鲍曼不动杆菌分泌物对铜绿假单胞菌及其生物膜的作用

Effect of Acinetobacter baumannii secretions on Pseudomonas aeruginosa cell growth and biofilm formation

目的探讨鲍曼不动杆菌培养上清对铜绿假单胞菌的浮游菌生长及生物膜形成的影响。方法收集鲍曼不动杆菌标准菌株(ATCC 19606和ATCC 1195)和临床菌株(AB23、AB39和AB53),提取6、12、16、24和48 h培养上清液。利用96孔板结合结晶紫染色法检测其培养上清液对铜绿假单胞菌标准菌株PAO1及其生物膜形成的影响;配制2×LB培养基,排除营养消耗对铜绿假单胞菌的影响;并进一步利用相对分子质量3 000蛋白质浓缩管对其培养上清液进行分离浓缩,初步探讨鲍曼不动杆菌培养上清液中有效成分的相对分子质量。结果 12~24 h内的鲍曼不动杆菌培养上清液,抑制铜绿假单胞菌增殖的效果最为显著,为便于操作,本实验采用16 h培养上清液进行后续实验。50%ATCC 1195和ATCC 19606培养上清液能显著抑制铜绿假单胞菌标准菌株PAO1浮游菌的增殖,可分别使其630 nm处的吸光度从0.688±0.014和0.692±0.014减少至0.431±0.023和0.428±0.020(t=16.780,P<0.05;t=18.500,P<0.05);且50%ATCC 1195和ATCC 19606培养上清液能显著抑制铜绿假单胞菌PAO1生物膜的形成,可使生物膜的形成量(A_(570 nm))从2.071±0.068和1.986±0.023减少至1.639±0.042和1.525±0.202(t=9.358,P<0.05;t=3.924,P<0.05);此外,与50%鲍曼不动杆菌培养上清液组相比,培养上清液中相对分子质量<3 000的成分抑制作用显著,可使生物膜的形成量从1.177±0.040减少至1.056±0.030(t=4.192,P<0.05),而>3 000的成分并无抑制作用。结论鲍曼不动杆菌分泌物能有效抑制铜绿假单胞菌浮游菌的增殖和生物膜的形成,其有效蛋白质成分相对分子质量<3 000。

Objective To investigate the effects of Acinetobacter baumannii culture supernatants on planktonic cell growth and biofilm formation of Pseudomonas aeruginosa. Methods The standard isolates（ ATCC 19606,ATCC 1195） and clinical isolates（ AB23,AB39,AB53） of Acinetobacter baumannii were collected and the 6,12,16,24 and 48 hour-cultured supernatants were extracted. The effects of the culture supernatants on the biofilm formation of Pseudomonas aeruginosa PAO1 were detected on the 96-well plate combined with crystal violet staining. Two-fold concentration of LB medium was prepared to eliminate the effects of nutrition consumption of Acinetobacter baumannii during culture on Pseudomonas aeruginosa growth. The active ingredients in the supernatant of Acinetobacter baumannii culture medium were investigated by using the concentrated tube containing protein with relative molecular mass 3 000. Results The most suitable period for Acinetobacter baumannii culture supernatant extraction was between 12 to 24 hours,so the 16 hourcultured supernatant was chosen for next experiments. The 50% culture supernatant of Acinetobacter baumannii ATCC 1195 and ATCC19606 significantly inhibited the planktonic cell growth of Pseudomonas aeruginosa PAO1,in which the absorbance at 630 nm reduced from（ 0. 688 ± 0. 014） and（ 0. 692 ± 0. 014） to（ 0. 431 ± 0. 023） and（ 0. 428 ± 0. 020） respectively（ t = 16. 780,P 〈 0. 05; t =18. 500,P 〈 0. 05）. The 50% culture supernatant of Acinetobacter baumannii ATCC 1195 and ATCC 19606 also significantly inhibited the biofilm formation of Pseudomonas aeruginosa PAO1 with decreased absorbance at 570 nm from（ 2. 071 ± 0. 068） and（ 1. 986 ±0. 023） to（ 1. 639 ± 0. 042） and（ 1. 525 ± 0. 202） respectively（ t = 9. 358,P 〈 0. 05; t = 3. 924,P 〈 0. 05）. The biofilm inhibitory effect of the protein with relative molecular mass less than 3 000 was obviously observed by reducing amount of biofilm formation from（ 1. 177 ± 0. 040） to（ 1. 056 ± 0. 030）（ t = 4. 192,P 〈 0. 05）,while there was no inhibitory effect of the proteins with relative molecular mass more than 3 000 in the composition. Conclusion Acinetobacter baumannii culture supernatant could effectively inhibit the planktonic cell growth and biofilm formation of Pseudomonas aeruginosa and the relative molecular mass of active ingredients in the culture supernatant may be less than 3 000.