本文選題：不動桿菌 + 多重耐藥　； 參考：《河北醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的：探討ICU患者下呼吸道檢出不動桿菌屬耐藥情況、不同致病狀態(tài)的檢出菌包膜蛋白產(chǎn)生情況及其與致病狀態(tài)的關(guān)系。我們共分離出10株定植菌株，32株感染菌株。通過分析其感染高危因素，探討定植菌與感染菌宿主在臨床方面差異。根據(jù)CPIS評分和定量培養(yǎng)的結(jié)果來判定細菌的定植與感染狀態(tài)。CPIS評分包括：體溫，外周血白細胞分類及計數(shù)，，氣道膿性分泌物的量，氧合指數(shù)，胸部影像檢查結(jié)果和呼吸道抽吸物革蘭染色鏡檢及培養(yǎng)結(jié)果。當(dāng)CPIS評分大于6分，同時定量培養(yǎng)結(jié)果大于菌落計數(shù)106CFU/ml診斷為呼吸機相關(guān)性肺炎，即患者為感染狀態(tài)。并研究耐藥的鮑曼不動桿菌在定植及感染狀態(tài)下外膜毒力蛋白（OmpA/BauA）的表達與其致病性的關(guān)系。 方法： 1菌株來源：收集2011-2012年來自河北省4家醫(yī)院的42株鮑曼不動桿菌菌株。 2藥敏試驗：采用K-B法鑒定分離的鮑曼不動桿菌對頭孢哌酮/舒巴坦、頭孢哌酮、頭孢噻肟、哌拉西林、頭孢他啶、頭孢吡肟、比阿培南、美羅培南、阿米卡星、環(huán)丙沙星、左氧氟沙星、慶大霉素、氯霉素、氨曲南、米諾環(huán)素、復(fù)方新諾明16種抗菌素的敏感性。并根據(jù)美國臨床實驗室標(biāo)準(zhǔn)化委員會（NCCLS）2008年版要求進行抗菌藥物敏感性判斷。 3分析患者臨床高危因素：通過藥敏試驗從中篩選出多重耐藥，泛耐藥及全耐藥的菌株，根據(jù)CPIS評分和患者分泌物定量培養(yǎng)的結(jié)果分離出定植菌株10株，感染菌株32株，并收集患者的臨床資料分析其兩組檢出菌宿主之間感染高危因素差別。 4PCR擴增外膜蛋白Ompa及BauA基因：根據(jù)GenBank公布的序列，設(shè)計Ompa(AY485227)及BauA(AY571146)基因的特異性引物。Ompa基因上游引物OMP1-5’ATTACGACGGAAATTGTAAGTAATTT-3’定位于Ompa基因+1一+26bp位堿基，下游引物OMP25’-CGACTCGTTAGAGTCGCTTTTTTATG-3’，定位于Ompa基因+1291一+1317bp位堿基，BauA基因上游引物OMP15’-TGCAAGCCCAACCTCACT-3’定位于BauA基因+4060一+4078bp位堿基；下游引物OMP25’-TTCTCCAGACTTAGGGAT-3’，定位于BauA基因+6642一+6660bp位堿基進行PCR擴增，將產(chǎn)物在0.8%瓊脂糖凝膠電泳。用凝膠成像系統(tǒng)攝像并保存。 5PCR產(chǎn)物序列分析。 結(jié)果： 1藥敏試驗應(yīng)用K-B法檢測42株鮑曼不動桿菌均表現(xiàn)為多重耐藥，其中對頭孢哌酮/舒巴坦耐藥率最低為30.95%，對頭孢噻肟的耐藥率95.23%。頭孢哌酮為88.10%，氨曲南及米諾環(huán)素分別為97.6%和78.57%；頭孢吡肟、頭孢他啶均為76.20%，慶大霉素、阿米卡星均為85.72%；哌拉西林與左氧氟沙星均為83.34%；復(fù)方新諾明為73.81%，對亞胺培南及比阿培南耐藥率分別為73.81%、92.86%，氯霉素的耐藥性69.05%，環(huán)丙沙星為90.48%。 2分析患者臨床高危因素：通過復(fù)習(xí)病歷收集多重耐藥鮑曼不動桿菌定植或感染患者的臨床資料（年齡、性別、醫(yī)學(xué)合并癥、ICU住院時間、侵入性操作及其持續(xù)時間、預(yù)后等資料）、結(jié)合臨床干預(yù)效果42株菌株分為定植組10例、感染組32例。最常見的暴露因素為：入住ICU，機械通氣，其他侵入性操作。最常見的基礎(chǔ)疾病為：腦血管病，其次為慢性阻塞性肺疾病。對于定植組與感染組臨床收集的資料無明顯統(tǒng)計學(xué)意義（P0.05）。感染組的死亡率大于定植組，但是兩組的死亡率差異無統(tǒng)計學(xué)意義（15.66%vs10%，P0.05）。但是對于ICU患者的定植與感染組中，感染組患者ICU的住院時間明顯大于定植組（P0.05）有統(tǒng)計學(xué)意義。 3PCR法細菌包膜蛋白測定：BauA定植組（陽性率70%，陰性率30%)，感染組（陽性率53.13%，陰性率46.87%）。通過卡方檢驗統(tǒng)計學(xué)方法分析其兩者的構(gòu)成比，P＞0.05，OmpA定植組（陽性率80%，陰性率20%)，感染組（陽性率87.5%，陰性率12.5%）。通過卡方檢驗統(tǒng)計學(xué)方法分析其兩者的構(gòu)成比，P＞0.05，說明兩者之間包膜蛋白構(gòu)成比差異無統(tǒng)計學(xué)意義，尚不能認(rèn)為定植組與感染組之間細菌的致病力與這兩種蛋白之間有關(guān)。 結(jié)論： 1本實驗通過藥敏分析了河北省4家醫(yī)院收集的鮑曼不動桿菌的耐藥情況。對頭孢哌酮\舒巴坦耐藥率最低為30.95%，對氨曲南的耐藥率最高，達到了97.60%，對碳青酶烯類耐藥率也在73.81%到92.86%之間。 2定植組與感染組在臨床高危因素方面沒有明顯區(qū)別，在死亡率上也沒有區(qū)別。但ICU入住時間在定植與感染組中有統(tǒng)計學(xué)差異，說明入住ICU時間越長造成菌株侵襲性機會越高。 3通過PCR分子生物學(xué)技術(shù)分析包膜蛋白OmpA在兩組之間無統(tǒng)計學(xué)差異，與鐵相關(guān)的外膜轉(zhuǎn)運蛋白BauA在定植組與感染組之間也無統(tǒng)計學(xué)差異。尚不能認(rèn)為這兩種蛋白產(chǎn)生水平與泛耐藥鮑曼不動桿菌致病性相關(guān)�？赡芘c其他毒力蛋白及患者免疫狀態(tài)的關(guān)系可能更密切。
[Abstract]:Objective : To investigate the relationship between bacterial resistance and pathogenic state in patients with lower respiratory tract infection in ICU . 10 strains of colonization and 32 strains of infection were isolated .

Method :

1 . Source : Collection of 42 strains of acinetobacter baumanii from 4 hospitals in Hebei Province in 2011 - 2012 .

2 Drug sensitivity test : The susceptibility of isolated baumanii was identified by K - B method . The susceptibility to antibiotics was determined according to the 2008 edition of American Clinical Laboratory Standardization Committee ( NCCLS ) .

3 . The clinical and high risk factors of the patients were analyzed . The strains with multiple drug resistance , pan - resistance and total resistance were screened from the drug - sensitive test , 10 strains were isolated according to CPIS score and quantitative culture of the patient ' s secretions , 32 strains were infected , and the clinical data of the patients were collected to analyze the difference of the high risk factors between the two groups .

Ompa ( AY485227 ) and BauA ( AY571146 ) gene were amplified by 4PCR . Ompa ( AY485227 ) and BauA ( AY571146 ) gene specific primers were designed according to the published sequence . The upstream primer of Ompa gene was located at the base of Ompa gene + 1 + 26bp . The upstream primer OMP15 ' - TGCAAGCCCAACCTCACT - 3 ' was located at the base of BauA gene + 4060 + 4078bp .
The downstream primer OMP25 ' - TTCTCCAGACT TAGGGAT - 3 ' was subjected to PCR amplification at the base of the BauA gene + 6642 I + 6660bp , and the product was subjected to gel electrophoresis of 0.8 % agarose gel .

5PCR product sequence analysis .

Results :

The drug resistance rate was 30.95 % , the drug resistance rate was 95.23 % , 80.10 % , 97.6 % and 78.57 % respectively .
Cefepime and ceftazidine were 76.20 % and 85.72 % respectively .
Piperacillin and levofloxacin were 83.34 % ;
The drug resistance rates were 73.81 % , 73.81 % , 92.86 % , 69.05 % and 90.48 % respectively .

The clinical data ( age , sex , medical complications , ICU admission time , invasive operation , duration and prognosis ) of patients with multiple drug resistance were analyzed . The most common underlying diseases were cerebrovascular disease , followed by chronic obstructive pulmonary disease . The most common underlying diseases were : cerebrovascular disease , followed by chronic obstructive pulmonary disease . The mortality rate in the infection group was higher than that in the colonization group , but there was no significant difference between the two groups ( 15.66 % vs 10 % , P0.05 ) . However , in the patients with ICU and infection group , the hospitalization time of ICU in the infection group was significantly higher than that in the colonization group ( P0.05 ) .

Detection of bacterial envelope protein by 3PCR method : BauA colonization group ( positive rate 70 % , negative rate 30 % ) , infection group ( positive rate 53.13 % , negative rate 46.87 % ) . By means of Chi - square test , the composition ratio , P & gt ; 0.05 , OmpA colonization group ( positive rate 80 % , negative rate 20 % ) , infection group ( positive rate 87.5 % , negative rate 12.5 % ) were analyzed . By means of Chi - square test , the composition ratio of the two proteins was analyzed by means of Chi - square test , P > 0.05 . The difference between the two proteins was not considered statistically significant , and it was not considered that the pathogenicity of bacteria between the colonization group and the infection group was related to the two proteins .

Conclusion :

1 . The drug resistance of baumanii collected by 4 hospitals in Hebei Province was analyzed by drug sensitivity . The drug resistance rate was 30 . 95 % , and the drug resistance rate to aztreonam was the highest , reaching 97.60 % , and the drug resistance rate was 73.81 % to 92.86 % .

There was no significant difference between the 2 colonization group and the infection group in the clinical high risk factors , but there was no difference in mortality rate . However , the ICU stay time had statistical difference in the colonization and infection group , indicating that the longer the ICU time , the higher the chance of invasive ability of the strain .

3 . There was no statistical difference between the two groups by PCR molecular biology technique . There was no statistical difference between the two groups .

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R516;R459.7

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