【摘要】：抗生素在水產(chǎn)養(yǎng)殖中有著不可取代的作用,但抗生素的長期濫用導(dǎo)致的細(xì)菌耐藥性問題愈發(fā)引起人們的關(guān)注�？股乜剐曰�(antibiotic resistance genes,ARGs)是細(xì)菌耐藥性傳播擴(kuò)散的關(guān)鍵因子。它可以通過可移動(dòng)遺傳元件在細(xì)菌間傳播,進(jìn)而會(huì)對(duì)食品安全和人類健康產(chǎn)生嚴(yán)重威脅。然而,目前關(guān)于ARGs的污染特征研究多集中于水產(chǎn)養(yǎng)殖的養(yǎng)殖用水和底泥中,水產(chǎn)品作為進(jìn)入人體的直接載體,水產(chǎn)品中的致病菌的耐藥性會(huì)對(duì)人體產(chǎn)生直接影響。然而,目前水產(chǎn)品批發(fā)市場(chǎng)中水產(chǎn)品中ARGs的污染特征卻并不清楚。因此,為了更清楚的了解水產(chǎn)品中ARGs的污染特征,更好的解決食源性致病菌的耐藥問題和臨床用藥問題,本研究做了以下3部分工作:(1)上海市水產(chǎn)品批發(fā)市場(chǎng)排污溝淤泥中抗生素抗性基因的污染分析;(2)中國東南沿海地區(qū)水產(chǎn)品中抗生素抗性基因的特征及與抗生素殘留的關(guān)聯(lián)分析;(3)副溶血性弧菌鏈霉素耐藥表型和基因型的不對(duì)應(yīng)性原因分析。(1)上海市水產(chǎn)品批發(fā)市場(chǎng)排污溝淤泥中抗生素抗性基因的污染分析為了探究待售水產(chǎn)品中抗生素抗性基因(Antibiotic resistance genes,ARGs)在水產(chǎn)品批發(fā)市場(chǎng)中的擴(kuò)散污染情況,選取上海市3個(gè)大型水產(chǎn)品批發(fā)市場(chǎng)作為研究對(duì)象,采用PCR的方法,對(duì)市場(chǎng)內(nèi)部和市場(chǎng)附近的排污溝表層淤泥中5大類(4種磺胺類、8種四環(huán)素類、6種β-內(nèi)酰胺類、5種氯霉素類、4種鏈霉素類)共27種ARGs進(jìn)行了檢測(cè),并利用PCR-DGGE技術(shù)分析了細(xì)菌種群多樣性。結(jié)果顯示共有17種ARGs在批發(fā)市場(chǎng)淤泥中被檢出,其中磺胺類、四環(huán)素類和鏈霉素類ARGs的檢出率較高且檢出種類較多,表明水產(chǎn)品批發(fā)市場(chǎng)含有種類豐富的ARGs,水產(chǎn)品以磺胺類、四環(huán)素類和鏈霉素類ARGs污染為主。對(duì)市場(chǎng)外圍的檢測(cè)顯示市場(chǎng)外圍區(qū)域ARGs的種類分布略低于市場(chǎng)內(nèi)部,表明水產(chǎn)品批發(fā)市場(chǎng)中ARGs的存在會(huì)通過污水排放途徑擴(kuò)散到周邊環(huán)境。DGGE圖譜的分析結(jié)果顯示,3個(gè)批發(fā)市場(chǎng)污泥樣品細(xì)菌種類差異性較大,表明環(huán)境中ARGs的種類分布與細(xì)菌的種類并無直接關(guān)聯(lián)。本研究首次通過調(diào)查水產(chǎn)品批發(fā)市場(chǎng)中飯排污溝淤泥中ARGs的污染證實(shí)水產(chǎn)品批發(fā)市場(chǎng)已經(jīng)成為ARGs的重要儲(chǔ)存庫,以此造成的污染會(huì)進(jìn)一步傳播到周邊環(huán)境,此外,這些種類豐富的ARGs重新擴(kuò)散到待售水產(chǎn)品中的風(fēng)險(xiǎn)很大,需引起人們足夠的重視。(2)中國東南沿海地區(qū)水產(chǎn)品中抗生素抗性基因的特征及與抗生素殘留的關(guān)聯(lián)分析抗生素抗性基因(Antibiotic resistance genes,ARGs)作為一種新型環(huán)境污染物,對(duì)人體健康構(gòu)成了潛在的威脅。本研究通過PCR和q PCR定性定量檢測(cè)94個(gè)水產(chǎn)品(其中18個(gè)為抗生素檢測(cè)陽性)魚皮上37種抗性基因的污染特征。此外,本研究分離出10株副溶血性弧菌,以驗(yàn)證水產(chǎn)品中副溶血性弧菌ARGs的存在性。結(jié)果顯示,相比與海水魚,淡水魚中含有的ARGs的種類更多,豐度更高。tet S、str A、str B、aad A、flo R、sul I和sul II被認(rèn)為是中國東南沿海地區(qū)水產(chǎn)品中的主導(dǎo)基因,它們的檢出率均大于30%并且在五個(gè)城市中均有檢出。分離的副溶血性弧菌抗性基因檢出了水產(chǎn)品中這7個(gè)主導(dǎo)基因,表明這7大ARGs在副溶血性弧菌中也已普遍存在�？股貦z測(cè)陽性的水產(chǎn)品中含有的ARGs明顯高于其他水產(chǎn)品,表明抗生素的使用不僅僅可能會(huì)誘導(dǎo)該種類下的抗性基因,還可能對(duì)誘導(dǎo)其他類ARGs的產(chǎn)生。本次研究并無磺胺類抗生素檢出,表明在沒有磺胺類抗生素的選擇性壓力下,sul I和sul II在水產(chǎn)品中已普遍存在。本研究表明淡水魚已成為ARGs的重要儲(chǔ)存庫,抗生素的使用可能不僅僅誘導(dǎo)該種類下的抗性基因,還可能誘導(dǎo)其他類ARGs的產(chǎn)生,需引起人們足夠的重視。本研究首次監(jiān)控了東南沿海地區(qū)水產(chǎn)品中ARGs的污染特征,為東南沿海地區(qū)水產(chǎn)品及水產(chǎn)品致病菌中抗性基因提供數(shù)據(jù)支持,為臨床醫(yī)療診斷提供基礎(chǔ)數(shù)據(jù)。(3)副溶血性弧菌鏈霉素耐藥表型和基因型的不對(duì)應(yīng)性分析近些年來,由于抗生素的濫用,細(xì)菌耐藥性帶來的臨床治療難題引起人們愈發(fā)的關(guān)注。本課題組之前的調(diào)查發(fā)現(xiàn),副溶血性弧菌耐藥表型和對(duì)應(yīng)的基因型并不一致,具體表現(xiàn)為有些副溶血性弧菌攜帶有抗性基因,而藥敏實(shí)驗(yàn)表現(xiàn)為對(duì)抗生素敏感。結(jié)合水產(chǎn)品中ARGs污染的主導(dǎo)基因和本課題組副溶血性弧菌的耐藥情況,選取抗鏈霉素類的aad A、sul I和sul II作為目標(biāo)基因。實(shí)驗(yàn)材料選取課題組10株攜帶有aad A卻對(duì)鏈霉素敏感的副溶血性弧菌菌株以及46株含有sul I或sul II卻對(duì)復(fù)方新諾明表現(xiàn)為敏感的菌株。結(jié)果發(fā)現(xiàn),副溶血性弧菌aad A基因有包括3大類8種多種突變形式。而sulⅠ突變主要發(fā)生在231位點(diǎn)缺失A,sulⅡ主要發(fā)生的是106位點(diǎn)缺失A。這些突變都有可能導(dǎo)致基因不能正常編碼。本實(shí)驗(yàn)表明在抗性基因(ARGs)擴(kuò)散傳播的過程中,由于各種原因造成的抗性基因突變?nèi)缛笔�、重�?fù)、移碼、插入等可能是造成細(xì)菌耐藥菌株表型缺失的一個(gè)重要原因。本實(shí)驗(yàn)首次通過分析抗性基因序列中堿基突變的方法闡述副溶血性弧菌耐藥表型和基因型不一致現(xiàn)象背后的原因。
[Abstract]:Antibiotics play an irreplaceable role in aquaculture, but the long-term abuse of antibiotics leads to the problem of bacterial resistance. Antibiotic resistance genes (ARGs) are a key factor in the spread of bacterial resistance. However, the current study on the pollution characteristics of ARGs is mostly concentrated in aquaculture water and sediment. Aquatic products as a direct carrier into the human body, the drug resistance of pathogenic bacteria in aquatic products will have a direct impact on the human body. However, the current wholesale market of aquatic products. The contamination characteristics of ARGs in aquatic products are not clear. Therefore, in order to better understand the contamination characteristics of ARGs in aquatic products, and to better solve the problem of drug resistance of foodborne pathogens and clinical drug use, the following three parts of work were done in this study: (1) The contamination fraction of antibiotic resistance genes in sewage silt of Shanghai aquatic products wholesale market. Analysis; (2) Characteristics of antibiotic resistance genes in aquatic products from southeastern coastal areas of China and their association with antibiotic residues; (3) Analysis of phenotype and genotype of streptomycin resistance of Vibrio parahaemolyticus. (1) Pollution analysis of antibiotic resistance genes in sewage sludge of Shanghai aquatic products wholesale market The spread of antibiotic resistance genes (ARGs) in aquatic products wholesale markets was investigated. Three large-scale aquatic products wholesale markets in Shanghai were selected as the research objects. PCR was used to detect 5 major types (4 sulfonamides, 8 tetracyclines, 6 tetracyclines) in the surface mud of sewage drains in and near the market. 27 kinds of ARGs including beta-lactams, 5 kinds of Chloramphenicols and 4 kinds of streptomycins were detected, and the diversity of bacterial population was analyzed by PCR-DGGE. The results showed that 17 kinds of ARGs were detected in the sludge of wholesale market, among which sulfonamides, tetracyclines and streptomycins were detected more frequently, indicating that there were many kinds of ARGs in aquatic products. The wholesale market contains abundant ARGs, and the aquatic products are mainly polluted by sulfonamides, tetracyclines and streptomycins. The detection of the periphery of the market shows that the distribution of ARGs in the periphery of the market is slightly lower than that in the interior of the market. The results showed that the bacterial species of sludge samples from the three wholesale markets were different, indicating that the distribution of ARGs in the environment was not directly related to the bacterial species. In addition, the risk of these ARGs reappearing in aquatic products for sale is very high, which needs to be paid enough attention. (2) Characteristics of antibiotic resistance genes in aquatic products in southeastern coastal areas of China and their association with antibiotic residues Resistance genes, ARGs, as a new type of environmental pollutants, pose a potential threat to human health. In this study, 37 resistance genes in 94 aquatic products (18 of which were positive for antibiotics) were detected qualitatively and quantitatively by PCR and q-PCR. In addition, 10 strains of Vibrio parahaemolyticus were isolated to verify the contamination characteristics of aquatic products. The results showed that freshwater fish contained more ARGs than seawater fish. Tet S, str A, str B, aad A, flo R, Sul I and sul II were considered to be the dominant genes in aquatic products in southeastern coastal areas of China. The detection rates of ARGs in freshwater fish were more than 30% and were detected in five cities. Seven major ARGs were found in aquatic products, indicating that the seven major ARGs were also widespread in Vibrio parahaemolyticus. Antibiotic-positive aquatic products contained significantly higher ARGs than other aquatic products, suggesting that the use of antibiotics may not only induce resistance genes in this species. No sulfonamide antibiotics were detected in this study, suggesting that Sul I and sul II were common in aquatic products without the selective pressure of sulfonamide antibiotics. This study monitored the pollution characteristics of ARGs in aquatic products in southeastern coastal areas for the first time, providing data support for the resistance genes in pathogenic bacteria of aquatic products and aquatic products in southeastern coastal areas, and providing basic data for clinical diagnosis. (3) Vibrio parahaemolyticus In recent years, due to the abuse of antibiotics, the problem of clinical treatment caused by bacterial drug resistance has attracted more and more attention. Previous investigations by our team found that the phenotype and genotype of V. parahaemolyticus were not consistent, and some V. parahaemolyticus were specific. Combining the dominant gene of ARGs contamination in aquatic products and the drug resistance of Vibrio parahaemolyticus in our research group, we selected aad-A, sul-I and sul-II as the target genes. Vibrio parahaemolyticus strains and 46 strains containing Sul I or Sul II but susceptible to compound trimethoprim were found. The results showed that the AAD A gene of Vibrio parahaemolyticus had three major classes and eight kinds of mutations. The Sul I mutation mainly occurred at 231 locus deletion A, and the Sul II mutation mainly occurred at 106 locus deletion A. This experiment shows that the phenotypic deletion of resistant strains may be caused by mutations such as deletion, repetition, code shifting and insertion in the diffusion of resistant genes (ARGs). The reasons behind the drug resistance phenotypes and genotype incompatibility of Vibrio parahaemolyticus are discussed.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS254.1;TS201.6

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