abaR基因对鲍氏不动杆菌生物膜形成的影响

Influences of abaR gene on biofilm formation of Acinetobacter baumannii

目的 检测鲍氏不动杆菌的耐药表型及abaR基因并探讨该基因对鲍氏不动杆菌生物膜形成的影响。方法 2014年2—7月，从上海交通大学医学院附属瑞金医院临床微生物科收集到159株鲍氏不动杆菌，按收集顺序从1开始进行编号。（1）通过检测鲍氏不动杆菌16S核糖体DNA序列对上述159株鲍氏不动杆菌进行菌种鉴定，根据药物敏感试验结果挑选出泛耐药菌株和敏感菌株，记录其菌株数及其标本来源。（2）取鲍氏不动杆菌泛耐药菌株和敏感菌株，培养12、24、48、72 h，通过噻唑蓝法测定鲍氏不动杆菌生物膜形成情况（以吸光度值表示）。（3）通过美国国家生物技术信息中心基因数据库进行基因序列分析鲍氏不动杆菌ATCC 17978中abaR基因序列，并将其与嗜油不动杆菌DR1菌株的LuxR型受体AqsR基因序列进行比对。以鲍氏不动杆菌ATCC 17978 abaR为目的基因序列，PCR扩增87和96号鲍氏不动杆菌菌株并测序。将测序结果与鲍氏不动杆菌ATCC 17978 abaR基因序列进行比对。（4）取87号和96号鲍氏不动杆菌菌株，每株细菌均分为0.1%二甲基亚砜（DMSO）组、10 μmol/L N－庚酰－L－高丝氨酸内酯（C7－HSL）组、10 μmol/L N－（3－氢氧化十二酰基）－DL－高丝氨酸内酯（OH－dDHL）组、1%DMSO组、100 μmol/L C7－HSL组、100 μmol/L OH－dDHL组，每组3孔，分别用对应终体积分数的DMSO或对应终物质的量浓度的C7－HSL和OH－dDHL处理。通过噻唑蓝法检测培养12、X4、48 h，87号菌株和96号菌株的生物膜形式情况（以吸光度值表示）。对数据行析因设计方差分析、单因素方差分析、LSD检验及Bonferroni校正。结果 （1）共收集18株泛耐药菌株和5株敏感菌株，其中泛耐药菌株主要分布于本院急诊ICU、烧伤整形科，标本以痰液、血液及创面分泌物为主；敏感菌株来源分散，标本以痰液为主。（2）鲍氏不动杆菌泛耐药菌株培养各时相点吸光度值与敏感菌株相近（P值均大于0.05）。鲍氏不动杆菌泛耐药菌株培养24 h吸光度值均显著高于该类菌培养12、48、72 h（P值均小于0.01）；鲍氏不动杆菌敏感菌株培养24 h吸光度值显著高于该类菌培养12 h（P〈0.01）。（3）鲍氏不动杆菌中存在LuxR型受体abaR基因序列，与嗜油不动杆菌DR1菌株中的LuxR型受体AqsR相似度为87%。87号菌株及96号菌株中的abaR基因序列与鲍氏不动杆菌ATCC 17978的相似度分别为98%、99%。（4）87号菌株0.1%DMSO组培养各时相点吸光度值和1%DMSO组相近（P值均大于0.05）。96号菌株0.1%DMSO组培养12 h吸光度值显著低于1%DMSO组（P〈0.01），培养24 h吸光度值显著高于1%DMSO组（P〈0.01）。87号菌株及96号菌株10 μmol/L C7－HSL组培养24 h吸光度值均显著低于各自对应的0.1%DMSO组（P值均小于0.01）。87号菌株100 μmol/L C7－HSL组培养各时相点吸光度值与1%DMSO组相近（P值均大于0.05），96号菌株100 μmol/L C7－HSL组培养12 h吸光度值显著低于1%DMSO组（P〈0.01）。87号菌株和96号菌株10 μmol/L OH－dDHL组培养各时相点吸光度值与0.1%DMSO组相近（P值均大于0.05）。87号菌株100 μmol/L OH－dDHL组培养各时相点吸光度值与1%DMSO组相近（P值均大于0.05）。96号菌株100 μmol/L OH－dDHL组培养12 h吸光度值显著高于1%DMSO组（P〈0.01）。87号菌株及96号菌株0.1%DMSO组和1%DMSO组培养24 h吸光度值均显著高于培养12、48 h（P值均小于0.01）。结论 鲍氏不动杆菌泛耐药菌株普遍存在。鲍氏不动杆菌中存在abaR基因，与生物膜形成相关。

Objective To detect drug-resistant phenotype and abaR gene of Acinetobacter baumannii （AB） and investigate influences of abaR gene on biofilm formation of AB.Methods From February to July 2014, 159 strains AB were collected from Department of Clinical Microbiology of Ruijin Hospital of School of Medicine of Shanghai JiaoTong University and numbered starting from 1 according time when they were collected. （1） The above-mentioned 159 strains of AB were identified by detecting gene sequence of 16S ribosomal DNA. According to results of drug sensitivity test, extensively drug-resistant strains and sensitive strains of AB were selected and counted, and their sources were recorded. （2） Extensively drug-resistant strains and sensitive strains of AB were collected to measure biofilm formation （denoted as absorbance value） by methyl thiazolyl tetrazolium method when strains at culture hour 12, 24, 48 and 72. （3） The abaR gene sequence of ATCC 17978 of AB was analyzed through Gene banks of National Center for Biotechnology Information and compared with AqsR gene sequence of LuxR type receptor of Acinetobacter oleivorans DR1. No. 87 and No. 96 AB strains were amplified and sequenced by polymerase chain reaction according to target gene sequence of abaR of ATCC 17978 of AB. The sequencing result was compared with abaR gene sequence of ATCC 17978. （4） No. 87 and No. 96 AB strains were collected and divided into 0.1% dimethyl sulfoxide （DMSO） group, 10 μmol/L N-heptanoyl-L-Homoserine lactone （C7-HSL） group, 10 μmol/L N-（3-Hydroxydodecanoyl）-DL-homoserine lactone （OH-dDHL） group, 1% DMSO group, 100 μmol/L C7-HSL group, and 100 μmol/L OH-dDHL, with 3 wells of each group. AB strains in the above groups were respectively dealt with DMSO of corresponding final volume fraction, C7-HSL and OH-dDHL of corresponding final amount-of-substance concentration. Biofilm formation （denoted as absorbance value） of AB was measured by methyl thiazolyl tetrazolium method at culture hour 12, 24, 48 and 72. Data were processed with analysis of variance of factorial design, one-way analysis of variance, LSD test and Bonferroni correction.Results （1） There were 18 extensively drug-resistant strains and 5 sensitive strains of AB. Samples of extensively drug-resistant strains were mainly collected from Emergency ICU and Department of Burns and Plastic Surgery of our hospital and were mainly from sputum, blood, and wound exudate. Samples of sensitive strains were collected dispersedly and were mainly from sputum. （2） Absorbance values of extensively drug-resistant strains and sensitive strains of AB at all culture time points were similar （with P values above 0.05）. Absorbance value of extensively drug-resistant strains of AB at culture hour 24 was obviously higher than that of these strains at culture hour 12, 48, or 72 （with P values below 0.01）. Absorbance value of sensitive strains of AB at culture hour 24 was obviously higher than that of these strains at culture hour 12 （P〈0.01）. （3） AbaR gene sequence of LuxR type receptor existed in AB. Similarity ratio between abaR gene sequence and LuxR type receptor AqsR gene sequence in Acinetobacter oleivorans DR1 was 87%. Similarity ratios between abaR gene sequence of No. 87 and No. 96 strains and ATCC 17978 of AB were 98% and 99%, respectively. （4） Absorbance values of 0.1% DMSO group of No. 87 strain at all culture time points were similar to those of 1% DMSO group （with P values above 0.05）. Absorbance value of 0.1% DMSO group of No. 96 strain at culture hour 12 was obviously lower than that of 1% DMSO group （P〈0.01）, while that at culture hour 24 was obviously lower than that of 1% DMSO group （P〈0.01）. Absorbance values of 10 μmol/L C7-HSL group of No. 87 and No. 96 strains at culture hour 24 were obviously lower than those of 0.1% DMSO group （with P values below 0.01）. Absorbance values of 100 μmol/L C7-HSL group of No. 87 strain at all culture time points were similar to those of 1% DMSO group, respectively （with P values above 0.05）. Absorbance value of 100 μmol/L C7-HSL group of No. 96 strain at culture hour 12 was lower than that of 1% DMSO group （P〈0.01）. Absorbance values of 10 μmol/L OH-dDHL group of No. 87 and No. 96 strains were similar to those of 0.1% DMSO group （with P values above 0.05）. Absorbance values of 100 μmol/L OH-dDHL group of No. 87 strain at all culture time points were similar to those of 1% DMSO group （with P values above 0.05）. Absorbance value of 100 μmol/L OH-dDHL group of No. 96 strain at culture hour 12 was obviously higher than that of 1% DMSO group （P〈0.01）. Absorbance values of 0.1% DMSO group and 1% DMSO group of No. 87 and No. 96 strains at culture hour 24 were obviously higher than those at culture hour 12 and 48 （with P values below 0.01）.Conclusions Extensively drug-resistant strains of AB exist commonly. AbaR gene exists in AB has relation with biofilm formation of AB.