there has been a worldwide epidemic caused by community-associated (CA)-MRSA strains, which can cause severe infections that can result in necrotizing fasciitis or even death in otherwise healthy children or adults outside of healthcare settings. People have made great progress to explain the reasons for enhanced virulence of CA-MRSA, here we review the recent progress made towards four virulence determinants, which are Panton-Valentine leukocidin (PVL), α-Hemolysin (Hla),α-type phenol-soluble modulin ( PSMα) and arginine catabolic mobile element (ACM E) .