氨溴索對(duì)流感嗜血桿菌生物膜的作用研究
發(fā)布時(shí)間：2018-12-07 07:01
【摘要】：細(xì)菌生物膜（Biofilm，BF）最早由1987年美國(guó)Costerton教授提出，是細(xì)菌在某些惰性實(shí)體表面形成的多細(xì)胞復(fù)合體的生存方式,是具有一定空間構(gòu)型的細(xì)菌群體,由被覆細(xì)菌、細(xì)菌脂多糖、纖維蛋白、DNA、RNA等成分組成。學(xué)者普遍認(rèn)為，超過(guò)80%的臨床感染和BF有關(guān)。流感嗜血桿菌是一種革蘭陰性桿菌，寄生在人上呼吸道，在人體免疫力低下時(shí)，引起感染。研究顯示，流感嗜血桿菌（Haemophilusinfluenzae）BF和多種疾病有關(guān)，如：慢性阻塞性肺疾病、肺纖維化、慢性鼻-鼻竇炎、分泌性中耳炎、兒童腺樣體肥大等。BF形成后，細(xì)菌對(duì)抗生素耐藥性可增強(qiáng)10-1000倍。氨溴索（ambroxol，AMB）是一種常用的祛痰劑，臨床常用于呼吸道痰液的稀釋，改善通氣狀況；近年研究發(fā)現(xiàn)，AMB對(duì)銅綠假單胞菌（Pseudomonas aeruginosa）、金黃色葡萄球菌（Staphylococcus aureus）等細(xì)菌BF具有清除和殺菌作用，但AMB是否對(duì)流感嗜血桿菌BF具有相同的作用，還未見(jiàn)報(bào)道。本課題從腺樣體肥大患兒離體腺樣體組織分離流感嗜血桿菌，體外培養(yǎng)BF，測(cè)定各臨床株的成膜能力，測(cè)定流感嗜血桿菌成膜前后對(duì)常用抗生素敏感性變化；然后從各流感嗜血桿菌臨床株中選取成膜能力最強(qiáng)的2株細(xì)菌，觀察AMB對(duì)流感嗜血桿菌BF清除作用以及對(duì)生物膜菌的殺菌作用，最后觀察AMB聯(lián)合抗生素對(duì)生物膜菌的殺菌作用。 首先收集腺樣體肥大患兒術(shù)中離體腺樣體標(biāo)本，經(jīng)過(guò)分離、培養(yǎng)和鑒定，將鑒定的流感嗜血桿菌菌株保存。96孔板結(jié)晶紫染色測(cè)定各臨床分離株形成BF能力；培養(yǎng)BF形成后，掃描電鏡（scanning electronmicroscope，SEM）觀察BF結(jié)構(gòu)，并比較BF形成前后對(duì)氨芐西林（ampicillin, AMPC）、頭孢曲松（ceftriaxone, CRO）、左氧沙星（levofloxacin, LVFX）、阿奇霉素（azithromycin, AZM）4種類別抗生素敏感性變化；從流感嗜血桿菌臨床株中選取2株成膜能力最強(qiáng)的流感嗜血桿菌菌株，結(jié)晶紫染色法觀察AMB對(duì)2株流感嗜血桿菌BF的清除作用，菌落計(jì)數(shù)法觀察AMB對(duì)其生物膜菌殺菌作用，最后用二苯基四氮唑溴鹽（MTT）減低法檢測(cè)AMB和AMPC聯(lián)合應(yīng)用對(duì)其生物膜菌殺菌作用。 共收集36份離體腺樣體組織，經(jīng)分離、培養(yǎng)、鑒定共獲得30株流感嗜血桿菌，體外培養(yǎng)72h發(fā)現(xiàn)30株流感嗜血桿菌均能形成BF，但成膜能力大小不一,HI12和HI23成膜最強(qiáng)。經(jīng)SEM觀察，流感嗜血桿菌BF菌體清晰,結(jié)構(gòu)明顯。相對(duì)浮游菌，BF形成后，生物膜菌對(duì)AMPC，CRO，，LVFX，AZM敏感性降低，AMPC的最低生物膜清除濃度（minimal biofilm eradication concentration，MBEC）值最高，最高可達(dá)其最低抑菌濃度（minimal inhibitory concentration，MIC）和最低殺菌濃度（minimal bactericidal concentration，MBC）值100倍之多；CRO的MBEC值是其MIC和MBC值的十幾倍到數(shù)十倍；LVFX的MBEC值是其MIC、MBC值的幾倍大小；AZM的MBEC值最接近于其MBC和MIC值。結(jié)晶紫法示，AMB對(duì)流感嗜血桿菌BF具有清除作用，當(dāng)AMB分別達(dá)到0.24mg ml-1和0.47mg ml-1時(shí)，分別對(duì)2株細(xì)菌HI23和HI12BF具有清除作用（P0.05)，SEM發(fā)現(xiàn)，AMB處理后BF結(jié)構(gòu)變薄，密度減小。菌落計(jì)數(shù)示，AMB具有殺菌作用，該殺菌作用呈現(xiàn)劑量依賴效應(yīng)。統(tǒng)計(jì)學(xué)顯示，AMB聯(lián)合AMPC對(duì)流感嗜血桿菌生物膜菌具有協(xié)同殺菌作用(P0.05)。 綜上實(shí)驗(yàn)得出：流感嗜血桿菌臨床株都可以形成BF，BF形成后，對(duì)AMPC、CRO、LVFX、AZM藥物敏感性降低；AMB對(duì)流感嗜血桿菌BF具有清除和殺菌作用，當(dāng)與AMPC聯(lián)合應(yīng)用時(shí)具有協(xié)同殺菌作用。本課題為AMB在臨床的應(yīng)用提供了理論基礎(chǔ)，可以為臨床治療流感嗜血桿菌BF感染性疾病提供有益的用藥指導(dǎo)。
[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.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R446.5
本文編號(hào)：2366775
[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.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R446.5
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