【摘要】：不動桿菌屬(Acinetobacter spp.)是非發(fā)酵條件致病菌,通常在機體抵抗力降低時引起感染,是院內(nèi)感染的重要機會致病菌之一,其中鮑曼不動桿菌生命力尤其頑強,可廣泛分布于醫(yī)院環(huán)境之中。近年來,隨著臨床上廣譜抗生素的大量應(yīng)用,出現(xiàn)了對大部分抗生素耐藥的多重耐藥及超耐藥鮑曼不動桿菌,并時常造成院內(nèi)感染的暴發(fā)流行,給臨床治療帶來極大挑戰(zhàn)。特別是,隨著產(chǎn)NDM-1超耐藥菌的出現(xiàn)與流行,全球已對抗生素濫用問題及細(xì)菌耐藥問題產(chǎn)生極大關(guān)注。本研究基于軍隊傳染病病原監(jiān)測平臺,2010~2014年間,本實驗室在我國北京、沈陽、蘇州、甘肅、廈門、南京等不同地區(qū),收集300株來自臨床病人、醫(yī)院環(huán)境的疑似不動桿菌屬細(xì)菌,利用16S r RNA測序技術(shù)對其進行檢測,共確認(rèn)不動桿菌均屬細(xì)菌292株,其中鮑曼不動桿菌251株、醋酸鈣不動桿菌19株、瓊氏不動桿菌19株、洛菲不動桿菌3株,蘇州地區(qū)分離117株、沈陽117株、甘肅33株、北京12株、濟南2株,廈門3株、廣州4株、南京4株。為了解我國不動桿菌屬細(xì)菌的耐藥特點,本研究對實驗室現(xiàn)有的226株細(xì)菌進行了抗生素敏感性試驗。藥敏結(jié)果顯示,來自北京、沈陽、蘇州3個地區(qū)的不動桿菌屬細(xì)菌,對臨床上8種首選治療鮑曼不動桿菌感染藥物產(chǎn)生了不同程度的耐藥。耐藥率從高到低依次為頭孢他啶96%、諾氟沙星/頭孢吡肟92%、亞胺培南88%、頭孢哌酮87%、哌拉西林84%、替卡西林/克拉維酸83%、阿米卡星77%。此外,多重耐藥不動桿菌共有211株、占測試菌株的97%,其中北京7株、占總數(shù)的3%,沈陽94株、占總數(shù)的45%,蘇州110株、占總數(shù)的52%。在這些多重耐藥不動桿菌屬細(xì)菌中,有150株菌對5類以上抗生素全部耐藥,其中沈陽57株、蘇州93株。以上結(jié)果表明,我國醫(yī)院內(nèi)流行的不動桿菌屬細(xì)菌耐藥情況目前十分嚴(yán)重。同時還可以發(fā)現(xiàn),這些耐藥不動桿菌對內(nèi)酰胺酶抑制劑和氨基糖苷類藥物的敏感性也比較高。所以,在臨床上可以考慮使用以上兩類藥物治療由不動桿菌屬細(xì)菌引起的感染,對已產(chǎn)生耐藥的藥物應(yīng)暫停使用,避免造成更嚴(yán)重的耐藥后果。為進一步探究我國不同地區(qū)不動桿菌的遺傳多態(tài)性及親緣關(guān)系,本研究利用脈沖場凝膠電泳(PFGE)方法,對292株不動桿菌屬細(xì)菌進行分子分型分析,顯示出較好的分型性、分辨力和重復(fù)性,能夠?qū)⒉≡w的流行病學(xué)相關(guān)性準(zhǔn)確地反映出來。使用Bio Numeries軟件對電泳成功的267株不動桿菌屬細(xì)菌進行聚類分析,按63%的cutoff值,可分成52個聚類群、共170種PFGE型別,相似性介于25%~100%之間。以上表明,我國不動桿菌屬細(xì)菌具有較高的遺傳多態(tài)性,存在多種不同型別的不動桿菌屬細(xì)菌的流行。其次,有一組數(shù)據(jù)顯示沈陽1株菌和甘肅1株菌的PFGE帶型相同,另一組數(shù)據(jù)顯示沈陽2株、甘肅4株、蘇州1株菌共7株菌的PFGE帶型相同,可見,在不同地區(qū)存在同一PFGE型別的菌株,可能是同一克隆不動桿菌屬細(xì)菌在不同地區(qū)之間的傳播。通過實驗結(jié)果發(fā)現(xiàn),甘肅地區(qū)醫(yī)院的ICU病房有9株菌PFGE帶型相同,同時ICU病房發(fā)現(xiàn)的9株菌與該醫(yī)院呼吸內(nèi)科分離的2株菌PFGE帶型形相同。以上表明,在醫(yī)院同科室內(nèi)流行的不動桿菌屬細(xì)菌存在著相同帶型,并且可以通過交叉感染的方式流行于不同病房之間。目前產(chǎn)NDM耐藥菌已經(jīng)在全球范圍內(nèi)播散,成為嚴(yán)重威脅公共健康的“超級細(xì)菌”。為了解目前我國不動桿菌屬細(xì)菌產(chǎn)NDM菌株的流行情況,本研究利用PCR擴增方法對實驗室在2010年至2014年期間收集自廈門、北京、廣州、南京、濟南、沈陽、蘇州、甘肅等8個地區(qū)的共572株不動桿菌屬細(xì)菌進行了bla NDM基因篩查,結(jié)果發(fā)現(xiàn)有45株菌為bla NDM陽性,占總數(shù)的8%,其中42株為產(chǎn)NDM-1菌株,北京地區(qū)產(chǎn)NDM-1陽性率最高,其中有7株產(chǎn)NDM-1不動桿菌分離自醫(yī)院污水。此外,蘇州地區(qū)還檢出1株菌為NDM-5陽性。以往報道NDM-5多被檢出于大腸桿菌中,在鮑曼不動桿菌中檢出的報道還很少。特別值得關(guān)注的是,本實驗還發(fā)現(xiàn)了2株鮑曼不動桿菌可能攜帶新型的bla NDM序列,其分別來自于廈門和北京,通過序列比對發(fā)現(xiàn)其第290位堿基由A變成C,導(dǎo)致其97位氨基酸由CAG變成CCG,即由谷氨酰胺(Gln/Q)變?yōu)楦彼?Pro/P)。從PFGE聚類圖來看,這兩株菌存在差異,其序列還有待于進一步驗證確定。選擇23株菌通過S1核酸酶-PFGE-Southern blot雜交法對bla NDM基因進行定位,發(fā)現(xiàn)有12株菌的bla NDM-1基因存在于質(zhì)粒上,且大小相同,約為30-50kb。此外,本研究還對我中心實驗室分離的國內(nèi)首株產(chǎn)NDM-1不動桿菌XM1570進行了全基因組序列分析。我們發(fā)現(xiàn)XM1570序列與之前報道的A.calcoaceticus PHEA-2相近,并且其質(zhì)粒p XM1上的bla NDM-1基因位于復(fù)合轉(zhuǎn)座子Tn125內(nèi),該轉(zhuǎn)座子包含兩個插入序列ISAba125,分別位于bla NDM-1基因的上下游,且兩者存在兩個堿基的差異。Tn125經(jīng)常與aph A6基因同時存在于質(zhì)粒上,并位于aph A6基因的下游。質(zhì)粒序列與目前國內(nèi)外報道的攜帶bla NDM-1基因質(zhì)粒序列相似性高。通過以上分析本研究獲得了以下有意義的結(jié)果:1、根據(jù)藥敏結(jié)果指導(dǎo)臨床用藥,制定合理的、有效的治療方案;2、合理使用抗生素,有效降低多重耐藥不動桿菌產(chǎn)生的幾率。3、應(yīng)用PFGE方法有助于發(fā)現(xiàn)菌株之間的遺傳關(guān)系,找到傳染源及時對其進行控制,防止不動桿菌引起院內(nèi)感染的流行。4、北京地區(qū)NDM-1的菌株在醫(yī)院污水內(nèi)檢出率高,醫(yī)院應(yīng)注意合理處置污水。5、發(fā)現(xiàn)了NDM新序列,暫定為NDM-15,NDM在我國流行過程中可能發(fā)生了變異,值得引起重視。6、我國流行的攜帶bla NDM-1基因的質(zhì)粒多定位于細(xì)菌的質(zhì)粒上。中國內(nèi)首株產(chǎn)NDM-1不動桿菌的bla NDM-1基因定位于質(zhì)粒上,并且該質(zhì)粒具有較高的轉(zhuǎn)移性,bla NDM-1基因極有可能通過接合作用擴散到其他強致病性的病原菌中,應(yīng)加強臨床產(chǎn)NDM細(xì)菌的監(jiān)測。
[Abstract]:Acinetobacter spp. ) The non-fermentation condition pathogenic bacteria, usually cause the infection when the body resistance is reduced, is one of the important opportunistic pathogens of the in-hospital infection, wherein the vitality of the Acinetobacter baumannii is particularly strong, and can be widely distributed in the hospital environment. In recent years, with the extensive application of broad-spectrum antibiotics, the multiple drug-resistant and super-drug-resistant Acinetobacter baumannii, which are resistant to most of the antibiotics, frequently cause the outbreak of in-hospital infection and bring great challenge to clinical treatment. In particular, with the emergence and prevalence of the NDM-1 super-resistant bacteria, the problem of the abuse of antibiotics and the drug resistance in the world have been greatly concerned. Based on the pathogen monitoring platform of the army infectious disease, in the period from 2010 to 2014, the laboratory collected 300 suspected Acinetobacter bacteria from the clinical patients and the hospital environment in different regions such as Beijing, Shenyang, Suzhou, Gansu, Xiamen, Nanjing and the like in China. The results showed that Acinetobacter baumannii was a bacterial 292 strain, including 251 strains of Acinetobacter baumannii,19 strains of Acinetobacter,19 strains of Acinetobacter,19 of Acinetobacter,3 strains of Acinetobacter,117 in Suzhou,117 in Shenyang and 33 in Gansu. 12 of Beijing,2 in Jinan,3 in Xiamen,4 in Guangzhou and 4 in Nanjing. In order to understand the drug-resistant characteristics of Acinetobacter spp., this study conducted the antibiotic sensitivity test for 226 bacteria in the laboratory. The results showed that the bacteria of Acinetobacter sp. from the three regions of Beijing, Shenyang and Suzhou had different degrees of drug resistance to the 8 kinds of drug of Acinetobacter baumannii. The resistance rate of ceftriaxone was 96%, norfloxacin/ cefixime 92%, imipenem 88%, ceftrione 87%, methicillin 84%, ticarcillin/ clavulanic acid 83%, and amikacin 77% from high to low. In addition,211 strains of multiple drug-resistant Acinetobacter, which accounted for 97% of the test strains,7 of Beijing,3% of the total,94 of Shenyang,45% of the total, and 110 in Suzhou, accounting for 52% of the total. Among these multiple drug-resistant Acinetobacter spp.,150 strains were all resistant to more than 5 antibiotics, including 57 of Shenyang and 93 in Suzhou. The above results show that the drug resistance of Acinetobacter is very serious in our hospital. It can also be found that the sensitivity of these drug-resistant Acinetobacter to the inner-aminidase inhibitor and the amino sugar-containing drug is also high. Therefore, it is clinically possible to consider the use of the above two types of drugs for the treatment of infections caused by the Acinetobacter, and the use of the drug that has been resistant to drug resistance should be suspended so as to avoid more serious drug-resistance consequences. In order to further explore the genetic polymorphism and the relationship of Acinetobacter baumannii in different regions of China, the molecular typing of 292 Acinetobacter sp. was carried out by using a pulse-field gel electrophoresis (PFGE) method to show good parting, resolution and repeatability. The epidemiological relevance of the pathogen can be accurately reflected. The results of cluster analysis of 267 strains of Acinetobacter, which were successful in electrophoresis using the Bio-Nueries software, can be divided into 52 groups according to the coutoff value of 63%. There are 170 PFGE types, and the similarity is between 25% and 100%. The above results show that the Acinetobacter sp. has a high genetic polymorphism, and there are many different types of Acinetobacter spp. Secondly, there was a group of data showing that the PFGE banding patterns of the 1 strains of Shenyang and 1 of the strains in Gansu were the same, and the other group of data showed that the PFGE banding patterns of 7 strains in Shenyang,4 and 1 of Suzhou were the same, and the same PFGE-type strains were found in different regions. The transmission of the same clone of Acinetobacter spp. in different regions may be the same. The results of the experiment show that there are 9 strains of PFGE in the ICU of the hospital in Gansu, and the 9 strains found in the intensive care unit are the same as those of the two strains of PFGE isolated from the respiratory department of the hospital. The above shows that there are the same banding patterns of the Acinetobacter species prevalent in the hospital and in the department, and can be popular among different wards by cross-infection. At present, the NDM-resistant bacteria have been spread worldwide and become a "super-bacterium which is a serious threat to public health". In order to understand the current situation of the NDM strain of Acinetobacter spp., the present study collected from Xiamen, Beijing, Guangzhou, Nanjing, Jinan, Shenyang and Suzhou by PCR amplification method from 2010 to 2014. The results showed that 45 strains were bla NDM positive and 8% of the total,42 of which were NDM-1 strains, and the NDM-1 in Beijing area was the highest, among which 7 strains of NDM-1 Acinetobacter were isolated from the hospital. In addition,1 strain of NDM-5 was detected in Suzhou. In the past, NDM-5 was detected in E. coli, and there were few reports in Acinetobacter baumannii. It is of particular interest to be concerned that 2 strains of Acinetobacter baumannii may carry a novel bla NDM sequence from Xiamen and Beijing, respectively, by a sequence comparison to the discovery that its position 290 base is changed from A to C, causing the 97-position amino acid to be changed from CAG to CCG, That is, Glutinamide (Gln/ Q) becomes proline (Pro/ P). From the PFGE cluster, there is a difference in the two strains, and the sequence of the two strains is still to be further verified and determined. The results showed that the bla NDM-1 gene of 12 strains was present on the plasmid and the size was the same, about 30-50 kb. In addition, the whole genome sequence analysis of NDM-1 Acinetobacter XM1570 from the domestic first strain isolated from the central laboratory was carried out. We found that the XM1570 sequence is similar to the previously reported A. calcoaceticus PHEA-2, and the bla NDM-1 gene on its plasmid p XM1 is located in the compound transposon Tn125, which contains two insertion sequences, ISAba125, located upstream and downstream of the bla NDM-1 gene, respectively, and there are two base differences. The Tn125 is often present on the plasmid at the same time as the aph A6 gene and is located downstream of the aph A6 gene. The sequence of the plasmid is similar to that of the plasmid sequence carrying the bla NDM-1 gene, which is reported both at home and abroad. Through the above analysis, the following results are obtained:1, according to the drug sensitivity result, the clinical medication is guided, a reasonable and effective treatment scheme is established;2, antibiotics are reasonably used, and the probability of multiple drug-resistant Acinetobacter can be effectively reduced. The application of the PFGE method can help to find the genetic relationship among the strains, to find the source of the infection to control it in time, to prevent the infection in the hospital by the Acinetobacter.4. The strain of the NDM-1 in the Beijing area is high in the hospital sewage, and the hospital should pay attention to the reasonable treatment of the sewage. NDM-15 and NDM were identified as NDM-15 and NDM in China. The bla NDM-1 gene of NDM-1 Acinetobacter, which is produced by the first plant in China, is located on the plasmid, and the plasmid has higher metastatic, and the bla NDM-1 gene is highly likely to spread to other highly pathogenic pathogenic bacteria through the joint action, and the monitoring of the clinical NDM bacteria should be enhanced.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.5

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