Molecular Epidemiological Investigation And Study On Resistant Mechanism Of Carbapenems-resistant Pseudomonas Aeruginosa

Objectives:1 In order to understand molecular epidemiology and genetic background of carbapenems-resistant Pseudomonas aeruginosa(CRPA)strains,molecular epidemiological investigation of CRPA strains isolated from the Clinical Microbiology Department of the First and Second Affiliated Hospital of Kunming Medical University from January 2017 to February 2021 were conducted.Those results will be useful for clinical prevention and treatment of Pseudomonas aeruginosa infection.2 Analyzing in vitro antimicrobial susceptibility test of carbapenems-resistant Pseudomonas aeruginosa strains to commonly used antimicrobial drugs to understand their drug resistance characteristics provides an important reference for clinical treatment of Pseudomonas aeruginosa infection.3 Through a variety of molecular biology and bioinformatics technology,the carbapenemases gene carrying,the mutation of outer membrane protein oprD gene,the expression of drug efflux pump gene mexA and the production of biofilm were analyzed,so as to understand the drug resistance mechanism of Pseudomonas aeruginosa isolates to carbapenems antibiotics.Those results will provide theoretical basis and guidance for prevention and control of nosocomial infection of Pseudomonas aeruginosa.Methods:Section 1:Antimicrobial susceptibility test and molecular epidemiological investigation of CRPA isolates1.Investigation of in vitro antimicrobial susceptibility test of CRPA strainIsolates isolated and preserved in the Clinical Microbiology Department of the First and Second Affiliated Hospital of Kunming Medical University from January 2017 to February 2021 were collected and identified by Bruker Biotyper automatic microbiological mass spectrometry system.K-B diffusion method was used to detect the susceptibility of all strains to Aztreonam(ATM).The Vitek 2 Compact automatic microbial identification drug sensitivity instrument was used to detect the sensitivity of 12 antibiotics:Piperacillin(PIP),ceftazidime(CAZ),cefepime(FEP),piperacillin/tazobactam(TZP),imipenem(IPM),meropenem(MEM),gentamicin(GM),tobramycin(TM),amikacin(AN),ciprofloxacin(CIP),levofloxacin(LEV),polymyxin B(PB).A total of 81 strains of CRPA isolated from urine,blood,secretions,sterile body fluids and alveolar lavage fluid were collected.The first carbapenem-resistant Pseudomonas aeruginosa strain from the infected site of patients was selected as the tested strain,and all of them were non-repeating strains.2.MLST typing investigation of CRPA strainsPseudomonas aeruginosa MLST typing scheme was used to amplify seven housekeeping genes of CRPA strain by polymerase chain reaction(PCR).The sequence type(ST)of CRPA strain was sequenced and confirmed.Cloning relationship and genetic information of each ST type were analyzed by Bionumerics software.Section 2:Study on the resistance mechanism of Pseudomonas aeruginosa to carbapenems1.Genotype detection of carbapenemase genePolymerase chain reaction(PCR)was used to amplify carbapenemase genes commonly found in 7 species of Pseudomonas aeruginosa,including blaKPC、blaGES、blaIMP、blaVIM、blaSPM、blaNDM and blaOXA-40.The amplified products were subjected to agarose gel electrophoresis,and the positive amplicons were sequenced by Sanger sequencing and aligned by BLAST.2.Detection and mutation analysis of oprD gene of outer membrane porinThe full length of Pseudomonas aeruginosa oprD gene was amplified by PCR and sequenced.The gene sequence was compared with the oprD sequence of the standard strain of Pseudomonas aeruginosa PAO1 to analyze drug resistance mutations.The comparison software was Mega-X.3.Detection of mexA gene expression level of efflux pump MexAB-OprMReal-time qPCR was used to detect the expression levels of mexA gene and rpoD gene of the efflux pump MexAB-OprM,and the expression levels of mexA gene and rpoD gene of Pseudomonas aeruginosa ATCC27853 were used as control.The efflux pump regulating genes mexR、nalC and nalD of the high expression strain of MexAB-OprM were amplified by PCR and sequenced,and the corresponding gene sequences of Pseudomonas aeruginosa PAO1 were aligned and analyzed.4.Detection of biofilm formationThe biofilm formation of Pseudomonas aeruginosa was detected by microtitration plate formation test,and the biofilm formation related gene pslA was detected by PCR.Results:Section 1:Antimicrobial susceptibility test and molecular epidemiological investigation of CRPA isolates1)The 81 CRPA strains were mainly isolated from urine,secretions,blood and drainage fluid,accounting for 43.20%,18.52%,16.05%and 11.11%,respectively.The other strains were alveolar lavage fluid,bile,cerebrospinal fluid and ascites,accounting for 4.94%,2.47%,2.47%and 1.23%,respectively.The main departments were burn department and emergency department.2)All the 81 CRPA strains showed high resistance rates to other antibiotics except polymyxin B;No strain resistant to polymyxin B was found.The drug resistance rate of 34 ST3390 strains to all antibiotics except polymyxin B was higher than 94%.3)MLST showed that 81 isolates belonged to 36 different ST types,of which 31 were known from database species and the other 5 were newly discovered in this study.ST3390 was the most common ST type,accounting for 41.9%(34/81).The sequence types of Pseudomonas aeruginosa in different periods were different from the dominant sequence types,so the monitoring of Pseudomonas aeruginosa infection should be strengthened.Section 2:Study on the resistance mechanism of Pseudomonas aeruginosa to carbapenems1)Only IMP,VIM and NDM type gene were detected among the 7 carbenenemase genes,and the detection rates were 37.04%,27.16%and 1.23%,respectively.No KPC,GES,SPM and OXA-40 carbenenemase genes were detected,and no strains co-expressing multiple carbenenemase genes were found.2)Sequence alignment analysis of oprD gene showed that 78(96.29%)of the 81 strains of CRPA had various types of mutations,among which 68 strains had different types of mutations of oprD gene and were dominated by insertion or deletion of single or multiple bases(76.54%),The deletion of A base at the nt109 of oprD gene was the main one.Other types of mutations include base mutations leading to premature termination codon and amino acid point mutations;The PCR results of 5 strains were negative.Three other CRPA strains did not detect any form of mutation.3)There were 17 strains with mexA gene overexpression,accounting for 20.99%.The amplification and sequencing of the regulatory genes mexR、nalC and nalD of the 17 overexpressed strains were performed.The sequence comparison with the standard strain Pseudomonas aeruginosa PAO1 showed that 5 strains of CRPA had point mutation of mexR gene or MexR protein,and 3 strains had point mutation of the amino acid of MexR protein V126E.One strain had amino acid mutation of MexR protein V115E,L119K,V126E and L135H,and one strain had small fragment deletion(nt 135-146 had deletion of 11 bases TATCGACGAAC).17 strains had amino acid point mutation of NalC protein,all strains had amino acid point mutation of G71E,one strain also had amino acid point mutation of A145 V,Q182K and L206V,and eight strains had amino acid point mutation of Q182K,L206 and VS209R simultaneously.7 strains simultaneously had S209R amino acid point mutation,1 strain simultaneously had L206V and S209R amino acid point mutation.10 strains had amino acid point mutation of NalD protein,including 1 strain had amino acid point mutation of N130S and 9 strains had amino acid point mutation of W49S.Among all the mutant types,G71E and S209R amino acid substitutions of NalC protein were the most common.In this study,new mutation types of MexR V115E,L119K and L13 5H were found.New mutation types of NalC Q182K,L206V;New mutant types of NalD,W49S and N130S.4)The microtitration plate assay showed that 91.35%(74/81)of the strains formed biofilm,while the positive rate of pslA detected by PCR was only 75.3%(61/81).Biofilm formation related gene pslA was detected in 86.79%of the carbapenemase-producing strains and 53.57%of the non-carbapenemase-producing strains.Conclusions:1)81 CRPA strains had higher resistance rate to antibiotics except polymyxin B(>50%).A total of 36 ST types were detected by MLST typing,of which ST3390 was the main type(41.97%),and five new sequence types,including ST3645,ST3646,ST3647,ST3648 and ST3649,were detected.2)The resistance mechanism of Pseudomonas aeruginosa to carbapenems antibiotics is complex and varied,and there are great differences among different regions.In this study,the main mechanism of the resistance of 81 PA strains to carbapenems antibiotics is the mutation of OprD protein in various forms accompanied by the formation of biofilm.Among them,the detection rate of carbapenase gene was 65.43%,which was much higher than other studies in China,which might be related to the different sources of the selected strains.The overexpression of the efflux pump MexAB-OprM is not the main mechanism of drug resistance,and the new mutation types of its regulatory gene proteins MexR,NalC and NalD found in this study may be the cause of its overexpression,which requires further study.3)The drug resistance rate of all ST3390 strains to other antimicrobial agents except polymyxin B was higher than 94%,and all of its oprD gene had A deletion of base A at nt109,and this mutation was rarely reported in the previous study,so whether this mutation was the cause of its high level of drug resistance needs to be further confirmed.