Antibacterial Effects And Mechanisms Of The Eugenol And Cinnamaldehyde On Legionella Pneumophila

Objective1.To investigate the antibacterial activity of eugenol and cinnamaldehyde against Legionella pneumophila and the inhibition of biofilm formation of Legionella pneumophila?2.To investigate the in vivo antibacterial activity of eugenol and cinnamaldehyde in L.pneumophila animal models.3.To investigate the antibacterial mechanism of eugenol and cinnamaldehyde against Legionella pneumophilaMethodsDrugs and StrainsLegionella pneumophila(serogroup 1,ATCC 33152)was presentation from Department of Respiratory Medicine,Shengjing Hospital of China Medical University,Eugenol and cinnamaldehyde Standard(purity 99%)was purchased from Shanghai YuanYe Biotechnology Co.,Ltd.AnimalsFemale-specific pathogen-free 6-8-week-old female A/J mice were purchased from Jackson Laboratories,They were housed in cages under constant temperature(22±2)?with a 12-h light/dark cycle(lights on from 8:00 to 20:00;20:00 8:00 lights)and given standard laboratory food and water ad libitum.Antibacterial activity assay in vitroDetermination of MIC and MBC was done by broth dilution method.Time-kill curve:different doses of the drug were added to the mixed broth to reach a final concentration of 1/2MIC,1MIC,2MIC,4MIC,8MIC,16MIC,32MIC,adding Legionella pneumophila(5×10~5CFU/ml),then it was incubated at 37?,taking 10 microliters of broth at 0h,0.5h,3h,6h,9h,24h respectively.After tenfold dilution it was inoculated on BCYE plate and incubated at 37?,colony counting was conducted 72 hours later.Time as the abscissa,the logarithm of the number of bacteria as the vertical axis,time-kill curve was drawn.Checkerboard method-microdilution method was used to determine the combined effect.FIC index=A drug MIC of combination/A drug MIC of alone+B drug MIC of combination/B drug MIC of alone.FIC index?0.5,>0.5~1,>1~2,>2 represents synergistic,additive,independent,antagonism respectively.Preparing biofilm,addition of different concentrations of drug at the beginning of the incubation was used to determine the effect of drugs on the formation of biofilm,addition of drugs after biofilm formation was to measure the bactericidal effect on the formed biofilm.Antibacterial activity assay in vivoL.pneumophila infection model:2×10~6CFU L pneumophila was injected in the trachea each mouse as the infected model.The same volume saline was injected as control group.One hour later,eugenol(160mg/kg/d)and cinnamaldehyde(120mg/kg/d)was injected intraperitoneally two consecutive days as treatment group.Each group comprised ten mice.L.pneumonia infected model of 10~8CFU dose(five mice each group)was used to evaluate the survival rate for seven days observation.Sample collection:At 48h point,blood,liver,spleen,kidney,lung were aseptically removed,after homogenation of organ,10 microliter was inoculated on BCYE plate to count L.pneumophila colony,supernatant was assayed for inflammatory cytokines.Plasma was measured for endotoxin using LAL kit.Each left lung tissue was taken,4%paraformaldehyde fixed,paraffin-embedded,sections-sliced,then HE staining was done.Investigation of the mechanismDifferent concentrations of the drugs were incubated with the L.Pneumophila at37?for 60 minutes,then the OD260 of the supernatant was determined.Through different treatment,different concentrations of the drugs was incubated with the L.Pneumophila at37?for 30 minutes,then the supernatant was analysed by SDS-PAGE electrophoresis,silver staining and coomassie Brilliant Blue staining.Integrity of the bacterial envelope was assessed by the Fluorescence-based LIVE/DEAD Baclight Bacterial Viability kit.Transmission electron microscope was used to observe the morphology changes of L.Pneumophila.After the 20h growth of L.Pneumophila,its concentration reached about10~9CFU/mL,adding eugenol,cinnamaldehyde,37?incubated for 30min,centrifuged,and then the bacterial pellet was fixed in 2.5%glutaraldehyde.Sending to the Electron Microscopy Center of China Medical University for observation.ResultsAntibacterial activity assay in vitroDetermination of MIC and MBC:The MIC was equal to MBC for eugenol with the concentration of 0.0125%,the MIC of cinnamaldehyd was between 15~30?g/ml,MBC was between 30~60?g/ml.Time-kill curve:Eugenol could completely kill Legionella at concentration of 0.1%(8MIC)and the value above of the concentration n half an hour;at concentration of0.05%(4MIC),it could kill the legionella at 1.5 hours;concentration 0.0125%(MIC),the slope was negative which proved eugenol as bactericides.Cinnamaldehyde used 1.5hours to completely kill Legionella at concentration of 1000?g/ml(32MIC),used 24hours at 2MIC,the slope was negative when at concentration of MIC which proved also as bactericide,but the antibacterial efficiency was inferiorer than eugenol under the same>2MIC value.Combination Experiment:The FIC index of the Combination Experiment of eugenol and cinnamaldehyde was 0.375,<0.5,the two drugs had a synergistic effect when used in combination.Effects on biofilms:Eugenol of 1/2MIC,1/4MIC concentration could effectively inhibit the formation of biofilms of Legionella pneumophila,was statistically significant.Cinnamaldehyde showed no significant inhibitory effect on biofilm formation.Antibacterial activity assay in vivoLP load in lungs:The LP load in lung tissue homogenates was decreased significantly in the group of eugenol 160mg/kg/d treatment compared with the N.S control group at 48hours point after injection of bacteria,eugenol accelerated the clearance of Legionella in the lungs.The LP load was not decreased significantly in the group of cinnamaldehyde120mg/kg/d treatment.Dissemination rate of LP to other organs:The rate of the dissemination to blood and spleen was significantly reduced in the eugenol treatment group compared with the N.S control group,while this effect was not observed in cinnamaldehyde treatment group.Lung pathology:The alveolar interval space was edema,thickened,with lots of neutrophil infiltration and disorder,consolidation of alveolar structural in Legionella pneumonia model.Alveolar septa edema,neutrophil infiltration was alleviated in the eugenol treatment group(p<0.05)but not in cinnamaldehyde treatment group.Plasma endotoxin levels measurement:The endotoxin level in eugenol treatment group was significantly lower than the model group,the endotoxin levels were not significantly decreased in the cinnamaldehyde treated groups.Cytokines of the lung tissue homogenates:The TNF-?,IL-1?levels were significantly lower(p<0.05)in eugenol treatment group compared with the model group.In the cinnamaldehyde treatment group,only TNF-?was decreased(p<0.05),IL-1?,despite agthe average declined,but with no statistically significance.Survival:In the lethal Legionella pneumophila pneumonia(10~8CFU/mice),the mice of the untreated group all died in three days,cinnamaldehyde treatment group died in five days,eugenol treatment group survived one(40%)on the seventh day,compared with the untreated group it was significantly different.Investigation of the mechanismOD260 absorbed substance:the OD260 was significantly higher in the Legionella solution with eugenol at 0.4%,0.8%concentrations than the normal group.There was no significant increase in cinnamialdehyde,levofloxacin treated group.SDS-PAGE electrophoresis of the supernants:the supernants of various groups was performed SDS-PAGE electrophoresis,silver staining and,the results showed eugenol at0.4%,0.8%concentrations displayed multiple protein bands,the prominent band was38kDa,Coomassie blue staining also displayed the band,which illustrated that the 38kDa molecular was the earliest and most leaked protein when treated by eugenol.LIVE/DEAD Baclight bacterial viability:the bacteria with intact envelope was decreased significantly in the eugenol group than the cinnamaldehyde group at the same concentration of 4MIC,8MIC and 32MIC.TEM for morphology:The normal Legionella pneumophila grown on plate was rod,with typical Gram-negative bacilli envelope,wavy outer membrane.After 20h broth culture,the LP entered the postexponential growth phase with more evident wavy outer membrane,rich cytoplasm with uniform density.Moreover,inclusions appeared in the cytoplasma,which displayed as multiple empty holes in TEM.The outer membrane of Cinnamaldehyde(1mg/ml)treated LP is still visible,with high cytoplasmic density.The wavy outer membrane of LP with eugenol(0.4%)treated disappeared,with thin cytoplasmic,lower density,and visible leakage from damaged cytoplasmic membrane.Conclusions1.Eugenol,cinnamaldehyde had excellent antibacterial effect on Legionella pneumophila,eugenol demonstrated a strong inhibitory effect against the formation of Legionella.2.Eugenol demonstrated good antibacterial activity against Legionella pneumophila pneumonia in vivo.3.Eugenol exerted activity on the envelope of Legionella pneumophila,leading to cell membrane damage and bacterial death.Cinnamaldehyde did not significantly alter the permeability of the envolope of Legionella pneumophila.