Studies On The Resistance Mechanism And The Resistance Gene Related Sequence Structures Of A Multi-drug Resistant Klebsiella Pneumoniae Strain KP1276

Objective:Klebsiella pneumoniae(Klebsiella pneumoniae,Kpn)is an important opportunistic pathogen causing iatrogenic infection.It can cause typical primary pneumonia and other infectious diseases.To study the drug resistance of Klebsiella pneumoniae and its molecular mechanism of dissemination is of great significance for controlling the drug resistance and dissemination of clinical Klebsiella pneumoniae as well as guiding clinical rational drug use.Method:The genome of Klebsiella pneumoniae was sequenced by using the next generation sequencing technologies(HiSeq-2500 andPacBio RS single molecule real-time sequencing platform).The comparative genomics method was used to analyze the variation and evolution of drug-resistant plasmids and drug-resistant gene-related mobile genetic elements.The molecular cloning technology was conducted to clone the resistance genes.The agar dilution method was used to detect the resistance levels(minimum inhibitory concentration,MIC)of the strains(including the cloned genes).The tranferability of the plasmid was detected through conjugation experiment and by pulse field gel electrophoresis(PFGE).Result:The results of this work include the followings:1.The Klebsiella pneumoniae genome consists of a chromosome and three plasmids,the chromosome is 5.4 Mb in length and encodes 4,998 coding sequences,and the three plasmids were about 228 kb(pKP 1276-228),182 kb(pKP1276-182)and 40 kb(pKP 1276-40)in length,encoding 264,222 and 59 genes,respectively.2.The KP1276 genome encodes 12 resistance-related genes.The chromosome encodes four resistance genes(fosA,blaLEN,oqxA and oqxB),and pKP 1276-182 encodes seven resistance genes(blaKLUC,blacARB,aadA1,aadA2,cmlAl,dfrA1 and sul2),while pKP 1276-40 encodes one resistance gene(aph(3’)-Ia).It was the first time to identify blaKLUC and blaCARB in Klebsiella pneumoniae.3.Compared with genomic sequence data collected from the NCBI database,we found that pKP 1276-182 had highly similar bone sequence with 8 plasmids previously isolated from different species of bacteria,including pIP1202,pP91278,pp99-018,R222,pVC1447,psrc119-a/C and 2 unnamed plasmids.4.pKP 1276-182 was a conjugative plasmid.All the resistance genes encoded on pKP1276-182 were located in the region between 43kb to 90kb.These resistance genes,together with transposase or integrase genes,constitute transposons and integrons(including a type 1 integron and a type 2 integron).5.klebsiella pneumoniae KP1276 was a multi-drug resistant bacterium.Cloning and functional testing of some known resistance genes showed that the blaLEN,6laCARB-2,blaKUC,aadA2 and cmlA1 genes had antibacterial activities.Conclusion:The resistance of klebsiella pneumoniae KP1276 to beta-lactams,aminoglycosides and Chloramphenicols is closely related with the resistance genes encoded on the conjugative plasmid pKP 1276-182.blaCARB and blaKLUC emerge in Klebsiella pneumonia and they are related with the mobile genetic elements and encoded on conjugative plasmid.These findings indicated that the resistance genes in klebsiella pneumoniae may be transferred horizontally between the same and different species of Enterobacteria,that might cause the spread of drug resistance.Attentions should be paied to monitor and prevent the spread of the resistance genes.