Molecular Characterization Of ESBLs Producing E.Coli And K.pneumoniae From Pulmonary Infection,as Well As Extensively-drug-resistant P.aeruginosa In Wuhan Union Hospital,with A Preliminary Mechanism Exploration Of ESBLs Producing Bacterial Heterogeneity

Part I Molecular characterization of extended-spectrum-β-lactamases(ESBLs)producing Escherichia coli,Klebsiella pneumoniae isolated from patients with Pneumonia in Wuhan union hospitalObjective To detect the molecular epidemiology of extended-spectrum β-lactamases’ genes from Klebsiella pneumoniae and Escherichia coli isolated from Pneumonia,identify their sub-types,and discuss the homology of resistant bacteria as well as to investigate the distribution,resistance mechanism,and approach of transmission so,that can provide evidence for the clinical therapy and control of nosocomial infection.Methods 263 specimens from Pneumonia were collected in the first affiliated hospital of Huazhong University of Science and Technology from December 2015 to June 2016.Total 59 samples were collected after excluding unqualified cases.ESBLs phenotype was ascertained by composite disk method after bacteria cultured in blood agar medium.Firstly,CTX-M,SHV,and TEM genes were detected by PCR.Then,we had sequenced PCR products along with upstream ISEcp1 and IS26,as ISEcp1 and IS26 upstream sequences contribute to ESBLs expression.Homologous analysis were measured with the help of repetitive extragenicpalindromic PCR(Rep-PCR)and multilocus sequence typing(MLST).Results The percentage of ESBL-producing isolates from pneumonia was 22.43%, among them species producing ESBLs had the highest rate of cephalosporin resistance.E.coli and K.pneumoniae showed resistant to Fluoroquinolones and most of the β-lactams,with the exception of imipenem,ertapenem,amikacin,and piperacillin/tazobactam.31 strains of Escherichia coli and 28 strains of Klebsiella pneumoniae were isolated from 59 specimens.Among 31 strains of Escherichia coli,20 strains(64.51%)carried CTX-M genes predominately CTX-M-3,CTX-M-14,and CTX-M-27;17 strains(54.84%)carried SHV genes predominately SHV-12,SHV-28,SHV-77,and SHV-81;3 strain(9.68%)carried TEM-1,respectively.1 of the ESBLs phenotype positive strain carried two genes(SHV-77 and SHV-81),and 1 more strain(3.23%)carried 3 genes(CTX-M-14,SHV-77,TEM-1)at the same time.Among the 28 strains of Klebsiella pneumoniae,21strains(75.00%)carried CTX-M genes predominately CTX-M-3,CTX-M-14,CTX-M-27,and CTX-M-65;23 strains(82.14%)carried SHV genes predominately SHV-1,SHV-2,SHV-11,SHV-28,SHV-77,SHV-78,and SHV-119;16 strain(57.14%)carried TEM genes predominately TEM-1,TEM-104,and TEM-135;7 strain(25%)simultaneously carried CTX-M,SHV,and TEM genes.MLST analysis showed that ST131 was the predominant sub-types of E.coli strains whereas ST11 was predominant sub-types of K.pneumoniae strains.Only 4 Carbapenem-resistant K.pneumoniae with NDM gene was detected.Conclusions(1)The percentage of ESBL-producing E.coli and K.pneumoniae in our hospital is high,mainly distributed in surgery department and ICU.Among them ventilator-associated pneumonia was the commonest one.(2)E.coli and K.pneumoniae showed resistant to Fluoroquinolones and most of the β-lactams,with the exception of imipenem,ertapenem,amikacin,and piperacillin/tazobactam.,so,these four antibiotics might be appropriate alternatives for treating pneumonia due to ESBL-producing isolates(3)the CTX-M was the main drug-resistant gene in Escherichia coli,among them CTX-M-14 was the most common sub-type;but SHV was the main drug-resistant gene in Klebsiella pneumoniae,among them SHV-11 was the most common sub-type.The different sub-types of SHV might exist in the same strain.ESBLs phenotype positive Enterobacteriaceae can carry many types ESBLs genes.(4)ST131 E.coli and ST11 K.pneumoniae were highly invasive and hyper-virulent,spreading in hospitals,especially in surgery department.(5)in my study,the co-existing ESBLs and Carbapenem-resistant was only observed in K.pneumoniae,that also carry NDM gene.(6)ISEcp1 and IS26 contributed to the expression of ESBLs.Part II Clinical epidemiology and molecular characterization of extensively-drug-resistant Pseudomonas aeruginosa in Wuhan union hospitalObjective To investigate the epidemiology and molecular characterization of resistance mechanisms of extensively-drug-resistant Pseudomonas aeruginosa in Wuhan union hospitalMethods The database at our clinical microbiology laboratory was reviewed to identify patients with P.aeruginosa infection from January 2016 to June 2017 at the first affiliated hospital of Huazhong University of Science and Technology.Carbapenem resistance,Extended-Spectrum-β-Lactamase,Aminoglycoside resistance,and quinolone resistance genes were detected by PCR and these PCR products were sequenced.Homologous analysis was measured with the help of repetitive extragenicpalindromic PCR(Rep-PCR)and multilocus sequence typing(MLST).Results Only 7 isolates out of 816(0.86 %)were colistin-only-sensitive Pseudomonas aeruginosa strains displaying resistance to all antimicrobial agents except colistin.Multilocus Sequence typing(MLST)showed that the isolates belonged to six different STs,among them,two shared the same STs sequence type ST274.Among six different STs,there were two novel STs:ST2546 and ST3001.These 7 isolates who were extensively drug-resistant P.aeruginosa strains were also associated with multiple drug resistance genes.Conclusions 1)The percentage of colistin-only-sensitive P.aeruginosa in our hospital was lower,mainly distributed in Respiratory Department and ICU.Sputum and bile cultures were predominantly specimens.To treat patients with pulmonary infection and biliary stents history is great challenge for us.2)co-existence of multiple drug resistance genes in colistin-only-sensitive P.aeruginosa was universal in my research.Plasmid-mediated quinolone resistance genes(PMQR)and Quinolone Resistance Determining Regions genes(QRDR)were detected at the same time by PCR.3)ST274 P.aeruginosa along with colistin-only-sensitive characterization will be a great trouble for patients who suffered from chronic pulmonary infection.Part III A preliminary mechanism exploration of ESBLs producing bacterial heterogeneityObjective Previous study about bacteria resistance was based on the different clonal populations data with ignoring cell-to-cell variation.But,a small part of isolates surviving,whose resistance are obviously different from others,even though after complete antibiotics treatment.Therefore,my aim of this research is based on my previous work on the molecular characteristics of ESBLs to further investigate the cell-cell variation on single cell level,and also the mechanisms of diversification in antibiotic resistance from signal clonal cells,which can provide the basic knowledge for bacteria that can fight against the fluctuating stress of antibiotics.Methods A specific expression vector pUA66 carrying green fluorescent protein(GFP)gene and the transcriptional region of ESBL with their entire coding sequences was reconstructed previously.Fluorescence microscope were applied to analyze the expression pattern of the ESBL gene in single cell via GFP reporters,and flow cytometry was used to sort out the high and low fluorescence intensity isolates,along with analysis of the drug resistance difference between these two sub-groups(high intensity isolates and low intensity isolates).Results The flow cytometry analysis showed remarkably difference between two groups.Cells with high fluorescence intensity(FI)were more complex in the context of bacteria structure,grew slower,and was prone to drug resistance compared to cells with low fluorescence intensity(FI).Conclusions 1)Heterogeneous resistance levels exists in single clonal population 2)There might be existence of a key factor causing heterogeneous resistance via modulating bacteria growth rate.