阴沟杆菌外膜通透性改变与其耐药性关系的研究

Study on the outer membrane permeation in the cross resistance of Enterobacter cloacae

用头孢噻肟梯度平板法多步诱导一株阴沟杆菌达稳定耐药 ,经药敏测定表明其对氟喹诺酮类、头孢菌素类、青霉素类交叉耐药。对耐药株外膜蛋白行 SDS- PAGE电泳 ,结果发现 36、39、40 k D等外膜蛋白带含量下降 ,与文献报告一致。用扫描透射电镜分别观察了阴沟杆菌的敏感株与耐药株 ,结果诱导耐药菌菌体变短 ,外膜皱褶明显 ,间隙加宽 ,胞内空泡减少 ,提示外膜屏障功能加强 ,细菌代谢不活跃 ,进一步以 HPL C测定细菌耐药前后对环丙沙星的摄取量 ,耐药菌与敏感菌摄取曲线峰浓度比 1∶ 1.2 8且耐药株中环丙沙星浓度低于临界耐药浓度 1mg/ L,这和药敏试验结果一致。本实验结果提示阴沟杆菌外膜通透性的下降可导致菌株对各类抗生素的交叉耐药 。

The relationship between the resistance to antibiotics and the outer membrane of a strain of Enterobacter cloacae were studied. Laboratory derived cefotaxime resistant mutant was induced by serial passages of clinical isolated susceptible strain on cefotaxime containing agar. Antibiotics susceptibility tests showed that the mutant was cross resistant to quinolones, cephalosporins and penicillins respectively. SDS PAGE electrophoresis revealed that major outer membrane protein bands of the mutant was reduced, including the 36,39 and 40kD. Morphological analysis was performed by scanning and transmission electron microscopy. The results showed that the mutant lost the rod like shape and had a more round appearance, more distinctive membrane fold, wide periplasmic space and less nucleiods. These changes may indicate outer membrane barrier of the mutant was stronger, and the bacterial metabolism was less active. The uptake of ciprofloxacin (CPLX) in the two strains with HPLC was also determined. Compared with the parent strain, the uptake of CPLX of the mutant was reduced: the ratio of peak concentration was 1∶1.28. These data suggests that the changes of the outer membrane of Enterobacter cloacae will lead to cross resistance to antibiotics, and the morphological changes were consistent with its function.