| Objective: To study the antibacterial effect of American peppermint essential oil on carbapenem-resistant Acinetobacter baumannii(CRAB)and its possible mechanism,so as to provide reliable treatment basis for the outbreak of CRAB.Method:1.The clinical isolated CRAB strains were collected and screened for CRAB by VITEK-2 Compact automatic microbiological analyzer and matching identification cards and drug sensitivity cards.According to the 2020 CLSI standard,the strains with low sensitivity to carbapenem antibiotics(imipenem,meropenem,ertapenem)were selected and checked by disk method as experimental strains. The tested strains were analyzed by mass spectrometry with automatic biomass spectrum detection system.2.The sensitivity of essential oil to CRAB was determined by Kmurb method,and the MIC value and MBC value of American peppermint essential oil were determined by microbroth dilution method.3.The chessboard titration experiment determined the synergistic effect of American mint and polymyxin B on CRAB,and the concentration of essential oil used in combination was 8-2 times of the MIC value of essential oil.4.Time growth curve to monitor the germicidal efficacy of American peppermint on CRAB at different time points within 24 hours.5.The inhibitory effect of essential oil on CRAB biofilm was determined by crystal violet staining biofilm inhibition experiment.6.The change of intracellular ATP content was measured by bioluminescence method.7.The changes of PH value and membrane potential of bacteria treated with essential oil were measured by flow cytometry.8.The morphological changes of cells treated with essential oil were observed under scanning electron microscope.Results:1.Distribution of strains: 48 strains of Acinetobacter baumannii: 38 strains of sputum(79.1%),1 strain of secretion(2.1%),1 strain of blood(2.1%),1 strain of bile(2.1%)and 7 strains of other specimens(14.6%).28 strains(58%)from intensive care units(Intensive Care Unit,ICU),8 strains(17%)from neurosurgery,9 strains(19%)from respiratory and critical care units,2 strains(4%)from plastic burns and cosmetology,and 1 strain(2%)from emergency medicine.2.The main ingredients of American peppermint essential oil: thymol,Yterpinene,o-cymene,1-octene-3-ol,2-methoxy-5-methyl-cumene,linalool,4-carene,3-cyperene,myrcene.The content of thymol can reach 69.75%.3.Analysis of the identification results of mass spectrometry detection system:48 test strains were further identified as Acinetobacter baumannii after mass spectrometry analysis.4.48 strains of CRAB were sensitive to American peppermint essential oil,and the paper containing 5%DMSO had no bacteriostatic effect,so using DMSO as cosolvent to dissolve essential oil had no effect on this experiment.5.The minimum inhibitory MIC of 48 strains of CRAB essential oil was 0.625μ l / ml,while the control group containing 2.5%DMSO had no inhibitory effect on bacterial growth.The MBC range is 0.625 μ l / ml～ 1.25 μ l / ml.6.Effect of essential oil on the growth kinetics of CRAB: CRAB did not grow at all concentrations within 0-2 h.From 2 h,CRAB grew gradually at the concentration of 4MIC and 16 MIC,CRAB grew slowly at the concentration of1/2MIC at 4h,and CRAB was completely inhibited under MIC,2MIC.7.Analysis of intracellular ATP concentration: the content of ATP in CRAB treated with peppermint MIC was lower than that in untreated CRAB,and the content of ATP in CRAB after 2MIC treatment was further lower than that in negative control group.8.PH value result analysis: The average p H of untreated CRAB cells was 6.27±0.12,and the p H value of cells treated with American mint MIC decreased from 6.27 ±0.12 to 4.33 ±0.31.After adding 2MIC American mint,the p H value further decreased from 6.27 ±0.12 to 3.23 ±0.21 compared with the negative control group.9.Analysis of the change of membrane potential: the cells treated with American peppermint showed membrane hyperpolarization and decreased fluorescence intensity(negative value).10.The effect of American mint on CRAB biofilm formation: the biofilm formation of CRAB was slightly inhibited under the action of 1max 4MIC and8 MIC and 16 MIC,and the decrease of biofilm formation under the action of2 MIC MIC was larger than that under 2MIC.11.The effect of American peppermint on the morphology of CRAB cells:FESEM analysis showed that the CRAB without American peppermint treatment was ball-rod-shaped and smooth.After being treated with American mint MIC and 2MIC,the surface of CRAB was collapsed.12.The results of combined antibacterial experiment showed that 23 strains(48%)had synergistic effect,13 strains(27%)had additive effect,12 strains(25%)had unrelated effect,and 0 strain(0%)had antagonistic effect.Conclusion:1.American peppermint essential oil has good antibacterial activity against CRAB and combined with polymyxin B,it has synergistic antibacterial effect on most CRAB,and greatly reduces the dosage of polymyxin B,which provides an effective basis for the development of new drug use measures.2.American peppermint essential oil can destroy the integrity of CRAB cell membrane,resulting in the decrease of intracellular ATP concentration,PH and membrane potential in CRAB cells.3.American peppermint essential oil can inhibit the formation of CRAB biofilm,and the stronger the anti-biofilm activity is with the increase of its concentration.4.Prospect: American peppermint contains too many different types of compounds,which can not be studied in detail because of the limited experimental conditions and time.In the future,we will make an in-depth study on the role of its main components or the synergistic antibacterial activity of some components,so as to provide more specific data for the development of traditional natural products against drug-resistant bacteria. |
PDF Full Text Request