| ObjectiveTo evaluate the in vitro antibacterial activity of tigecycline(TGC),tigecycline combined with amikacin(AK)and tigecycline combined with polymyxin B(PB)against carbapenem resistant Klebsiella pneumoniae(CRKP).To investigate the epidemic distribution of related drug resistance genes and to explore the effect of resensitization solution on tigecycline susceptibility test by disk method.The efficacy and in vitro drug sensitivity test of different anti-infective regimens in 4 cases of CRKP bloodstream infection were analyzed in order to provide reference for clinical treatment of carbapenem-resistant Klebsiella pneumoniae.MethodsPart Ⅰ:A total of 71 non-repetitive CRKP strains isolated from the clinical departmentof our hospital from February 2018 to October 2018 were collected.n vitro drug sensitivity test of antimicrobial agents contained in GN-09 drug sensitive cards by VITEK 2 Compact instrument method.The drug sensitivity of tigecycline and amikacin in vitro was determined by disk method.The MIC values of tigecycline,tigecycline combined with amikacin and tigecycline combined with polymyxin B were detected by microbroth dilution method.Different concentrations of drug sensitivity groups were designed by checkerboard combined drug sensitivity test,and the fractional inhibitory concentration index(FICI)was calculated.The reliability of disk method for the detection of tigecycline was studied by dripping resensitization solution.The resistance genes of tigecycline,amikacin and polymyxin B in CRKP were screened by PCR(including tet(A),tet(X),rmt A,rmt B,arm A,aac(3)-I,aac(3)-II,aac(6￠)-Ib,ant(3￠￠)-Ia,MCR-1).Some of the sequencing results were compared with BLAST in Gene Bank database to determine drug resistance genes.Part Ⅱ:Four strains of CRKP isolated from clinical blood infection in our hospital from February 2019 to March 2020 and their clinical data were collected.VITEK 2Compact method was used to detect the MIC of common antimicrobials(same as the first part),and checkerboard combined drug sensitivity test was used to detect the MIC of tigecycline,polymyxin B and tigecycline combined with polymyxin B,and the FIC index was calculated.PCR method was used to screen the common related drug resistance genes of tigecycline and polymyxin B(same as the first part),and the association between combined anti-infective regimen,inflammatory index and in vitro drug sensitivity test was analyzed with clinical cases.ResultPart Ⅰ:The results of VITEK 2 Compact method showed that the resistance rates ofCRKP to ceftazidime,ceftriaxone,cefepime,ampicillin/sulbactam and piperacillin/tazobactam were all 100%(71/71),to imipenem and meropenem were98.6%(70/71),to ciprofloxacin and levofloxacin were 93.0%(66/71),the resistance rates of aztreonam and amikacin were 97.2%(69/71)and 87.3%(62/71),respectively.The results of disk method showed that the resistance rates of CRKP to amikacin and tigecycline were 76.1%(54/71)and 5.6%(4/71),respectively,and the results of microbroth dilution method showed that the resistance rates of CRKP to amikacin,tigecycline and polymyxin B were 85.9%(61/71),7.0%(5/71)and 1.4%(1/71),respectively.When tigecycline combined with amikacin acted on CRKP,the synergistic effect,additive effect,irrelevant effect and antagonistic effect of checkerboard combined drug sensitivity test were 8.5%(6/71),15.5%(11/71),70.4%(50/71)and5.6%(4/71),respectively.When tigecycline was combined with polymyxin B,the synergistic,additive and unrelated effects of checkerboard combined drug sensitivity test were 4.2%(3/71),78.9%(56/71)and 16.9%(12/71),respectively,and there was no antagonistic effect.Methodological evaluation:The sensitivity of CRKP to amikacin detected by microbroth dilution method and VITEK 2 Compact instrument method was the same(c~2<0.01,P>0.99),and the difference between the results of disk method and the former two methods was statistically significant(c~2=13.56,P<0.01;c~2=10.33,P<0.01).The difference between microbroth dilution method and disk method in detecting the sensitivity of tigecycline was statistically significant(c~2=34.33,P<0.01).After the combined action of tigecycline and amikacin,the MIC value of the two drugs decreased,and the MIC value of tigecycline was significantly different from that of the single drug(t=2.39,P<0.05),the MIC value of amikacin in combined drug sensitivity decreased more significantly than that of single drug,and the difference was statistically significant(t=16.61,P<0.01).After adding resensitization solution,the sensitive rate of tigecycline(97.2%)was significantly higher than that of 66.2%before dripping resensitization solution(c~2=8.90,P<0.01)and 77.5%of microbroth dilution method(c~2=11.22,P<0.01).PCR amplification of 71 strains of CRKP and 23 strains carrying aminoglycoside resistance genes,those carrying a single drug resistance gene are:the carrying rates of rmt B gene,aac(3)-I gene,aac(3)-II gene and aac(6￠)-Ib gene were 9.9%(7/71),2.8%(2/71),4.2%(3/71)and 14.1%(10/71),respectively.Those who carry two kinds of drug resistance genes at the same time are:the carrying rate of rmt B gene and aac(6￠)-Ib gene was 1.4%(1/71).No rmt A drug resistance gene,arm A drug resistance gene and ant(3￠￠)-Ia drug resistance gene were detected.The detection of tigecycline resistance gene showed that the carrying rate of tet(A)gene of efflux pump was 81.7%(58/71).The carrying rates of tet(A)gene and rmt B gene,tet(A)gene and aac(6￠)-Ib gene,tet(A)gene and aac(3)-I gene were 5.6%(4/71),14.1%(10/71)and2.8%(2/71),respectively.No tigecycline inactivating enzyme tet(X)gene was detected.No MCR-1 gene resistant to polymyxin B was detected in 71 strains of CRKP.Part Ⅱ:The efficacy of different anti-infective regimens in the treatment of CRKP bloodstream infection was different.In 2 cases of CRKP,the inflammatory indexes(PCT,CRP,IL-6)decreased significantly after meropenem combined with sultopenem and tigecycline combined with sultopenem,respectively.Data deficiency in 2 cases after meropenem combined with vancomycin and tigecycline combined with polymyxin B,respectively,and the follow-up antibacterial efficacy could not be observed for the time being.The results of VITEK 2 Compact assay showed that 4 strains of CRKP were resistant to cephalosporins,imipenem,ampicillin/sulbactam and aztreonam,3 strains were resistant to meropenem,ciprofloxacin,levofloxacin and piperacillin/tazobactam,and 2 strains were resistant to amikacin.Four strains of CRKP were detected by microbroth dilution method.The results showed that 2 strains of CRKP were sensitive to tigecycline,2 strains were mediated to tigecycline,and 4 strains were sensitive to polymyxin B.The results of combined drug sensitivity of tigecycline and polymyxin B showed that 3 strains of CRKP had additive effect and only 1 strain had synergistic effect.PCR assay showed that all the 4 strains of blood CRKP carried the drug resistance gene of efflux pump tet(A),but no drug resistance genes of tet(X)and MCR-1 were found.ConclusionPart Ⅰ:The drug resistance of CRKP is serious.The sensitivity of microbroth dilution method and VITEK 2 Compact instrument method for detection of amikacin is the same,however,there were differences in the sensitivity of amikacin and tigecycline by disk method,and the laboratory should take microbroth dilution method as the gold standard.The combined drug sensitivity test showed that the synergistic effect of tigecycline combined with amikacin and polymyxin B was not good,respectly,but the MIC decreased in varying degrees after combined action,and the decrease of MIC of amikacin was more obvious.Therefore,the dose of amikacin can be reduced properly when tigecycline combined with amikacin is selected in clinical treatment.The tigecycline resistance gene is mainly tet(A)gene,and the amikacin resistance gene is mainly rmt B gene and aac(6￠)-Ib gene.It is not possible to confirm the relationship between the drug resistance gene and the drug sensitivity of tigecycline combined with amikacin.Tet(X)gene and MCR-1 gene have not been detected in this study,but further attention should be paid to it.The sensitivity of resensitization solution to tigecycline is different from that of microbroth dilution method,and whether it is consistent with the clinical effect needs to be further confirmed.Part Ⅱ:PCT and CRP decreased progressively after effective treatment of CRKP bloodstream infection,and PCT may decrease first.When CRKP bloodstream infection is suspected clinically,a combination therapy based on tigecycline can be considered.In this study,the effect of tigecycline dose on anti-infective therapy was not observed.The combined antibacterial effect of tigecycline and polymyxin B in vitro is mainly additive,which can be used in the treatment of CRKP bloodstream infection.At the same time,4strains of CRKP drug resistance genes are found to carry tet(A)gene,but no tet(X)and MCR-1 resistance genes. |
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