Identification And Mechanism Of Novel Trimethoprim Resistance Genes And Research On Extensively Drug Resistant Bacteria

The rapid growth of antibiotic use and bacterial resistance has become one of the urgent problems in the field of medicine and health in the 21st century.Trimethoprim(TMP),synthesized for clinical use in early 1960s,is a competitive inhibitor for bacterial enzyme dihydrofolate reductase(DHFR),which can prevent the reduction of dihydrofolate(DHF)to tetrahydrofolate(THF).Trimethoprim inhibits bacterial growth by interfering with the bacterial folate metabolism pathway.As a vital precursor substance for cellular metabolism,THF takes part in DNA,RNA and protein synthesis.DHFR is necessary for the de novo synthesis of THF,which cannot be transported from extracellular space by bacteria.Therefore,TMP and other antifolate drugs targeting DHFRs are effective against protozoa and bacteria,while having little impact on human.However,with the extended and overuse of TMP in human and livestock,the number and proportion of TMP-resistant bacteria found in clinical and even natural environments are gradually increasing.Over 40 different dfr genes have been known to encode TMP resistant DHFRs.These dfr genes,usually found in mobile genetic elements including integrons,plasmids and transposons,are easily transmissible between bacteria which leads to the formation of multi-,extensively-,and pan-drug resistant bacteria.Thus,the identification and characterization of novel dfr genes found in clinical and environmental samples have realistic significance for determining the molecular mechanism and the risk of dissemination of TMP resistance.The systematic analysis of novel TMP-resistant DHFRs aims to provide strategic and directional support for the development of new drugs as well as reduction of high-level TMP resistance.Aquatic environments harbor numerous unidentified antibiotic resistance genes(ARGs)and genetic arrays,and have become a regnant pool for breeding novel ARGs and antibiotic resistant bacteria(ARB).In this study,four novel DHFR-coding genes were found in four aquatic opportunistic pathogens:one Aeromonas hydrophila and three Proteus spp.A series of structural,bioinformatical,phenotypical,genetic analyses were performed for these four novel dfr genes which leads to elucidation of TMP resistance mechanisms and further proposal for structural basis for TMP resistance.In addition,the antibiotic resistance properties and their genetic basis of an extensively drug(including TMP)resistant Klebsiella pneumoniae isolated from clinical stool samples.The main work and findings are summarized as below:1.Four novel dfr genes lead to high-level resistance to TMP and co-trimoxazole.Minimum inhibitory concentrations for TMP and co-trimoxazole were measured for Proteus vulgaris LK4,Proteus penneri LTe9,Proteus vulgaris LK8 and A.hydrophila Sam7-TMC1 with the broth microdilution method recommended by the Clinical and Laboratory Standards Institute(CLSI).High level of TMP(>1024 g/L)and co-trimoxazole resistance(>64/1216 g/L)were found for these strains.To further confirm that these dfr genes can indeed lead to TMP resistance,we cloned the four novel dfr genes into pACYC184 vector(low copy number,cloning site downstream of the call gene)using the GB/dir homologous recombination based cloning method and transformed the recombinant vectors into Escherichia coli DH5?.The same high level of TMP and co-trimoxazole resistance were found in the four dfr-harboring E.coli strains in comparison with E.coli DH5?,confirming that they can indeed lead to TMP resistance.2.Sequence alignments and phylogenetic analyses for novel and known DHFRs determined that three novel dfr genes associated with complex class 1 integrons encode Family A DHFRs,while dfrB 7 gene in class 1 integron encodes the 7th Family B DHFR.The genetic structure of three complex class 1 integrons,harboring three new gene arrays and three novel ISCR1-associated dfr genes,were analyzed by sequencing.The protein sequences encoded by these genes were subject to phylogenetic analysis along with known DHFRs using MEGA 7.0.The products of the three novel dfr genes from P.vulgaris LK4,P.penneri LTe9 and P.vulgaris LK8 were found to co-cluster with other Family A DHFRs,and subsequently named DfrA42,DfrA43 and DfrA44.DfrB7,encoded by a new gene dfrB7 in class one integron from A.hydrophila Sam7-TMC1,clusters with other Family B DHFRs.3.Enzymatic parameters of novel TMP resistant DHFRs are quite close to the E.coli chromosomal DHFR EcDHFR that does not confer TMP resistance.On the other hand,Family B DHFRs DfrBl and DfrB7 have drastically lower catalytic efficiencies.Therefore,TMP resistance conferred by these DHFRs is not due to significantly higher catalytic activities or efficiencies.In order to determine the enzymatic parameters of TMP resistance conferring DHFRs,recombinant EcDHFR,DfrA1,DfrB1,DfrA42,DfrA43 and DfrA44 proteins were overexpressed using the pET15b(+)vector and purified to apparent homogeneity.NADPH:dihydrofolate(DHF)oxidoreductase enzymatic parameters were determined by nonlinear regression analysis of rates of decreasing A340 absorbance at different concentrations of DHF.The kinetic constants Km and kcat were calculated,showing no significant difference between TMP-resistant DHFRs and TMP-sensitive EcDHFR.Family B DHFRs DfrBl and DfrB7 have similar substrate affinities(Km)with EcDHFR,but have drastically(200-400 fold)lower activities(kcat)leading to significantly lower catalytic efficiencies(kcat/Km).With these results,a conclusion that TMP resistance conferred by these DHFRs is due to significantly higher catalytic activities or efficiencies cannot be drawn.4.Three hypotheses are proposed for the mechanism of acquired TMP resistance conferred by DHFRs:DHFRs may be overexpressed by a strong promoter to overcome loss caused by TMP binding;DHFRs may have a high activity so that unbound DHFRs may still have enough activity for cell metabolism;DHFRs may also show a decreased affinity with TMP so that the level of inhibition is decreased.With binding analysis,the mechanism of TMP resistance conferred by novel DHFRs is confirmed to be reduction of binding to TMP.Isothermal titration calorimetry(ITC)experiments were performed on a PEAQ-ITC isothermal calorimeter from MicroCal at 298 K to compare the binding of TMP to resistant DHFRs and sensitive EcDHFR.A strong(10-40 fold)reduction of affinity with TMP(KD)was found for DfrA42,DfrA43 and DfrA44 in comparison with EcDHFR,while all Family B DHFRs were unable to bind with TMP.These results strongly suggest that although showing significant differences,both Family A and Family B DHFRs showed a sharp reduction in the binding with TMP.Therefore,these experimental results suggest that reduced affinity for TMP is the primary cause for TMP resistant Family A and B DHFRs,in agreement with the third hypothesis.5.The protein crystals of binary complexes DfrA44·NADPH,DfrA44·TMP and ternary complex DfrA44·NADPH·TMP were screened and optimized.After X-ray diffraction,the structural data of DfrA44 was collected and analyzed.DfrA44 has a typical Rossman fold structure containing 6 ?-helices and 8 ?-strands.Crystal Screen Kit,PEGRx Screen Kit and PEG/Lon Kit were used to screen and optimize by the hanging drop method.Protein structures were calculated with X-ray diffraction data.The interactions between TMP and DfrA44 can be divided into three groups:a strong hydrogen bond network between the bottom of the binding pocket and the pyrimidine ring,moderate hydrophobic interactions between the middle of the binding pocket and the benzene ring,and weak hydrogen bond interactions between methoxy groups connecting the benzene ring and the opening of the binding pocket.The pyrimidine ring also forms ?-? interaction at the bottom of the binding pocket.Binding with NADPH leads to slight twisting of the structures,shrinking the opening of the substrate-binding tunnel by 2.9 A.Binding with NADPH leads to the inversion of the direction for the 2-methoxy group on the benzene ring.This is caused by the change of the shape of TMP binding site:after NADPH binding,the distance between P52 and the 2-methoxy group decreased from 4.9 A to 1.9 A,leading to strong steric hindrance,forcing the 2-methoxy group to invert,resulting in an increased distance of 3.0 A.6.Changes of binding tunnel entrances may be responsible for TMP resistance for DfrA44.Comparison of the ternary DfrA44·NADPH·TMP complex structure with other reported TMP-resistant and TMP-sensitive DHFR structures lead to the finding that the binding schemes between TMP and DHFRs are similar among all DHFRs,while the entrances did appear to be different between DfrA44 and TMP-sensitive DHFR,in that a smaller and more hydrophobic entrance was found for DfrA44.It is therefore postulated that the size and hydrophobicity of substrate binding tunnel entrances may play an essential role in the TMP resistance for DHFRs.Further experimental evidence is needed to confirm this suggestion.7.An extensively drug resistant(XDR)Klebsiella pneumoniae 2-1 strain was isolated from the stool sample of a patient diagnosed of colorectal cancer.Whole geneome sequencing of the complete genome of K.pneumoniae 2-1 was performed to determine the genetic structure and molecular mechanisms of resistance.Two multidrug resistance circular plasmids were found to account for the extensively drug resistance phenotype.K.pneumoniae 2-1,isolated from the stool sample of a patient diagnosed of colorectal cancer,was found to be resistant to all the antibiotics tested except for cefepime(a fourth generation cephalosporin),tigecycline(a last line of defense antibiotic),and ceftazidime-avibactam.By sequencing the complete genome of K pneumoniae 2-1,we found it contains a 5.23 Mb chromosome and two circular plasmids with the sizes of 246 kb and 90 kb.These two plasmids were,respectively,classified as IncHI1B and IncA/C2 plasmids,based on the typing of replicon sequences and the PlasmidFinder database.The larger plasmid,pKP21HI1 was found to be a new conjugation-defective plasmid belonging to incompatibility group HI1B and a new sequence type.Further comparative genomics analysis and antimicrobial resistance gene analysis showed that although a great deal of changes took place on the chromosome of K.pneumoniae 2-1 in comparison with the reference genome of K.pneumoniae HS11286,the extensively drug resistance phenotype of K.pneumoniae 2-1 is primarily due to the two multidrug resistant plasmids it contains.This work explains the genetic and mechanistic basis of the extensive drug resistance of K.pneumoniae 2-1,and found that plasmids play key roles in the strong antibiotic resistance of bacteria.In conclusion,the study of novel ARGs and strong ARBs isolated from environmental and clinical samples has a great significance for identifying the causes for antimicrobial resistance,and analyzing the status and prospect of antibiotic resistance.In this work,four novel dfr resistant genes in TMP resistant bacteria were identified.With a series of biochemical experiments,including the identification,classification,expression and purification,enzymatic activity analysis,binding with TMP for DHFRs,we explored the specific mechanism of TMP resistance with biochemical and structural relevance.These results indicate that TMP resistance mechanism is complicated and probably affected by many residues involved in NADPH binding,TMP binding,or other functions.Nevertheless,reduced binding with TMP was shown to be responsible for TMP resistance for the DHFRs identified in this thesis.In addition,we identified a TMP-resistant K.pneumoniae 2-1 strain isolated from clinical stool sample.Through the study of molecular mechanism in resistant phenotypes and genetic structure,we can conclude that multiple-drug resistance plasmids with abundant resistant genes can lead to extensively resistant phenotype,and put forward a proposed strategy of reducing resistance by inhibiting multiple-drug resistant plasmids and their dissemition.