本文選題：質(zhì)粒 + 抗生素��； 參考：《河南師范大學》2015年碩士論文

【摘要】：由于抗生素在禽畜養(yǎng)殖、臨床醫(yī)藥等方面的大量使用,近年來出現(xiàn)了大量的多重耐藥細菌,但是,多重耐藥菌產(chǎn)生的機制并不完全清楚。研究表明,質(zhì)粒經(jīng)常攜帶一些可移動的元件(如轉(zhuǎn)座子),捕捉不同的抗性基因介導抗生素抗性基因的擴散,這被認為是多重耐藥細菌產(chǎn)生的主要機制之一。本論文圍繞質(zhì)粒在環(huán)境抗生素抗性基因水平傳播中所起的作用進行以下幾方面的研究:一、雞糞中多重耐藥細菌的分離鑒定及抗性檢測。用含有慶大霉素、氯霉素、阿莫西林、頭孢氨芐、環(huán)丙沙星、四環(huán)素、紅霉素、卡那霉素這八種抗生素的培養(yǎng)基(LB培養(yǎng)基、SS培養(yǎng)基),我們從一規(guī)�；B(yǎng)雞場的雞糞中篩選出35株多重耐藥細菌。這些多重耐藥細菌分布在13個不同的屬,以革蘭氏陰性細菌為主導。在LB培養(yǎng)基中G-細菌與G+細菌分別占63%和37%,在SS培養(yǎng)基中G-細菌與G+細菌分別占87%和13%。這些細菌中對紅霉素具有耐藥性的最多,對阿莫西林具有耐藥性的最少,它們分布于變形菌門、放線菌門以及厚壁菌門�？股乜剐曰虺Ｅc重金屬抗性基因定位在同一個遺傳元件上,我們將沒有抗生素抗性的大腸桿菌與2株耐8種抗生素細菌進行對比實驗。結(jié)果表明,這些多重耐藥菌對Zn、Pb、Cu、Mn的抗性比沒有抗生素抗性的細菌強。二、多重耐藥細菌質(zhì)粒的檢測及遺傳特性研究。我們提取了各多抗菌的抗性質(zhì)粒并轉(zhuǎn)化入模式菌株DH5a中。進一步檢測發(fā)現(xiàn),這些多抗菌中均有一大小在15kb左右的質(zhì)粒被成功轉(zhuǎn)入DH5a中。攜帶該質(zhì)粒的DH5a可以在加有四環(huán)素、卡那霉素、頭孢氨芐的LB固體培養(yǎng)基上生長,這表明該質(zhì)粒上可能攜帶這三種抗生素抗性基因。測序結(jié)果顯示,該質(zhì)粒大小為14591bp,將其與NCBI中現(xiàn)有質(zhì)粒進行同源性對比分析,發(fā)現(xiàn)其與一植物雙元穿梭表達載體p ZGA22的相似度達到99%。通過生物信息學分析,我們發(fā)現(xiàn)其攜帶編碼四環(huán)素類(tet R_C、tet R_N)、磷酸轉(zhuǎn)移酶類(NTPII、NTPIII)抗性基因以及一個能同時控制多種抗生素抗性和重金屬抗性的超家族域Glo_EDI_BRP。這些結(jié)果表明,該質(zhì)粒很可能具有介導環(huán)境中多種耐藥基因(包括多種重金屬抗性基因)發(fā)生水平轉(zhuǎn)移的能力。三、質(zhì)粒的水平轉(zhuǎn)移情況研究。首先,我們對不同多抗菌中15Kb大小質(zhì)粒進行膠回收后酶切。酶切后的電泳圖譜顯示,這些質(zhì)粒很可能是同一個質(zhì)粒。接下來,我們根據(jù)該質(zhì)粒的全序列設計12對引物,對其進行PCR擴增測序與拼接。結(jié)果顯示,存在于干燥棒狀桿菌Corynebacterium xerosis、河流漫游球菌Vagococcus fluvialis、大腸桿菌Escherichia coli、變形桿菌Proteus sp.、小鱒魚大洋芽孢桿菌Oceanobacillus oncorhynchi、檸檬酸桿菌Citrobacter sp.中的15Kb大小質(zhì)粒確為同一質(zhì)粒。因此,這一15Kb大小質(zhì)粒是環(huán)境中介導抗生素抗性基因發(fā)生水平轉(zhuǎn)移的一個重要載體,它可能攜帶多種抗性基因在不同種屬細菌中進行水平轉(zhuǎn)移。
[Abstract]:Due to the extensive use of antibiotics in livestock breeding and clinical medicine, a large number of multidrug resistant bacteria have appeared in recent years, but the mechanism of multidrug resistance bacteria is not completely clear. Studies have shown that plasmids often carry some mobile elements (such as transposons) to capture different resistance genes to mediate the diffusion of antibiotic resistance genes, which is considered to be one of the main mechanisms for the production of multidrug resistant bacteria. In this paper, the role of plasmids in the horizontal transmission of environmental antibiotic resistance genes was studied in the following aspects: 1. Isolation, identification and detection of multidrug resistant bacteria in chicken manure. With gentamicin, chloramphenicol, amoxicillin, cefalexin, ciprofloxacin, tetracycline, erythromycin, Kanamycin (LB medium / SS medium) was used to isolate 35 strains of multidrug resistant bacteria from chicken dung of a large scale chicken farm. These multidrug resistant bacteria were distributed in 13 different genera, mainly Gram-negative bacteria. In LB medium, G- bacteria and G bacteria accounted for 63% and 37%, respectively. In SS medium, G- bacteria and G bacteria accounted for 87% and 13%, respectively. Among these bacteria, the most resistant to erythromycin and the least to amoxicillin are found in Proteus, actinomycetes and phylum thunbergiformis. Antibiotic resistance genes are often located on the same genetic element as heavy metal resistance genes. Two strains of Escherichia coli with no antibiotic resistance were compared with two strains of antibiotic resistant bacteria. The results showed that the resistance of these multidrug resistant bacteria to ZnPbCU mn was stronger than that of bacteria with no antibiotic resistance. Second, the detection and genetic characteristics of multidrug resistant bacteria plasmids. We extracted various antimicrobial resistant plasmids and transformed them into model strain DH 5a. Further detection showed that one of the plasmids about 15kb was successfully transferred into DH 5a. DH 5a carrying the plasmid could grow on LB solid medium containing tetracycline, kanamycin and cefalexin, which suggested that the plasmid might carry these three antibiotic resistance genes. The results of sequencing showed that the size of the plasmid was 14591bp.The homology analysis between the plasmid and the existing plasmid in NCBI showed that the similarity between the plasmid and a plant shuttle expression vector pZGA22 was 99%. By bioinformatics analysis, we found that it carries the tet (tet), phosphotransferases (NTPII), NTPIII resistance genes and a superfamily domain GloEDI _ (BRP), which can control both antibiotic resistance and heavy metal resistance. These results suggest that the plasmid may be capable of mediating horizontal transfer of many drug resistant genes (including multiple heavy metal resistance genes) in the environment. Third, the horizontal transfer of plasmids. Firstly, 15 KB plasmids of different antimicrobial species were recovered by gel and digested by enzyme. The electrophoretic patterns of the digested plasmids showed that the plasmids were probably the same plasmids. Then, we designed 12 pairs of primers according to the whole sequence of the plasmid, sequenced and sequenced them by PCR. The results showed that it existed in Corynebacterium xerosis, Vagococcus fluvialisi, Escherichia coli, Proteus sp., Oceanobacillus oncorhynchiand Citrobacter sp. The size of 15 kb plasmids was the same. Therefore, this 15Kb plasmid is an important vector that mediates the horizontal transfer of antibiotic resistance genes in the environment, and it may carry a variety of resistance genes for horizontal transfer in different species of bacteria.
【學位授予單位】：河南師范大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：X172;Q78

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