Evaluation Of Five Systems For Identification Of Anaerobic Bacteria,Analysis Of Antimicrobial Susceptibilities Of Bacteriodes Fragilis Group Isolates And Resistant Mechanisms Of These Isolates

Background:Anaerobic bacteria are often involved in the intra-abdominal infections,pelvic infections,biliary tract infections,complicated skin and soft tissue infections and blood stream infections.Technical complexity of the isolation,identification and susceptibility test for anerobes make it a challenge for most clinical microbiology laboratories.The introduction of Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry(MALDI-TOF MS)had made a revolution in the identification of microorganisms.Besides foreign mainstream equipment,Chinese local brands of MALDI-TOF MS systems were also been developed and applicated in clinic.However,the system evalution of their identification for anerobes haven t been performed.For the reason of the limitation of isolation and identification ability of anerobes,empirical antimicrobial therapy for the anerobic bacterial infections were often conducted.Antimicobial susceptilibities data and resistant mechanism of anerobic bacterial are very limited in China.The purpose of this study is to evaluate the identification abilities of four MALDI-TOF MS systems and VITEK 2 ANC card for clinical related anerobic bacteria isolates,and analyze the the antimicrobial susceptibilities of Bacterioaes fragilis group isolates and resistant mechanisms of them.Methods:Clinical isolated anaerobic bacteria and reference isolates were collected from 2010 to 2017.16S rRNA gene sequencing was used as the reference method for the identification.All the isolates were identified by VITEK MS?Clin-ToF-? MS?Autof MS 1000,M-Discover 100 and VITEK 2 ANC card.Antimicrobial susceptibilities of 138 isolates of B.fragilis group against 19 antibiotics were determined by agar dilution method recommended by Clinical and Laboratory Standard Institute(CLSI).CfiA,IS,cepA,cfxA,nim,ermF,mefA,tetQ,tetX and B.fragilis enterotoxin gene bft were amplificated by PCR.Whole genomes sequencing were performed for 14 isolates of carbapenem-resistant strain and 3 isolates of carbapenem-susceptible strain.Antimicrobial resistant genes,toxin genes and phylogeny analysis by SNPs were conducted.Results:479 isolates of anaerobic bacteria were collected,which were identified as 25 genera and 71 species by 16S rRNA gene sequencing.The accurate identification rate at species and mixed-species level of VITEK MS,Clin-ToF-? MS,Autof MS 1000,M-Discover 100 and VITEK 2 ANC card were 86.0%,83.7%,92.5%,89.4%and 88.6%,respectively,and 89.1%,93.9%,99.2%,97.1%and 94.2%at genus level.For the accurate identification rate at species and mixed-species level,there were no statistical differences among VITEK MS,Clin-ToF-? MS and M-Discover 100,and between Autof MS 1000 and M-Discover 100.The accurate identification rate of Autof MS 1000 was higher than VITEK MS,Clin-ToF-? MS and VITEK 2 ANC(P<0.005),and that of M-Discover 100 was also much higher than VITEK 2 ANC(P<0.005).Based on CLSI breakpoints,metronidazole and chloramphenicol were the most susceptible agents tested for 138 isolates of 8.fragilis group(99.3%and 92.8%,respectively),followed by carbapenems and piperacillin/tazobactam(76.8%-79.0%).Ampicillin,tetracycline and clindamycin were the most resistant agents,against which the resistant rates were 89.9%-99.3%.The resistant rates of ertapenem,imipenem and meropenem ranged from 19.65to 22.5%.The positive rates of cfiA,IS,cepA,cfxA,nim,ermF,mefA,tetQ,tetX and bft were 37.7%,13.8%,55.1%,33.3%0%,77.4%,8.8%,92.8%,11.6%and 0%respectiely.By whole genome sequencing,2-7 resistant genes were detected per isolate.The 14 isolates of carbapenem-resistant strain were all cfiA positive.The positive isolates number of cepA,ermF,tetQ and tetX was 5,15,16 and 1 in 17.No bft positive isolates were found.In phylogeny tree,14 isolates of carbapenem-resistant strains and 3 isolates of carbapenem-susceptible strains were assembly in two clusters respectively.Conclusions:The identification abilities of all the five systems can satisfy the clinical requirement.Compared to the international mainstream brand equipment,VITEK MS,the three systems of MALDI-TOF MS of Chinese local brands showed equal or better abilities for anerobes identification.For clinical isolated strains of B.fragilis group,metronidazole and chloramphenicol were the most susceptible agents tested.High attention should be paied for the emergency of multi-drug resistance to antibiotics including carbapenems,the higher resistant rates and resisant gene positive rates than the reports from other counties and past reports in China.No isolates of nim or bft positive isolates were found in our study.