D-酪氨酸与3,3'-二氨基二丙基胺对多重耐药鲍曼不动杆菌生物膜形成与分散的影响

Effects of D-tyrosine and norspermidine on the biofilm formation and dispersion of multi-drug resistant Acinetobacter baumannii

目的探讨生物膜分散物质D-酪氨酸与3,3＇-二氨基二丙基胺对多重耐药鲍曼不动杆菌生物膜的影响。方法从住院患者痰标本中分离并筛选出40株多重耐药鲍曼不动杆菌,用96孔板培养结合结晶紫染色法观察其生物膜形成能力并绘制生物膜生长曲线,另分析D-酪氨酸与3,3＇-二氨基二丙基胺两种分散物质对多重耐药鲍曼不动杆菌生物膜的影响,再进行活菌计数以评估两种分散物质的杀菌效果。结果 40株多重耐药鲍曼不动杆菌有37株成膜能力阳性。生物膜的生长经历指数生长期（0~24 h）、平台期（24~42 h）和衰退期（42~54 h）3个阶段。与对照组相比,0.1 mmol/L 3,3＇-二氨基二丙基胺作用JYK-0301、JYK-0302和JYK-0303号菌株24 h后,吸光度（A570 nm）显著降低（t值分别为6.92、9.63和9.01,P值均〈0.05）;且用0.1 mmol/L的3,3＇-二氨基二丙基胺处理JYK-0301菌株24 h后可使平均活菌计数由3.10×108±8.83×107CFU/m L减至21.6±10.78 CFU/m L（t=4.31,P〈0.05）。与对照组相比,0.1 mmol/L 3,3＇-二氨基二丙基胺作用JYK-0301、JYK-0302和JYK-0303的成熟生物膜24 h后A570 nm显著减少,差异有统计学意义（t值分别为3.76、5.67和3.98,P均〈0.05）;且1 mmol/L的3,3＇-二氨基二丙基胺作用JYK-0301成熟生物膜24 h后可使平均活菌计数由2.83×109±1.02×109CFU/m L减少至1.07×103±8.20×102CFU/m L（t=6.34,P〈0.05）。与未加D-酪氨酸的对照组比较,不同浓度D-酪氨酸作用JYK-0301、JYK-0302、JYK-0303及其成熟生物膜24 h后,A570 nm差异均无统计学意义（P均〉0.05）。结论 3,3＇-二氨基二丙基胺能有效抑制多重耐药鲍曼不动杆菌生物膜的形成,且能分散成熟生物膜,具有一定杀菌作用。而D-酪氨酸对多重耐药鲍曼不动杆菌生物膜无类似能力。

Objective To investigate the effects of D-tyrosine and norspermidine on the biofilm of multi-drug resistant Acinetobacter baumannii（ MDRAB）. Methods Forty MDRAB isolates were obtained from sputum samples of inpatients. Then,their biofilm formation ability was evaluated by cell culture and crystal violet staining,and the biofilm growth curve was drawn. Next,the effects of Dtyrosine and norspermidine on the biofilm formation and dispersion of these isolates were analyzed,and their bactericidal ability was determined by count plate. Results Thirty-seven of the 40 MDRAB isolates displayed a biofilm-positive phenotype. The biofilm-forming process of MDRAB on a 96-well cell culture plate included exponential phase（ 0-24 h）,stationary phase（ 24-42 h） and decline phase（ 42-54 h）. Compared with the control,0. 1 mmol/L of norspermidine could significantly reduce the A570 nmvalue of JYK-0301,JYK-0302 and JYK-0303 isolates at 24 h（ t = 6. 92,9. 63 and 9. 01,respectively,P〈0. 05）. Moreover,after the JYK-0301 isolate was incubated with 0. 1 mmol / L of norspermidine for 24 hours,the mean plate count was reduced from 3. 10 × 108± 8. 83 × 107 CFU / m L to21. 6 ± 10. 78 CFU / m L（ t = 4. 31,P〈0. 05）. Similarly,compared with the control,0. 1 mmol / L of norspermidine could also reduce the A570 nmvalue of JYK-0301,JYK-0302 and JYK-0303 mature biofilm at 24 h（ t = 3. 76,5. 67 and 3. 98,respectively,P〈0. 05）.Moreover,after the JYK-0301 mature biofilm was incubated with 1 mmol / L of norspermidine for 24 hours,the mean plate count was reduced from 2. 83 × 109± 1. 02 × 109 CFU / m L to 1. 07 × 103± 8. 20 × 102 CFU / m L（ t = 6. 34,P〈0. 05）. However,all of different concentrations of D-tyrosine showed no any effect on the growth and mature biofilm of JYK-0301,JYK-0302 and JYK-0303 isolates（ P〈0. 05）. Conclusion Norspermidine but not D-tyrosine may inhibit the biofilm formation of MDRAB,disperse the mature biofilm and show bactericidal ability.