Identification And MLST Typing Of Carbapenem-Resistant Klebsiella Pneumoniae By Tandem 16S RRNA Gene And Housekeeping Genes

Objectives:Carbapenem-resistant Klebsiella pneumoniae is an important clinical pathogen,often causing serious infection and even threatening the life of patients.Bacterial species identification and MLST typing are crucial for the diagnosis of clinical carbapenem-resistant Klebsiella pneumoniae infection and whether there is in-hospital transmission.The identification of bacterial species was mainly accomplished by the identification of 16S rRNA gene.As for whether there was in-hospital transmission,MLST typing method was used.Seven housekeeping genes of Klebsiella pneumoniae were amplified and sequenced,and ST types of the bacteria were obtained by comparing their allele numbers,so as to determine whether there was clonal transmission among clinical isolates.The aim of this study is to establish an experimental method--Multi+Overlap PCR method,which series 16S rRNA gene and housekeeping genes,and conduct MLST typing while identifying carbapenem-resistant Klebsiella pneumoniae strains.In addition,the molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae isolates from Yunnan Province in recent years were investigated.Methods:1.Establishment of Multi+Overlap PCR method:Using carbapename-resistant Klebsiella pneumoniae genomic DNA as template,the 16SrRNA gene and seven housekeeping genes of Klebsiella pneumoniae（pgi,mdh,gap A,Pho E,inf B,rpo B and ton B）were amplified and connected into large fragments by three rounds of PCR.Through sequencing and comparison of large fragments,The16SrRNA gene of bacteria can be identified and its MLST typing can be obtained.The specific operations are as follows:the first round of octuple PCR was performed to simultaneously amplify 16SrRNA gene and seven housekeeping genes of Klebsiella pneumoniae in the same system.Magnetic bead purification was performed by nucleic acid purification kit to remove redundant primers and primer dimers in reaction products.In the second round of fragment linking PCR,the recovered product of magnetic bead purification was used as the template,and the principle of overlapping extension PCR was used to connect each gene fragment in the eightfold PCR product,and the large fragment formed by the interconnection of each fragment was obtained.The third round of full-length fragment PCR uses the large fragment obtained by fragment linking PCR as the template,and uses a pair of peripheral primer to specifically amplify the full-length fragment connected by eight target genes in the set order.The full-length fragment of about 4.5kb was obtained,which contains the 16S rRNA gene and seven housekeeping genes of Klebsiella pneumoniae.By sequencing the full-length fragment,the bacteria strains and MLST typing could be identified simultaneously.2.Verification of Multi+Overlap PCR method:The 16S rRNA gene and seven housekeeping genes of carbapenem-resistant Klebsiella pneumoniae were amplified one by one used the conventional PCR method,and then sequenced and compared to obtain the 16S rRNA gene sequence and MLST typing of the bacteria.Meanwhile,the Multi+Overlap PCR method established in this study was used for strain identification and typing,and the results of the two methods was compared respectively to verify the accuracy of results of Multi+Overlap PCR method.3.Clinical isolates of carbapenem-resistant Klebsiella pneumoniae detected in hospitals in Yunnan were collected,basic clinical data were analyzed,and drug-resistant genotypes,16S rRNA genes and MLST genotypes were identified by conventional PCR method,and molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae in Yunnan were studied.Results:1.This study established an experimental method,which can connect16S rRNA gene and seven housekeeping genes of Klebsiella pneumoniae into a large fragment sequence.Through sequencing,carbapenem-resistant Klebsiella pneumoniae was identified and MLST typing was conducted at the same time.The specific method was as follows.The first step was eightfold PCR.25μL amplification system was set as mix=12.5μL,genome DNA=1μL,H₂0=11.04μL,primers corresponding to each gene were 16S rRNA=0.08μL,pgi=0.05μL,mdh=0.12μL,gap A=0.10μL,Pho E=0.08μL,inf B=0.08μL,rpo B=0.05μL and ton B=0.17μL.The amplification procedure was predenaturation 98℃-2 min,denaturation 98℃-10sec,annealing and extension 70℃-30 sec（35 cycles）,final extension 70℃-5 min,The multiple amplified product was purified and recovered by magnetic bead purification kit.The second step was fragment connection PCR,the 25μL amplification system was set as mix=12.5μL,the recovered eightfold PCR product=8μL,H₂0=4.5μL,and the amplification procedure was as follows:Predenaturation 98℃-2 min,denaturation 98℃-20 sec,annealing 52℃-15 sec,extension 72℃-90 sec（40 cycles）,final extension 72℃-5 min.The third step was full-length fragment PCR,25μL amplification system was set as mix=12.5μL,fragment link PCR product=1μL,peripheral primers=0.125μL,H₂0=11.25μL,and the amplification procedure was as follows:Predenaturation 98℃-2 min,denaturation 98℃-10 sec,annealing and extension 70℃-30 sec（35 cycles）,final extension 70℃-5 min.2.For 8 carbapenem-resistant Klebsiella pneumoniae clinical isolates,strain identification and MLST typing were carried out using the method established in this study and the conventional PCR method,and the results obtained by the two methods were completely consistent after comparison,confirming that the method established in this study has the accuracy required for laboratory research.3.From September 2015 to August 2022,a total of 55 carbapenem-resistant Klebsiella pneumoniae isolates were collected in Yunnan.Among them,52 strains carried bla _KPC-2,which was the main drug-resistant gene,and 2 strains carried bla _NDM-1.Among all the samples,one strain was found to carry both bla _KPC-2and bla _NDM-1,while 2 strains were not detected to have bla _KPC-2and/or bla _NDM-1,and 55 samples were identified as Klebsiella pneumoniae by 16S rRNA gene.A total of eight ST types were detected by MLST.They were ST11,ST23,ST231,ST65,ST86,ST107,ST375 and ST37,among which ST11 was the main type.Conclusions:1.In this study,a laboratory method--Multi+Overlap PCR method was successfully established,which can connect 16S rRNA gene and housekeeper genes in series,and conduct 16S rRNA gene identification and MLST typing for carbapenems Klebsiella Pneumoniae simultaneously.However,there was still some defects in this method,and further details need to be perfected.2.In the past eight years,bla _KPC-2and ST 11 were the main prevalent resistance genes and ST type of carbapenem-resistant Klebsiella Pneumoniae in Yunnan.3.In this study,we found a carbapenem-resistant Klebsiella pneumoniae strain in Yunnan,which carrys two carbapenem-resistant genes,bla _KPC-2and bla _NDM-1.