RND外排泵对多重耐药鲍曼不动杆菌AYE菌株生物被膜形成的影响

Effect of RND efflux pump on biofilm formation of multidrug-resistant Acinetobacter baumannii AYE strain

摘要:

目的 探讨多重耐药鲍曼不动杆菌AYE菌株RND外排泵基因影响生物被膜形成的作用机制。 方法 采用无标记基因敲除方法分别敲除AYE菌株的RND外排泵基因（adeB、adeFGH、adeIJK），采用实时荧光定量聚合酶链式反应（qRT-PCR）验证菌株外排泵基因敲除情况，通过结晶紫染色法检测菌株基因敲除前后生物被膜形成情况，比较4种外排泵抑制剂（PAβN、奥美拉唑、维拉帕米、CCCP）对AYE菌株生物被膜形成的影响。 结果 qRT-PCR结果显示，AYE菌株RND外排泵基因（adeB、adeFGH、adeIJK基因）成功敲除，获得AYE△ adeB、AYE△ adeFGH、AYE△ adeIJK菌株，生物被膜形成量分别为1.59±0.06、2.15±0.19、1.91±0.02，其中adeB和adeIJK基因敲除后菌株生物被膜形成量低于敲除前（AYE：2.31±0.01），差异均有统计学意义（均P < 0.05）。在PAβN、奥美拉唑、维拉帕米、CCCP作用下，鲍曼不动杆菌标准菌株AYE的生物被膜形成量分别为2.14±0.03、2.24±0.02、1.93±0.05、2.09±0.04，均低于对照组（AYE：2.31±0.01），差异均有统计学意义（均P < 0.05），其中PAβN形成量最少。 结论 外排泵基因adeB、adeFGH、adeIJK对多重耐药鲍曼不动杆菌AYE生物被膜的形成有重要作用，外排泵抑制剂PAβN、奥美拉唑、维拉帕米、CCCP对鲍曼不动杆菌AYE菌株生物被膜的形成有抑制作用。

Abstract:

Objective To explore the mechanism of resistance-nodulation-cell division (RND) efflux pump genes of multidrug-resistant Acinetobacter baumannii (MDR-AB) AYE strain in influencing biofilm formation. Methods RND efflux pump genes(adeB, adeFGH, adeIJK) of Acinetobacter baumannii AYE strain were knocked out by marker-free gene knockout method, real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to verify the gene knockout of strain efflux pump genes, biofilm formation before and after gene knockout was detected by crystal violet staining, effect of 4 efflux pump inhibitors (PAβN, omeprazole, verapamil and CCCP) on biofilm formation of AYE strain was compared. Results qRT-PCR showed that RND efflux pump genes (adeB, adeFGH, adeIJK genes) of AYE strain were successfully knocked out, AYE△ adeB, AYE△ adeFGH, and AYE△ adeIJK strains were obtained, the amount of biofilm formation was 1.59±0.06, 2.15±0.19 and 1.91±0.02 respectively, the amount of biofilm formation after adeB and adeIJK gene knockout was lower than that before knockout (AYE: 2.31±0.01), difference were all statistically significant (all P < 0.05). Under the effect of PAβN, omeprazole, verapamil and CCCP, the amount of biofilm formation of standard strain AYE of Acinetobacter baumannii were 2.14±0.03, 2.24±0.02, 1.93±0.05 and 2.09±0.04 respectively, which were all lower than those in the control group (AYE: 2.31±0.01), difference was statistically significant (all P < 0.05), PAβN formation was the least. Conclusion Efflux pump genes adeB, adeFGH and adeIJK play important role in the formation of biofilm of MDR-AB AYE, efflux pump inhibitors PAβN, omeprazole, verapamil and CCCP have an inhibitory effect on biofilm formation of Acinetobacter baumannii AYE strain.