我国不动杆菌的耐药性、遗传多态性及产NDM耐药菌研究

发布时间：2019-05-21 23:49

【摘要】：不动杆菌属(Acinetobacter spp.)是非发酵条件致病菌,通常在机体抵抗力降低时引起感染,是院内感染的重要机会致病菌之一,其中鲍曼不动杆菌生命力尤其顽强,可广泛分布于医院环境之中。近年来,随着临床上广谱抗生素的大量应用,出现了对大部分抗生素耐药的多重耐药及超耐药鲍曼不动杆菌,并时常造成院内感染的暴发流行,给临床治疗带来极大挑战。特别是,随着产NDM-1超耐药菌的出现与流行,全球已对抗生素滥用问题及细菌耐药问题产生极大关注。本研究基于军队传染病病原监测平台,2010~2014年间,本实验室在我国北京、沈阳、苏州、甘肃、厦门、南京等不同地区,收集300株来自临床病人、医院环境的疑似不动杆菌属细菌,利用16S r RNA测序技术对其进行检测,共确认不动杆菌均属细菌292株,其中鲍曼不动杆菌251株、醋酸钙不动杆菌19株、琼氏不动杆菌19株、洛菲不动杆菌3株,苏州地区分离117株、沈阳117株、甘肃33株、北京12株、济南2株,厦门3株、广州4株、南京4株。为了解我国不动杆菌属细菌的耐药特点,本研究对实验室现有的226株细菌进行了抗生素敏感性试验。药敏结果显示,来自北京、沈阳、苏州3个地区的不动杆菌属细菌,对临床上8种首选治疗鲍曼不动杆菌感染药物产生了不同程度的耐药。耐药率从高到低依次为头孢他啶96%、诺氟沙星/头孢吡肟92%、亚胺培南88%、头孢哌酮87%、哌拉西林84%、替卡西林/克拉维酸83%、阿米卡星77%。此外,多重耐药不动杆菌共有211株、占测试菌株的97%,其中北京7株、占总数的3%,沈阳94株、占总数的45%,苏州110株、占总数的52%。在这些多重耐药不动杆菌属细菌中,有150株菌对5类以上抗生素全部耐药,其中沈阳57株、苏州93株。以上结果表明,我国医院内流行的不动杆菌属细菌耐药情况目前十分严重。同时还可以发现,这些耐药不动杆菌对内酰胺酶抑制剂和氨基糖苷类药物的敏感性也比较高。所以,在临床上可以考虑使用以上两类药物治疗由不动杆菌属细菌引起的感染,对已产生耐药的药物应暂停使用,避免造成更严重的耐药后果。为进一步探究我国不同地区不动杆菌的遗传多态性及亲缘关系,本研究利用脉冲场凝胶电泳(PFGE)方法,对292株不动杆菌属细菌进行分子分型分析,显示出较好的分型性、分辨力和重复性,能够将病原体的流行病学相关性准确地反映出来。使用Bio Numeries软件对电泳成功的267株不动杆菌属细菌进行聚类分析,按63%的cutoff值,可分成52个聚类群、共170种PFGE型别,相似性介于25%~100%之间。以上表明,我国不动杆菌属细菌具有较高的遗传多态性,存在多种不同型别的不动杆菌属细菌的流行。其次,有一组数据显示沈阳1株菌和甘肃1株菌的PFGE带型相同,另一组数据显示沈阳2株、甘肃4株、苏州1株菌共7株菌的PFGE带型相同,可见,在不同地区存在同一PFGE型别的菌株,可能是同一克隆不动杆菌属细菌在不同地区之间的传播。通过实验结果发现,甘肃地区医院的ICU病房有9株菌PFGE带型相同,同时ICU病房发现的9株菌与该医院呼吸内科分离的2株菌PFGE带型形相同。以上表明,在医院同科室内流行的不动杆菌属细菌存在着相同带型,并且可以通过交叉感染的方式流行于不同病房之间。目前产NDM耐药菌已经在全球范围内播散,成为严重威胁公共健康的“超级细菌”。为了解目前我国不动杆菌属细菌产NDM菌株的流行情况,本研究利用PCR扩增方法对实验室在2010年至2014年期间收集自厦门、北京、广州、南京、济南、沈阳、苏州、甘肃等8个地区的共572株不动杆菌属细菌进行了bla NDM基因筛查,结果发现有45株菌为bla NDM阳性,占总数的8%,其中42株为产NDM-1菌株,北京地区产NDM-1阳性率最高,其中有7株产NDM-1不动杆菌分离自医院污水。此外,苏州地区还检出1株菌为NDM-5阳性。以往报道NDM-5多被检出于大肠杆菌中,在鲍曼不动杆菌中检出的报道还很少。特别值得关注的是,本实验还发现了2株鲍曼不动杆菌可能携带新型的bla NDM序列,其分别来自于厦门和北京,通过序列比对发现其第290位碱基由A变成C,导致其97位氨基酸由CAG变成CCG,即由谷氨酰胺(Gln/Q)变为脯氨酸(Pro/P)。从PFGE聚类图来看,这两株菌存在差异,其序列还有待于进一步验证确定。选择23株菌通过S1核酸酶-PFGE-Southern blot杂交法对bla NDM基因进行定位,发现有12株菌的bla NDM-1基因存在于质粒上,且大小相同,约为30-50kb。此外,本研究还对我中心实验室分离的国内首株产NDM-1不动杆菌XM1570进行了全基因组序列分析。我们发现XM1570序列与之前报道的A.calcoaceticus PHEA-2相近,并且其质粒p XM1上的bla NDM-1基因位于复合转座子Tn125内,该转座子包含两个插入序列ISAba125,分别位于bla NDM-1基因的上下游,且两者存在两个碱基的差异。Tn125经常与aph A6基因同时存在于质粒上,并位于aph A6基因的下游。质粒序列与目前国内外报道的携带bla NDM-1基因质粒序列相似性高。通过以上分析本研究获得了以下有意义的结果:1、根据药敏结果指导临床用药,制定合理的、有效的治疗方案;2、合理使用抗生素,有效降低多重耐药不动杆菌产生的几率。3、应用PFGE方法有助于发现菌株之间的遗传关系,找到传染源及时对其进行控制,防止不动杆菌引起院内感染的流行。4、北京地区NDM-1的菌株在医院污水内检出率高,医院应注意合理处置污水。5、发现了NDM新序列,暂定为NDM-15,NDM在我国流行过程中可能发生了变异,值得引起重视。6、我国流行的携带bla NDM-1基因的质粒多定位于细菌的质粒上。中国内首株产NDM-1不动杆菌的bla NDM-1基因定位于质粒上,并且该质粒具有较高的转移性,bla NDM-1基因极有可能通过接合作用扩散到其他强致病性的病原菌中,应加强临床产NDM细菌的监测。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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