The Antibacterial Effects And Mechanisms Of Thanatin Against Multi-drug Resistant NDM-1 Producing Bacteria

ObjectiveGram-negative bacteria carrying New Delhi metallo-β-lactamase（NDM-1）are a class of multi-drug resistant superbugs,which can hydrolyze almost allβ-lactam antibiotics,including carbapenems.After the discovery,these superbugs are emerging worldwide and associated with high mortality in clinic.Meanwhile,NDM-1-producing bacteria can secrete the OMVs carrying NDM-1 and the bla_NDM-1 gene,which favor the horizontal transfer of bla_NDM-1 among various types of Gram-negative strains and accelerate the severe global spread of the resistant gene.However,facing the ever-increasing,fast-spreading and highly lethal NDM-1-producing strains,the optimal treatment options for controlling the infection and transmission caused by NDM-1-producing bacteria are still limited.Therefore,it is imminent to develop the effective drugs to combat these pathogens.Thanatin is a cationic antimicrobial peptide isolated from insect.Previous studies revealed that thanatin exhibits strong bactericidal effects against Gram-positive bacteria,Gram-negative bacteria and fungi.The already known antibacterial mechanism of thanatin is membrane permeabilization.In the present study,our initial findings showed that thanatin not only exhibited potent antibacterial activity against NDM-1-producing strains by disrupting OM integrity but also inhibited the activity of NDM-1 enzyme.Remarkably,sub-MICs of thanatin could reverse carbapenem resistance.These results indicated thanatin may combate the NDM-1-producing pathogens via a special and unknown mechanism.Thus,in the next study,we plan to verify the antibacterial property of thanatin against NDM-1-producing strains,explore the mechanism of thanatin in inhibiting the enzymatic activity of NDM-1,and reveal the mechanism of thanatin in blocking outer membrane vesicle（OMV）-mediated resistant transmission.Elucidating these above problems is helpful to reveal the new mechanisms of thanatin against NDM-1-producing bacteria,and provide a new idea and strategy for developing anti-NDM-1 drugs.Methods1.The antibacterial activity and safety of thanatin in vitro and in vivoClinically isolated E.coli and K.pneumoniae strains were obtained from Xijing Hospital,and the presence of bla_NDM-1 was confirmed by PCR.The minimum inhibitory concentration（MIC）of thanatin and the time-kill curves for clinically isolated strains were identified according to basic microbiological protocol.Next,the sepsis and pneumonia models induced by NDM-1-producing bacteria were established,the antimicrobial effects of thanatin in vivo were observed by measuring the survival rate and bacterial loads in organs,and the protective effect of thanatin on the organs was also examed by HE staining.To evaluate the safety of thanatin,HUVEC,HPAEpi C,and mouse neuron cells were used to examine the cytotoxicity of thanatin.The acute toxicity experiment in vivo was also performed by treating BABL/c mice with 60 mg/kg thanatin via intraperitoneal injection.2.The mechanism of thanatin in disrupting OM integrityThe outer and inner membrane permeability of thanatin was measured by the uptake of NPN and PI respectively.The OM integrity was directly observed by scanning electron microscope（SEM）.To further verify the mechanism of thanatin in competitively replacing the divalent cations from LPS and disrupting the integrity of OM,the release of Ca²⁺and LPS from OM was detected by the kits,the effects of divalent cations and metal-chelating agents on the antibacterial activity of thanatin were investigated via MIC assay,the affinities of thanatin,Ca²⁺,and Mg²⁺to LPS were directly examined by isothermal titration calorimetry（ITC）.The outer membrane permeability of thanatin was also measured by detecting the NDM-1 protein levels in culture supernatants and bacterial precipitates after thanatin treatment.3.The mechanism of thanatin in inhibiting the enzymatic activity of NDM-1To preliminarily examine whether thanatin can inhibit the enzymatic activity of NDM-1 in the culture supernatant of E.coli XJ141026,imipenem hydrolysis in the supernatant was monitored via a microplate spectrophotometer.To determine the direct interaction between NDM-1 and thanatin,microscale thermophoresis（MST）was used to detect the binding affinities of purified NDM-1 to thanatin,colistin and Zn²⁺.To further define the inhibitory activity and manner of thanatin,the purified NDM-1 protein was incubated with thanatin,and then imipenem hydrolysis was examined.To directly confirm whether thanatin could inhibit NDM-1 by removing Zn²⁺from NDM-1,ICP-MS and Zn²⁺restoration assays were performed.To define thanatin could potentially protect imipenem and meropenem from hydrolysis and restore the antibiotic susceptibility of NDM-1-producing E.coli and K.pneumoniae strains as an NDM-1 inhibitor,the antimicrobial effects of the combination of carbapenems and thanatin were determined in vitro and in vivo.4.The mechanism of thanatin in blocking OMV mediated resistant transmissionOMV were isolated from the culture supernatant of NDM-1-producing E.coli XJ141026,the size distribution and morphology of OMV was determined by NTA and transmission electron microscopy（TEM）.The presence of bla_NDM-1 and NDM-1 protein in OMV was confirmed by PCR and Western-Blot.Then,the membrane-permeabilizing effect of thanatin on OMV was detected by membrane permeability assay and Western-Blot.To directly determine whether thanatin could block the OMV-mediated transformation,the MICs of imipenem and meropenem were determined in the transformants,the presence of bla_NDM-1 and NDM-1 protein was confirmed in the transformants by PCR and Western-Blot.Results1.Thanatin exhibits potent antibacterial activity against NDM-1-producing E.coli and K.pneumoniae strains in vitro and in vivo with low toxicityThanatin exhibited potent inhibitory effect on the growth of all NDM-1-producing E.coli and K.pneumoniae strains with the MIC values at 2-8μg/m L.The results of the time-kill curves indicated that thanatin rapidly reduced the bacterial population in a time-dependent manner.In the mouse sepsis and pneumonia model induced by NDM-1-producing E.coli,6 mg/kg thanatin could markedly increase the survival rate,decrease the bacterial titers in tissues,and protect the organs.In the mouse pneumonia model induced by NDM-1-producing K.pneumoniae,9 mg/kg thanatin could markedly increase the survival rate,decrease the bacterial titers in lungs,and protect the lungs.Thanatin had lower toxicity toward HUVEC,HPAEpi C,and mouse neuron cells.The safe concentrations of thanatin were 100 times higher than the MICs of thanatin against NDM-1-producing strains.All mice survived for 15 days after i.p.injection of 60 mg/kg thanatin.2.Thanatin competitively replaces divalent cations from LPS,thereby disrupting the integrity of OM and leading to bacterial deathThe results of membrane permeability assay showed that the outer and inner membrane integrity was damaged by thanatin in a time-dependent manner.SEM image also showed that thanatin could directly disrupte OM integrity.Then we found that thanatin could release the Ca²⁺and LPS from bacterial OM in a time-and concentration-dependent manner.These effects could be significantly inhibited by increasing the concentrations of divalent cations;as a result,the antibacterial efficacy of thanatin was adversely affected.Consistently,the chelation of the divalent cations by metal-chelating agents facilitated the bactericidal activity of thanatin.The direct evidence of ITC showed that the affinity of thanatin was about 100 times higher than that of Ca²⁺or Mg²⁺to LPS.All these data suggested that thanatin competitively replaced the divalent cations to bind to LPS,thereby disrupting the integrity of OM and leading to bacterial death.Meanwhile,thanatin could release the NDM-1 into the culture supernatant in a time-and concentration-dependent manner by permeabilization.3.Thanatin inactivates NDM-1 by displacing Zn²⁺and reverses carbapenem resistance in vitro and in vivoWe found the hydrolysis of the supernatant to imipenem was decreased with increasing thanatin concentration.MST results showed that thanatin exhibited approximately 10times higher affinity to NDM-1 than Zn²⁺,indicating the direct interaction between NDM-1 and thanatin.Then,we proved that thanatin could directly inhibit enzymatic activity of NDM-1 as a competitive inhibitor.The results of ICP-MS and Zn²⁺restoration assays proved that thanatin inactivated NDM-1 by displacing Zn²⁺.Finally,we found that thanatin,as an NDM-1 inhibitor,could effectively restore the susceptibility of NDM-1-producing bacteria to imipenem and meropenem in vitro and in vivo.4.Thanatin disrupts the integrity of OMV and blocks the horizontal transfer of blaNDM-1Classical ball-like structure of the OMVs obtained from NDM-1-producing E.coli XJ141026 was observed under TEM.Size analysis revealed that the diameter of OMV was about 146.2 nm.PCR and Western-Blot analysis also proved that OMV contained bla_NDM-1 and NDM-1 protein.Then,the result of membrane permeability assay showed that the membrane integrity of OMV was damaged by thanatin in a concentration-and time-dependent manner.What’s more,NDM-1 protein resisted treatment with proteinase K in intact OMVs,whereas the NDM-1 was readily degraded in thanatin-treated vesicles.Then,we found that transformants showed elevated MIC values to imipenem and meropenem after incubated with intact OMVs,and the high expression of bla_NDM-1 and NDM-1 in transformants was confirmed by PCR and Western-Blot.These results indicated that OMVs could serve as a delivery system for antibiotic resistance.However,the OMV-mediated transformation was blocked by thanatin.The transformants were still sensitive to imipenem and meropenem with lower expression levels of bla_NDM-1 and NDM-1 protein after incubated with thanatin-treated OMVs.These results indicated that thanatin could rupture OMVs carrying bla_NDM-1 and cut off the resistant transmission.Conclusion1.Thanatin exhibits potent antibacterial activity against NDM-1-producing E.coli and K.pneumoniae strains in vitro and in vivo with low toxicity.2.Thanatin competitively replaces the divalent cations to bind to LPS,thereby disrupting the integrity of OM and leading to bacterial death.3.Thanatin inhibits the enzymatic activity of NDM-1 by displacing Zn²⁺as a competitive inhibitor.4.Sub-MIC of thanatin restores the susceptibility of NDM-1-producing E.coli and K. pneumoniae strains to imipenem and meropenem in vitro and in vivo.5.Thanatin ruptures outer membrane vesicles carrying bla_NDM-1 and blocks the resistant transmission.