左氧氟沙星对绿脓杆菌生物膜诱导作用及药物治疗方法研究
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摘要
绿脓杆菌(P.Aeruginosa)也称铜绿假单胞菌,是目前引起医院内感染的重要病原菌之一,它的耐药机制很多,而生物膜的形成是一个很重要的耐药机制,也是引起临床治疗失败的很重要原因。
经过对铜绿假单胞菌生物膜形成机制的研究发现,铜绿假单胞菌形成生物膜的过程中除了群体感应系统(QS系统)之外,还存在着另一种调节机制-喹诺酮信号系统(PQS系统),喹诺酮类信号分子在生物膜的形成机制中则是作为PQS调控系统的主要的信号分子。
喹诺酮类药物的结构与喹诺酮信号分子相似,都是以喹啉环为主体的结构,那么喹诺酮类药物在生物膜的形成中到底充当什么角色?它能否诱导生物膜的形成?临床上有哪些药物能够穿透生物膜,从而更有效的治疗铜绿假单胞菌生物膜引起的感染?
围绕着上面的问题,本课题展开了以下研究。
1.左氧对生物膜诱导作用及其它药物对生物膜形成的影响
实验目的：用左氧氟沙星体外诱导铜绿假单胞菌标准菌株与临床分离菌株,观察左氧氟沙星对铜绿假单胞菌形成生物膜的诱导作用；在实验里加入阿奇霉素、亚胺培南与氨溴索,观察这些药物对生物膜的作用,观察是否能破坏细菌生物膜结构或延迟生物膜的形成。
实验用菌株：三株铜绿假单胞菌,标准菌株ATCC27853为本室保存菌株,其余两株为野生菌株,临床分离获得。
实验方法：
(1)左氧对生物膜诱导作用
使用左氧氟沙星诱导铜绿假单胞菌,细菌经四十代的诱导后,用银染与电镜两种方法观察生物膜,与诱导前的菌株进行对比,观察左氧氟沙星在诱导铜绿假单胞菌生物膜形成上的作用。
(2)阿奇霉素、亚胺培南、氨溴索对生物膜形成的影响
取六株铜绿假单胞菌(使用左氧氟沙星诱导前后的各3株),进行以下实验,放入培养液不经培养直接加入阿奇霉素、亚胺培南、氨溴索,观察同一时间内生物膜的形成情况。另取同样的六株铜绿假单胞菌,培养足够的时间,形成生物膜后,加入阿奇霉素、亚胺培南、氨溴索,通过振荡和高温高压的方法,比较已形成生物膜的变化。
取使用左氧氟沙星诱导前后的铜绿假单胞菌菌株各3株,培养形成生物膜后,加入氨溴索,电镜观察它们的区别。
实验结果：
(1)标准菌株编号H007,野生菌株编号P012、P033,诱导四十代后产生菌株编号：H031、H038、H039。从银染和电镜的结果中可以看出在同一时间内经过左氧氟沙星诱导的铜绿假单胞菌,与未经使用左氧氟沙星诱导的铜绿假单胞菌形成的生物膜差别明显。
(2)从实验结果中加入阿奇霉素、亚胺培南后可以看出经左氧氟沙星诱导后的菌株,加入阿奇霉素和亚胺培南后生物膜形成的时间比未经过诱导的菌株产生生物膜的时间早。未加入阿奇霉素与亚胺培南的菌株比加入阿奇霉素、亚胺培南的菌株形成生物膜的时间早。加入阿奇霉素、亚胺培南的菌株振荡后生物膜恢复的速度比不加入阿奇霉素、亚胺培南的菌株慢。高温高压之后被左氧氟沙星诱导过的铜绿假单胞菌生物膜经过125℃、0.15MPa的高压20min,生物膜仍然完好无损,加亚胺培南的管经过高压后生物膜消失。加入氨溴索后,电镜下观察经左氧氟沙星诱导和未经左氧氟沙星诱导的铜绿假单胞菌菌株生物膜表面都被打上孔洞。
2.建立体外铜绿假单胞菌生物膜形成的实验模型装置
实验目的：按照文献报导的方法进行改进,用0.22μm微孔滤膜建立一个体外生物膜模型,并且用此装置进行能透过生物膜的药物筛选,用高效液相色谱仪检测透过生物膜的药物的含量。
实验用菌株：两株铜绿假单胞菌,一株为标准菌株H007,另一株为第一部分通过左氧氟沙星诱导而得到的H031。
实验方法：建立装置,使用氨溴索促进环丙沙星透过实验用于检验装置是否建立成功。用精密度实验检测装置使用的可重复性。
实验结果：未使用左氧氟沙星诱导的铜绿假单胞菌中,未加入氨溴索的菌株环丙沙星透过的量为3.136μg·ml-1和3.270μg·ml-1,透过率为3.136%和3.270%,加入氨溴索后,环丙沙星的透过量为3.911μg·ml-1和4.3796μg·ml-1,透过率为3.911%和4.379%；使用左氧氟沙星诱导的铜绿假单胞菌,未加入氨溴索的菌株,环丙沙星的透过量为1.6557μg·ml-1和1.6825μg·ml-1,环丙沙星的透过率为1.6557%和1.6825%,加入氨溴索的菌株中,环丙沙星的透过量为3.4660μg·m1-1和3.4206μg·ml-1,透过率为3.4660%和3.4206%。
3.氨溴索分别与四种抗生素联合使用对铜绿假单胞菌生物膜的透过作用
实验目的：筛选能够透过铜绿假单胞菌生物膜的抗生素,为临床上治疗生物膜感染提供科学依据。
实验用菌株：三株铜绿假单胞菌,一株为标准菌株H007,另两株为实验一通过左氧氟沙星诱导而得到的H031和H039。
实验方法：用氨溴索分别与亚胺培南、头孢哌酮／舒巴坦、哌拉西林／他唑巴坦、头孢他啶作为透过生物膜的药物,分别用这几种药单独使用作为对照,使用5%-100%MeOH/H2O,40min浓度梯度,用岛津20A高效液相DAD检测仪LC-Solution工作站进行检测。
实验结果：头孢哌酮／舒巴坦有峰,并且峰的紫外吸收特征峰与纯品在此处的紫外特征吸收相似,说明头孢哌酮／舒巴坦有可能透过生物膜,对于加入氨溴索后的作用,头孢哌酮／舒巴坦还是能透过生物膜,且透过量增加。
4.验证实验
实验目的：在不同的高效液相条件下(岛津LC-10A)检测氨溴索对头孢哌酮／舒巴坦的透过作用。
实验用菌株：两株铜绿假单胞菌,一株为标准菌株H007,另一株为左氧氟沙星诱导得到的H031。实验方法：分别使用头孢哌酮／舒巴坦与头孢哌酮／舒巴坦+氨溴索作为透过生物膜的药物,用岛津LC-10A高效液相SPD-10AD紫外检测器、浙大N2000工作站检测其透过峰高度。
实验结果：只加入头孢哌酮／舒巴坦的装置,透过头孢哌酮／舒巴坦色谱吸收峰高为185.091mAU,同时加入头孢哌酮／舒巴坦和氨溴索的装置,透过头孢哌酮／舒巴坦色谱吸收峰高为207.233mAU。
以上研究结论
1.左氧氟沙星能诱导生物膜的形成。
2.阿奇霉素、亚胺培南有抑制生物膜生长的作用,但是对于已形成的生物膜无效。氨溴索可以在形成生物膜的表面上打孔。
3.头孢哌酮／舒巴坦可以透过生物膜,加入氨溴索之后头孢哌酮／舒巴坦透过生物膜的浓度增加,此实验提示这两种药合用可以治疗临床上形成生物膜的铜绿假单胞菌感染。
Pseudomonas aeruginosa is an important sources of nosocomial infection caused by pathogenic bacteria. In all the mechanisms of drug-resistant, biofilm formation in Pseudomonas aeruginosa is not only a major resistance mechanisms, but also very important reason for Treatment failure in clinic. In study of forming mechanism about biofilm of Pseudomonas aeruginosa, the forming process about biofilm of Pseudomonas aeruginosa has QS systems in addition, there is also another adjustment mechanism:PQS system. Quinolone signal molecules is the main signal molecule as a molecule of PQS control system in the biofilm forming mechanism, the structure of Quinolones is similar to the structure of quinolone signal molecule, both of them are based on quinoline ring as the main structure.
What roles does Quinolones play in the formation of biofilm of Pseudomonas aeruginosa? Whether can it induce the formation of biofilms? What medicines do they have to penetrate the biofilm in clinical which can treat infections caused by biofilms of Pseudomonas aeruginosa more effective?
Due to the problems above, we doing the following research.
1.Levofloxacin-induced effects on biofilm of Pseudomonas aeruginosa and the effects on biofilm by other medicine.
Purpose:Induce standard strains of Pseudomonas aeruginosa and wild in vitro using Levofloxacin, Observe the effect of Levofloxacin in the forming biofilm of Pseudomonas aeruginosa. Add Azithromycin, Imipenem and Ambroxol in the experiment, Observe effects of them to the formation of biofilms, Observe whether they can destroy the structure of biofilm or delay the Forming time of biofilm.
Strain Source:3 Strains of Pseudomonas aeruginosa, the Standard strain ATCC27853(H007) was preserved by our laboratory, the other two wild strains is isolated from the clinical.
Methods:(1)The effects on biofilm of Pseudomonas aeruginosa by Levofloxacin-induced.
Use levofloxacin the concentrations of which is MIC to induce Pseudomonas aeruginosa, we obsere the Results in two way-Silver staining and Scanning electron microscope(SEM) after 40 generations-induced by using Levofloxacin, compare to the strain before Levofloxacin-induced, Observe the Levofloxacin-induced effects on biofilm of Pseudomonas aeruginosa.
(2) The effects of Azithromycin, Imipenem and Ambroxol on biofilm.
Use the 6 strains of Pseudomonas aeruginosa(3 was Levofloxacin-induced and the other 3 was the strains before Levofloxacin-induced) to engage the experiments which were put into the culture medium with the directly dosing(the medicine is Azithromycin, Imipenem and Ambroxol).Take another 6 same strains which were put into the culture medium with cultivating Sufficient time,let the Biofilm form. Add Azithromycin, Imipenem and Ambroxol into the one which were put into the culture medium with the directly dosing.Observe the formation of biofilm at same time. Add Azithromycin, Imipenem and Ambroxol into the one which were put into the culture medium with cultivating,use the means of oscillation and high temperature with high pressure, Observe the changes about biofilm which was formed in the environment of different medicine.
Use the 6 strains of Pseudomonas aeruginosa(3 was Levofloxacin-induced and the other 3 was the strains before Levofloxacin-induced, which were put into the culture medium with cultivating Sufficient time,let the Biofilm form, Add Ambroxol into the culture medium of Pseudomonas aeruginosa, Observe what was the differences between them.
Results:(1) Standard strain number H007,Wild strain numbers P012, P033,the strain after 40 generations Levofloxacin-induced number H031、H038、H039.We can found obvious difference of biofilm between Levofloxacin-induced strain and Levofloxacin-noinduced strain which were formed at same time by using the means of Silver staining and SEM.
(2) From the experimental results by adding azithromycin or imipenem we can see that the forming time of biofilm with Levofloxacin-induced which added azithromycin, imipenem is more earlier than the forming time of biofilm with Levofloxacin-noinduced, the forming time of biofilm with adding azithromycin or imipenem is more earlier than that without adding azithromycin and imipenem one.The speed which biofilm recover with adding azithromycin or imipenem is more slowly than that of the one without adding azithromycin or imipenem.Biofilms Levofloxacin-induced are still intact after high temperature125℃and high pressure last 20min,but the biofilms added imipenem was gone.The biofilm of Pseudomonas aeruginosa made lots of holes in the surface both Levofloxacin-induced one and Levofloxacin-noinduced one.
2. The establishment experimental model of device which biofilm formation of Pseudomonas aeruginosa in vitro
Purpose:Improve the experimental model of device according to literature reported methods, use 0.22μm Millipore which biofilm grow on it to establish a experimental model of device in vitro,and use this device to Screen medicine which can through the biofilm,use HPLC to detect the concentration after the medicine through the biofilm.
Strain Source:2 Strains of Pseudomonas aeruginosa, Standard strain ATCC27853 and the strain number H031 which induced by using Levofloxacin from Standard strain ATCC27853.
Methods:Establish the device, repeat the experiments which Ambroxol increase the concentration of Ciprofloxacin which Penetrate the biofilm to test whether the device is created successfully.Use precision experiments to test the repeatability of the experiment.
Results:In Pseudomonas aeruginosa with Levofloxacin-noinduced and without adding Ambroxol,the concentration of Ciprofloxacin is 3.136μg·ml-1 and 3.270μg·ml-1,the transmittance is 3.136 and 3.270. In Pseudomonas aeruginosa with Levofloxacin-noinduced and with adding Ambroxol,the concentration of Ciprofloxacin is 3.911μg·ml-1 and 4.3796μg·ml-1,the transmittance is 3.911 and 4.3796. In Pseudomonas aeruginosa with Levofloxacin-induced and without adding Ambroxol,the concentration of Ciprofloxacin is 1.6557μg·ml-1 and 1.6825μg·ml-1,the transmittance is 1.6557 and 1.6825. In Pseudomonas aeruginosa with Levofloxacin-induced and with adding Ambroxol,the concentration of Ciprofloxacin is 3.4660μg·ml-1 and 3.4206μg·ml-1,the transmittance is 3.4660 and 3.4206.
3.The effect of penetration to use Ambroxol combining with four kinds of antibiotics on biofilm of Pseudomonas aeruginosa
Purpose:Screen the antibiotic which can penetrate biofilm of Pseudomonas aeruginosa. Provid laboratory support for treating biofilm infection.
Strain Source:3 Strains of Pseudomonas aeruginosa,the Standard strain ATCC27853(H007), the other are H031and H039 which were Levofloxacin-induced strain.
Methods:Use Ambroxol combining with Thailand, cefoperazone/sulbactam, piperacillin/tazobactam, ceftazidime respectively for the medicine to penetrate the biofilm of Pseudomonas aeruginosa, Use these medicine as controls respectively, Use conditions of HPLC are 5%-100%MeOH/H2O,40min, Shimadzu HPLC-20A, detector with DAD, Workstation software of LC-Solution.
Results:Cefoperazone/Sulbactam has peak,and the UV of peak is similar to the pure in this position, it means that Cefoperazone/Sulbactam penetrate the biofilm of Pseudomonas aeruginosa, and the quantity which penetrate the biofilm of Pseudomonas aeruginosa become larger.
4. Verification experiment
Purpose:Use different HPLC,In different conditions (Shimadzu LC-10A), detect the quantity of Cefoperazone/Sulbactam to verify the effect that Ambroxol combining with Cefoperazone/Sulbactam.
Strain Source:2 Strains of Pseudomonas aeruginosa, Standard strain ATCC27853 and the strain number H031 which induced by using Levofloxacin from Standard strain ATCC27853.
Methods:Use cefoperazone/sulbactam and cefoperazone/sulbactam+Ambroxol as the medicine through the biofilm respectively, detect the height of the peak with HPLC(Shimadzu LC-10A), UV detector is SPD-10AD, Workstation software of Zhejiang University N2000.
Results:In the devices add cefoperazone/sulbactam only, the height of peak is 185.091. In the devices add cefoperazone/sulbactam+Ambroxol, the height of peak is 207.233.
Research above have shown that
1.Levofloxacin can induce the formation of biofilm.
2.Azithromycin, imipenem has the effect to inhibit the growth of biofilm, However, it is useless to the biofilm which is grown. Ambroxol can dig holes on the surface of biofilm.
3.The experiment which Ambroxol accelerates the Penetration of Ciprofloxacin is repeat successfully.
4.Cefoperazone/Sulbactam can penetrate biofilm of Pseudomonas aeruginosa, after adding Ambroxol, the concentration of Cefoperazone/Sulbactam is larger. This experiment shown that this two medicine can use together to treat the infection by biofilm of Pseudomonas aeruginosa in clinic.
经过对铜绿假单胞菌生物膜形成机制的研究发现,铜绿假单胞菌形成生物膜的过程中除了群体感应系统(QS系统)之外,还存在着另一种调节机制-喹诺酮信号系统(PQS系统),喹诺酮类信号分子在生物膜的形成机制中则是作为PQS调控系统的主要的信号分子。
喹诺酮类药物的结构与喹诺酮信号分子相似,都是以喹啉环为主体的结构,那么喹诺酮类药物在生物膜的形成中到底充当什么角色?它能否诱导生物膜的形成?临床上有哪些药物能够穿透生物膜,从而更有效的治疗铜绿假单胞菌生物膜引起的感染?
围绕着上面的问题,本课题展开了以下研究。
1.左氧对生物膜诱导作用及其它药物对生物膜形成的影响
实验目的：用左氧氟沙星体外诱导铜绿假单胞菌标准菌株与临床分离菌株,观察左氧氟沙星对铜绿假单胞菌形成生物膜的诱导作用；在实验里加入阿奇霉素、亚胺培南与氨溴索,观察这些药物对生物膜的作用,观察是否能破坏细菌生物膜结构或延迟生物膜的形成。
实验用菌株：三株铜绿假单胞菌,标准菌株ATCC27853为本室保存菌株,其余两株为野生菌株,临床分离获得。
实验方法：
(1)左氧对生物膜诱导作用
使用左氧氟沙星诱导铜绿假单胞菌,细菌经四十代的诱导后,用银染与电镜两种方法观察生物膜,与诱导前的菌株进行对比,观察左氧氟沙星在诱导铜绿假单胞菌生物膜形成上的作用。
(2)阿奇霉素、亚胺培南、氨溴索对生物膜形成的影响
取六株铜绿假单胞菌(使用左氧氟沙星诱导前后的各3株),进行以下实验,放入培养液不经培养直接加入阿奇霉素、亚胺培南、氨溴索,观察同一时间内生物膜的形成情况。另取同样的六株铜绿假单胞菌,培养足够的时间,形成生物膜后,加入阿奇霉素、亚胺培南、氨溴索,通过振荡和高温高压的方法,比较已形成生物膜的变化。
取使用左氧氟沙星诱导前后的铜绿假单胞菌菌株各3株,培养形成生物膜后,加入氨溴索,电镜观察它们的区别。
实验结果：
(1)标准菌株编号H007,野生菌株编号P012、P033,诱导四十代后产生菌株编号：H031、H038、H039。从银染和电镜的结果中可以看出在同一时间内经过左氧氟沙星诱导的铜绿假单胞菌,与未经使用左氧氟沙星诱导的铜绿假单胞菌形成的生物膜差别明显。
(2)从实验结果中加入阿奇霉素、亚胺培南后可以看出经左氧氟沙星诱导后的菌株,加入阿奇霉素和亚胺培南后生物膜形成的时间比未经过诱导的菌株产生生物膜的时间早。未加入阿奇霉素与亚胺培南的菌株比加入阿奇霉素、亚胺培南的菌株形成生物膜的时间早。加入阿奇霉素、亚胺培南的菌株振荡后生物膜恢复的速度比不加入阿奇霉素、亚胺培南的菌株慢。高温高压之后被左氧氟沙星诱导过的铜绿假单胞菌生物膜经过125℃、0.15MPa的高压20min,生物膜仍然完好无损,加亚胺培南的管经过高压后生物膜消失。加入氨溴索后,电镜下观察经左氧氟沙星诱导和未经左氧氟沙星诱导的铜绿假单胞菌菌株生物膜表面都被打上孔洞。
2.建立体外铜绿假单胞菌生物膜形成的实验模型装置
实验目的：按照文献报导的方法进行改进,用0.22μm微孔滤膜建立一个体外生物膜模型,并且用此装置进行能透过生物膜的药物筛选,用高效液相色谱仪检测透过生物膜的药物的含量。
实验用菌株：两株铜绿假单胞菌,一株为标准菌株H007,另一株为第一部分通过左氧氟沙星诱导而得到的H031。
实验方法：建立装置,使用氨溴索促进环丙沙星透过实验用于检验装置是否建立成功。用精密度实验检测装置使用的可重复性。
实验结果：未使用左氧氟沙星诱导的铜绿假单胞菌中,未加入氨溴索的菌株环丙沙星透过的量为3.136μg·ml-1和3.270μg·ml-1,透过率为3.136%和3.270%,加入氨溴索后,环丙沙星的透过量为3.911μg·ml-1和4.3796μg·ml-1,透过率为3.911%和4.379%；使用左氧氟沙星诱导的铜绿假单胞菌,未加入氨溴索的菌株,环丙沙星的透过量为1.6557μg·ml-1和1.6825μg·ml-1,环丙沙星的透过率为1.6557%和1.6825%,加入氨溴索的菌株中,环丙沙星的透过量为3.4660μg·m1-1和3.4206μg·ml-1,透过率为3.4660%和3.4206%。
3.氨溴索分别与四种抗生素联合使用对铜绿假单胞菌生物膜的透过作用
实验目的：筛选能够透过铜绿假单胞菌生物膜的抗生素,为临床上治疗生物膜感染提供科学依据。
实验用菌株：三株铜绿假单胞菌,一株为标准菌株H007,另两株为实验一通过左氧氟沙星诱导而得到的H031和H039。
实验方法：用氨溴索分别与亚胺培南、头孢哌酮／舒巴坦、哌拉西林／他唑巴坦、头孢他啶作为透过生物膜的药物,分别用这几种药单独使用作为对照,使用5%-100%MeOH/H2O,40min浓度梯度,用岛津20A高效液相DAD检测仪LC-Solution工作站进行检测。
实验结果：头孢哌酮／舒巴坦有峰,并且峰的紫外吸收特征峰与纯品在此处的紫外特征吸收相似,说明头孢哌酮／舒巴坦有可能透过生物膜,对于加入氨溴索后的作用,头孢哌酮／舒巴坦还是能透过生物膜,且透过量增加。
4.验证实验
实验目的：在不同的高效液相条件下(岛津LC-10A)检测氨溴索对头孢哌酮／舒巴坦的透过作用。
实验用菌株：两株铜绿假单胞菌,一株为标准菌株H007,另一株为左氧氟沙星诱导得到的H031。实验方法：分别使用头孢哌酮／舒巴坦与头孢哌酮／舒巴坦+氨溴索作为透过生物膜的药物,用岛津LC-10A高效液相SPD-10AD紫外检测器、浙大N2000工作站检测其透过峰高度。
实验结果：只加入头孢哌酮／舒巴坦的装置,透过头孢哌酮／舒巴坦色谱吸收峰高为185.091mAU,同时加入头孢哌酮／舒巴坦和氨溴索的装置,透过头孢哌酮／舒巴坦色谱吸收峰高为207.233mAU。
以上研究结论
1.左氧氟沙星能诱导生物膜的形成。
2.阿奇霉素、亚胺培南有抑制生物膜生长的作用,但是对于已形成的生物膜无效。氨溴索可以在形成生物膜的表面上打孔。
3.头孢哌酮／舒巴坦可以透过生物膜,加入氨溴索之后头孢哌酮／舒巴坦透过生物膜的浓度增加,此实验提示这两种药合用可以治疗临床上形成生物膜的铜绿假单胞菌感染。
Pseudomonas aeruginosa is an important sources of nosocomial infection caused by pathogenic bacteria. In all the mechanisms of drug-resistant, biofilm formation in Pseudomonas aeruginosa is not only a major resistance mechanisms, but also very important reason for Treatment failure in clinic. In study of forming mechanism about biofilm of Pseudomonas aeruginosa, the forming process about biofilm of Pseudomonas aeruginosa has QS systems in addition, there is also another adjustment mechanism:PQS system. Quinolone signal molecules is the main signal molecule as a molecule of PQS control system in the biofilm forming mechanism, the structure of Quinolones is similar to the structure of quinolone signal molecule, both of them are based on quinoline ring as the main structure.
What roles does Quinolones play in the formation of biofilm of Pseudomonas aeruginosa? Whether can it induce the formation of biofilms? What medicines do they have to penetrate the biofilm in clinical which can treat infections caused by biofilms of Pseudomonas aeruginosa more effective?
Due to the problems above, we doing the following research.
1.Levofloxacin-induced effects on biofilm of Pseudomonas aeruginosa and the effects on biofilm by other medicine.
Purpose:Induce standard strains of Pseudomonas aeruginosa and wild in vitro using Levofloxacin, Observe the effect of Levofloxacin in the forming biofilm of Pseudomonas aeruginosa. Add Azithromycin, Imipenem and Ambroxol in the experiment, Observe effects of them to the formation of biofilms, Observe whether they can destroy the structure of biofilm or delay the Forming time of biofilm.
Strain Source:3 Strains of Pseudomonas aeruginosa, the Standard strain ATCC27853(H007) was preserved by our laboratory, the other two wild strains is isolated from the clinical.
Methods:(1)The effects on biofilm of Pseudomonas aeruginosa by Levofloxacin-induced.
Use levofloxacin the concentrations of which is MIC to induce Pseudomonas aeruginosa, we obsere the Results in two way-Silver staining and Scanning electron microscope(SEM) after 40 generations-induced by using Levofloxacin, compare to the strain before Levofloxacin-induced, Observe the Levofloxacin-induced effects on biofilm of Pseudomonas aeruginosa.
(2) The effects of Azithromycin, Imipenem and Ambroxol on biofilm.
Use the 6 strains of Pseudomonas aeruginosa(3 was Levofloxacin-induced and the other 3 was the strains before Levofloxacin-induced) to engage the experiments which were put into the culture medium with the directly dosing(the medicine is Azithromycin, Imipenem and Ambroxol).Take another 6 same strains which were put into the culture medium with cultivating Sufficient time,let the Biofilm form. Add Azithromycin, Imipenem and Ambroxol into the one which were put into the culture medium with the directly dosing.Observe the formation of biofilm at same time. Add Azithromycin, Imipenem and Ambroxol into the one which were put into the culture medium with cultivating,use the means of oscillation and high temperature with high pressure, Observe the changes about biofilm which was formed in the environment of different medicine.
Use the 6 strains of Pseudomonas aeruginosa(3 was Levofloxacin-induced and the other 3 was the strains before Levofloxacin-induced, which were put into the culture medium with cultivating Sufficient time,let the Biofilm form, Add Ambroxol into the culture medium of Pseudomonas aeruginosa, Observe what was the differences between them.
Results:(1) Standard strain number H007,Wild strain numbers P012, P033,the strain after 40 generations Levofloxacin-induced number H031、H038、H039.We can found obvious difference of biofilm between Levofloxacin-induced strain and Levofloxacin-noinduced strain which were formed at same time by using the means of Silver staining and SEM.
(2) From the experimental results by adding azithromycin or imipenem we can see that the forming time of biofilm with Levofloxacin-induced which added azithromycin, imipenem is more earlier than the forming time of biofilm with Levofloxacin-noinduced, the forming time of biofilm with adding azithromycin or imipenem is more earlier than that without adding azithromycin and imipenem one.The speed which biofilm recover with adding azithromycin or imipenem is more slowly than that of the one without adding azithromycin or imipenem.Biofilms Levofloxacin-induced are still intact after high temperature125℃and high pressure last 20min,but the biofilms added imipenem was gone.The biofilm of Pseudomonas aeruginosa made lots of holes in the surface both Levofloxacin-induced one and Levofloxacin-noinduced one.
2. The establishment experimental model of device which biofilm formation of Pseudomonas aeruginosa in vitro
Purpose:Improve the experimental model of device according to literature reported methods, use 0.22μm Millipore which biofilm grow on it to establish a experimental model of device in vitro,and use this device to Screen medicine which can through the biofilm,use HPLC to detect the concentration after the medicine through the biofilm.
Strain Source:2 Strains of Pseudomonas aeruginosa, Standard strain ATCC27853 and the strain number H031 which induced by using Levofloxacin from Standard strain ATCC27853.
Methods:Establish the device, repeat the experiments which Ambroxol increase the concentration of Ciprofloxacin which Penetrate the biofilm to test whether the device is created successfully.Use precision experiments to test the repeatability of the experiment.
Results:In Pseudomonas aeruginosa with Levofloxacin-noinduced and without adding Ambroxol,the concentration of Ciprofloxacin is 3.136μg·ml-1 and 3.270μg·ml-1,the transmittance is 3.136 and 3.270. In Pseudomonas aeruginosa with Levofloxacin-noinduced and with adding Ambroxol,the concentration of Ciprofloxacin is 3.911μg·ml-1 and 4.3796μg·ml-1,the transmittance is 3.911 and 4.3796. In Pseudomonas aeruginosa with Levofloxacin-induced and without adding Ambroxol,the concentration of Ciprofloxacin is 1.6557μg·ml-1 and 1.6825μg·ml-1,the transmittance is 1.6557 and 1.6825. In Pseudomonas aeruginosa with Levofloxacin-induced and with adding Ambroxol,the concentration of Ciprofloxacin is 3.4660μg·ml-1 and 3.4206μg·ml-1,the transmittance is 3.4660 and 3.4206.
3.The effect of penetration to use Ambroxol combining with four kinds of antibiotics on biofilm of Pseudomonas aeruginosa
Purpose:Screen the antibiotic which can penetrate biofilm of Pseudomonas aeruginosa. Provid laboratory support for treating biofilm infection.
Strain Source:3 Strains of Pseudomonas aeruginosa,the Standard strain ATCC27853(H007), the other are H031and H039 which were Levofloxacin-induced strain.
Methods:Use Ambroxol combining with Thailand, cefoperazone/sulbactam, piperacillin/tazobactam, ceftazidime respectively for the medicine to penetrate the biofilm of Pseudomonas aeruginosa, Use these medicine as controls respectively, Use conditions of HPLC are 5%-100%MeOH/H2O,40min, Shimadzu HPLC-20A, detector with DAD, Workstation software of LC-Solution.
Results:Cefoperazone/Sulbactam has peak,and the UV of peak is similar to the pure in this position, it means that Cefoperazone/Sulbactam penetrate the biofilm of Pseudomonas aeruginosa, and the quantity which penetrate the biofilm of Pseudomonas aeruginosa become larger.
4. Verification experiment
Purpose:Use different HPLC,In different conditions (Shimadzu LC-10A), detect the quantity of Cefoperazone/Sulbactam to verify the effect that Ambroxol combining with Cefoperazone/Sulbactam.
Strain Source:2 Strains of Pseudomonas aeruginosa, Standard strain ATCC27853 and the strain number H031 which induced by using Levofloxacin from Standard strain ATCC27853.
Methods:Use cefoperazone/sulbactam and cefoperazone/sulbactam+Ambroxol as the medicine through the biofilm respectively, detect the height of the peak with HPLC(Shimadzu LC-10A), UV detector is SPD-10AD, Workstation software of Zhejiang University N2000.
Results:In the devices add cefoperazone/sulbactam only, the height of peak is 185.091. In the devices add cefoperazone/sulbactam+Ambroxol, the height of peak is 207.233.
Research above have shown that
1.Levofloxacin can induce the formation of biofilm.
2.Azithromycin, imipenem has the effect to inhibit the growth of biofilm, However, it is useless to the biofilm which is grown. Ambroxol can dig holes on the surface of biofilm.
3.The experiment which Ambroxol accelerates the Penetration of Ciprofloxacin is repeat successfully.
4.Cefoperazone/Sulbactam can penetrate biofilm of Pseudomonas aeruginosa, after adding Ambroxol, the concentration of Cefoperazone/Sulbactam is larger. This experiment shown that this two medicine can use together to treat the infection by biofilm of Pseudomonas aeruginosa in clinic.
引文
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[1]范柏.喹诺酮类抗菌药的作用机制及细菌耐药性的研究进展[J].国外医药抗生素分册,2004,25(1)：27-29,48
[2]Narcisa Mesarosl, Youri Glupczynsk,A combined phenotypic and genotypic method for the detection of Mex efflux pumps in Pseudomonas aeruginosa[J]. Journal of Antimicrobial Chemotherapy, (2007) 59,378-386
[3]Eric Deziel, Francois Lepine, Sylvain Milot,et al. Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell communication[J]. Microbiology,2004,101(5):1339-1344
[4]Stephen P. Diggle, Pierre Cornelisb, Paul Williamsa, Miguel Ca'mara.4-Quinolone signalling in Pseudomonas aeruginosa:Old molecules, new perspectives[J]. International Journal of Medical Microbiology,2006,296:83-91
[5]Don Monroe. Looking for Chinks in the Armor of Bacterial Biofilms[J]. PLoS Biology,2007,5(11): 2458-2461
[6]Youai Hao, Stephen C. Winans, Bernard R. Glick. Identification and characterization of new LuxR/LuxⅠ-type quorum sensing systems from metagenomic libraries[J]. Environmental Microbiology, 12(1):105-117
[7]Gulgun Bosgelmez-Tinaz, Seyhan Ulusoy. Characterization of N-butanoyl-L--homoserine lactone (C4-HSL) deficient clinical isolates of Pseudomonas aeruginosa[J]. Microbial Pathogenesis,2008,44: 13-19
[8]Stephen P. Diggle, Sandra Matthijs, Victoria J. Wright, et al. The contribution of MvfR to Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regulation:multiple quorum sensing-regulated genes are modulated without affecting lasRI rhlRIor the production of N-acyl-L-homoserine lactones[J]. Chemistry and Biology,2007,14:87-96
[9]Paul Williams, Miguel Camara. Quorum sensing and environmental adaptation in Pseudomonas aeruginosa:a tale of regulatory networks and multifunctional signal molecules[J].Current Opinion in Microbiology,2009(12):182-191
[10]Eric Deziel,Suresh Gopalan,Anastasia P. Tampakaki, et al. The contribution of MvfR to Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regulation:multiple quorum sensing-regulated genes are modulated without affecting lasRI rhlRI or the production of N-acyl-L-homoserine lactones[J]. Molecular Microbiology,2005,55(4):998-1014
[11]马睿,东山康仁,泉川公一.铜绿假单胞菌感应分子对照质粒及整合突变株的构建[J].上海交通大学学报,2006,26(9)：1027-1031
[12]Severine Aendekerk, Stephen P. Diggle, Zhijun Song, et al. The MexGHI-OpmD multidrug efflux pump controls growth, antibiotic susceptibility and virulence in Pseudomonas aeruginosa via 4-quinolone-dependent cell-to-cell communication[J]. Microbiology,2005,151:1113-1125
[13]Severine Aendekerk, Bart Ghysels, Pierre Cornelis et al. Characterization of a new efflux pump, MexGHI-OpmD, from Pseudomonas aeruginosa hat confers resistance to vanadium[J]. Microbiology, 2002,148:2371-2381
[2]Lewis K. Riedle of Biofilm Resistanle[J]. Antimicrob Agents Chemother,2001,45:999-1007
[3]王燕.铜绿假单胞菌的耐药机制及抗生素应用[J].实用医技杂志,2008,15(11)：1408-1409
[4]李冬梅,赵秀媛.细菌生物膜在临床中的研究进展[J].实用医技杂志,2007,14(16)：2251-2253
[5]Xie Yi, Zeng Wei, Jia Wen-Xiang,et al. Functional Effects of LasR/RhlR on Pseudomonas aeruginosa Biofilm Development and Lung Infections in Mice[J]. Progress in Biochemistry and Biophysics,2006,33(1):31-38
[6]杨朵,,张正..细菌生物膜及其相关研究进展[J]. China Academic Journal Electronic Publishing House.2007,10(11):1416-1422
[7]常桂娇,王艾琳.细菌生物膜及其与细菌耐药性关系的研究进展[J].山东医药,2007,47(5)：81-82
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