氨溴索对流感嗜血杆菌生物膜的作用研究

发布时间：2018-12-07 07:01

【摘要】：细菌生物膜（Biofilm，BF）最早由1987年美国Costerton教授提出，是细菌在某些惰性实体表面形成的多细胞复合体的生存方式,是具有一定空间构型的细菌群体,由被覆细菌、细菌脂多糖、纤维蛋白、DNA、RNA等成分组成。学者普遍认为，超过80%的临床感染和BF有关。流感嗜血杆菌是一种革兰阴性杆菌，寄生在人上呼吸道，在人体免疫力低下时，引起感染。研究显示，流感嗜血杆菌（Haemophilusinfluenzae）BF和多种疾病有关，如：慢性阻塞性肺疾病、肺纤维化、慢性鼻-鼻窦炎、分泌性中耳炎、儿童腺样体肥大等。BF形成后，细菌对抗生素耐药性可增强10-1000倍。氨溴索（ambroxol，AMB）是一种常用的祛痰剂，临床常用于呼吸道痰液的稀释，改善通气状况；近年研究发现，AMB对铜绿假单胞菌（Pseudomonas aeruginosa）、金黄色葡萄球菌（Staphylococcus aureus）等细菌BF具有清除和杀菌作用，但AMB是否对流感嗜血杆菌BF具有相同的作用，还未见报道。本课题从腺样体肥大患儿离体腺样体组织分离流感嗜血杆菌，体外培养BF，测定各临床株的成膜能力，测定流感嗜血杆菌成膜前后对常用抗生素敏感性变化；然后从各流感嗜血杆菌临床株中选取成膜能力最强的2株细菌，观察AMB对流感嗜血杆菌BF清除作用以及对生物膜菌的杀菌作用，最后观察AMB联合抗生素对生物膜菌的杀菌作用。首先收集腺样体肥大患儿术中离体腺样体标本，经过分离、培养和鉴定，将鉴定的流感嗜血杆菌菌株保存。96孔板结晶紫染色测定各临床分离株形成BF能力；培养BF形成后，扫描电镜（scanning electronmicroscope，SEM）观察BF结构，并比较BF形成前后对氨苄西林（ampicillin, AMPC）、头孢曲松（ceftriaxone, CRO）、左氧沙星（levofloxacin, LVFX）、阿奇霉素（azithromycin, AZM）4种类别抗生素敏感性变化；从流感嗜血杆菌临床株中选取2株成膜能力最强的流感嗜血杆菌菌株，结晶紫染色法观察AMB对2株流感嗜血杆菌BF的清除作用，菌落计数法观察AMB对其生物膜菌杀菌作用，最后用二苯基四氮唑溴盐（MTT）减低法检测AMB和AMPC联合应用对其生物膜菌杀菌作用。共收集36份离体腺样体组织，经分离、培养、鉴定共获得30株流感嗜血杆菌，体外培养72h发现30株流感嗜血杆菌均能形成BF，但成膜能力大小不一,HI12和HI23成膜最强。经SEM观察，流感嗜血杆菌BF菌体清晰,结构明显。相对浮游菌，BF形成后，生物膜菌对AMPC，CRO，，LVFX，AZM敏感性降低，AMPC的最低生物膜清除浓度（minimal biofilm eradication concentration，MBEC）值最高，最高可达其最低抑菌浓度（minimal inhibitory concentration，MIC）和最低杀菌浓度（minimal bactericidal concentration，MBC）值100倍之多；CRO的MBEC值是其MIC和MBC值的十几倍到数十倍；LVFX的MBEC值是其MIC、MBC值的几倍大小；AZM的MBEC值最接近于其MBC和MIC值。结晶紫法示，AMB对流感嗜血杆菌BF具有清除作用，当AMB分别达到0.24mg ml-1和0.47mg ml-1时，分别对2株细菌HI23和HI12BF具有清除作用（P0.05)，SEM发现，AMB处理后BF结构变薄，密度减小。菌落计数示，AMB具有杀菌作用，该杀菌作用呈现剂量依赖效应。统计学显示，AMB联合AMPC对流感嗜血杆菌生物膜菌具有协同杀菌作用(P0.05)。综上实验得出：流感嗜血杆菌临床株都可以形成BF，BF形成后，对AMPC、CRO、LVFX、AZM药物敏感性降低；AMB对流感嗜血杆菌BF具有清除和杀菌作用，当与AMPC联合应用时具有协同杀菌作用。本课题为AMB在临床的应用提供了理论基础，可以为临床治疗流感嗜血杆菌BF感染性疾病提供有益的用药指导。
[Abstract]:The bacterial biofilm (Biofilm, BF) was first presented by Professor Costellton, the United States in 1987, a way of living of a multi-cell complex formed on the surface of certain inert entities, a bacterial population with a certain spatial configuration, consisting of coated bacteria, bacterial lipopolysaccharides, fibrin, DNA, and the like. It is widely believed that more than 80% of clinical infection and BF are related. Haemophilus influenzae is a Gram-negative bacterium, which is parasitic on the upper respiratory tract of the human body and causes infection when the immunity of the human body is low. Studies have shown that Haemophilus influenzae (Haemophilus influenzae) BF is associated with various diseases, such as chronic obstructive pulmonary disease, pulmonary fibrosis, chronic nasal-nasal inflammation, secretory otitis media, and adenoids of children. After BF formation, the antibiotic resistance of bacteria can be increased by 10-1000 times. Ambroxol (Ambroxol, AMB) is a commonly used expectorant, which is commonly used in the dilution of the sputum of the respiratory tract and improves the ventilation condition. In recent years, it has been found that the AMB has the functions of clearing and sterilizing the bacteria BF of Pseudomonas aeruginosa and Staphylococcus aureus. However, whether AMB has the same effect on Haemophilus influenzae BF is not reported. In this study, Haemophilus influenzae was isolated from the body of the body of the body of the gland-like body, the BF was cultured in vitro, the film-forming ability of each clinical strain was determined, and the sensitivity of the common antibiotics was determined before and after the formation of the Haemophilus influenzae. Two strains of bacteria with the strongest film-forming ability were selected from the clinical strains of Haemophilus influenzae, and the effect of AMB on the BF of Haemophilus influenzae and the sterilization of the biofilm was observed, and the bactericidal effect of the AMB combined with antibiotics on the biofilm was observed. The samples of the body-like body were collected, isolated, cultured and identified, and the identified Haemophilus influenzae strains were preserved. The BF capacity of each clinical isolates was determined by the crystal violet staining of 96-well plates, and the scanning electron microscopy (SEM) was performed after the formation of the BF. e. The BF structure was observed by SEM, and the sensitivity of the antibiotic to the four kinds of antibiotics was changed, such as ampicillin (AMPC), ceftriaxone (CRO), levofloxacin (LVFX), aziramycin (AZM) and 4 kinds of antibiotics, before and after BF formation. 2 strains of Haemophilus influenzae with the strongest film-forming ability were selected from the clinical strains of Haemophilus influenzae, and the removal of 2 strains of Haemophilus influenzae BF was observed by the crystal violet staining method. The application of the combination of AMB and AMPC to the sterilization of the biofilm was detected by using the method of MTT reduction. 30 strains of Haemophilus influenzae were isolated and cultured, 30 strains of Haemophilus influenzae were isolated and cultured, and 30 strains of Haemophilus influenzae were found to be able to form BF, but the film-forming capacity was different in size, HI12 and H23. The membrane was the strongest. The bacteria of Haemophilus influenzae BF were clear and knotted by SEM. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of AMPC were the highest, and the minimum inhibitory concentration (MBC) of AMPC was the highest, and the minimum inhibitory concentration (MBC) of AMPC was the highest. The MBEC value of the CRO is several times to tens of times the MIC and MBC values; the MBEC value of the LVFX is several times the MIC, MBC value; the MBEC value of the AZM is the closest to its MBC and M The results showed that AMB had a clear effect on H. influenzae BF when AMB reached 0.24mg/ ml-1 and 0.47mg ml-1, respectively. The degree of reduction is reduced. The colony count shows that the AMB has a bactericidal action which presents a dose in accordance with the present invention. The results showed that AMPC combined with AMPC had a synergistic bactericidal effect on Haemophilus influenzae biofilm (P0. The results showed that the clinical strains of Haemophilus influenzae can form BF and BF, and the sensitivity of AMPC, CRO, LVFX and AZM is reduced. AMB has a clear and bactericidal effect on Haemophilus influenzae BF. The purpose of this study is to provide a theoretical basis for the clinical application of AMB, which can be used for the treatment of Haemophilus influenzae BF infectious diseases.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R446.5

【参考文献】