| Enterobacteriaceae are the most important pathogen of bacterial infections in community and hospital,accounting for about 80%of all gram-negative bacilli infections.Enterobacteriaceae can cause urinary tract,intestinal tract,biliary tract and abdominal infection,as well as septicemia.Carbapenems are broad-spectrumβ-lactam antibiotics which against gram-negative bacteria.They are considered as the last line of defense against gram-negative bacteria since 1980s.However,overuse of carbapenems have led to the emergence of carbapenem-resistant Enterobacteriaceae（CRE）.At present,the rapid prevalence and dissemination of CRE in medical institutions has become a major public health problem in the world.In 2013,CRE was listed as the top priority of"emergency threat"in the"bacterial resistance threat report"issued by the Centers for Disease Control and Prevention.CRE can cause a high mortality and huge threat,however the treatment drugs for CRE infections remain limited.The treatment drugs of CRE include polymyxin,tegafycline,fosfomycin and aminoglycoside.In clinic,two or even three drugs are used for combined treatment.However,the combination of multi-drugs can enhance the bactericidal effect and also increase the incidence of adverse reactions in patients.Therefore,it is significance to study on the mechanism of carbapenem resistance to take effective measures to block or delay the spread of CRE in clinic.There are three main mechanisms of carbapenem resistance in Enterobacteriaceae.（1）Production of carbapenemase enzymes.（2）The change of penicillin binding protein of target structure.（3）Combination of AmpC and/or extended-spectrumβ-lactamases（ESBL）and mutation of membrane porin.Carbapenemase is the main cause of resistance to carbapenems in Enterobacteriaceae.The common carbapenemases include KPC,NDM,IMP,OXA,etc.Carbapenemase resistance genes can be carried by chromosomes or plasmids,wherein plasmids are the main carrier of the transmission of carbapenemase genes.The plasmid can be spread resistance genes by means of conjugation,transformation and transduction between different strains and genera,which results in rapid dissemination of carbapenemase-producing resistant bacteria.Although there are a large number of studies reported the spread of drug-resistant plasmids,it is not clear that the specific mechanism due to the numerous influencing factors and complex mechanisms.Therefore,this study collected the clinical separation Enterobacteriaceae with low sensitivity to carbapenems were collected from four provinces in China.We screened the carbapenemase-producing CRE,analyzing environment of carbapenemase-resistant genes and fine structure of plasmids,exploring the influential factors of plasmids transmission,which provided theoretical basis for clarifing the transmission mechanism of carbapenemase resistance genes.This study consists of three parts:Part one:Screening of clinical isolates of CRE.In this part,172 Enterobacteriaceae isolates with low sensitivity to carbapenems（imipenem MIC≥1μg/mL）were collected from four provinces in China.Carbapenemase-producing CRE were screened by antimicrobial susceptibility testing,modified Carba NP Test and PCR.And then the carbapenem-resistant Klebsiella pneumoniae were analyzed by MLST.172 Enterobacteriaceae isolates consisted of 9 Escherichia coli isolates,98 Klebsiella pneumoniae isolates,7 Citrobacter freundii isolates,57 Enterobacter cloacae isolates and 1non-decarboxylic Lecteria isolates.The results of antimicrobial susceptibility testing showed that 69 isolates from 172 low sensitivity Enterobacteriaceae strains were resistant to imipenem and meropenem,namely CRE.The 69 CRE isolates showed higher drug resistance rate and MIC than carbapenem-sensitive strains.The resistance rate ofβ-lactam antibiotics（ampicillin,ceftazidime,cefepime and aztreonam）were all over 70%,and the resistance rate of amikacin was the lowest（34.78%）.The carbapenemase was detected by modified Carba NP Test in 69 CRE isolates.The results showed that 44 CRE isolates produced carbapenemase,of which 33 isolates produced A enzyme,9 isolates produced B enzyme,and 2 isolates produced both A enzyme and B enzyme.The mechanism of the other 25 isolates might be non-carbapenem enzyme.Carbapenemase genes of the 44 carbapenemase-producing CRE isolates were amplified by PCR,40 isolates were positive.It was suggested that the other 4 isolates might have new resistance genotypes or some resistance genes had mutations.Among the 40 CRE strains included 32 blaKPC positive strains,7 blaNDM positive strains and 4 blaIMP positive strains.Notably,there were two strains that produced more than two kinds of carbapenemase,among the blaKPC and blaNDMDM of 15K323 strain（Klebsiella pneumoniae）were both positive,and the blaKPC,blaNDM and blaIMPMP of P10159（Citrobacter franseri）were all positive.26 carbapenem-resistant Klebsiella pneumoniae isolates were analyzed by MLST.6genotypes were identified,the most common genotype was ST11（14 strains）,the other genotypes included ST133（5 strains）,ST37（3 strains）,ST686（2 strains）,ST107（1 strain）and ST215（1 strain）.Part two:The genetic basis study of the transmission of carbapenem-resistant genes.In the previous part,we screened the carbapenemase-producing CRE isolates,learned about their distribution,and preliminarily analyzed their genetic background.In this part,we studied the mechanism of resistance through the highly resistant strains which produced multiple carbapenemases.Firstly,the plasmids were isolated,identified and sequenced.If the isolated plasmids were inconsistent with the bacterial resistance,there might be multiple plasmids presumably,and then the bacterial genome and plasmids were sequenced and all the sequences of plasmids were spliced.We understood the environment of drug-resistant genes and the fine structure of plasmids by sequencing,which laed a foundation for the transmission mechanism of carbapenem-resistant genes in plasmids in the further research.Plasmids with carbapenem-resistant genes were found in all 7 Enterobacteriaceae isolates.The results of sequence analysis showed that the carbapenem-resistant genes were located in the insertion and/or transposition sequence.Except for pP10164-3,the similarity of skeleton structure and nucleotide sequence between other plasmids and the reported plasmids were about 90%and 99%,respectively.Most of plasmids showed the change of the drug-resistant variable regions.Although these plasmids had been reported,they were the first time to find in Citrobacillus freundi and Leclercia adecarboxylata.The five resistant plasmids including pP10159-1,pP10159-2,pP10159-3,pP10159-4and pP10159-5 from Citrobacillus freundi P10159 were determined through high-throughput genome sequencing.pP10159-1 carried blaNDM-1,and was highly similar to the IncX3 type plasmid pNDM-HN380.pP10159-2 carried blaIMP-4MP-4 and qnrS1,which was highly similar to the IncN1 type plasmid pP378-IMP.pP10159-3 carried blaKPC-2,which was very similar to the pHS062105-3 and pECN49-KPC gene structures.They belonged to a novel plasmid in the unknown incompatible group.The skeleton region of multidrug resistance plasmids pP10159-4 and pP10159-5 were highly similar to IncHI4 plasmid pNDM-CIT and type 2 IncC plasmid pR55,respectively,but their accessory resistant regions differed from pNDM-CIT and pR55.The five plasmids from the P10159 isolate contained a total of 24 different genes or gene loci,which contributed to resistance to 13distinct antibiotic molecules or toxic compounds.The results of high throughput sequencing of Leclercia adecarboxylata pP10164showed that there were 3 resistant plasmids including pP10164-NDM,pP10164-2 and pP10164-3.Among them,pP10164-NDM was highly similar to IncF IIY plasmid pKOXNDM1,and blaNDM-1 gene was located in compound transposon Tn125.pP10164-2was similar to IncHI2 plasmid R478 of Serratia marcescens.However,it was no sequence similarity between pP10164-3 and known DNA sequence.pP10164-2 and pP10164-3carried a large of drug-resistant genes to resist to 10 kinds of antibiotics and 7 kinds of heavy metal.The other 5 Enterobacteriaceae isolates found only a drug-resistant plasmid.The results of high throughput sequencing showed that the 14E509 plasmid p14E509-NDM of Escherichia coli carried blaNDM-1,which was highly similar to IncX3 plasmid pNDM-HN380.Klebsiella pneumonia HB27,ZJ121,15K169 and 15K366 carried a plasmid with KPC gene,which were pHB27-KPC,pZJ121-KPC,p15K169-KPC and p15K366-KPC.The genetic structure of the four plasmids was very similar to each other.And they were homologous with the IncFII type plasmid p0716-KPC and p0716-KPC of Klebsiella pneumonia.Their insertion regions of resistant genes included MDR,blaKPC and Tn6029 regions.This study screened a special isolate（P10159）which produced 3 kinds of carbapenemases（NDM,IMP and KPC）,loaded 5 different kinds of resistant plasmids carrying different resistant genes.And it hinted that plasmids coexistence might be important condition for high and pan-drug resistant.This kind of isolates was very rare and valuable for in-depth study.In addition,we established the"small fragment library the second generation high-throughput sequencing+large fragment library the second generation high-throughput sequencing"and"small fragment library second generation high-throughput sequencing+the third generation high-throughput sequencing"assembled accurately chromosome and plasmids sequences,established technology foundation for studying high resistance caused by plasmids coexist.In the next part,we would focus on the key links that affect the transmission of plasmids carrying carbapenemase genes.Part three:Study of the mechanism of carbapenem-resistant genes horizontal transfer in plasmidsIn this part,the highly resistant carbapenemase-producing isolates were used as the research object.Plasmids of isolates were lost by natural passage,non-plasmids isolates were obtained plasmids by transformation and combination.RNA-seq transcriptome was used to analyze the differences of plasmid before and after plasmid loss or transfer,which was verificated by RT-PCR.Key genes were analyzed through bioinformatics,which were confirmed by the method of gene knockout and so on.4 highly resistant carbapenemase-producing isolates were tested for plasmid passage loss.Thereinto 14E509(containing blaNDM plasmid)was lost after 35 passages,and plasmid-eliminating strain 14E509(blaNDM-)was obtained.The other three strains of carbapenemase-producing strains were not lost after 100 passages.As a strain without plasmid,E259 was transformed into p14E509 to obtain plasmid into strain 14E509NDME259.RNA-seq transcriptome was used to analyze the difference of plasmid before and after plasmid loss and transfer.The results showed that there were 514 up-regulated genes and767 down-regulated genes in the expression of 14E509 vs 14E509(blaNDM-).The expression of E259 vs 14E509NDME259 was up-regulated in 24 genes and down-regulated in 22genes.It was showed that loss and acquisition of plasmid may be related to multiple factors and pathways by GO analysis,pathway enrichment analysis KEGG metabolic pathway analysis,and significant differentially expressed genes analysis.Propionate metabolic pathway and quorum sensing pathway were the most important factors affecting the loss and acquisition of plasmids.But significant differences expression of entrizid:947500（geneid:mqsR）was concentrated repeatedly to bioinformatics signaling pathways.It was suggested that mqsR might be the key link of plasmid transmission.It was found that the biofilm formation ability of mqsR deleted strains was weakened by knocking out mqsR,and the frequency of bacteria in biofilm to uptake exogenous drug-resistant plasmids was significantly reduced(3.33×10-7±9.41×10-88 vs1.57×10-8±1.38×10-8,P<0.05).However,although the plasmid uptake ability of mqsR deleted strains was decreased in phytoplankton(2.37×10-9±7.85×10-1010 vs1.59×10-9±1.01×10-9,P>0.05),there was no statistical difference.It was suggested that mqsR played an important role in promoting the transmission of plasmids in the biofilm,but there was no significant influence on the uptake of plasmids for plankton bacterium,and its mechanism still need to be further studied.In conclusion,this part studies found that loss and gain of plasmid carrying carbapenemase genes may be associated with multiple factors and pathways.The most influential pathways were propionic acid metabolism pathway and the quorum sensing system.And,for the first time,we found mqsR played an important role in promoting the spread of plasmid in biofilm.However,besides mqsR,other signaling pathways still need to be further verified.Main conclusions:1.In the study area,the main types of carbapenemase isolated from Enterobacteriaceae were KPC,NDM and IMP,and the main type of Klebsiella pneumoniae was KPC.2.The most common genotype of carbapenem-resistant Klebsiella pneumoniae was ST11,suggesting that ST11 was easier to acquire resistant genes and plasmids,or its genes were more suitable for stable replication and retention of plasmids.3.The main cause of carbapenemase-resistant in Enterobacteriaceae was resistant plasmids.The spread of plasmids lead to the rapid increase of Enterobacteriacea.The coexistence of multiple plasmids was an important condition for high resistance and pan-resistance in bacteria.4.In this study,propionate metabolic pathway and quorum sensing pathway were the most important factors affecting the loss and acquisition of plasmids.5.The loss and acquisition of plasmids might be related to multiple factors and pathways.mqsR played an important role in promoting the transmission of plasmids in biofilm,but there was no significant effect on the uptake of plasmids in planktonic bacteria.The mechanism still needs to be studied in the future. |
PDF Full Text Request