烧伤患者鲍氏不动杆菌pgaABC基因簇表达及生物膜表型变化

Expressions of pgaABC gene clusters and changes in biofiim phenotype of Acinetobacter baumannii in burn patients

目的 观察并探讨烧伤患者鲍氏不动杆菌pgaABC基因簇转录表达水平及生物膜形成过程中表型变化.方法收集2009年1月-2010年10月笔者单位住院烧伤患者创面、血液和静脉导管分离的鲍氏不动杆菌24株,以其标准菌株ATCC 19606为对照.采用实时荧光定量RT-PCR技术检测各菌株pgaABC基因簇表达;分别采用改良微孔法及试管法在静置（静态）和摇菌（动态）状态下体外培养各菌株16 h,进行细菌生物膜半定量检测;各菌株体外培养48 h后荧光染色,激光扫描共聚焦显微镜观察并测定生物膜厚度.对数据行t检验.结果 （1）鲍氏不动杆菌临床菌株pgaB基因转录相对表达量为27.91±7.93,明显高于设定基准为1.00的标准菌株（t=5.77,P＜0.05）;pgaA和pgaC的表达量分别为1.01±0.28、1.15±0.38,与标准菌株比较差异无统计学意义（t值分别为0.04、0.64,P值均大于0.05）.（2）静态培养16 h,鲍氏不动杆菌临床菌株与标准菌株的生物膜半定量结晶紫染色吸光度值相近;而临床菌株动态培养16 h,生物膜半定量结晶紫染色形成明显的紫色环,吸光度值为1.25±0.31,显著高于标准菌株（0.76±0.03,t=2.67,P＜0.05）.（3）体外培养48 h,临床菌株ECM中绿色荧光强度及分布均多于标准菌株,临床菌株生物膜厚度为（27.3±9.4）μm,明显大于标准菌株的（15.6±1.7）μm,t=2.09,P＜0.05.结论烧伤患者鲍氏不动杆菌pgaABC基因簇中pgaB转录水平增高,从而导致细菌ECM增多,这可能与鲍氏不动杆菌临床菌株生物膜形成能力和厚度增加有关.

Objective To observe expressions of pgaABC gene clusters and changes in biofilm （BF） phenotype in Acinetobacter baumannii （AB） isolated from burn patients. Methods From January 2009 to October 2010, 24 strains of AB isolated from burn patients hospitalized in our burn wards were collected for the study, while the standard strain ATCC 19606 was used as control. Expressions of pgaABC gene clusters were detected by real time fluorescence quantitative RT-PCR. All strains were cultured for 16 hours in vitro, BF with semi-quantitative detection was respectively evaluated by modified microtiter-plate test under stable condition and tube test under shaking condition for expression of absorbance value. All strains were cultured for 48 hours in vitro, then stained with fluorescent agent and collected for measurement of BF thickness with confocal laser scanning microscopy （ CLSM ）. Data were processed with t test. Results （ 1 ） The expression of pgaB gene （27.91 ± 7.93 ） in clinical AB strains was much higher than that of standard strain ATCC 19606 （1.00, t = 5.77, P 〈 0.05 ）. There was no statistical difference in expression of pgaA and pagC genes between standard strain ATCC 19606 （1.00） and clinical AB strains （1.01 ± 0.28,1.15 ±0.38, with t value respectively 0.04, 0.64, P values all above 0.05）. （2） After being cultured for 16 hours, BF of clinical AB strains cultured under shaking condition formed distinct ＂purple circle＂ , and its absorbance value （ 1.25 ±0.31 ） was higher than that in standard strain ATCC 19606 （0.76 ± 0.03, t =2.67, P 〈 0.05 ）. There was no obvious difference in absorbance value between clinical AB strains and standard strain ATCC19606 cultured under stable condition. （3） After being culture for 48 hours, green fluorescence intensity and distribution in extracellular matrix of clinical AB strains were stronger as compared with those of standard strain ATCC 19606, and BF thickness in clinical AB strains [（27.3 ± 9.4） μm] was thicker than that in standard strain ATCC 19606 [（ 15.6 ± 1.7） μm, t = 2.09, P 〈 0. 05]. Conclusions The high expression of pgaB gene in AB strains isolated from burn patients can induce production of extracellular matrix, which may be related to increase in the ability and thickness of BF formation.