葡萄糖对水性可降解聚氨酯装载抗菌肽LL-37抑制体外尿源性ESBLs大肠埃希菌生物膜形成的影响

Influence of glucose on biodegradable waterborne polyurethane blend together with LL-37 in inhibiting ESBLs producing Escherichia coli biofilm in vitro

摘要:

目的：探讨葡萄糖浓度变化对水性可降解聚氨酯（biodegradable waterborne polyurethane，BWPU）装载人源抗菌肽LL-37体外抑制ESBLs大肠埃希菌（Escherichia coli，E.coli）生物膜活性的影响。方法：采用尿源性ESBLs E.coli临床株（E44）为实验株，E.coli标准菌株ATCC25922（E0）做质控。构建胱细菌生物膜动态孵育模拟系统。“两步法”制备新型LDI－ＢＷＰＵ乳液PCLPU33。“物理溶解和常温干燥法”制备ＬＤＩ－ＢＷＰＵ装载不同LL-37浓度的载肽膜。设立4个实验分组：H组：高肽组（LL-37，2 000 ?滋g/mL）、L组：低肽组（LL-37，250 ?滋g/mL）、P组：阳性对照（亚胺培南，8 ?滋g/mL）、N组：阴性对照（无抗菌药），持续感染人工尿液环境下孵育48 h，观察不同葡萄糖浓度（0、11.1、33.3 mmol/L）及控制葡萄糖浓度（33.3→0 mmol/L）对载肽膜抑制生物膜的影响。检测方法包括：生物膜细菌活菌计数；Syto-9/PI荧光染色结合激光共聚焦显微镜构建立体图测量生物膜厚度；扫描电镜观察生物膜结构的细微观。生物膜细菌活菌计数和生物膜厚度的多组间均数比较采用单因素方差分析，α=0.01。结果：基于胱细菌生物膜动态孵育模拟系统，在持续感染人工尿液环境下不同葡萄糖浓度观测点，P、H、L组生物膜活菌计数和厚度均明显低于N组（aP=0.000）。H组生物膜活菌计数在无糖环境（G0 mmol/L）明显低于L组（bP=0.000），在含糖环境下（G11.1，33.3 mmol/L）与L组差异无统计学意义（P＞0.01）；H组生物膜厚度在不同葡萄糖观测点均明显低于L组（bP=0.000）。与无糖环境（G0 mmol/L）相比，含糖环境（G11.1，33.3 mmol/L）H、L组生物膜活菌计数和生物膜厚度均明显增加（cP=0.000）。调控葡萄糖浓度后（33.3→0 mmol/L），H、L组BF细菌活菌计数和生物膜厚度均明显降低（dP=0.000）。结论：持续感染人工尿液环境下不同葡萄糖浓度时，载肽膜通过抑制生物膜生长、杀灭生物膜细菌明显抑制细菌生物膜形成。

Abstract:

Objective：To investigate the influence of the change of glucose concentration on ‘LL-37 sustained-release BWPU mem－brane’ in inhibiting the formation of the BF of urinary pathogenic ESBLs producing E.coli. Methods：Urinary pathogenic ESBLs pro－ducing E.coli isolates（E44） and standard strains ATCC25922（E0） were collected. Dynamic simulated bladder BF model was adopted. ‘Two-step approach’ was adopted to prepare new LDI-BWPU emulsion PCLPU33. ‘Physical dissolution and air-drying’ loading technique was adopted to prepare LL-37 sustained-release BWPU membrane.H group：high peptide（LL-37，2000 ?滋g/mL），L group：low peptide group（LL-37，250 ?滋g/mL），P group：positive control（Imipenem，8 ?滋g/mL），and N group：negative control（without antibacterial drug） were set up to incubate under artificial urine environment for 48hours. The influence of different glucose concen－trations（0，11.1，33.3 mmol/L），control of glucose concentration（33.3→0 mmol/L） on LL-37 sustained-release BWPU membrane in inhibiting BF were observed. The detection methods included viable bacteria count method. Syto-9/P fluorescent staining，combined with laser scanning confocal microscope，was applied to construct a space diagram to measure the BF thickness. Scan－ning electron microscope was adopted to observe the microscopic view of BF structure. One-way analysis of variance（ANOVA） was adopted in viable bacteria count，BF thickness and comparison of means of multiple groups. Results：For the observation point of different glucose concentration under the constantly infected artificial urine environment，the viable bacteria count and BF thickness of P group，H group，and L group were significantly decreased than that of N group（aP=0.000）. The viable bacteria count of group H was significantly decreased than that of L group（bP=0.000）in the glucose-free environment（G0 mmol/L）,and there was no statistical dif－ference between the H and L group in the glucose-containing environment（G11.1，33.3 mmol/L）（P＞0.01）；The BF thickness of the H group was significantly decreased than that of L group at the observation point of different glucose concentration（bP=0.000）. With the increase of glucose concentration，the viable bacteria count and BF thickness of the H and L groups，increased significantly（cP=0.000）. When glucose concentration was controlled（33.3→0 mmol/L） in the H and L groups，the viable bacteria count and BF thickness were were significantly decreased（dP=0.000）. Conclusion：When the glucose concentration is different under the constantly infected artificial urine environment，LL-37 sustained-release BWPU membrane can inhibit BF growth，kill BF bacteria and signifi－cantly inhibit BF formation.