Genotype Of Carbapenem Resistant Enterobacteriaceae And Antibacterial Activity Of Ceftazidime-Avibactam In Vitro

Background:Enterobacteriaceae is widely distributed in the human intestinal tract and nature environment,when the body’s mechanical or immune barrier is destroyed,it can cause serious diseases.Carbapenems are the primary choice for the treatment of infection caused by persistent,high-yielding AmpC enzyme and/or ESBLs gram-negative bacillus.While the emergence of carbapenem resistant enterobacteriaceae(CRE)poses an unprecedented threat to human life and health.The main mechanism of enterobacteriaceae resistance to carbapenem is the production of carbapenemases.KPC,NDM,VIM,IMP and OXA-48 are the five most common carbapenemases.Not only does CRE exhibit pan-resistance to commonly used clinical antibiotics,but also when they carried carbapenenase genes,it can be widely spread through a variety of genetic components.Rapid detection of CRE and molecular epidemiology are of great significance.Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry(MALDI-TOF MS)has become a revolutionary detection technology applied in clinical microbiology laboratories.Its extended functions of drug resistance detection and strain typing in epidemiological studies are of great significance for nosocomial infection control of CRE.Ceftazidime-Avibactam(CZA)is a new drug composed of Ceftazidime(CAZ)and a novel β-lactamase inhibitor Avibactam(AVI)for CRE that inhibits class A,C and some class D carbapenemases.The sensitivity of CRE to Ceftazidime-Avibactam in China is still lacking corresponding data.Therefore,it is worthy of further study on how to conduct accurate treatment of CRE and extend the life of the new drug.Objective:1.To investigate the clinical distribution and drug resistance characteristics of CRE in Renmin Hospital of Wuhan University from 2006 to 2018.2.To study the phenotype and genotype of carbapenemases in CRE.3.To evaluate the application value of MALDI-TOF MS in rapid detection and molecular typing of Klebsiella pneumoniae producing KPC.4.To investigate the antibacterial activity of Ceftazidime-Avibactam against CRE in vitro and the combined effect with other antibiotics.Methods:1.The research collected the susceptibility information of the CRE strain isolated from Renmin Hospital of Wuhan University from January 2006 to December 2018.The source,species distribution,characteristics of the infected population,sensitivity of different CRE strains to commonly used clinical antibiotics and changes of drug resistance in 13 years were analyzed.2.101 strains of CRE isolated from clinical specimens of our hospital from January 2017 to December 2018 were collected.The identification results were reviewed by Bruker MALDI-TOF MS,and the drug sensitivity was reviewed by BD phoenixtm-100.PCR screening was conducted for five common carbapenems encoding genes:blaKPC,blaNDM,blaIMP,blaVIM,and blaOXA-48.To evaluate the efficacy of carbapenase inactivation test(mCIM test and eCIM test)and inhibitor based combined disk test(CDT)to detect and distinguish metallo-carbapenemase and serine-carbapenemase.3.30 Klebsiella pneumoniae producing and not producing KPC were used as model strains respectively.An experimental model for rapid detection of Klebsiella pneumoniae producing KPC was established by MALDI-TOF MS clinprotool,and 48 clinical isolates were used for model verification(including Internal evaluation and external evaluation).4.MALDI-TOF MS Biotyper 3.0 was used to perform dendrogran on 54 strains of KPC-producing Klebsiella pneumoniae in this part,and the results were compared with MLST results to evaluate the molecular typing efficiency of MALDI-TOF MS.5.The antimicrobial susceptibility test of Ceftazidime-Avibactam to all CRE isolations collected in our study was carried out by KB(Kirby-Bauer)paper diffusion method.And the antibacterial effect of Ceftazidime-Avibactam combined with Tigecycline,Amikacin,Levofloxacin,SXT,Aztreonam,Imipenem,Cefoperazone-sulbactam and Fosfomycin were evaluated.Results:1.During the 13 years from 2006 to 2018,a total of 545 strains of CRE were isolated in the hospital,accounting for 28.5‰ of the total enterobacteriaceae,and 10.5‰ of the total bacteria.The specimens of the hospital were mainly composed of sputum(38.5%).Klebsiella pneumoniae(50.3%)was the dominant strain,and the detection rate increased year by year.And 96.3%strains came from adults,3.7%came from children(<16),1.3%came from neonatal.ICU(18.5%),Neurology(14.7%),Neurosurgery(11.2%),respiratory(9.5%),urology(8.3%)were the five main department.The resistance rate of CRE to Imipenem and Meropenem were 95.4%and 85.4%,respectively.The resistant rates of the otherβ-lactam antibiotics were higher than 92.8%,amd the drug resistance rates to Amikacin was 45.2%.2.A total of 79 Carbapenemase-producing strains(CPE)were detected from 101 CRE,with a detection rate of 78.2%(79/101).The carbapenemase genes included:38 blaKPC,32 blaNDM,5 blaIMP,1 blaVIM,2 blaKPC+NDM,1 blaNDM+IMP,and no blaOXA-48 was detected.Klebsiella pneumoniae mainly carried blaKPC,while Escherichia coli mainly carried blaNDM.The coincidence rate of mCIM combined with eCIM and CDT were 97.5%(77/79)and 96.2%(76/79),respectively,compared with gene detection.The enzyme producers identified by the two phenotypes were mostly blaKPC and blaNDM.Both assays had a misclassification in two blaKPC+NDM-producing isolates of Klebsiella oxytoca.3.The cross validity of the three algorithms of MALDI-TOF MS clinprotool QC,GA and SNN was 88.72%,90.71%and 92.85%,respectively.The recognition ability was 91.67%,96.67%and 98.89%,respectively.Two characteristic peaks 8372.66m/z and 11222.8m/z were obtained by QC algorithm.Using SNN algorithm,a total of 10 characteristic peaks were obtained,respectively:4520.58m/z,10382.17m/z,5070.3m/z,11880.37m/z,2329.78m/z,10883.34m/z,11522.6m/z,2534.97m/z,4110.6m/z and 4547.9m/z.Using GA algorithm,10 characteristic peaks were obtained,3097.76m/z,6098.21m/z,9095.43m/z,5281.54m/z,7745.39m/z,3181.5m/z,7306.82m/z,4547.9m/z,3636.85m/z and 8879.47m/z,respectively.The sensitivity,specificity,positive predictive value and negative predictive value of the three algorithms were all more than 90%.4.54 strains of KPC-producing Klebsiella pneumoniae were divided into three categories by MSP cluster analysis:type A 1(1.85%),type B 53(98.15%).The MLST strains were ST11 52(96.3%),ST265 1(1.85%)and ST230 1(1.85%).The two typing methods have a well degree of consistency.5.113 CRE strains showed an overall sensitivity of 49.6%to Ceftazidime-Avibactam,84.6%of CRKPN was sensitive to Ceftazidime-Avibactam.According to the results of combined drug sensitivity test,no antagonism effect was observed for all the combinations.For different strains,the combination of Ceftazidime-Avibactam with Aztreonam had synergistic effects on 100%CRKP,76.5%CRCEO,62.5%CRECL,80%CEKOX,respectively.Ceftazidime-Avibactam combined with Imipenem and Cefoperazone-sulbactam had synergistic effects on 86.2%CRKP,while Ceftazidime-Avibactam combined with fosfomycin had cumulative effects on 4.6%CRKP and 36.7%CRECO.For different enzyme types,the combination of Ceftazidime-Avibactam with Aztreonam had synergistic effects on 100%KPC-producing strain,77.8%metalloenzyme producing strain and 100%KPC+NDM producing strain,and cumulative effect on 22.2%metalloenzyme producing strain.Ceftazidime-Avibactam combined with Imipenem and Cefoperazone-sulbactam had synergistic effects on 100%KPC-producing strains.Conclusion:1.The distribution of CRE specimens shows that they are common in respiratory.Infection is more common in the elderly,ICU,neurology,neurosurgery,respiratory and urology are the five major areas of CRE infection.At the same time,the younger age of CRE infection must be taken seriously.Most of CRE present pan-resistance.Clinicians should focus on the drug resistance of Carbapenem-resistant Klebsiella pneumoniae.2.Carbapenem-resistant Klebsiella pneumoniae in the hospital mainly carried blaKPC,Carbapenem-resistant Escherichia coli mainly carried blaNDM,and other CRE strains mainly carried metalloenzymes.Both phenotypic tests(mCIM test and eCIM test)and inhibitor based combined disk test(CDT)can effectively identify and distinguish the single and the same type carbapenemases,which are easy to operate,accurate,low-cost and suitable for basic hospital laboratories.3.KPC-producing Klebsiella pneumoniae could be identified quickly and accurately by MALDI-TOF MS clinprotool.ST11 is the main ST type in the laboratory.The cluster analysis of KPC-producing Klebsiella pneumoniae was analyzed by MALDI-TOF MS,which was well consistent with MLST.4.Ceftazidime-Avibactam is only sensitive to serine-carbapenemases and should not be used indiscriminately as the initial treatment for patients.The combination of Ceftazidime-Avibactam with Aztreonam may be an option for treatment of CRE infection,whether serine-carbapenemases or metallo-carbapenemases.