| The infections of multidrug-resistant Acinetobacter baumannii have become a worldwide public health problem with high mortality rate and huge medical burden.In recent years,mucoid multidrug-resistant A.baumannii(MDRAB)has gradually increased in clinical practice.As to antimicrobial therapy,colistin and tigecycline are generally regarded as the last line of the therapy for multidrug-resistant A.baumannii infections.However,during the antibiotics therapy,colistin-resistant and tigecycline-resistant A.baumannii have emerged.Eravacycline is a novel synthetic fluorocycline of the tetracycline class and has greate activity against multi-drug resistant bacterium.So this study will be divided into two parts to study the biological characteristics of mucoid multi-drug-resistant A.baumannii and the resistance mechanisms of multi-drug resistant A.baumannii to Eravacycline.In the first part,we collected 60 strains of A.baumannii from 56 clinical patients,including 56 mucoid-type isolates and 4 matt-type isolates.The clinical data of the patients were collected and analyzed retrospectively.The morphology of the colonies and capsules were observed.The antibiotics susceptibilities were tested,and the biofilm formation ability was determined by crystal violet staining.The whole genome sequencing(WGS)was performed on all the strains,and then the core genome multilocus sequence typing(cgMLST)and drug resistance gene analysis were performed.Finally a part of isolates were selected to test virulence in a Galleria mellonella model.The results revealed that mucoid-type A.baumannii infections led to a serious course of diseases,difficult treatment and poor prognosis.The colonies of mucoid A.baumannii were moist and viscous,easy to fuse,and can be drawn.Almost all mucus-type A.baumannii(98.2%)were multi-drug resistant isolates,and the resistance rates of cefoperazone/sulbactam,tigecycline and colistin were relatively lower,15.0%,35.0%and 3.3%respectively.And Eravacycline had lowest resistance rate of 1.7%.Compared with the matt-type A.baumannii,the mucoid-type A.baumannii had a distinct capsular structure,but there were no difference in growth rate and biofilm-forming ability.According to cgMLST analysis,the main ST types of mucoid-type A.baumannii were ST191 and ST195,of which ST191 isolates were more virulence,while ST 195 isolates were weaker.In the second part,a series of in vitro eraccycline passage experiments were carried out on multidrug-resistant A.baumannii MDR-ZJ06,and two Eravacycline-resistant strains ZJ06-1-E5 and ZJ06-2-E6 were obtained.The whole genome sequences of resistant isolates were sequenced and putative mutations were verified via PCR and Sanger sequencing.It was found that the gene adeS had a three-base deletion mutation in ZJ06-2-E6,which may be related to Eravacycline resistance.The efflux pump inhibition experiment proved that ZJ06-2-E6 was positive for the efflux pump,and further analysis of the relative expression of the efflux pump by qRT-PCR revealed that the efflux pump adeABC was overexpressed in the resistant strain.The expression level of efflux pump adeABC was restored to normal and the MIC of,eravcycline in ZJ06-2-E6 reduced 4-fold when complemented ZJ06-2-E6 with wild-type adeS.These results indicated the deletion mutation in adeS,leading to the overexpression of efflux system adeABC,had played a role in decreased susceptibility to Eravacycline in A.baumannii.In summary,the mucoid-type A.baumannii had a distinct capsular structure,but its growth rate and biofilm-forming ability were similar to matt-type A.baumannii.The main ST types of mucoid-type A.baumannii were ST191 and ST195,of which ST191 isolates were more virulence,while ST 195 strain is weaker.Almost all mucoid-type A.baumannii were multi-drug resistant bacteria with the lowest resistance rate to eravacycline.The deletion mutation of adeS mediated the up-regulation of efflux pump adeABC,which may be one of the important mechanisms of eravacycline resistance in MDRAB. |
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