敲除abaR基因对鲍氏不动杆菌生长代谢和生物膜形成的影响

Influence of abaR gene knockout on growth metabolism and biofilm formation of Acinetobacter baumannii

目的探讨敲除abaR基因对鲍氏不动杆菌生长代谢和生物膜形成的影响。方法应用同源重组方法敲除鲍氏不动杆菌ATCC 17978(野生株)abaR基因,获得ATCC 17978abaR敲除株(ATCC 17978/ΔabaR::Kn),通过PCR电泳和测序验证。应用酶标仪测定鲍氏不动杆菌野生株和敲除株培养18 h内的生长曲线,同时应用结晶紫染色法分别于培养8、24、48 h,测定生物膜形成能力,结果均以吸光度值表示,每个时间点样本数为3。对数据进行析因设计方差分析、单因素方差分析、t检验、LSD检验。结果(1)打靶片段ΔabaR::Kn的PCR产物大小为3029 bp。ATCC 17978野生株成功敲除abaR基因后获得ATCC 17978敲除株(ATCC 17978/ΔabaR::Kn),敲除株PCR产物大小3300 bp,说明abaR基因被成功敲除。(2)培养2、3、4 h,鲍氏不动杆菌野生株吸光度值稍高于敲除株;培养5~18 h,鲍氏不动杆菌野生株和敲除株各时间点吸光度值相近。(3)培养8、24 h,鲍氏不动杆菌野生株生物膜形成能力(0.644±0.066、0.574±0.184)与敲除株(0.559±0.008、0.394±0.030)相近(t=2.209、1.167,P>0.05);培养48 h,鲍氏不动杆菌野生株生物膜形成能力(1.157±0.259)明显强于鲍氏不动杆菌敲除株(0.576±0.026,t=3.865,P<0.05)。鲍氏不动杆菌野生株培养48 h的生物膜形成能力明显强于培养8、24 h(P<0.05),鲍氏不动杆菌敲除株培养24 h的生物膜形成能力明显弱于培养8、48 h(P<0.05)。结论利用同源重组方案,可成功敲除鲍氏不动杆菌ATCC 17978 abaR基因,获得鲍氏不动杆菌敲除株ATCC 17978/ΔabaR::Kn。敲除abaR基因后,鲍氏不动杆菌自身的生长代谢不受影响,但其生物膜形成能力明显减弱。

Objective To investigate the influence of abaR gene knockout on growth metabolism and biofilm formation of Acinetobacter baumannii.Methods The abaR gene was knocked out from Acinetobacter baumannii standard strain ATCC 17978(wild strain)by homologous recombination method,and then the ATCC 17978 abaR knockout strain(ATCC 17978/ΔabaR::Kn)was obtained and verified by polymerase chain reaction(PCR)electrophoresis and sequencing.The growth curves of Acinetobacter baumannii wild strain and Acinetobacter baumannii knockout strain were determined by microplate reader within cultivation hour(CH)18,and the biofilm formation ability was measured by crystal violet staining at CH 8,24,and 48,respectively.The sample number at each time point was 3.The results were denoted as absorbance value.Data were processed with analysis of variance of factorial design,one-way analysis of variance,t test,and least-significant difference test.Results(1)The length of PCR product of target fragmentΔabaR::Kn was 3029 bp.The abaR gene was knocked out to obtain the knockout strain ATCC 17978/ΔabaR::Kn.The length of PCR product of the knockout strain was 3300 bp.The abaR gene was successfully knocked out.(2)At CH 2,3,and 4,the absorbance values of Acinetobacter baumannii wild strain were slightly higher than those of the knockout strain.The absorbance values of Acinetobacter baumannii wild strain and knockout strain were similar from CH 5 to 18.(3)At CH 8 and 24,the biofilm formation ability of Acinetobacter baumannii wild strains(0.644±0.066,0.574±0.184)was similar to that of knockout strains(0.559±0.008,0.394±0.030,t=2.209,1.167,P>0.05).At CH 48,the biofilm formation ability of Acinetobacter baumannii wild strains(1.157±0.259)was significantly stronger than that of Acinetobacter baumannii knockout strains(0.576±0.026,t=3.865,P<0.05).The biofilm formation ability of Acinetobacter baumannii wild strains at CH 48 was significantly stronger than that at CH 8 and 24(P<0.05).The biofilm formation ability of Acinetobacter baumannii knockout strains at CH 24 was significantly weaker than that at CH 8 and 48(P<0.05).Conclusions The abaR gene of Acinetobacter baumannii ATCC 17978 can be successfully knocked out by homologous recombination to obtain its knockout strain ATCC 17978/ΔabaR::Kn.The abaR gene does not affect the growth and metabolism of Acinetobacter baumanniibut can weaken its biofilm formation ability.