Resistance Mechanisms Agaisnt Polymyxin B In Multidrug-resistant Acinetobacter Baumannii

Objectives: Detection of common ?-lactamase genes in multi-drug resistant Acinetobacter baumannii,and induction of Acinetobacter baumannii resistance to polymyxin B in vitro,to investigate the resistant mechanism among multiple-drug resistant Acinetobacter baumannii strains against polymyxin B.Methods: 1.Six-three clinical strains of multi-drug resistant Acinetobacter baumannii were collected in Southwest Medical University Affiliated Hospital,the minimum inhibitory concentrations(MICs)of Imipenem,Meropenem,Ceftazidime,Cefepim,Levofloxacin,Ciprofloxacin,Amikacin,Polymyxin B and Cefoperazone/ Sulbactam against the experimental strains were determined by micro-broth dilution method.2.The homology of those isolates was analyzed by enterobacteriaceae repeat gene conserved sequence-PCR(ERIC-PCR).3.The ?-lactamase genes including TEM,AmpC,OXA-23,KPC,IMP,VIM,efflux pump genes including adeB,adeG,adeJ,adeR,adeS,two-component regulatory systems genes including pmrA and pmrB were detected by PCR.4.Derivative clones resistance against polymyxin B were obtained by using in vitro induced experiment.5.The expression of the efflux pump genes adeB,adeJ,adeG,adeR,adeS and two-component regulatory systems genes Pmr A,pmrB in the clinically isolated strains and the induced polymoxin B resistant strains were detected by real-time quantitative polymerase chain reaction(RT-qPCR).6.The sequence of cell membrane lipid A biosynthesis gene lpxA/lpxC/lpxD and two-component regulatory systems genes pmrA,pmrB of the clinically isolated strains and the induced polymoxin B resistant strains were studied by using PCR amplification and sequencing of the relevant genes.7.The protein fingerprint profiles of the clinically isolated strains and the induced polymoxin B resistant strains were detected by MALDI-TOF MS.Results: 1.Among the 63 Acinetobacter baumannii isolates,the resistant rates of Imipenem,Meropenem,Ciprofloxacin and Amikacin were 100%;the resistant rate to ceftazidime and cefepime were 92%,97% respectively;and the resistant rate to cefoperazone sulbactam was 43%,while the susceptible rate to polymyxin B was 100%.2.After ERIC-PCR genotyping,63 strains of multi-drug resistant Acinetobacter baumannii were divided into 5 types,mainly for type A(58 strains,26 strains for type A1,32 strains for type A2,type B(2 strains),type C,D and E were 1 strain each;and most of the 63 strains were came from Intensive Care Unit and Respiratory Medicine Department.3.Among the 63 strains of multi-drug resistant Acinetobacter baumannii,50 strains were positive for TEM gene,58 strains were positive for AmpC gene,62 strains were positive for OXA-23 gene,none for KPC,IMP and VIM.Mreover,efflux pump genes adeB,adeJ,adeG,adeR,adeS were found in 61,62,62,60,60 strains respectively;and two-component regulatory systems genes PmrA,pmrB were found in 61,59 respectively.4.Eighteen polymoxin B resistant strains were induced and screened from thirty-three clinically mufti-drug resistant strains.MICs against polymoxin B of the induced strains arose from 0.5?g/ml to 256?g/ml,while the MICs of other antibiotics did not change.5.The expression of mRNA in the efflux pump genes showed the expression of adeB,adeG,adeJ were no significant difference in the clinically isolated strains and the induced polymoxin B resistant strains(P= 0.586,0.983,0.948 respectively);the expression of PmrA was increased in polymoxin B resistant strains(p=0.014),while the expression of Pmr B have no difference in the clinically isolated strains and polymoxin B resistant strains(p=0.472).6.PCR amplification and DNA sequencing of cell membrane lipid A biosynthesis gene lpxA/lpxC/ lpxD and two-component regulatory systems genes pmrA,pmrB identified a mutation of G to A in lpxC gene,this mutation will lead to an amino acid change(glutamic acid to lysine);and no other mutations were identified in lpxA,lpxD,pmrA and pmrB.7.In MALDI-TOF MS analysis,compared with the clinically isolated strains,the polymyxin B resistant strains were found a specific peak enhancement at 5177 Da and 6093 Da,and a specific peak weaken at 8049 Da.Conclusions: 1.While most clinically isolated Acinetobacter baumannii strains are Multidrug-resistant against commonly used antibiotics,but they are still susceptible to polymyxin B.2.The resistant mechanism of Acinetobacter baumannii to common antibiotics is related to the production of ?-lactamase and the expression of efflux pumps.3.The resistant strains against polymix B can be induced in vitro.The resistance mechanism of the induced strains may be related with high expression of PmrAB,and point mutation in cell membrane lipid A biosynthesis gene lpxC.