| BACKGROUND AND AIMSPurpose:Acinetobacter baumannii is an important pathogen in the nosocomial infections worldwide.In recent years,this opportunistic pathogen has emerged as one of the main causes of hospital-acquired infections,such as ventilator-associated pneumonia,urinary tract infections,bloodstream infections and surgical wound infections.The risk factors associated with A.baumannii infections include invasive medical procedure,mechanical ventilation,immune suppression,burns and trauma.Additionally,A.baumannii is one of the most frequently isolated clinical pathogens in China.Carbapenems,such as imipenem and meropenem,either alone or in combination with other antibiotics are currently the most effective therapeutic options for A.baumannii infections.However,carbapenem-resistant clinical isolates of A.baumannii have notably increased in recent years,and have resulted in a delay in treatment.It is significant elucidate the molecular mechanism of resistance to carbapenem of A.baumannii for solving the prevailing issue.Acquired or endogenous carbapenemase activity,together with decreased outer membrane permeability and overproduction of efflux pumps constitute the causes of carbapenem resistance in A.baumannii clinical strains.Recently,efflux pumps resistance mechanism research in A.baumannii has generated considerable interest.To date,five super families of efflux pumps have been found associated with antibiotic resistance in A.baumannii:the resistance-nodulation-cell division(RND)family,16-18 the ATP-binding cassette(ABC)transporters family,the multidrug and toxic compound extrusion(MATE)family,19 the major facilitator super(MFS)family,and the small multidrug resistance(SMR)family.20,21 Among these pumps,three members of the RND family.AdeABC,AdeFGH and AdeIJK,are regarded as the most important for extruding a quite broad range of substrates.Carbapenemases combined with active efflux pumps and decreased permeability of membrane have been illustrated to be main mechanisms of carbapenem resistance.However,under the stress of carbapenem,how a clinical strain developed carbapenem resistance in A.baumannii remains unclear and few investigations have focused on the efflux pump mechanism as an independent contributor to carbapenem resistance in A.baumannii.This study aimed to investigate the contributions of different efflux pumps and outer membrane proteins in developing carbapenem resistance in a clinical isolate of A.baumannii and reveal the possible mechanism of overproduction of main efflux pumps.METHODS AND RESULTSMethods:Firstly,an imipenem-susceptible multidrug-resistant clinical strain was identified as A,baumannii and named SZE In this study.Then imipenem-selected mutants were selected from SZE strain using a serial sub-cultivation method on Muller Hinton(MH)plate through consecutive 58-subcultivations.Several common carbapenemases were detected by poly-merase chain reaction(PCR).And the Antimicrobial Susceptibility Testings(ASTs)were detected with and without the efflux pump inhibitor CCCP.Secondly,Gene expressions of four families of efflux pumps,five outer membrane proteins and blaOXA-51-like were determined by reverse transcription-quantitative PCR(RT-qPCR),and comparisons were made between SZE strain and the imipenem-selected mutants at transcriptional level.The efflux pump genes were adeB,adeG and adeJ(RND family),abeM(MATE family),abeS(SMR family),and cracA,amvA and tetB(MFS family).The outer membrane protein genes were carO,omp33,ompA,ompW,and oprD.Regulatory genes of RND type efflux pumps,including adeRS,adeL and adeN,were sequenced and aligned in SZE and its mutant.Last,expressions of efflux pumps and membrane proteins were compared at the protein level through iTRAQ(isobaric tags for relative and absolute quantitation)and 2D LC-MS/MS(two-dimensional liquid chromatography-tandem mass spectrometry).Results:Firstly,under consecutive imipenem-selected stress,the MIC to imipenem increased gradually from 0.125μg/ml to 8μg/ml in mutants of SZE strain.And the result of Antimicrobial Susceptibility Testing changed from "sensitive,S" to"intermediate,I".The effect of resistance inducement was almost neutralized when treated with an efflux pump inhibitor.And the acquisition of impinem-resistance might be attributed to efflux pump mechanism.Secondly,this isolate was both susceptible to imipenem and meropenem,without carrying any blaOXA-23-like,blaOXA-24-like,blaOXA-58-like and blaNDM-1 carbapenemase genes.And no ISAbal/blaOXA-51-like gene combination was found in this strain.And the expression of efflux pumps adeB,adeG,and adeJ increased 6.9-,4.0-and 2.1-fold in mutants,respectively compared to SZE strain.Under stress of the antimicrobial,a single mutation(G to A)in adeR at nucleotide 58 was detected in imipenem-selected mutant(G45).Such a replacement would lead to a substitution of aspartic acid to asparagine at position 20 of AdeR at the protein level and possibly up-regulated the adeB expression,and then affected the carbapenem resistance in A.baumannii strains.On the other hand,the relative expression of the selected outer membrane protein genes,including carO,omp33,ompA,ompW and oprD,did not changed significantly in the inducement.And lastly,through iTRAQ and 2D LC-MS/MS,several proteins of efflux pumps and membrance porions were identified and the relative expressions were compared in SZE strain and its impenem-selected mutans,and we found respectively five efflux pump proteins increased theis expressions and seven mebrane proteins decreased their expression significantly.CONCLUSIONUnder consecutive imipenem-selected stress in vitro,A.baumannii strain evolved the ability to reduce susceptibility to a variety of antimicrobials by overproduction of efflux pumps.It has been reported that imipenem is a potent inducer of multidrug resistance in A.baumannii.Especially,the resistance-nodulation-cell division(RND)super family and a nucleotide mutant in adeRS regulating system might cause the overexpression of adeABC. |
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