| Objectives:Enterococcus bacteria in the nosocomial infection often cause urinary t ract,soft tissue,blood stream and other infections,with the use of antibiotics,the emerg ence of multidrug-resistant enterococcus and its new drug-resistant genes.Linezolid is a new class of oxazolidinone antibiotics,which has a unique antiba cterial mechanism and is also one of the last lines of defense in the treatment of multid rug-resistant Enterococcus.The resistance mechanism varies according to the type,de gree and region of Enterococcus.In this study,the drug resistance mechanism of linezo lid-resistant Enterococcus(LRE)in our hospital from September 2018 to December2020 was studied,aiming to clarify the main drug resistance mechanism of Enterococ cus,and provide a basis for clinical treatment and drug resistance supervisi on.As a new microbe identification tool,the Matrix-assisted Laserdesorption Time-of-fl ight Mass Spectrometry(MALDI-TOF MS)has the advantages of accuracy,speed and low cost.Through the screening and verification of the specific peak of Linezolid resistant Enterococcus Faecalis(LREfs),To evaluate the rapid identification ability of MALDI-TOF MS for LREfs.Methods:1.Clinical isolates of non-repetitive linazolid-resistant enterococcus from theSecond Affi liated Hospital of Kunming Medical University were collected from September 2018 to December 2020 and stored in a refrigerator at-80℃.Bacteria species identif ication and drug sensitivity test were carried out by Vitek-2 Compact automatic syste m.Minimum inhibitory concentration(MIC)of strains identified as Enterococcus and resistant to linezolid was determined by broth dilution method,and30 strainswith M IC greater than or equal to 8ug/mL were selected as experimental strains.2.The Polymerase chain reaction(PCR)method was used to amplifying the resistant genes optrA,cfr,poxtA,23 S rRNA V gene and L3,L4 ribosomal locus gene(rplC,rplD).The positive products were sequenced and verified to understand the resist ance mechanism of 30 strains of linezolid-resistant Enterococcus.3.LREfs specific peak selection and validation:Among the 27 strains of linezoli d-resistant Enterococcus faecalis,the 15 strains with optrA gene only and the15 strain s of linezolid-sensitive Enterococcus faecalis collected at the same time were selected as the specific peak screening group,as a specific peak by MALDI-TOF MS identific ation of strains,use Flexcon trol 3.0 software,Flex analysis 3.0software analy sis and Clinpro Tools 3.0 software,obtained the map,the processing map and screenin g of LREfs specific peak;In order to evaluate the clinical application value of MALDI-TOF MS for rapid identification of LREFs,7 strains of linezolimid-resist ant Enterococcus faecalis carrying optrA gene and 15 strains of sensitive Enteroco ccus faecalis collected at the same period were identified by MALDI-TOF MS as th e specific peak validation group.Results:1.A tota l of 30 strains of linezolid-resistant Enterococcus were collected from September 2018 to D ecember 2020,including 27 strains of Enterococcus faecalis and 3 strains of Enteroc occus faecium.The sample sources included urine(18 strains),drainage fluid(8 st rains),pus(2 strains),blood(1 strain),and wound secretion(1 strain).Drug resistant bacteria were mainly distributed in urology,ICU and hepatobiliary surgery.Th e drug resistance rate of linezolid resistant Enterococcus faecalis to erythromycin,tetracycline,chloramphenicol and minocycline was more than 80%,while the drug re sistance rate of linezolid resistant Enterococcus faecalis to tegacycline,teicolanin,vanc omycin,ampicillin,furantoin and penicillin was relatively low.The main strains of linezolid-resistant enterococcus were nosocomial infection(17 strains)and comm unity infection(13 strains).The main source of community infection samples was urine specimen(11 strains),and the distribution was concentrated in the urologica l department outpatient department(11 strains).2.PCR resistance gene detection results showed that 23 strains carrying optrA g enes,9 strains exist L4 mutation,5 strains of cfr gene,3 strains exist L3 mutation,23 Sr RNA mutation was detected,all strains poxtA genes are all negative,there is a single strain of 20 strains of resistant mechanism,double strain of 10 strains resistant mechani sm,including optrA+L4mutations(6)strains,cfr+L4 mutations(2)strains,cfr+optrA(1)strains,optrA+L3 mutant strain(1)strains.The community-acquired drug-resistant strains(13 strains)mainly carried optrA gene,including optrA(8strains),optrA+L4 mutation(2 strains),optrA+L3 mutation(1 strain),L4 mutation(1 strain),and cfr+L4 mutation(1 strain).3.Among the algorithm models of MALDI-TOF MS to identify LREfs carrying only optrA gene,the Sup port Vector machine-3(SVM-3)model had the best ability to identify LREfs,with a sp ecificity of 81.71% and sensitivity of 95.56%.Three specific peaks were screened by Cl inPro Tools 3.0 software:peak 6,peak 11,peak 14(mass/charge ratio:2083.97m/z,2188.58m/z,2223.54m/z),and the correct identification rate of linezolid resistance carrying optrA gene was 28.6% in the subsequent verification based on the model established according to the specific peaks.The above 3 differential p roteins peaks could not be used as differential protein peaks to distinguish LREfs from LSEfs,and no specific protein peaks were found in this study to distinguish linezol id resistant and linezolid sensitive Enterococcus faecalis.Conclusions:The main r esistance m echanism of linezolid resistant Enterococcus is mediated by optrA gene.The horizon tal transfer of resistant genes is a potential threat to the outbreak or epidemic of infe ction.Therefore,it is necessary to strengthen antimicrobial management and nosocomi al infection control,further investigate the risk factors and transmission routes o f community infection,and prevent and control the outbreak or epidemic of drug-resist ant bacteria.MALDI-TOF MS as a rapid,accurate and low cost of microbial iden tification tool,but in this study to carry optrA dung enterococcus resistance mechanism o f drug resistance gene identification ability is low,linezolid resistant Enterococcus faecalis carrying optrA gene has not been effectively identified.experimental sampl e size is limited and the drug-resistant bacteria in the validation group were optrA gene combined with other drug-resistant mechanisms.this study further research needs to be expanded sample size. |
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