基于pDS132的基因敲除中创伤弧菌氯霉素的耐药性变化及影响

Changes and effects of chloramphenicol resistance of Vibrio vulnificus in gene knockout with pDS132

【背景】基于自杀载体的基因敲除在单基因敲除上的应用较为常见,但在多基因敲除过程中细菌耐药性的变化及对后续敲除的影响尚未明确。【目的】探究基于自杀载体pDS132的创伤弧菌vvhA与rtxA1双基因敲除株构建过程中,创伤弧菌对氯霉素耐药性的变化及对后续基因敲除的影响。【方法】基于自杀载体pDS132的同源重组法构建创伤弧菌YJ016的单基因敲除株YJ016-ΔvvhA、YJ016-ΔrtxA1和双基因敲除株YJ016-ΔvvhAΔrtxA1,记录单交换筛选时的氯霉素浓度与筛选平板上成功重组的菌株所占比例。琼脂稀释法测定野生型与敲除株的氯霉素最低抑菌浓度(Minimum InhibitoryConcentration,MIC)和抗性突变频率,纸片扩散法测定菌株对其他药物的敏感性,分析其对单交换筛选的影响。【结果】单基因敲除株的氯霉素MIC及氯霉素抗性突变频率高于野生型;双基因敲除株的庆大霉素抑菌圈直径小于野生型。单基因敲除时,单交换重组菌株的占比为100%(20/20);在YJ016-ΔvvhA上敲除rtxA1基因,氯霉素筛选浓度为2、4μg/mL时,单交换重组菌株的占比分别为40%(8/20)、5%(1/20);在YJ016-ΔrtxA1上敲除vvhA基因,氯霉素筛选浓度为2、4μg/mL时,单交换重组菌株的占比均为0%(0/20)。【结论】基于自杀载体pDS132的基因敲除中创伤弧菌氯霉素的耐药性升高,可能影响后续单交换重组的筛选,该结果为基于自杀载体的同源重组技术应用于多基因敲除提供了参考。

[Background]Many domestic studies have reported in detail the application of gene knockout with suicide vectors in single gene knockout.However,the changes in bacterial resistance after gene knockout and the impact on subsequent gene knockout remain unclear.[Objective]In order to explore the changes in the resistance of Vibrio vulnificus(V.vulnificus)to chloramphenicol and its effect on subsequent gene knockout in the process of gene knockout with pDS132,the vvhA and rtxA1 gene knockout strains were constructed.[Methods]The single gene knockout strains YJ016-ΔvvhA,YJ016-ΔrtxA1 and double gene knockout strains YJ016-ΔvvhAΔrtxA1 of V.vulnificus YJ016 were constructed by the homologous recombination with suicide vector pDS132.The concentrations of chloramphenicol and the proportions of strains successfully recombined on the screening plate were recorded.The minimum inhibitory concentration(MIC)of each strain to chloramphenicol and other antibiotics,and the mutation frequency of chloramphenicol resistance were determined and then analyzed.[Results]The MIC and the mutation frequency of chloramphenicol of the single gene knockout strains were higher than that of the wild type;the diameter of the gentamicin inhibition zone of the double gene knockout strains was smaller than that of the wild type.When the rtxA1 gene was knocked out on YJ016-ΔvvhA and the chloramphenicol concentration of was 2,4μg/mL,the proportions of strains with single-crossover recombination were 40%(8/20)and 5%(1/20),respectively;when the vvhA gene was knocked out on YJ016-ΔrtxA1,the proportion was 0%(0/20).[Conclusion]In the process of gene knockout with the suicide vector pDS132,the resistance of V.vulnificus to chloramphenicol increased,which may affect the subsequent screening in single-crossover recombination.The result is helpful for the application of the homologous recombination technique with suicide vector to multiple gene knockout.