細(xì)菌生物被膜耐藥及形成相關(guān)基因調(diào)控機(jī)制的研究
發(fā)布時間：2018-09-09 12:17
【摘要】：目的 研究機(jī)械通氣患者氣管導(dǎo)管細(xì)菌生物被膜的病原學(xué)分布,通過生物被膜菌與相應(yīng)浮游菌在體外培養(yǎng)的藥敏試驗進(jìn)行比較,進(jìn)而探討細(xì)菌生物被膜對抗生素的耐藥性。進(jìn)一步以臨床分離的表皮葡萄球菌生物被膜菌和對應(yīng)的浮游菌為研究對象,探討表皮葡萄球菌生物被膜形成相關(guān)的基因(操縱子—icaA基因,轉(zhuǎn)錄調(diào)控子一sigB基因,操縱子調(diào)節(jié)基因—icaR基因,毒力因子調(diào)節(jié)系統(tǒng)—agrA基因)的分布及與生物被膜形成的關(guān)系。 方法 (1)研究了110例施行人工氣道(氣管插管和氣管切開)并機(jī)械通氣的患者拔除的氣管導(dǎo)管,用剛果紅培養(yǎng)基篩選出生物被膜菌株和相應(yīng)的浮游菌株,并對生物被膜菌進(jìn)行半定量檢測,藥敏實驗分析生物被膜菌株和相應(yīng)的浮游菌在普通MH培養(yǎng)基上耐藥差異以及生物被膜陽性菌在泊洛沙姆(Poloxamer, F-127)培養(yǎng)基和普通MH培養(yǎng)基上耐藥性差異進(jìn)行分析。 (2)收集氣管導(dǎo)管中分離的表皮葡萄球菌,按生物被膜陽性表皮葡萄球菌及相應(yīng)的浮游菌表皮葡萄球菌分為2組。使用RT-PCR分別對生物被膜菌組和浮游菌組細(xì)菌的icaA、sigB、icaR、agrA基因輝度比進(jìn)行檢測；利用Real-time PCR分別檢測生物被膜菌組和浮游菌組細(xì)菌的icaA、sigB、icaR、agrA四種與生物被膜形成相關(guān)的基因的表達(dá),并通過2-△△Ct方法進(jìn)行相對定量。分析表皮葡萄球菌臨床菌株中icaA、sigB、icaR、agrA基因轉(zhuǎn)錄水平與表皮葡萄球菌生物被膜形成的相關(guān)性。 結(jié)果 (1)110例機(jī)械通氣患者氣管導(dǎo)管中篩選出生物被膜陽性菌61株,陽性率為55.45%。以葡萄球菌屬,糞腸球菌及銅綠假單胞菌最常見,生物被膜菌株與相應(yīng)浮游菌株在普通MH培養(yǎng)基上藥敏結(jié)果相似,差異無統(tǒng)計學(xué)意義(P0.05)；生物被膜菌株在普通MH培養(yǎng)基上和在Poloxamer培養(yǎng)基上藥敏結(jié)果差異有統(tǒng)計學(xué)意義(P0.05)且后者耐藥性更強(qiáng)。 (2) RT-PCR中icaA、sigB、icaR、agrA基因的輝度測定值統(tǒng)計分析結(jié)果顯示生物被膜陽性表皮葡萄球菌及相應(yīng)的浮游菌表皮葡萄球菌兩組細(xì)菌間差異無顯著性(P0.05),但icaA、sigB基因生物被膜菌組平均秩及秩和明顯高于浮游菌組；而icaR、agrA基因生物被膜菌組平均秩及秩和明顯小于浮游菌組。 (3) Real-time PCR檢測顯示表皮葡萄球菌生物被膜菌組icaA, sigB基因表達(dá)量分別是浮游菌組的418.8倍與81倍,而icaR, agrA基因在生物被膜菌組中表達(dá)量僅為浮游菌組的1/145與1/270。 結(jié)論 (1)本院機(jī)械通氣患者氣管導(dǎo)管生物被膜病原學(xué)分布以葡萄球菌屬,糞腸球菌和銅綠假單胞菌為主,生物被膜菌與浮游菌在普通MH培養(yǎng)基上耐藥性分析未見明顯差異,生物被膜菌在Poloxamer培養(yǎng)基上和在MH培養(yǎng)基上耐藥性有明顯差異,推測Poloxamer培養(yǎng)基有可能反映生物被膜菌較真實的對藥物的耐受情況,其耐受性更強(qiáng)。 (2) icaA、sigB、icaR、agrA基因與表皮葡萄球菌生物被膜形成關(guān)系密切,icaA, sigB基因能促進(jìn)表皮葡萄球菌生物被膜的形成,而icaR, agrA基因能抑制表皮葡萄球菌生物被膜的形成。提示agr系統(tǒng)能調(diào)節(jié)icaA、sigB、icaR基因的表達(dá),agr系統(tǒng)可以抑制icaA和.sigB這兩種基因的表達(dá)；促進(jìn)icaR基因的表達(dá)。
[Abstract]:objective
To study the Pathogenic Distribution of bacterial biofilm in tracheal tube of patients with mechanical ventilation, and compare the antibiotic susceptibility test of biofilm bacteria and corresponding plankton bacteria cultured in vitro to explore the antibiotic resistance of bacterial biofilm. Objectives: To investigate the distribution of genes related to biofilm formation of Staphylococcus epidermidis (operon-icaA gene, transcription regulator sigB gene, operon regulator-icaR gene, virulence factor regulator-agrA gene) and their relationship with biofilm formation.
Method
(1) 110 patients with artificial airway (tracheal intubation and tracheotomy) and mechanical ventilation were studied. Biofilm strains and corresponding plankton strains were screened by Congo red medium. Semi-quantitative detection of biofilm bacteria was carried out. Drug susceptibility test showed that biofilm strains and corresponding plankton bacteria were cultured in MH. The difference of drug resistance in culture medium and the difference of drug resistance of biofilm-positive bacteria in Poloxamer (F-127) medium and MH medium were analyzed.
(2) Staphylococcus epidermidis isolated from tracheal cannula was divided into two groups according to biofilm-positive Staphylococcus epidermidis and corresponding planktonic bacteria. The expression of icaA, sigB, icaR and agrA genes in S. epidermidis and S. epidermidis was quantified by 2-delta CT. The correlation between the transcription levels of icaA, sigB, icaR and agrA genes and the biofilm formation of S. epidermidis was analyzed.
Result
(1) 61 strains of biofilm-positive bacteria were screened out from 110 patients with mechanical ventilation, and the positive rate was 55.45%. Staphylococcus, Enterococcus faecalis and Pseudomonas aeruginosa were the most common strains. The drug sensitivity of biofilm-positive bacteria was similar to that of corresponding planktonic bacteria on the common MH medium, but the difference was not statistically significant (P 0.05). The results of susceptibility test on MH medium and Poloxamer medium were significantly different (P 0.05), and the latter was more resistant.
(2) Statistical analysis of the luminosity of icaA, sigB, icaR, agrA genes in RT-PCR showed that there was no significant difference between the two groups (P 0.05), but the average rank and rank of icaA, sigB gene biofilm group were significantly higher than those of plankton group. The average rank and rank of the membranous bacteria group were significantly lower than that of the planktonic bacteria group.
(3) Real-time PCR showed that the expression levels of icaA and sigB genes in S. epidermidis biofilm group were 418.8 and 81 times higher than those in plankton group, while the expression levels of icaR and agrA genes in biofilm group were only 1/145 and 1/270 of that in plankton group.
conclusion
(1) Staphylococcus, Enterococcus faecalis and Pseudomonas aeruginosa were the main pathogens of tracheal tube biofilm in patients with mechanical ventilation in our hospital. There was no significant difference in drug resistance between biofilm bacteria and plankton bacteria on ordinary MH medium. The drug resistance of biofilm bacteria on Poloxamer medium and MH medium was significantly different. Poloxamer medium may reflect the true tolerance of biofilm bacteria to drugs, and it is more tolerant.
(2) icaA, sigB, icaR and agrA genes are closely related to the biofilm formation of S. epidermidis. icaA and sigB genes can promote the biofilm formation of S. epidermidis, while icaR and agrA genes can inhibit the biofilm formation of S. epidermidis. It suggests that AGR system can regulate the expression of icaA, sigB and icaR genes, and agr system can inhibit the formation of icaA and si. GB expression of these two genes promotes icaR gene expression.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378
本文編號：2232350
[Abstract]:objective
To study the Pathogenic Distribution of bacterial biofilm in tracheal tube of patients with mechanical ventilation, and compare the antibiotic susceptibility test of biofilm bacteria and corresponding plankton bacteria cultured in vitro to explore the antibiotic resistance of bacterial biofilm. Objectives: To investigate the distribution of genes related to biofilm formation of Staphylococcus epidermidis (operon-icaA gene, transcription regulator sigB gene, operon regulator-icaR gene, virulence factor regulator-agrA gene) and their relationship with biofilm formation.
Method
(1) 110 patients with artificial airway (tracheal intubation and tracheotomy) and mechanical ventilation were studied. Biofilm strains and corresponding plankton strains were screened by Congo red medium. Semi-quantitative detection of biofilm bacteria was carried out. Drug susceptibility test showed that biofilm strains and corresponding plankton bacteria were cultured in MH. The difference of drug resistance in culture medium and the difference of drug resistance of biofilm-positive bacteria in Poloxamer (F-127) medium and MH medium were analyzed.
(2) Staphylococcus epidermidis isolated from tracheal cannula was divided into two groups according to biofilm-positive Staphylococcus epidermidis and corresponding planktonic bacteria. The expression of icaA, sigB, icaR and agrA genes in S. epidermidis and S. epidermidis was quantified by 2-delta CT. The correlation between the transcription levels of icaA, sigB, icaR and agrA genes and the biofilm formation of S. epidermidis was analyzed.
Result
(1) 61 strains of biofilm-positive bacteria were screened out from 110 patients with mechanical ventilation, and the positive rate was 55.45%. Staphylococcus, Enterococcus faecalis and Pseudomonas aeruginosa were the most common strains. The drug sensitivity of biofilm-positive bacteria was similar to that of corresponding planktonic bacteria on the common MH medium, but the difference was not statistically significant (P 0.05). The results of susceptibility test on MH medium and Poloxamer medium were significantly different (P 0.05), and the latter was more resistant.
(2) Statistical analysis of the luminosity of icaA, sigB, icaR, agrA genes in RT-PCR showed that there was no significant difference between the two groups (P 0.05), but the average rank and rank of icaA, sigB gene biofilm group were significantly higher than those of plankton group. The average rank and rank of the membranous bacteria group were significantly lower than that of the planktonic bacteria group.
(3) Real-time PCR showed that the expression levels of icaA and sigB genes in S. epidermidis biofilm group were 418.8 and 81 times higher than those in plankton group, while the expression levels of icaR and agrA genes in biofilm group were only 1/145 and 1/270 of that in plankton group.
conclusion
(1) Staphylococcus, Enterococcus faecalis and Pseudomonas aeruginosa were the main pathogens of tracheal tube biofilm in patients with mechanical ventilation in our hospital. There was no significant difference in drug resistance between biofilm bacteria and plankton bacteria on ordinary MH medium. The drug resistance of biofilm bacteria on Poloxamer medium and MH medium was significantly different. Poloxamer medium may reflect the true tolerance of biofilm bacteria to drugs, and it is more tolerant.
(2) icaA, sigB, icaR and agrA genes are closely related to the biofilm formation of S. epidermidis. icaA and sigB genes can promote the biofilm formation of S. epidermidis, while icaR and agrA genes can inhibit the biofilm formation of S. epidermidis. It suggests that AGR system can regulate the expression of icaA, sigB and icaR genes, and agr system can inhibit the formation of icaA and si. GB expression of these two genes promotes icaR gene expression.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 鄒彬彬;漆涌;伍勇;;尿路感染生物被膜菌臨床分布及耐藥性研究[J];國際檢驗醫(yī)學(xué)雜志;2009年05期
2 汪復(fù);;2005中國CHINET細(xì)菌耐藥性監(jiān)測結(jié)果[J];中國感染與化療雜志;2006年05期
3 汪復(fù);朱德妹;胡付品;阮斐怡;倪語星;孫景勇;徐英春;孫宏莉;胡云健;艾效曼;俞云松;楊青;孫自鏞;簡翠;賈蓓;黃文祥;卓超;蘇丹虹;魏蓮花;吳玲;張朝霞;季萍;王傳清;薛建昌;張泓;李萬華;;2007年中國CHINET細(xì)菌耐藥性監(jiān)測[J];中國感染與化療雜志;2008年05期
4 鄒彬彬;漆涌;伍勇;;細(xì)菌群體感應(yīng)系統(tǒng)與生物薄膜形成的研究進(jìn)展[J];臨床檢驗雜志;2009年01期
5 江娟;孫景勇;歐元祝;秦智強(qiáng);陳潔敏;瞿滌;;醫(yī)源性表皮葡萄球菌ica操縱子轉(zhuǎn)錄水平對生物膜表型的影響[J];上海醫(yī)學(xué);2006年01期
6 李睿明;;抗感染治療新靶點——細(xì)菌生物膜[J];醫(yī)學(xué)與哲學(xué)(臨床決策論壇版);2006年04期
7 李乃靜;何平;李勝岐;;老年機(jī)械通氣患者氣管內(nèi)導(dǎo)管生物被膜的觀察[J];中華老年醫(yī)學(xué)雜志;2006年02期
8 王勇翔,李華林,李平洋,倪語星,聞玉梅;表皮葡萄球菌生物膜形成與ica操縱子的相關(guān)性研究[J];中華微生物學(xué)和免疫學(xué)雜志;2003年06期
9 龐杏林;鄭桂麗;李孝權(quán);鄧志愛;莫自耀;;醫(yī)院感染凝固酶陰性葡萄球菌分布及耐藥性分析[J];中華醫(yī)院感染學(xué)雜志;2007年10期
10 蘭琳;;醫(yī)院感染凝固酶陰性葡萄球菌耐藥性研究[J];中國實用醫(yī)藥;2008年25期
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