抗生素M331的抗铜绿假单胞菌的作用及对其体外形成生物被膜影响的研究
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摘要
目的：本课题通过对铜绿假单胞菌临床株和标准株的生物学特性、其生物被膜形态学的研究,探讨了不同浓度的M331以及其联合大环内酯类抗生素对铜绿假单胞菌及其形成生物被膜的影响,以探讨新的治疗铜绿假单胞菌感染及它的生物被膜的方法；并初步探讨了药物M331的药物毒性。
方法：应用四区划线法分离纯化了菌株；通过对比其对细胞的粘附能力,来观察其毒力；采用改良的组织培养平板法,建立了临床株PA1和标准株PA27853生物被膜的模型；应用微量稀释法,测定了不同药物及其联合用药对PA1、PA3、PA5、PA2059, PA27853的MIC值,应用微量稀释法,测定了PA1、PA2059的MBEC值；应用MTT法测定了不同剂量M331及联合用药后PA1、PA27853生物被膜中的存活细菌数；应用银染法及扫描电镜鉴定生物被膜,并观察了不同剂量M331及其联合用药后PA1、PA27853的超微结构,对其菌落形态学进行描述。
结果：临床株和标准株不同菌株的产绿色素的情况不一；PA1、PA27853对Hela细胞的粘附性实验显示,PA1、PA27853对Hela细胞的粘附类型为弥散性粘附,且临床株PA1比标准株PA27853更易于粘附Hela细胞。银染法快速鉴定结果显示,都可见浓密黑染交织分布的物质,其间隙中可见聚集的短杆状菌体。扫描电镜观察,铜绿假单胞菌呈短杆状,聚集成团,为浓厚的粘液样物质紧密包绕,菌体之间相互以粘稠的纤维状粘液丝相连。MTT法测定不同剂量M331及联合罗红霉素PA1、PA27853生物被膜中存活的细菌数,结果显示,罗红霉素对M331有增效作用。应用银染法、扫描电镜观察不同剂量M331和联合罗红霉素处理的PA1、PA27853的生物被膜,可见随着M331及罗红霉素的剂量增大,PA1、PA27853生物被膜中的细菌逐渐被清除,细菌量逐渐减少。
结论：通过前期的实验研究,发现从贵州省耕土中分离的某种放线菌能产生一种抗生素,经前期的实验证实其对铜绿假单胞菌疗效较确定,对其它常见菌种无效,我们将此代谢产物命名为M331。本课题通过研究发现,不同抗菌药物对PA1、PA27853的浮游菌的杀菌活性存在明显差异,M331的疗效比庆大霉素弱,与多粘菌素B相当,强于羧苄青霉素。使用大环内酯类抗生素能显著增强M331的抗菌活性,酶抑制剂也能增强β-内酰胺酶类抗生素的抗菌活性。国内外研究发现,14、15元环的大环内酯类抗生素能抑制细菌藻酸盐的合成,其作用机制可能是抑制藻酸盐合成的关键酶——甘露糖脱氢酶(GMD)的活性,与其它药物联合应用能够抑制细菌生物被膜的形成。应用不同剂量的M331及联合罗红霉素在生物被膜培养过程中分别处理PA1、PA27853,显示了罗红霉素联合M331可有效抑制铜绿假单胞菌生物被膜的形成,其对Vero细胞无细胞毒性作用,是一个较为理想的针对铜绿假单胞菌的抗生素。
Objective:To study the biological characteristics as well as the biofilm morphology of PA1 and PA27853, and identify the sensitivity of PA1 and PA27853 in planktonic or in bacterial biofilms to different kinds of antibiotics. To investigate the influence of different concentration of M331 and the combination of M331 and Roxithromycin on the formation of Pseudomonas aeruginosa biofilm. And to examine the cytotoxicity of M331.
Method:The bacteria were purified by coloning them and then the colony forming unit was used to the bacteria counting. The modified plate culture method was used to establish biofilm model in vitro; The morphology of PA1 and PA27853 biofilms was observed by both optical microscopy and SEM. The minimal inhibitory concentrations(MICs) of M331, gentamycin, Carbenicillin, Polymyxin B, and Roxithromycin and of the combination of M331 and Roxithromycin, Piperacillin and tazobactam were determined by micro-dilution method. The minimal biofilm eradication concentration(MBEC) was also determined by micro-dilution method. The viable counts of bacteria in PA1 and PA27853 biofilms were carried out by MTT method after treated with different concentration of M331 or the combination of M331 and Roxithromycin. PA1 and PA27853 biofilms were observed by the optical microscopy after being stained by AgNO3, and by SEM after treated with different dosages of M331 and the combination of M331 and Roxithromycin. The cytotoxicity of M331 were monitered by the of MTT method.
Results:Microscopy of PA1 and PA27853 biofilms showed that cluster bacilli is surrounded by dense black-stained substance. The SEM results showed that bacteria were either the bacillus surrounded by accidented substance or the bacteria connected to each other by mucoid substance to form biofilms.The cell adhension of PAl and PA27853 were ubiquitous, and PA1-adhered cell outnumbered that of PA27853. The MTT assay results imply that M331 have no cytotoxicity. of PA27853. The MTT assay results imply that M331 have no cytotoxicity.
The MICs of M331 were lower than that of gentamycin and Polymyxin B, on the other hand, higher than Carbenicillin. Roxithromycin enhanced the penetration of M331 to PA1 and PA27853 strain through biofilms. PA1 and PA27853 biofilms were gradually cleared with the increased dosages of M331 or the combination of M331 and Roxithromycin.
Conclusion:Previous research work have been done to illustrate that a strain of actinomycetes desingated M331 which extracted from the soil in Guizhou Province has therapeutic effects on Pseudomonas aeruginosa, and macrolides can enhance the its biological effects on Pseudomonas aeruginosa and its biofilms. From this investigation, we provide evidence that Sub-MIC levels of Roxithromycin and M331 substantially inhibited the structure and architecture of the biofilm, and reduced the bacteria count accordingly.
方法：应用四区划线法分离纯化了菌株；通过对比其对细胞的粘附能力,来观察其毒力；采用改良的组织培养平板法,建立了临床株PA1和标准株PA27853生物被膜的模型；应用微量稀释法,测定了不同药物及其联合用药对PA1、PA3、PA5、PA2059, PA27853的MIC值,应用微量稀释法,测定了PA1、PA2059的MBEC值；应用MTT法测定了不同剂量M331及联合用药后PA1、PA27853生物被膜中的存活细菌数；应用银染法及扫描电镜鉴定生物被膜,并观察了不同剂量M331及其联合用药后PA1、PA27853的超微结构,对其菌落形态学进行描述。
结果：临床株和标准株不同菌株的产绿色素的情况不一；PA1、PA27853对Hela细胞的粘附性实验显示,PA1、PA27853对Hela细胞的粘附类型为弥散性粘附,且临床株PA1比标准株PA27853更易于粘附Hela细胞。银染法快速鉴定结果显示,都可见浓密黑染交织分布的物质,其间隙中可见聚集的短杆状菌体。扫描电镜观察,铜绿假单胞菌呈短杆状,聚集成团,为浓厚的粘液样物质紧密包绕,菌体之间相互以粘稠的纤维状粘液丝相连。MTT法测定不同剂量M331及联合罗红霉素PA1、PA27853生物被膜中存活的细菌数,结果显示,罗红霉素对M331有增效作用。应用银染法、扫描电镜观察不同剂量M331和联合罗红霉素处理的PA1、PA27853的生物被膜,可见随着M331及罗红霉素的剂量增大,PA1、PA27853生物被膜中的细菌逐渐被清除,细菌量逐渐减少。
结论：通过前期的实验研究,发现从贵州省耕土中分离的某种放线菌能产生一种抗生素,经前期的实验证实其对铜绿假单胞菌疗效较确定,对其它常见菌种无效,我们将此代谢产物命名为M331。本课题通过研究发现,不同抗菌药物对PA1、PA27853的浮游菌的杀菌活性存在明显差异,M331的疗效比庆大霉素弱,与多粘菌素B相当,强于羧苄青霉素。使用大环内酯类抗生素能显著增强M331的抗菌活性,酶抑制剂也能增强β-内酰胺酶类抗生素的抗菌活性。国内外研究发现,14、15元环的大环内酯类抗生素能抑制细菌藻酸盐的合成,其作用机制可能是抑制藻酸盐合成的关键酶——甘露糖脱氢酶(GMD)的活性,与其它药物联合应用能够抑制细菌生物被膜的形成。应用不同剂量的M331及联合罗红霉素在生物被膜培养过程中分别处理PA1、PA27853,显示了罗红霉素联合M331可有效抑制铜绿假单胞菌生物被膜的形成,其对Vero细胞无细胞毒性作用,是一个较为理想的针对铜绿假单胞菌的抗生素。
Objective:To study the biological characteristics as well as the biofilm morphology of PA1 and PA27853, and identify the sensitivity of PA1 and PA27853 in planktonic or in bacterial biofilms to different kinds of antibiotics. To investigate the influence of different concentration of M331 and the combination of M331 and Roxithromycin on the formation of Pseudomonas aeruginosa biofilm. And to examine the cytotoxicity of M331.
Method:The bacteria were purified by coloning them and then the colony forming unit was used to the bacteria counting. The modified plate culture method was used to establish biofilm model in vitro; The morphology of PA1 and PA27853 biofilms was observed by both optical microscopy and SEM. The minimal inhibitory concentrations(MICs) of M331, gentamycin, Carbenicillin, Polymyxin B, and Roxithromycin and of the combination of M331 and Roxithromycin, Piperacillin and tazobactam were determined by micro-dilution method. The minimal biofilm eradication concentration(MBEC) was also determined by micro-dilution method. The viable counts of bacteria in PA1 and PA27853 biofilms were carried out by MTT method after treated with different concentration of M331 or the combination of M331 and Roxithromycin. PA1 and PA27853 biofilms were observed by the optical microscopy after being stained by AgNO3, and by SEM after treated with different dosages of M331 and the combination of M331 and Roxithromycin. The cytotoxicity of M331 were monitered by the of MTT method.
Results:Microscopy of PA1 and PA27853 biofilms showed that cluster bacilli is surrounded by dense black-stained substance. The SEM results showed that bacteria were either the bacillus surrounded by accidented substance or the bacteria connected to each other by mucoid substance to form biofilms.The cell adhension of PAl and PA27853 were ubiquitous, and PA1-adhered cell outnumbered that of PA27853. The MTT assay results imply that M331 have no cytotoxicity. of PA27853. The MTT assay results imply that M331 have no cytotoxicity.
The MICs of M331 were lower than that of gentamycin and Polymyxin B, on the other hand, higher than Carbenicillin. Roxithromycin enhanced the penetration of M331 to PA1 and PA27853 strain through biofilms. PA1 and PA27853 biofilms were gradually cleared with the increased dosages of M331 or the combination of M331 and Roxithromycin.
Conclusion:Previous research work have been done to illustrate that a strain of actinomycetes desingated M331 which extracted from the soil in Guizhou Province has therapeutic effects on Pseudomonas aeruginosa, and macrolides can enhance the its biological effects on Pseudomonas aeruginosa and its biofilms. From this investigation, we provide evidence that Sub-MIC levels of Roxithromycin and M331 substantially inhibited the structure and architecture of the biofilm, and reduced the bacteria count accordingly.
引文
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