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中国图书资料分类法分类号R378.21
Active efflux of antibiotic in susceptible and mutiple-antibiotic-resistant Escherichia coli
ZHANG Xiao-Lin＃, WANG Fu, ZHU De-Mei
(＃Institute of Clinical Pharmacology, the First Hospital, Beijing Medical University, Beijing100083)
MeSHEscherichia coli／drug effTetracyclines/metabPenicillin G/metabDrug tolerance
ABSTRACTObjective: To investigate active efflux of drugs in clinical isolated strains of Escherichia coli.Methods: The accumulation of tetracycline and penicillin in multiple-antibiotic-resistance (Mar) and susceptible strains of Escherichia coli and ATCC25922 was determined using filitration method.Results: The steady concentration of tetracycline and penicillin in Mar and susceptible strains of Escherichia coli was decreased with supply of energy, with supply of inhibitor of energy; the steady concentration was remarkably increased, but the change in susceptible strain was less than in Mar strain. However, the concentration of tetracycline and penicillin in ATCC25922 was significantly decreased with supply of inhibitor of energy.Conclusion: There was active efflux of tetracycline and penicillin in clinical isolated strains of Escherichia coli, but the capability was less than in Mar strains.
(J Beijing Med Univ, 1999,31:67-70)
近年来对细菌耐药机制的研究发现,在大肠杆菌、绿脓杆菌、金黄色葡萄球菌等耐药菌株中存在主动外排系统,主动外排系统的存在是这些细菌产生耐药性的重要原因［1～4］。主动外排系统由于能主动将抗菌药物从细胞内泵出细胞外而使细菌获得耐药性。然而,我们的研究表明,在临床分离的大肠杆菌敏感株中亦可能存在主动外排系统。
1材料与方法
1.1试剂
3H-四环素，2.849×1010Bq*mmol－1，DuPont公司产品。3H-青霉素(penicillin, Pen)，7.77×1011Bq*mmol－1，Amshan公司产品。Luria-Bertani (LB)肉汤培养基，每升含NaCl 10 g、胰蛋白胨5 g、酵母抽提物10 g。
1.2抗生素
氯霉素(chloramphenicol, Chl)、四环素(tetracyclin, Tet)、哌拉西林(piperacillin, Pip)、庆大霉素(gentamicin, Gen)、诺氯沙星(norfloxacin, Nor)、环丙沙星(ciprofloxacin, Cip)均为卫生部药品生物制品检验所标准品。
1.3菌株
对上海医科大学华山医院1994～1995年度临床分离的大肠杆菌进行筛选。以对氯霉素、四环素、哌拉西林、庆大霉素、氟喹诺酮均耐药的菌株为多重耐药株,均敏感的菌株为敏感野生株。ATCC25922为药敏质控菌。LM313.1为野生株AG100经氯霉素诱导的耐药株,含多重耐药(multiple-antibiotic-resistance, Mar)操纵子。LM313.5是LM313.1经噬菌体将Tn5插入MarA并使之失活的突变株。该两株细菌由美国Tufts大学的Levy博士赠送。
1.4最低抑菌浓度(minium inhibation concentra-
tion, MIC)测定
以琼脂对倍稀释法测定抗菌药物对大肠杆菌的MIC。
1.5大肠杆菌对四环素和青霉素的积聚实验
大肠杆菌对四环素的积聚实验按文献［4］的方法并加改进。大肠杆菌对青霉素的积聚实验按文献［5］的方法并加以改进。大肠杆菌细胞内药物的稳态质量摩尔浓度以nmol*g－1表示。
2结果
2.1大肠杆菌对6种抗菌药物的敏感性(表1)
表1选择大肠杆菌对抗菌药物的敏感性(MIC)
Table 1The Susceptibility of Escherichia coli to 6 Antibiotics(MIC)
Strain ofE.coli ρ/mg.L－1 Pip Gen Tet Chl Nor Cip R12 ≥256 ≥16 ≥32 ≥64 ≥32 ≥8 R28 ≥256 ≥16 ≥32 ≥64 ≥32 ≥8 S36 ≤4 ≤0.5 ≤1 ≤2 ≤1 ≤0.25 LM313.1 4 0.25 4 ≥64 0.5 0.125 LM313.5 0.06 0.25 0.25 8 0.03 0.016 ATCC25922 1 1 0.5 2 0.5 0.008
从临床分离的菌株中筛选出多重耐药株R12、R28,敏感株S36。
2.2临床分离的大肠杆菌对四环素的积聚(图1)
CCCP was added at a final concentration of 0.25 mmol.L－1 at
the time indicated by the arrow.
1临床分离大肠杆菌对四环素的积聚
Figure 1The accumulation of tetracycline in clinical
isolates of Escherichia coli
在加入3H-四环素后1 min R28、R12、S36细胞内药物质量摩尔浓度均接近稳态。R28细胞内药物的稳态质量摩尔浓度大约为S36的40%,R12约为S36的50%。加入氰氯苯腙(carbonyl cyanide m-chlorphenyl-hydrazone, CCCP)后,R28、R12细胞内四环素稳态质量摩尔浓度上升了大约1.5倍,S36略有上升。
2.3临床分离大肠杆菌对青霉素的积聚(图2)
CCCP was added at a final concentration of 0.25 mmol.L－1 at the time indicated by the arrow and glucose was added at a final concentration of 0.2% at the time indicated by the black arrow.
2临床分离大肠杆菌对青霉素的积聚
Figure 2The accumulation of penicillin in clinical
isolates of Escherichia coli
当悬浮液中不含葡萄糖时,S36、R12细胞内稳态青霉素质量摩尔浓度相似。加入葡萄糖后,两者细胞内青霉素质量摩尔浓度均下降,尤以R12为甚。而当加入CCCP后,细胞内青霉素质量摩尔浓度均上升,R12上升幅度大于S36。R28中加入CCCP后,细胞内青霉素质量摩尔浓度上升了3倍。
2.4大肠杆菌LM313.1与LM313.5对四环素的积聚(图3)
CCCP was added at a final concentration of 0.25 mmol.L－1 at the time indicated by the arrow．
3大肠杆菌LM313.1和LM313.5对四环素的积聚
Figure 3The accumulation of tetracycline in Escherichiacoli
LM313.1 and LM313.5
LM313.1中Tet质量摩尔浓度约为LM313.5的60%,加入CCCP后,LM313.1中稳态四环素质量摩尔浓度上升了近60%,LM313.5仅轻度增加。
2.5大肠杆菌ATCC25922对四环素和青霉素的积聚(图4)
