| Objective: With the increase of bacterial resistance,carbapenem-resistant Klebsiella pneumoniae(CRKP)has been found more and more frequently in clinic.Tigecycline is a novel antibiotic and one of the few antibacterial agents that can treat CRKP.Since the clinical application of tigecycline,there have been reports of tigecycline-resistant CRKP.The purpose of this study was to determine the resistance of CRKP to tigecycline and other commonly used antibiotics,and to understand the carrying of the drug resistant genes such as acr A,acr B,tol C,oqx A,oqx B,tet X,tet X1 and tet X2 in tigecycline-insensitive CRKP.Methods: Take 40 not-repeated CRKP strains which saved in the microorganism room of the first affiliated hospital of China Medical University and collected in January 2016 to June 2017.The resistance of CRKP to common drugs was determined by the BD Phoenix 100.The tigecycline-resistance of CRKP was detected by MTS method,microbroth dilution method,BD Phoenix 100,and disk diffusion method.The tigecycline-resistant CRKP was acquired by tigecycline induced,and the tet X,tet X1,tet X2,acr A,acr B,tol C,oqx A and oqx B genes in tigecycline-insensitive CRKP were detected by PCR.Results: 1.The sensitive rates of CRKP to amikacin,ciprofloxacin,ofloxacin,norfloxacin,aztreonam were lower than 10%,to ampicillin/sulbactam,piperacillin/tazobactam,ceftazidime,cefoxitin,ceftriaxone,cefuroxime,cefepime,cefoperazone/sulbactam,gentamycin,tobramycin and nitrofurantoin were 0,to trimethoprim/sulfamethoxazole was less than 50%.2.Tigecycline-resistance was detected by four methods,and the results showed that the insensitivity rate of MTS method was 5.00%,and the MIC ranged from 0.19 to 2 ug/ml;BMD was 7.50%,and the MIC ranged from 0.25 to 2 ug/ml;BD Phoenix 100 was 100.00%,and the MIC ranged from 2 to 4ug/ml;and the disk diffusion method was 10.00%.The results of BD Phoenix100 and the disk diffusion method were compared with those of MTS method,P < 0.05,and the difference was statistically significant.Compared with the MTS method,the results of the microbroth dilution method showed no significant difference(P > 0.05).3.The MIC of two tigecycline-insensitive CRKP strains was 4 ug/ml after one week of induction,and the two tigecycline-insensitive CRKP strains was transformed into tigecycline-resistant strains.The disk diffusion method showed that the bacteriostatic circles of the induced strains(three repeated strains)of no.13 and no.25 were all disappeared.The diameter of the bacteriostatic circles of the original strain no.13 was16 mm,and the diameter of the bacteriostatic circles of the original strain no.25 was20 mm.4.The electrophoresis strip diagram of PCR products showed that there were acr A and acr B bands in all four strains,and the positions of bands were basically consistent with the size of the target product,indicating that the two genes were positive in all four strains.Oqx A,oqx B,tol C,tet X,tet X1 and tet X2 showed no bands,indicating that the four strains did not have these six genes.The product sequencing results were input into the NCBI database for comparison,showing that the similarity between acr A and acrB and the target gene was 97% and 99%,respectively.Conclusion: 1.The sensitivity of CRKP to commonly used antimicrobial agents is extremely low,but the sensitivity to tigecycline is high,and tigecycline can be used as the preferred drug for the treatment of CRKP.2.When the disk diffusion method and automated instrument are used to determine the sensitivity of tigecycline,the sensitivity results can be directly reported.If the insensitivity results are found,it is recommended to use MTS method or microbroth dilution method to review the results.3.In vitro,continuous stimulation with low concentration of tigecycline can induce sustained increase in MIC of CRKP against tegacycline.4.The acr A and acr B may be correlated with drug resistance of tigecycline. |
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