重组溶葡球菌酶抗菌药效学及耐药机制研究
摘要
溶葡球菌酶(Lysostaphin)是一种含Zn~(2+)的内(切)肽酶，为单链分子，相对分子量约27kDa，最早于六十年代由Schindler和Schuhardt从模仿葡萄球菌(S．simulan)的培养物中发现并分离。具有特异性水解细菌胞壁肽聚糖交联结构Gly五肽桥联(水解第2与第3位Gly形成的肽键)的作用。由于Gly五肽桥联结构主要存在于葡萄球菌细胞壁，其中又以金黄色葡萄球菌(S．aureus)细胞壁中分布最广，因而此酶主要对葡萄球菌尤其金葡菌表现很强的溶杀菌作用。此酶抗金葡菌感染的药用价值在六、七十年代开始得到很多开发人员的重视，并在大量动物试验和部分人体治疗中得到证明。但此后由于一些抗葡萄球菌效果良好的抗生素如万古霉素(vancomycin)、替考拉宁(teicoplanin)等相继出现，加之部分研究者对酶纯度和抗原性的顾虑，此酶的药用研究一度有所延缓。八十年代以后，随着生物工程技术的迅速发展，国内外研究人员利用基因重组的方法将此酶成功表达于多种工程菌并实现产业化，＞90％以上的高纯度重组溶葡球菌酶使实现临床应用成为可能。目前随着耐甲氧西林金葡菌(MRSA)等耐药葡萄球菌在医院和社区的日益流行，溶葡球菌酶的药用价值再次受到人们重视。近十年来重组溶葡球菌酶再次成为抗葡萄球菌新药开发的热点。此外，有研究表明金葡菌对溶葡球菌酶的耐药机制可揭示金葡菌对另一种重要抗生素——β-内酰胺类抗生素的敏感性相关因子的作用，因此本研究从体外、体内抗菌活性以及体外诱导金葡菌耐药机制等方面对此酶进行了研究。
一、体外抗菌药效学研究
本研究中，首次大量采用国内临床分离菌，通过测定MIC、MBC、杀菌曲线、联合药敏、抗菌活性影响因素以及扫描电镜观察，考查重组溶葡球菌酶的体外抗菌活性。结果表明，重组溶葡球菌酶抗菌谱单一，仅对葡萄球菌有效，且对金葡菌的抗菌活性优于凝固酶阴性葡萄球菌。结合MBC、杀菌曲线和扫描电镜结果，可以判断此酶为杀菌药，且作用迅速，呈浓度依赖性杀菌活性。棋盘法联合药敏试验结果显示此酶与两种青霉素和奈替米星呈协同抗菌作用。
二、体内抗菌药效学研究
通过小鼠腹腔感染保护试验、兔皮肤开放性创伤感染多次治疗试验、兔皮肤烫伤感染单次治疗试验、小型猪烫伤感染多次治疗试验等考查了重组溶葡球菌酶在动物体内及局部病灶的抗金葡菌感染治疗作用。结果表明，对腹腔注射2.3×10~5CFU／
Lysostaphin, a monomer of 27kD endopeptidase with Zinc as cofactor, was first isolated and extracted from culture of Staphylococcus simulans by Schindler and Schuhardt in 1964. The enzyme kills the organism by hydrolyzing a pentaglycine cross-bridge structure unique to staphylococcus cell wall. Since the cell wall cross-bridges structrue of S. aureus is composed of a high proportion of pentaglycine, lysostaphin is very effective in lysing the organism cells. Lysostaphin was studied in the 1960s and 1970s as a potential therapeutic agent in numerous animal models and in some human patients. However, although its antimicrobial properties appeared promising, development of lysostaphin as a new therapeutic agent was abandoned. Some of the reasons for failure to pursue the clinical use of lysostaphin included subsequent availability of some effective anti-staphylococcal antibiotics such as vancomycin and teicoplanin, fears concerning the potential immunogenicity of a parenterally administered protein, and the impurity of lysostaphin preparations. When time came into 1980s, more purified and higher producible recombinant lysostaphin was emerged with the rapid development of biological engineering technology in the world. The availability of recombinant lysostaphin that is more than 90% pure makes it feasible to be used in clinical. More recently, with the increasing emergence of antimicrobal resistant staphylococcus especially methicillin resistant Staphylococcus aureus (MRSA) in hospital and community, much work has focused on the development of lysostaphin as a new drug. In addition, previous studies showed that resistance to lysotaphin in S. aureus mutant (induced in vitro or in vivo) was associated with the characteristics of increased susceptibility to β-lactams and mutations in femA gene. In this study, the in vitro and in vivo antibacterial activity of the recombinant lysostaphin was evaluated, and the mechanism of in vitro induced resistance to lysostaphin in 5. aureus was investigated. A. The in vitro antibacterial activity of recombinant lysostaphin
To evaluated the in vitro activity of recombinant lysostahin against pathogen, numerous bacteria isolated from hospitals was used, the minimum inhibitory concentrations (MICs), the minimum bactericidal concentrations (MBCs), the time-kill curves, effects of culture
一、体外抗菌药效学研究
本研究中，首次大量采用国内临床分离菌，通过测定MIC、MBC、杀菌曲线、联合药敏、抗菌活性影响因素以及扫描电镜观察，考查重组溶葡球菌酶的体外抗菌活性。结果表明，重组溶葡球菌酶抗菌谱单一，仅对葡萄球菌有效，且对金葡菌的抗菌活性优于凝固酶阴性葡萄球菌。结合MBC、杀菌曲线和扫描电镜结果，可以判断此酶为杀菌药，且作用迅速，呈浓度依赖性杀菌活性。棋盘法联合药敏试验结果显示此酶与两种青霉素和奈替米星呈协同抗菌作用。
二、体内抗菌药效学研究
通过小鼠腹腔感染保护试验、兔皮肤开放性创伤感染多次治疗试验、兔皮肤烫伤感染单次治疗试验、小型猪烫伤感染多次治疗试验等考查了重组溶葡球菌酶在动物体内及局部病灶的抗金葡菌感染治疗作用。结果表明，对腹腔注射2.3×10~5CFU／
Lysostaphin, a monomer of 27kD endopeptidase with Zinc as cofactor, was first isolated and extracted from culture of Staphylococcus simulans by Schindler and Schuhardt in 1964. The enzyme kills the organism by hydrolyzing a pentaglycine cross-bridge structure unique to staphylococcus cell wall. Since the cell wall cross-bridges structrue of S. aureus is composed of a high proportion of pentaglycine, lysostaphin is very effective in lysing the organism cells. Lysostaphin was studied in the 1960s and 1970s as a potential therapeutic agent in numerous animal models and in some human patients. However, although its antimicrobial properties appeared promising, development of lysostaphin as a new therapeutic agent was abandoned. Some of the reasons for failure to pursue the clinical use of lysostaphin included subsequent availability of some effective anti-staphylococcal antibiotics such as vancomycin and teicoplanin, fears concerning the potential immunogenicity of a parenterally administered protein, and the impurity of lysostaphin preparations. When time came into 1980s, more purified and higher producible recombinant lysostaphin was emerged with the rapid development of biological engineering technology in the world. The availability of recombinant lysostaphin that is more than 90% pure makes it feasible to be used in clinical. More recently, with the increasing emergence of antimicrobal resistant staphylococcus especially methicillin resistant Staphylococcus aureus (MRSA) in hospital and community, much work has focused on the development of lysostaphin as a new drug. In addition, previous studies showed that resistance to lysotaphin in S. aureus mutant (induced in vitro or in vivo) was associated with the characteristics of increased susceptibility to β-lactams and mutations in femA gene. In this study, the in vitro and in vivo antibacterial activity of the recombinant lysostaphin was evaluated, and the mechanism of in vitro induced resistance to lysostaphin in 5. aureus was investigated. A. The in vitro antibacterial activity of recombinant lysostaphin
To evaluated the in vitro activity of recombinant lysostahin against pathogen, numerous bacteria isolated from hospitals was used, the minimum inhibitory concentrations (MICs), the minimum bactericidal concentrations (MBCs), the time-kill curves, effects of culture
引文
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