鲍曼不动杆菌基因点突变与耐药性及生物膜特征的相关性研究

Correlation study between gene point mutation of Acinetobacter baumannii and drug resistance and biofilm characteristics

目的研究鲍曼不动杆菌基因组上3个基因ptk^(D569N),shlB^(R403H)及iclR^(Y49H)分别与基因pmrA^(I13M)双位点突变,对鲍曼不动杆菌的耐药性和生物膜形成的影响。方法在突变菌株AT196011的基础上,通过同源重组,构建3株双突变菌株AT196012、AT196013和AT196014;通过生长曲线考察不同点突变对细菌生长的影响;采用微量肉汤稀释法测定突变菌株对多黏菌素的最低抑菌浓度;通过时间-杀菌曲线动态分析,比较各突变菌株对多黏菌素的耐药性差异;采用结晶紫染色法对生物膜进行半定量测定,并用CCK-8试剂对生物膜内活菌数进行相对定量测定。结果对PCR产物进行电泳检测与测序表明三株双突变菌株构建成功。各突变菌株对多黏菌素的最低抑菌浓度和时间-杀菌曲线表明,相较于AT196011单突变菌株,双突变株AT196012和AT196014对多黏菌素的耐药性增强,而AT196013的耐药性无明显变化;在生物膜形成方面,3株双突变菌株的生物膜生成量显著高于AT196011单突变菌株;1/2 MIC浓度的多黏菌素对AT196013与AT196014双突变菌株生物膜形成的抑制作用显著低于AT196011单突变菌株,其生物膜内活菌量也显著多于AT196011单突变菌株。结论基因ptk^(D569N)、iclR^(Y49H)和基因pmrA^(I13M)的双点突变,会增加鲍曼不动杆菌对多黏菌素的耐药性;基因ptk^(D569N),shlB^(R403H)H和iclR^(Y49H)和pmrA^(I13M)的双点突变,会协同促进生物膜的形成,且shlB^(R403H)H和iclR^(Y49H)的促进作用更强,相应双突变菌株对多黏菌素的耐受性更强。

Objective To investigate the effects of three point mutations in three different genes(ptk^(D569N),shlB^(R403H)H and iclR^(Y49H))on drug resistance and biofilm formation in Acinetobacter baumannii.Methods Homologous recombination was used to create three double mutant strains,AT196012,AT196013,and AT196014,based on the previously created mutant strain AT196011;growth curves were used to examine the effects of various point mutations on bacterial growth,and the micro-broth dilution method was used to estimate the mutant strain’s lowest inhibitory concentration for colistin;the differences in the resistance of each mutant strain to colistin were compared using dynamic analysis of the time-kill curve;the biofilms were semi-quantitatively quantified by crystal violet staining,and CCK-8 reagent was used to measure relative viable bacterial counts in the biofilms.Results Three double mutant strains were successfully created,according to the electrophoresis and sequencing results of the PCR products.The double mutant strains AT196012 and AT196014 had stronger drug resistance than the single mutant strain AT196011,with no discernible difference in resistance between AT196013 double mutant strain and AT196011 single mutant strain;In terms of biofilm formation,the three double mutant strains produced significantly more biofilm than the single mutant strain AT196011;the inhibitory effect of 1/2 MIC colistin on the formation of biofilm in the double mutant strains AT196013 and AT196014 was significantly lower than in the single mutant strain AT196011 and the amount of viable bacteria in the biofilm was significantly higher than that of AT196011 single mutant strain.Conclusions Genes ptk^(D569N) and iclR^(Y49H),and the gene pmrA^(I13M),have a synergistic effect in drug resistance;the gene ptk^(D569N),shlB^(R403H)H and iclR^(Y49H),and the gene pmrA^(I13M),also have synergistic effects in promoting the formation of biofilms.shlB^(R403H)H and iclR^(Y49H) had stronger promoting effects,and the corresponding double mutant strains had stronger tolerance to colistin.