Study Of Effects And Mechanisms Of Sub-MIC Antimicrobial Agents On Etiological Behaviors Of Acinetobacter Baumannii In Vitro

BackgroundAcinetobacter baumannii is one of the opportunistic pathogens which can live in the environment.It can also live in the human bodies.Under certain conditions,A.baumannii could cause severe infection through entering human tissues such as lung and blood,especially in immunocompromised patients in ICU.Unlike most Gram-negative bacteria,A.baumannii has a strong adhesive ability to colonize the infected site and can form bacterial biofilms or invasion into cells,resulting in higher drug resistance and evade from host immunity.On the one hand,the incidence of A.baumannii infection has been high in recent years and has become one of the most common clinical pathogens.On the other hand,the outcome of A.baumannii infection is poor and the choice of medication is very limited.A.baumannii mainly causes nosocomial infections,most often involving the respiratory tract.Its infections often were secondary to the compromised immune system,when most patients were treated with antimicrobial agents.Currently,clinical isolations of A.baumannii have a high antibiotic resistance rates for most antimicrobials over 50%.The conventional treatment of A.baumannii make the pathogen exposure to sub-inhibitory concentrations(Sub-minimum inhibitory concentration,sub-MIC)levels.Even for sensitive drugs,there is an inevitable sub-inhibitory concentration phase before the therapeutic concentration is reached.However,there are few studies on the influence of sub-MIC drugs on clinical isolates of A.baumannii.Numerous studies have suggested that antimicrobial agents is origin from small molecules released by bacteria,which can affect the bacterial on a series of physiological and pathological behavior,such as proliferation,adhesion and bacterial biofilm formation Etc.These regulations can significantly affect the occurrence and process of infection.However,the sub-MIC drug on A.baumannii infection remain unknown.Is it good or bad? Can the medicine option help prevention or successful treatment of infection? At present,there is no enough researches to give answers.Combined with the research progress on the influence of sub-MICs on bacterial etiology at home and abroad,this study aimed to study the effects of sub-MIC drugs on adhesion colonization,biofilm formation and invasion of A.baumannii And its mechanism.The results of this study can enrich the phenomenon and theory that sub-MIC affects the physiological and pathological behaviors of pathogen,and contribute to the rational use of antimicrobial agents in medical institutions that widely disperse A.baumannii such as ICU.ObjectiveTo study the effect of antimicrobial agents on the pathogenicity of biofilm formation,adhesion and invasion of A.baumannii at the sub-inhibitory concentration and its mechanismMethods1.Clinical study of drug resistance and epidemiology of A.baumanniiA total of 773 clinical isolates of A.baumannii were collected,of which 697 were from Henan Provincial People's Hospital,and 75 clinical isolates were from the Xinqiao Hospital of People's Liberation Army Military Medical University(formerly the Third Military Medical University).The minimum inhibitory concentration of clinical isolates was determined by agar plate dilution method.Due to the limitation of samples,the epidemiological study was conducted to select the isolates of Henan Provincial People's Hospital,which met the needs of sample size and high resistance.The methods included the identification of main resistance genes and multi-site sequence typing(MLST).The detection of carbapenemase resistance genes were performed using the PCR amplification,and MLST typing was performed to obtain alleles and ST by seven housekeeping genes amplification,sequencing and sequence alignment.2 Effects of sub-MIC antimicrobial agents on the biofilm formation,adhesion and invasion of A.baumannii to human respiratory epithelial cells(HBE)Based on the drug resistance,10 strains of representative clinical isolates of MDR were selected,which had strong drug resistance and significant biofilm formation.The effects of sub-antibacterial concentrations on the biofilm formations of A.baumannii were studied by 15 antibiotics.The agents which showed significant inhibitory effects were used to be further tested with A.baumannii ATCC 17978 strain.Microtitle-plate assay(24 h)was used to test biofilm formation,and the biofilm study model Flow-Chamber system,which was closer to the in vivo environment,was used to study the inhibition effect on biofilm formation(72 h)through the standard strain ATCC 17978 and representative clinical strainWB1549 by using azithromycin,which was found to have significant biofilm inhibition in the early screening.It was confirmed that the inhibitory concentration of azithromycin had a significant inhibitory effect.HBE cells were cultured to establish a model of cell infection of A.baumannii in vitro.The effects of sub-MIC antimicrobials on the adhesion and invasion of A.baumannii to HBE cells were investigated.The determination and observation of the amount of adherent bacteria were carried out by plate colony counting method and laser confocal fluorescence staining(CLSM)method.The determination of the amount of invasive bacteria was carried out by plate colony counting method.3 The mechanisms of the effect of sub-MIC antimicrobial agents on the pathogenicity of A.baumanniiThe whole genome expression profile was analyzed by RNA-seq method.The changes of all-gene transcriptase of ATCC 17978 treated with or without AZM were compared.The selected 9 key genes were identified by QPCR method in ATCC 17978 and WB1549 clinical isolate,respectively.The effects of AZM on the pathogenic behavior of biofilm formation,adhesion and invasion of host cells were investigated by bacteria swimming test,scanning and transmission electron microscopy.The effects of AZM on the expression of IL-8,TNF?,and Toll-like receptor 2,4 in HBE were studied by ELISA or Western Blot,and then the changes of bacterial invasion behavior treated with or without AZM by specific inhibition were further studied,so as to explore the mechanism of its movement and adhesion.Results1.The resistance of A.baumannii has a tendency to accelerate make the sub-MIC condition is more common when the treatment carried.(1)Drug resistance study showed that except for minocycline and other less-used antimicrobials,the resistance rate of A.baumannii in Henan Provincial People's Hospital was increased significantly in recent 10 years.Meanwhile,clinical isolates in Xinqiao Hospital had high sensitivity to tigecycline,which may contribute to their strict administration in tigecycline use.(2)Among the 697 A.baumannii strains,49 ST types were found,including both common genotypes in China and uncommon genotypes.And it is a rare situation that seven NDM-positive strains were found in the same hospital.2.Some sub-MIC antibiotics including AZM showed inhibitory effects on biofilm formations of MDR clinical isolates,which had similar effects of adhesion and invasion to HBE cells.(1)Generally,different sub-MIC antimicrobial agents showed different effects on specify strains.But nine antibiotics including azithromycin and amikacin showed the inhibitory effect on biofilm formation of MDR A.baumannii in more strains.Azithromycin showed the most common inhibitory effects onbacterial biofilm formation.Similar effect was observed by azithromycin on ATCC 17978.The inhibition of the biofilm was further verified in the classical flow chamber system(including ATCC 17978 and clinical strain WB1549).(2)Infection model of A.baumannii in vitro was established by incubating HBE cells in vitro.The effects of amikacin,azithromycin and cefepime on ATCC 17978 and WB1549 were observed by plate counting and laser confocal observation,which showed significant inhibitory effects.(3)Amikacin and azithromycin showed a significantly inhibitory effect on ATCC 17978 and WB1549 clinical isolates invading HBE cells,and the effect of azithromycin was relatively stronger.3.Possible mechanisms of altering of pathogenicity of A.baumannii by AZM.(1)Genome-wide expression analysis showed that the expression of A.baumannii was significantly changed when treated with 0.25 ?g/mL AZM,of which 264 were significantly up-regulated and 422 were significantly down-regulated(P < 0.05).GO analysis showed that the genes that were significantly inhibited were mainly composed of outer membrane proteins such as movement,adhesion,biofilm formation and maintenance of cell structure,protein synthesis,protein translation and oxidative stress.(2)Genes of movement,adhesion,biofilm formation related to the outer membrane,were verified in this study,which were reported to have significant changes.Both in ATCC 17978 and in clinical isolates of WB1549,OmpA,bmfs and other genes showed a significant difference in expression.(3)Swimming test showed that the mobility of A.baumannii was inhibited by AZM.The results of gene expression suggested that it was related to the inhibition of type ? pili synthesis.(4)Morphological observation of adhesion and invasion of A.baumannii after AZM-treated suggested that the mode of interaction between host cells and bacteria may change."Zipper-like" model of invasion into HBE by A.baumanni was inhibited."Zipper-like" model was host cell actin-mediated bacterial internalization,and the use of act in inhibitor cytochalasin could make the inhibition of AZM-treated bacterial invasion disappeared,suggesting that ligands in the outer membrane of A.baumannii that bind to HBE cell surface receptors were reduced when treated with AZM.(5)Compared with AZM-untreated A.baumannii,AZM-treated A.baumannii showed a significant inhibition effect on HBE cells in releasing IL-8,while no significant changes were found in TNF?.AZM-treated A.baumannii showed a lower Toll-like receptor 2 expression after being infected with HBEs for 6 h,suggesting that the ligand which can be recognized by the Toll-like receptor was decreased after AZM treatment.Since the gene expression of LPS biosynthesis has not been observed to be significantly changed,and due to the host cell immune response time delay,we speculate that AZM indirectly reduces the agonism of Toll-like receptors by inhibiting the biosynthesis of the outer membrane proteins associated with the movement and adhesion of type ? pilus,OMPA,PBPs and reducing the direct effect of bacterial-host cells,and then inhibit its bacterial adhesion and invasion to host cells.ConclusionsIn recent years,the resistance of A.baumannii has rapidly increased,and the resistance genes spread fast,so the options of treatment on A.baumannii infections were limited.In the study,we find that sub-MIC azithromycin,amikacin and some other antimicrobial agents are difficult to clear A.baumannii,but significantly inhibit the formation of their biofilms.And further studies show that azithromycin inhibit ATCC 17978 and clinical isolates to adhere or invade into HBE cells.Azithromycin significantly inhibits bacterial adhesion,biofilm formation,and invasion related gene,attenuates mobility of A.baumannii,reduces the activation of Toll-like receptors in host cells,and weakens the actin-mediated HBEs' internalization of bacteria.Results of this study suggests that AZM can greatly affect the membrane structure and related functions of A.baumannii,such as the expression of outer membrane proteins type ? pilus,OMPA and PBPs,and can significantly inhibit pathogenic behaviors of biofilm formation,adherence and invasion.Azithromycin is one of the most commonly used drugs for the treatment of respiratory infections,and its exposure to Acinetobacter baumannii is often at the level of sub-MIC,which will inevitably affect the pathogenic behavior of the bacteria and even affect the progress of infection treatment.The results of this study may provide a good foundation for the further study of azithromycin in sub-MIC,and provide useful references for the clinical use of antimicrobial agents more rationally and effectively.