Study On Efflux Pump Of Multi-drug Resistant Pseudomonas Aeruginosa And The Homology Analysis In Taiyuan

Object:In order to understand the distribution and drug resistance of Pseudomonas aeruginosa（PA）in Taiyuan,the pathogenic bacteria isolated from positive blood culture patient in the Second Hospital of Shanxi Medical University over the last 11 years were analyzed.Furthermore,the resistance mechanism on efflux pump of multi-drug resistant PA（MDRP）and the homology of MDRP in Taiyuan region were studied.To investigate further mechanisms,the relationship between high expressed Mex AB-OprM gene and the mutations of mex R,nalC as well as nalD were researched.This study will provide scientific basis on the control of hospital infection and the rational use of antibiotics in clinical practice.Part 1.Analysis of the distribution and drug resistance of blood cultured-positive pathogens from 2008 to 2018Methods:Firstly,we collected the clinical blood culture specimens in the Second Hospital of Shanxi Medical University from January 2008 to December 2018.The pathogens were cultivated using automatic blood culture instrument BACTEC^TMFX.Then,the identification of pathogen and the susceptibility test were conducted by VITEK 2 COMPACT automatic identification of susceptibility analyzer.All data were analyzed by WHONET 5.6 and SPSS21.0 software.A value of P<0.05 is considered as statistically significant.ResultA total of 3195 strains pathogenic bacteria were isolated,of which the gram-negative bacteria accounted for 63.38%（2025 strains）,gram positive bacteria accounted for 36.62%（1170 strains）.The top five detection rates were followed by e.coli（966 strains,30.23%）,coagulase negative staphylococcus（537 strains,16.81%）,klebsiella pneumoniae（324strains,10.14%）,pseudomonas aeruginosa（151 strains,4.73%）and staphylococcus aureus（151 strains,4.73%）.According to the age group of pathogenic bacteria,the detection rate of Enterococcus faecalis in the 41-65 years group and >65 years group was higher than that in the 18-40 years group.The detection rates of Escherichia coli,Klebsiella pneumoniae and Staphylococcus aureus in different age groups were significantly different（P<0.05）.The results of drug resistance analysis showed that meropenem,imipenem and amikacin had good antibacterial activity against Enterobacteriaceae pathogens（sensitivity rate>75%）.The susceptibility rate of PA to the studied antibiotics was higher than that of Acinetobacter baumannii,and the difference was statistically significant（P<0.05）.The isolation rate of methicillin-resistant Staphylococcus aureus（MRSA）in Staphylococcus was 14.3%.The detection rate of methicillin-resistant coagulase-negative staphylococcus（MRCNS） was 78%.The susceptibility rate of Enterococcus faecium was lower than Enterococcus faecalis,andpositive cocci resistant to Deicolanin and Vancomycin were unfound.Part 2.Studies the efflux pump of MDRP and the gene mutation analysis.Method Thirty-two strains of non-repetitive MDRP isolated from three general hospitals in Taiyuan,Shanxi Province from June 2018 to June 2019 were collected.The minimum inhibitory concentrations（MIC）of the strains to ceftazidime,cefoperazone and sulbactam,imipenem,ciprofloxacin,amikacin and amtronam were determined by broth dilution method.Efflux pump inhibitors phenylalanine-arginine-β-naphthalamide combined with ceftazidime,cefoperazone sulbactam,imipenem,ciprofloxacin,amikacin and amtronam were used to screen the phenotype of efflux pump.The mRNA expressions of MEXA,MEXC,MEXE and MEXX were performed by RT-PCR and calculated using the 2^-（?）Ct method.The results were shown as the relative amount normalized to GAPDH.The mex R,nal C and nal D genes which regulated the efflux pump Mex AB-opr M were amplified and sequenced.Result1.A total of 32 PA strains were completely resistant to ciprofloxacin（100%）,and lower resistance rate to aminoglycosides amikacin（64.5%）.The resistance rates of ceftazidime,amtronam,imipenem and cefoperazone and sulbactam were all more than 90%.2.By comparing the MIC values with and without the efflux pump inhibitor PAβN,the positive screening rates of PA efflux pump phenotypes resistant to ciprofloxacin,ceftazidime,amtronam,cefoperazone and sulbactam,imipenem,and amicacin were 96.8%,44.8%,41.4%,41.4%,40%,and 10%,respectively.3.Isolated clinically from the 32 strains of Pseudomonas aeruginosa,the relatively high expression rates of efflux pump membrane fusion proteins MexA,MexC,MexE and MexX were 34.4%,3.0%,18.8%and 32.3%,respectively.4.The mutation rates of 32 clinical isolates of Mex R,nal C and nal D,the regulatory genes of the PA pump MexAB-OPRM,were 15.6%,53.1% and 37.5%,respectively.The mutation rates of both genes were 43.8%,and the MexR,nalC and nal genes of strain T5 were mutated simultaneously.Part 3.The homology typing study of PA using Matrix-assisted laser desorption/ionization mass spectrometry（MALDI-TOF MS）.MethodThe 32 strains of PA which isolated clinically were resuscitated,and obtained the protein finger of PA.Then imported fingerprint spectrum into Maldi Biotyper software.PCA Dendrogram was selected after all the samples were selected,and the function of PCA cluster analysis was used for homology analysisResult1.As illustrated from the clustering analysis diagram,32 strains of bacteria were divided into two families（ⅠandⅡ）,and 3 subfamilies（Ⅰ,Ⅱ_a andⅡ_b）.The strain ofⅠandⅡ_a mainly drived from hospital S,strainsⅡ_b mainly drived from hospital F and hospital T.2.There was a significant difference of drug resistance rate between the strainsⅠ,Ⅱ_a andⅡ_bto amikacin;the rates were 50%,36.4%and 91.7%,respectively.3.The high-expressed membrane fusion proteins of the strainⅠwas mainly mex A,which expression rate was 50.0%.The high-expressed membrane fusion proteins of the strainⅡ_awere mexA,mexC and mexX,and the expression rates were all 36.4%.The high-expressed membrane fusion proteins of the strainⅡ_b were mexA and mexX,and the expression rates were 33.3%and 25% respectively.4.The deletion of CCGCTCACCCCG occurs at the 364 to 375-position of mexR gene in strain No.T4,T5 and S10 of the family I.Meanwhile,the same mutation also occurs on the147-position of nal C gene.The mutation of base G to base A represented the mutation of aspartic acid to asparagine.The base G at 147-position of nal C in strain F4,F8,F12,F13,T1 of the subfamilyⅡ_b mutated to base A.That means the mutation of aspartic acid to asparagine.Another mutation was the base G to base A,which means the the mutation of glutamic acid to arginine.The same mutation was occurred on the the 540-position of nal D gene,which means the the mutation of aspartic acid to glutamate.4.Conclusion1.The major bloodstream infections in hospital was Gram-negative bacilli infection.And Pseudomonas aeruginosa ranked fourth among the pathogenic bacteria of bloodstream infections.and Pseudomonas aeruginosa ranked first among the Non-fermenting bacteria of bloodstream infections.Antibacterial drugs should be used rationally in clinical anti-infection treatment according to the distribution of pathogenic bacteria,drug resistance characteristics and age of patients,so as to improve the effect.2.The drug resistance mechanism of ciprofloxacin on Pseudomonas aeruginosa is mainly related to the production of efflux pump.The base mutation of the regulatory genes mexR,nalC and nalD leads to the high expression of efflux pump Mex AB-Opr M.Therefore,the drug resistance of Pseudomonas aeruginosa is related to the high expression of efflux pump.3.The strains from different hospitals all had base deletions at positions 364 to 375 of mexR gene,and the mutation of base G to base A occurs at 147-position of nal C gene.This results suggested that the homology among multi-drug resistant Pseudomonas aeruginosa in Taiyuan.4.MALDI-TOF MS technology makes the analye simple and fast,and this technology provide us a new method for the prevention and control of nosocomial infection.