| Objective:Klebsiella pneumonaie(KP)has become the second largest pathogenic bacteria in Enterobacteriaceae after Escherichia coli.It is also an opportunistic pathogen associated with a variety of infections,resulting in relatively difficult to treat in clinic.Drug resistance and hypervirulence are generally considered as two non-overlapping evolutionary directions in KP,both of which will eventually lead to the successful epidemic of high-risk clone lineages.In recent years,due to the spread of resistant and virulent plasmid,KP integrated both two phenotypes have been reported and have gradually become a worldwide problem.In addition,more than 5000 ST types have been detected at present.With the continuous detection of new ST types,their drug resistance and hypervirulence are continuously enhanced,which is worthy of our attention.However,there are relatively few drugs for effective treatment at present,so it is particularly urgent to find new antibacterial drugs for treatment.However,there are relatively few drugs used to effectively treat,so it is particularly urgent to find new antibacterial drugs.Therefore,through the relevant research on KP resistance mechanism,virulence factors and molecular characteristics,real-time monitoring of the emergence of new ST type,and explore the anti-bacteria,anti-biofilm and anti-virulence ability of the synthetic compound MTEBT-3 on resistance and hypervirulence of KP in our study,and it does have a certain guiding significance for the development of new antibacterial drugs,but also provides a new method for clinical treatment of infection.Methods:1、Study on resistance,virulence and molecular characteristics of carbapenem-resistant Klebsiella pneumoniae CRKP in four hospitals in Taiyuan Strains of CRKP were collected from four Grade III Class A general hospitals in Taiyuan City from October 2019 to March 2021.The minimum inhibitory concentrations(MICs)against commonly used antibiotics were determined by agar dilution method or microbroth dilution method.The phenotype of carbapenemase production was identified by modified carbapenemase inactivation method(m CIM)and EDTA-modified carbapenemase inactivation method(e CIM).The hypermucoviscous phenotype of the strain was determined by the string test,and the biofilm production was determined by the crystal violet staining.The related resistance and virulence genes were amplified by polymerase chain reaction(PCR)to assess in determining the resistance and virulence characteristics of the strains.Multi-locus sequence typing(MLST)was compared to determine the sequence types(STs)of the strains and the serotypes were determined by wzi locus.MEGA was used to analyze the genetic relationship among these strains.2、Study on resistance,virulence and molecular characteristics of three new sequence types of the CRKP Three CRKP strains were collected from November 2019 to April 2021,among which the strain 1 and 2 were both isolated from the hospital A,and the strain 3 was from the hospital B.The clinical characteristics of these three patients were analyzed through His case information system.Serum resistance assay was regarded as the phenotypic assay to assist in the evaluation of virulence of CRKP strain.Detecting resistance genes and virulence genes carried by the three CRKP via Whole-Genome Sequencing(WGS).Software e-Burst was used to analyze the relationship between the three new sequence types and the popular clone lineage ST11.The other methods such as the antimicrobial sensitivity test,carbapenemase phenotype test,the string test,biofilm formation assay,STs and serotype identification were all described in the first part.3、Study on the efficacy of synthetic compound MTEBT-3 against CRKP strain in vitro A CRKP strain KPN 23 with relatively strong resistant and virulent was selected for the study.The MIC of the synthetic compound MTEBT-3 was determined by microbroth dilution method.The anti-bacteria ability of the synthetic compound MTEBT-3 was evaluated through the synergistic antimicrobial sensitivity test with commonly antibiotics MEM,IPM,TGC and the time-kill assay.The development of resistance of the synthetic compound MTEBT-3 was determined by continuous 15 generations,and further compared with common antibacterial drugs MEM and IPM.Biofilm elimination assay,the double immunofluorescence staining assay under laser confocal microscopy(CLSM)and the bacterial content release assay were used to determine the ability of the synthetic compound MTEBT-3 against biofilms.The change of the synthetic compound MTEBT-3in the level expression of virulence genes of the CRKP was determined by the fluorescence quantitative PCR.Results:1、Study on resistance,virulence and molecular characteristics of carbapenem-resistant Klebsiella pneumoniae CRKP in four hospitals in Taiyuan A total of 50 strains of CRKP were collected.Among them,12 were collected from the hospital A,23 were from the hospital B,12 were from the hospital C,and 3 were from the hospital D.The results of antimicrobial sensitivity tests showed that all strains were resistant to IPM and MEM,more than 80% were resistant to CSL,ATM and LEV,38% were resistant to AMK,and only 8% were resistant to TGC.The results of carbapenemase phenotype showed that 47 CRKP produced carbapenemas(94.00%).Carbapenase genes were carried in 50 CRKP: 32 carried bla KPC(64.00%),15 carried bla NDM(30.00%),4 carried bla SME(8.00%),3 carried bla IMI(6.00%),and 6 carried bla OXA-23(12.00%).9 CRKP carried two or more carbapenase genes,1 strain from hospital A,8 strains from hospital B,and the other two hospitals were not detected.In addition,50 CRKP were also detected to carry a large number of ESBLs genes,including21 strains carrying bla CTX-M(42.00%),48 strains carrying bla TEM(96.00%),46 strains carrying bla SHV(92.00%),and 10 strains carrying AmpC genes bla DHA(20.00%).The string test results showed that the four CRKP had hypermucoviscous phenotype.The results of biofilm experiment showed that the biofilm formation was different at different time points,with the biofilm formation of 38,17 and 26 strains at24 h,48h and 72 h,respectively.Virulence gene amplification results showed that 6 strains carried iuc A gene(12.00%),11 strains carried iut A gene(22.00%),3 strains carried iro N gene(6.00%),5 strains carried rmp A gene(10.00%),and 6 strains carried rmp A2 gene(12.00%),9 strains carried ure A gene(18.00%),4 strains carried mrk D gene(8.00%)and three carried kfu gene(6.00%).In addition,9 CRKP carried two or more virulence genes,4 from hospital A and 5 from hospital B,and the other two hospitals were not detected.The MLST results showed that 23 ST types were totally detected,of which ST11 type(22.00%)was the main type,including 7 strains detected in hospital A,7 strains detected in hospital B,1 strain detected in hospital C and 2 strains detected in hospital D.In addition,three new ST types(ST5365,ST5587 and ST5647)were diccovered.MEGA analysis showed that the 50 CRKP were divided into 6 main clusters,and there was a certain genetic relationship among the strains.Among 50 CRKP strains,KL64(22.00%)was the main serotypes detected by wzi locus,and next was K28(12.00%).2、Study on resistance,virulence and molecular characteristics of three new sequence types of the CRKP Three new STs(ST5365,ST5587 and ST5647)were identified.The patients who isolated strain 1 and strain 2 were hospitalized for 164 days and 56 days,respectively,and both improved before discharging from hospital.The patient who isolated strain 3was hospitalized for 21 days and was discharged still in critical condition.All the three CRKP isolates were multi-resistant,but all sensitive to TGC and COL.The string test showed that all the three strains were negative.All the three CRKP could form biofilms,among which strain 1 and strain 2 were moderate producers and strain 3 was weak producer.The results of serum resistance assay indicated that only strain 2 showed resistance.The WGS analysis showed that three CRKP isolates all carried multiple resistance genes,such as carbapenemase,sulfonamides,fluoroquinolones,aminoglycosides and tetracycline genes.In addition,they also carried several virulence genes encoding siderophore,fimbriae,capsule and lipopolysaccharide.The serotypes of strain 1 and strain 2 were K35 and KL47,respectively,while the serotype of strain 3 was not detected yet.3、Study on the efficacy of synthetic compound MTEBT-3 against CRKP strain in vitro The MIC of the synthetic compound MTEBT-3 was 8μg/m L.The results of the synergistic antimicrobial sensitivity test showed that the synthetic compound MTEBT-3had an additive effect with the commonly used antibiotics MEM and IPM,and had a synergistic effect with TGC at the concentration of 1*MIC;When the final concentration of the synthetic compound MTEBT-3 reached to 2*MIC or above,the results showed that the synthetic compound MTEBT-3 had no correlation with MEM and IPM,while had an additive effect with TGC.The time-kill assay results showed that the synthetic compound MTEBT-3 had the antibacterial effect at different concentration conditions(1*MIC,2*MIC,4*MIC,8*MIC and 16*MIC),and it was concentration-dependent.The results of resistance development studies showed that KPN 23 was less prone to develop resistance to the synthetic compound MTEBT-3 than the antimicrobial agents MEM and IPM.The biofilm elimination experiment showed that the inhibition ability of the synthetic compound MTEBT-3 was the strongest at 24 h,which could reach about 50%.While the inhibition ability of the synthetic compound MTEBT-3 was reduced at 48 h,which was about 37%,and even the inhibition rate of the biofilm was only about 5% at72 h.By observing the morphology of bacteria under CLSM,it could be seen that the synthetic compound MTEBT-3 had good bactericidal ability and biofilm destruction ability.The results of study on the release of bacterial contents showed that the release of bacterial contents was related to the concentration of the synthetic compound MTEBT-3,which is concentration-dependent.The q-PCR results showed that fim H gene expression level decreased significantly after the intervention of antibacterial agents,especially in the MTEBT-3 group,which reduced about 1 times(P<0.01).The expression level of mrk A gene was decreased significantly only in IPM group(P < 0.01).wbb M gene expression levels were significantly decreased in all three groups(P<0.01).Conclusion:1.All the 50 strains of CRKP were resistant to common antibiotics in varying degrees,and KPC-producing enzyme was the predominate resistance mechanism.In terms of virulence,the results of the string test showed that 6 strains of CRKP was positive.15 strains of CRKP simultaneously carried one or more virulence factors,and the predominate serotype was KL64.In addition,most of the strains could form biofilms.ST11 was the mainly endemic clone lineage in the four Grade III Class A general hospitals in Taiyuan.In addition,among the 4 hospitals,the distribution of resistance,virulence and ST types varied.2.Three brand-new STs were discovered in the study,namely ST5365,ST5587 and ST5647,which were not only multi-resistance,but also virulent.Among them,ST5365 is closely related to ST11,while ST5587 and ST5647 are far from ST11.3.The synthetic compound MTEBT-3 has excellent antibacterial ability,strong membrane-disruption ability and great anti-hypervirulence ability. |
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