近年来,抗生素在人类医疗以及养殖业方面滥用情况越发严重,越来越多的抗生素进入环境已成为不争的事实。环境中抗生素的存在除了导致化学药物污染外,更诱导细菌产生的抗生素抗性基因(Antibiotic Resistance Genes,ARGs)污染,并由此可能引发的生态问题亦令人关注。因为抗生素在环境中浓度的增长所带来的抗生素选择性压力的加大,将加速抗生素耐药性细菌以及ARGs在环境中的出现和传播。同时,也增加了人类致病菌获得ARGs、对抗生素产生抗性的几率。因此,ARGs的存在及传播将严重威胁人类的健康。 当ARGs存在于基因转移单位时,将可以通过水平基因转移(Horizontal Gene Transfer, HGT)在不同的细菌或细菌种群中传播,从而使更多的微生物获得对抗生素的抗性;同时,由于基因转移单位具有自我复制功能,因此ARG可以一直存在于微生物种群中。进入自然水环境中的ARGs,更可在不同的环境介质中迁移,使得自然环境成为多种抗生素抗性基因的储库,即,ARGs在环境中的存在具有“持久性”和“可复制性”,被认为是一种新型的环境污染物(Emerging pollutants)。 珠三角地区为抗性素污染的重要地区。为研究ARGs在该地区的存在,本研究以北江为主要研究对象,于2009年12月,沿北江流域一共采集20个河水样品,考查磺胺及四环素抗性基因污染情况。获得了一些很有意义的研究结果: 1)通过含有磺胺二甲嘧啶(280 mg/L)或四环素(20 mg/L)的抗性培养基,研究北江水样中带有耐药性基因的细菌存在情况。20个样品中,17个样品检出了对磺胺二甲嘧啶有抗性的细菌,14个样品检出了对四环素有抗性的细菌。阴性对照均没有细菌生长,说明该方法可用于检测水环境中细菌耐药性。耐药性结果表明该地区抗生素抗性细菌的存在十分普遍,特别下游地区受耐药性细菌影响更加严重。 2)我们比较了两种水样DNA提取方法的优劣。采用水样DNA提取试剂盒的提取效率及提取的DNA样品纯度均比使用传统SDS-CTAB提取法高。我们选用水样DNA提取试剂盒提取DNA样品,供后续分子生物学实验使用。 3)利用聚合酶链反应(PCR)对北江水样细菌DNA研究发现,本研究使用的2种磺胺类抗性基因(sulI和sulII)在北江水样DNA的检出率高达85%以上;9种四环素抗性基因中除tetE和tetS之外,编码排出泵抗性机制(tetA、tetC、tetE、tetG)、编码修饰作用位点抗性机制(tetM、tetO、tetQ)、编码酶法使抗生素失活抗性机制(tetX)的抗性基因均有检出。所有样品中均含有多种抗生素抗性基因,其中tetA,tetC,tetG和tetO的检出率超过了70%; 4)SYBR Green I实时定量PCR方法对检出的抗生素抗性基因进行定量分析,以ARGs的拷贝数与16S rDNA的比值代表ARGs占样品DNA中的丰度。结果表明,对于四环素抗性基因,在所定量的19个样品中,tet C和tet A的基因拷贝数与16S rDNA的比值在0-1.32和0-1.37×10~(-1)范围内,大大高于其他tet基因在样品中的含量,是主要的tet抗性基因。北江水域中两种磺胺类抗性基因含量水平分别在0-4.16×10~(-2)及0-7.09×10~(-3)范围内。与美国科罗拉多州北部河流相比,北江水域的磺胺类抗性基因水平均要高得多,说明北江水域受抗生素抗性基因污染更为严重。 5)磺胺类抗生素抗性基因与四环素类抗生素抗性基因普遍存在于北江水环境中,但其分布并不一致。研究也发现,sul I的分布与磺胺类抗生素含量有一定的相关性,表明抗性素的污染是其抗性基因存在的重要因素。尽管河水中未能定量检出四环素,但其抗性基因却普遍存在。这反映了抗性基因的存在并不完全取决于环境抗生素的污染程度,而是由于抗性基因污染物的“持久性”或“可复制性”的特点所决定。

In recent years, the abuse of antibiotics in human medical treatment and aquaculture has become more and more serious, and it has become an indisputable fact that more and more antibiotics enter the environment. The presence of antibiotics in the environment not only leads to the pollution of chemical drugs, but also induces the pollution of Antibiotic Resistance Genes (ARGs) produced by bacteria, and the ecological problems that may be caused by this are also cause for concern. The increase in antibiotic selective pressure brought about by the increase in the concentration of antibiotics in the environment will accelerate the emergence and spread of antibiotic-resistant bacteria and ARGs in the environment. At the same time, it also increases the probability of human pathogenic bacteria acquiring ARGs and becoming resistant to antibiotics. Therefore, the existence and spread of ARGs will seriously threaten human health. When ARGs exist in the gene transfer unit, they can be spread among different bacteria or bacterial populations through Horizontal Gene Transfer (HGT), so that more microorganisms can gain resistance to antibiotics; at the same time, due to gene transfer The unit has the function of self-replication, so ARG can always exist in the microbial population. The ARGs that enter the natural water environment can also migrate in different environmental media, making the natural environment a reservoir of multiple antibiotic resistance genes, that is, the existence of ARGs in the environment is "persistent" and "reproducible" ", is considered a new type of environmental pollutants (Emerging pollutants). The Pearl River Delta region is an important area for resistance to pollution. In order to study the existence of ARGs in this area, this study took Beijiang as the main research object. In December 2009, a total of 20 river water samples were collected along the Beijiang basin to investigate the pollution of sulfa and tetracycline resistance genes. Some very meaningful research results have been obtained: 1) Through the resistance medium containing sulfamethazine (280 mg/L) or tetracycline (20 mg/L), the study of the drug resistance genes in Beijiang water samples The presence of bacteria. Among the 20 samples, 17 samples were detected with bacteria resistant to sulfamethazine, and 14 samples were detected with bacteria resistant to tetracycline. There was no bacterial growth in the negative control, indicating that this method can be used to detect bacterial resistance in the water environment. The results of drug resistance showed that the existence of antibiotic-resistant bacteria in this area is very common, especially downstream areas are more severely affected by drug-resistant bacteria. 2) We compared the pros and cons of the two DNA extraction methods from water samples. The extraction efficiency and the purity of the extracted DNA sample using the water sample DNA extraction kit are higher than that of the traditional SDS-CTAB extraction method. We use water DNA extraction kits to extract DNA samples for subsequent molecular biology experiments. 3) The use of polymerase chain reaction (PCR) to study the bacterial DNA of Beijiang water samples found that the detection rate of the two sulfonamide resistance genes (sulI and sulII) used in this study in the DNA of Beijiang water samples was as high as 85%; 9 In addition to tetE and tetS, the tetracycline resistance genes encode the resistance mechanism of the discharge pump (tetA, tetC, tetE, tetG), the resistance mechanism of the modified site (tetM, tetO, tetQ), and the enzyme method to make antibiotics. The resistance genes of the inactivated resistance mechanism (tetX) were all detected. All samples contain a variety of antibiotic resistance genes, among which the detection rate of tetA, tetC, tetG and tetO exceeds 70%; 4) SYBR Green I real-time quantitative PCR method quantitatively analyzes the detected antibiotic resistance genes, The ratio of the copy number of ARGs to 16S rDNA represents the abundance of ARGs in the sample DNA. The results showed that for the tetracycline resistance gene, in the quantified 19 samples, the ratio of the gene copy number of tet C and tet A to 16S rDNA was in the range of 0-1.32 and 0-1.37×10~(-1). Much higher than the content of other tet genes in the sample, it is the main tet resistance gene. The levels of two sulfonamide resistance genes in Beijiang waters were in the range of 0-4.16×10~(-2) and 0-7.09×10~(-3). Compared with the northern rivers of Colorado in the United States, the level of sulfonamide resistance genes in the waters of the North River is much higher, indicating that the waters of the North River are more polluted by antibiotic resistance genes. 5) Sulfa antibiotic resistance genes and tetracycline antibiotic resistance genes are common in Beijiang water environment, but their distribution is not consistent. Studies have also found that the distribution of sul I has a certain correlation with the content of sulfa antibiotics, indicating that the contamination of resistance genes is an important factor in the existence of resistance genes. Although tetracycline could not be detected quantitatively in river water, its resistance genes are widespread. This reflects that the existence of resistance genes does not entirely depend on the pollution degree of environmental antibiotics, but is determined by the characteristics of "persistence" or "reproducibility" of the pollutants of resistance genes.

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