Study On Genotype Detection Of Carbapenem-resistant Enterobacterales And Antimicrobial Activities Of Combined Drug Susceptibility Test

Objective To investigate the clinical distribution,drug resistance analysis and genotypes of carbapenem-resistant Enterobacterales(CRE)in Fuyang Hospital of Anhui Medical University,and to explore the effects of imipenem(IPM)and meropenem(MEM)combined with ceftazidime(CAZ),cefoperazone/sulbactam(SCF),amikacin(AK),levofloxacin(LEV)and fosfomycin(FOS)against KPC-,NDM-,IMP-producing strains.The antibacterial effect of ceftazidime/avibactam(CZA)alone or combined with aztreonam(ATM)against KPC-,NDM-,IMP-producing strains.Methods 128 non-repetitive CRE strains were isolated from clinical samples from the clinical Laboratory of Fuyang Hospital of Anhui Medical University from August 2017 to March 2021.All CRE strains were identified and rechecked by VITEK 2 Compact and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS).WHONET 5.6 was used to analyze clinical distribution and drug resistance.All strains were analyzed by carbapenemase phenotypic screening testcarbapenemase phenotypic screening test(m CIM and e CIM)and PCR was used to detect the genotype,and the enzyme type of some strains were detected by the rapid detection method of carbapenemase(colloidal gold immunochromatography).The micro-broth dilution method was used to determine the minimal inhibitory concentration(MIC)of each antibiotic in 38 CRE strains.Combined antimicrobial susceptibility test was performed by the checkerboard dilution method.The Fractional inhibitory concentration(FIC)was calculated to determine the combined effect.The time-kill assays was used to study the bactericidal effect of CZA alone or combined with ATM.Results The bacterial composition analysis showed that Klebsiella pneumoniae accounted for the highest proportion(72.66%),followed by Escherichia coli(16.41%).CRE strains were mainly distributed in neurosurgery department,followed by ICU,with sputum specimens as the main type,followed by urine specimens.The resistance rates to ertapenem(ETP),IPM and MEM were 100%,83.6% and 80.5%,while the resistance rate to tigacycline was lower(2.5%).The results of m CIM and e CIM showed that 82 strains were positive for m CIM and negative for e CIM.22 strains were positive for m CIM and e CIM.m CIM negative,e CIM did not explain 24 strains.PCR results showed that 82 strains of KPC,21 strains of NDM and 1 strain of IMP.The results of enzyme type detection by colloidal gold immunochromatography were consistent with those of genetype detection by PCR.The synergy rate of IPM and MEM combined with SCF were the highest,which were 63.2% and 68.4%,and the sum of synergy rate and addition rate was 100%.While the sum of synergy and addition rate of IPM combined with FOS was the lowest(40.5%).In Klebsiella pneumoniae,Escherichia coli,KPC-positive strain and NDM-positive strains,the sum of synergy rate and addition rate of IPM and MEM combined with SCF were 100%,the sum of synergy rate and addition rate of IPM combined with FOS were the lowest,which were 24%,66.7%,22.7% and 64.3%.The MIC of CZA for the KPC-producing strains with Lower than or equal to 4μg/m L,while the MIC values of NDM and IPM strains against CZA were>128μg/m L.The synergy rate of CZA combined with ATM was 100%.All combined schemes had no antagonistic effect.The time-kill assays revealed that the strains regrew at CZA various concentrations of 0.5,1MIC at 10 hours and CZA at various concentrations of 2,4 and 8MIC showed signiffcant bactericidal efffciency to the KPC-producing strains.For NDM-,IMP-producing strains,no colony growth was detected after 8 hours of incubation with CZA in combination with ATM.Conclusion CRE strains were mainly distributed in neurosurgery department,mainly sputum specimens.The main drug-resistant strains were Klebsiella pneumoniae(72.66%),carrying KPC(78.8%),NDM(20.2%),IMP(1%)resistance genes.The synergy rate of IPM and MEM combined with SCF was the highest(63.2% and 68.4%,respectively),and the sum of the synergy rate and cumulative rate was 100%.CZA alone or in combination with ATM showed good synergistic bacteriostatic or bactericidal activity against KPC-,NDM-,IMP-producing strains.The antibacterial effect of different combination regimens of the same strain is different,and the effect of the same combination regimens of different strains is also different.The dosage ratio between the two drugs is also very important.These results have important reference value for strengthening clinical drug resistance monitoring and rational selection of antibiotics.