The Study Of The Role Of ?-lactamases In The Sulbactam Resistance Mechanisms Of Acinetobacter Baumannii

Multidrug-resistant Acinetobacter baumannii has been a serious challenge of anti-infections treatment in the world.Sulbactam should be a plausible option for treating multidrug-resistant Acinelobacter infections because of its intrinstic antibacterial activity,however,the rate of clinical Acinetobacter baumannii strain resistant to sulbactam or sulbactam-based compound has increased sharply in recent years,and the resistant mechanism of sulbactam has not been fully studied.The study aimed to reveal on the contribution of ?-lactamases in the sulbactam resistance of A.baumannii by divided three parts of contents.In the first part,PCR,Real-time fluorescence quantitative PCR,gene clone and whole-genome sequencing methods were used to study the(3-lactamase gene blaTEM-1 in the clinical A.baumannii strains in order to discover the contribution of the blaTEM-1 gene on the different sulbactam MICs of A.baumannii strains in aspects of the gene distribution,copy number and the expression level.A total of 2,197 nonduplicate clinical A.baumannii isolates were collected from 27 provinces in China from the nationwide survey program.Eighty-eight isolates were picked out by using multilocus sequence typing(MLST)and sulbactam MIC,all of which were from diverse genetic background.Among them,57 and 31 isolates were respectively distributed at the sulbactam-nonsusceptible and sulbactam-susceptible groups.When comparing positive rate of the blaTEM-1 gene,significant difference was found statistically between the sulbactam-nonsusceptible group and the sulbactam-susceptible group.The sulbactam MIC of transformant had increased to 32-fold after abtaining the cloned plasmid with blaTEM-1.In addition,a moderate association between the expression of and MIC of sulbactam(log2)was observed in the A.baumannii clinical strains(r=0.541;P<0.001).Promoter P4 was the only promoter ahead of the blaTEM-1 gene.Regarding the blaTEM-1 copy number,one strain,entitled ZS3,was the only one possessed multi-copy of blaTEM-1 with MICSUL=256 mg/L.ZS3 harbored four tandem copies of transposon structure(IS26-blaTEM-1D-Tn3-IS26)on chromosome with same direction.This part of result showed that blaTEM-1 played an important role in the development of sulbactam resistance in A.baumannii,and the increase of copy number might lead to development of sulbactam MIC.In Part ?,PCR,Real-time fluorescence quantitative PCR methods were used to study the distribution of gene type of ampC in the clinical A.baumannii strains and the effects of the the copy number,expression of ampCon the sulbactam MIC.The results showed that all the strains possessed the ampC gene,and the ampC-2(protein type:ADC-30)was the major gene type,and the level of increased sulbactam MIC conferred by different gene types of ampC was similar.In addition,the relative expression of ampC was significantly higher in the sulbactam-nonsusceptible group than in the sulbactam-susceptible group(P<0.001).The level of ampC expression was moderately correlated with the MIC of sulbactam(log2)in the A.baumannii isolates(r=0.553;P<0.001).All isolates possessed only one copy of ampC,the promoter ISAba1 was only located upstream of ampC-2(protein type:ADC-30)and ampC-20(protein type:ADC-73).The rate of ISAba1-ampC-positive strains in the sulbactam-nonsusceptible group was significantly increased compared with that in the sulbactam-susceptible group(78.95%vs.0%,P<0.001).The results illustrated that the role of ampC in sulbactam resistance is related to the presence of upstream promoter ISAba1,not the ampC gene type.ISAba1,as a strong promoter,conferred the sulbactam resisitance in A baumannii by upregulating ampC expression.The Part ? of this study mainly underwent whole genome sequencing method to explore novel ?-lactamases gene associated with the sulbactam resistance.Gene knockouts and complementational experiment,as well as Real-time fluorescence quantitative PCR were performed to verify the contribution of the novel mechanism.Comparing to sulbactam-susceptible group,the rate of blaOXA-23 was much higher than that in the sulbactam-nonsusceptible group.The blaOXA-23 gene knockouts(A2265?blaOXA-23)exhibited increased susceptibility to sulbactam(8-fold)compared to the previous strain(A2265),while the MIC of sulbactam recovered to previous level at or above of that of the complements.Furthermore,the sulbactam MIC did not correlate with the transposon types(Tn2006 and Tn2009)or copy number of blaOXA-23.Altogether,our result in this part proved that the ?-lactamase gene blaOXA-23 as a novel mechanism played an important role in the sulbactam resistance,but the level of sulbactam MIC did not depend on the transposon type or copy number of blaOXA-23.In conlusion,sulbactam resistance in A.baumannii is multifactorial and can be affected via blaTEM-1 ISAba1-ampC and blaOXA-23.This report is the first to identify four tandem copies of blaTEM-1D located on the A.baumannii chromosome,and the multiplication of blaTEM-1D may enhance the sulbactam resistance level by increasing the expression of the ?-lactamase TEM-1D.ISAba1,which is associated with ampC,plays a key role in sulbactam resistance by upregulating ampC expression.In addition,blaOXA-23 is the novel m echanism directly related to sulbactam resistance except blaTEM-1 or ampC.