The Role Of Whole Genome Sequencing In Predicting Antimicrobial Susceptibility Of Carbapenem-resistant Acinetobacter Baumannii

Objectives:The widespread of carbapenem-resistant Acinetobacter baumannii?CRAB?has deeply increased the pressure of clinical anti-infective treatments.With the decreasing in cost and turnaround time of whole genome sequencing?WGS?,genomic epidemiological study provides a new perspective for effective monitoring and preventing the spread of multi-drug resistant bacteria in clinical settings.The aim of this study is to invesitigate the genomic epidemiological characteristics of CRAB strains recovered from two tertiary hospitals in Hangzhou.The feasibility of whole genome sequencing in the prediction of antimicrobial susceptibility of CRAB isolates was also evaluated.Methods:A total of 68 A.baumannii isolates were collected from Zhejiang Provincial People's Hospital and the 117 Hospital of People's Liberation Army,from January 28,2015 to June 1,2018.The minimum inhibitory concentrations?MICs?for each strain against 13 antimicrobial agents were determined by agar dilution method and broth dilution method.Whole genome sequencing of these isolates were performed using Illumina Hi Seq X Ten.Following sequencing,shovill was used to trim low-quality bases and to perform the de novo assembly.The size of contigs less than250 bp was removed from the assembled genome.Phylogenetic relationship of 68 A.baumannii isolates were analyzed using Bac WGSTdb and Snippy,based on core genome multilocus sequence typing?cg MLST?and core genome single nucleotide polymorphism?cg SNP?strategies.Res Finder database was used to identify acquired antimicrobial resistance genes by Abricate.Further bioinformatics analyses,such as identification of IS elements and the copy number of each antimicrobial resistance gene,were predicted by using ISsaga and Ariba.Expression of antimicrobial resistance genes in each strain were detected by high-throughput real-time quantitative PCR.Finally,these data were analyzed by SPSS statistical software?Version 23.0?and visualized by ggpolt2 package in R script.Results:Antimicrobial susceptibility testing?AST?data showed that the resistance rate of CRAB against minocycline was 36.76%.Most of antibiotic resistance rates were higher than 75%,except for tigecycline and colistin.The strains can be classified into10 STs based on Oxford MLST scheme,which incorporate 7 existing STs and 3 novel STs.Among them,ST208 was the most populated sequence type,followed by ST191and ST195.A total of 65 strains belonged to A.baumannii CC92 clonal complex.We also found that the strains belong to rare STs were more susceptible to most commonly used antimicrobial agents than isolates belong to CC92?P<0.05?.Most of the strains were closely related,except for three isolates?A52,A42 and A22?,which indicated a high clonal homogeneity between the two hospitals.These isolates can be divided into two major clusters based on cg SNP analysis,which differed from each other by 1?630SNPs.The minimum spanning tree based on cg MLST allelic profiles showed that these isolates can be grouped into 4 cluster,which differed by a maximum of 10 alleles.The percentage consistencies between different antimicrobial resistance phenotypes and antimicrobial resistance determinants were quite different.According to the classification of antimicrobials,the percentage consistency was 97.06%for?-lactams,92.65%for aminoglycosides,73.53%for macrolides,77.94%for sulphonamides,and 77.94%for tetracyclines.The coefficient of agreements,such as the presence/absence of the antimicrobial resistance genes,were higher for the phenotype of S/NS than that of R/I/S.Some of them possesed a good prediction ability for antimicrobial resistance,i.e.S/NS of five?-lactams?IPM,FEP,CAZ,CTX and PIP?can be associated with bla OXA-23?1.000?.For tetracyclines,tet B performed well agreement?1.000?with S/NS of minocycline.The vast majority of antimicrobial resistance genes of CRAB maintained a single copy,and a few appeared two or three copies.The results of multiple linear regression analysis showed that the adjust R square of models,established by the expression of antimicrobial resistance gene of CRAB and the?MIC?of antibiotics,were the lowest in the three models.For example,the adjust R square of models,established by presence/absence,copy number,expression of antimicrobial resistance resistance genes and the?MIC?of imipenem,were 0.756,0.470 and 0.160,respectively.Conclusion:The A.baumannii clonal complex CC92 was prevalent in the two clinical settings.Our study confirmed the feasibility of WGS in predicting the antimicrobial susceptibility phenotype of CRAB;especially the presence/absence of antimicrobial resistance genes showed a good predictive ability of the S/NS phenotype,i.e.both bla _OXA-23 and tet B gene are promising markers for the prediction of?-lactam and minocycline resistance.