【摘要】：超級(jí)細(xì)菌的流行是當(dāng)前人類面臨的重大生存問(wèn)題。超級(jí)細(xì)菌的流行與抗生素抗性基因(ARGs,antibiotic resistance genes)通過(guò)的傳播與擴(kuò)散密切相關(guān)。目前對(duì)ARGs的研究主要集中在醫(yī)學(xué)領(lǐng)域(病原菌和醫(yī)院環(huán)境),而環(huán)境耐藥細(xì)菌的ARGs可通過(guò)質(zhì)粒等可移動(dòng)遺傳元件(MGEs,Mobile genetic elements)在環(huán)境細(xì)菌和人體病原菌之間傳播和擴(kuò)散,從而對(duì)人類健康和生態(tài)環(huán)境造成極大威脅,但目前對(duì)環(huán)境耐藥細(xì)菌的危害嚴(yán)重低估,認(rèn)識(shí)很少。質(zhì)粒除了攜帶ARGs外,多數(shù)重金屬抗性基因也主要定位于其中,ARGs和重金屬抗性基因在質(zhì)粒上的共分布可以導(dǎo)致重金屬對(duì)ARGs具有協(xié)同選擇作用。目前抗生素的濫用對(duì)耐藥細(xì)菌的選擇作用已成為微生物學(xué)研究的熱點(diǎn),但重金屬的使用對(duì)環(huán)境耐藥細(xì)菌及其攜帶ARGs的影響涉及較少。工業(yè)廢水的排放以及畜牧養(yǎng)殖業(yè)的廣泛使用導(dǎo)致水體和豬糞環(huán)境中重金屬污染嚴(yán)重,而這些環(huán)境系統(tǒng)與人類生活關(guān)系密切,因此有必要分析重金屬對(duì)這些環(huán)境中的耐藥細(xì)菌及其攜帶ARGs的選擇與遷移的影響及機(jī)制,為有效控制細(xì)菌耐藥性的轉(zhuǎn)移及超級(jí)細(xì)菌的蔓延提供理論基礎(chǔ)。為了研究重金屬對(duì)細(xì)菌耐藥性的影響,本研究具體分析了在重金屬脅迫條件下病原細(xì)菌,河水及豬糞環(huán)境細(xì)菌的類群、耐藥比例以及耐藥基因表達(dá)的情況,具體結(jié)果如下:1、經(jīng)重金屬銅(Cu)馴化得到的大腸埃希氏菌突變菌株對(duì)鹽酸四環(huán)素的最高耐受濃度提高了133%;銅綠假單胞菌突變株對(duì)鹽酸四環(huán)素最高耐受濃度提高了52%。經(jīng)重金屬鋅(Zn)馴化的金黃色葡萄球菌突變株對(duì)鹽酸四環(huán)素最高耐受濃度比出發(fā)菌提高了25%;銅綠假單胞菌突變株對(duì)鹽酸四環(huán)素最高耐受濃度提高了52%。2、通過(guò)重金屬Zn脅迫培養(yǎng)之后的河水樣本重金屬和抗生素抗性菌的數(shù)量和分離率均有顯著提高(p0.05),與對(duì)照組相比,Zn處理組Cu抗性菌分離率提高了0.9%;Zn抗性菌分離率提高了11.1%;鹽酸四環(huán)素抗性菌分離率提高了0.4%;氯霉素抗性菌分離率提高了4.6%;氨芐青霉素抗性菌分離率提高了2.3%;鏈霉素抗性菌的分離率提高了6.1%。3、通過(guò)高通量測(cè)序分析對(duì)照組(基礎(chǔ)飼料喂養(yǎng))和重金屬處理組(在基礎(chǔ)飼料中添加400mg/kg Cu和1000 mg/kg Zn)豬糞樣本細(xì)菌多樣性與物種豐度,重金屬處理組比對(duì)照組的豬糞菌群多樣性高。本研究分析了豐度前十的門、綱、目、科、屬各分類層級(jí)細(xì)菌群落:對(duì)照組的優(yōu)勢(shì)菌門為厚壁菌門(Firmicutes)和擬桿菌門(Bacteroidetes),分別占了55.7%和37.3%,而重金屬處理組的優(yōu)勢(shì)菌門為厚壁菌門(Firmicutes),占了92.12%。分析的豐度前十的菌屬中發(fā)現(xiàn),顫螺菌屬(Oscillospira)、梭菌屬(Clostridium)、SMB53、瘤胃球菌屬(Ruminococcus)、中間普氏菌(PrevoteLLa)、糞球菌屬(Coprococcus)等六個(gè)屬在對(duì)照組和重金屬處理組均有分布。而鏈球菌屬(Streptococcus)(6.78%)、密螺旋體屬(Treponema)(2.78%)、YRC22(1.94%)、巨球型菌屬(Megasphaera)(0.37%)僅在對(duì)照組分布。4、重金屬處理組的抗性菌數(shù)量和抗性菌分離率顯著高于對(duì)照組(P0.05)。重金屬抗性菌的分離率在重金屬處理組的比例為:Cu抗性菌29.58%,Zn抗性菌41.26%;在對(duì)照組的比例分別是:1.34%,1.52%。抗生素抗性菌在重金屬處理組的分離率為:Tet抗性菌33.00%,Chl抗性菌21.37%,Amp抗性菌24.37%,Str抗性菌25.37%;在對(duì)照組的比例分別是:2.84%,1.25%,8.79%,17.50%。銅藥協(xié)同抗性菌在重金屬處理組的分離率為15.11%;在對(duì)照組是0.82%。鋅藥協(xié)同抗性菌在重金屬處理組的分離率27.21%,在對(duì)照組是1.02%。5、通過(guò)qPCR檢測(cè)兩種不同養(yǎng)殖方式的豬場(chǎng)糞便核基因組DNA上的抗性基因(24個(gè)抗性基因)相對(duì)表達(dá)豐度,結(jié)果表明重金屬處理組抗性基因的表達(dá)多樣性和豐度明顯高于對(duì)照組:其中19個(gè)抗性基因在重金屬處理組樣本有表達(dá),而對(duì)照組樣本中有13個(gè)抗性基因擴(kuò)增成功,僅在重金屬處理組表達(dá)的基因有6個(gè)(str A、ampC、ermB、ermA、sul2和pcoA)。在兩組均表達(dá)的13個(gè)抗性基因中,有10個(gè)抗性基因(str B、mefA、sul1、tetX、tetQ、tetW、tetO、tetM、tetG、和zntA)在重金屬處理組的表達(dá)豐度明顯高于對(duì)照組(P0.05),而僅有3個(gè)抗生素抗性基因(ereA、ereB和tetA)在對(duì)照組的表達(dá)豐度高于重金屬處理組(P0.05)。6、規(guī)模化養(yǎng)豬場(chǎng)的豬糞樣本抗性菌具有較高的分離率。本研究從中分離到27株重金屬銅、鋅和抗生素協(xié)同抗性菌,對(duì)其進(jìn)行了分子鑒定,鑒定結(jié)果發(fā)現(xiàn)有7株為糞腸球菌(Enterococcus faecalis),7株為腸球菌屬(Enterococcus sp.),4株為變形菌屬(Proteus sp.),2株為摩根氏菌屬(Morganella sp.),2株為堅(jiān)強(qiáng)腸球菌(Enterococcus durans),2株為屎腸球菌(Enterococcus faecium),2株為克雷伯氏菌屬(KLebsiella sp.),1株為海氏腸球菌(Enterococcus hirae)。7、從規(guī)�；B(yǎng)殖場(chǎng)分離的共抗性菌株中提取到10個(gè)攜帶多種抗性基因的耐藥質(zhì)粒。攜帶的抗性基因PCR擴(kuò)增電泳圖顯示:質(zhì)粒P1攜帶8個(gè)耐藥基因(tetL、tetG、tetM、str A、strB、cmr、erm B、ere B),質(zhì)粒P10、P21、P26攜帶6個(gè)耐藥基因;P11、P12、P25、P27,P13則攜帶4個(gè)耐藥基因,而P3攜帶3個(gè)耐藥基因(tet L、tetM、erm B);具備鋅-藥共抗性基因的質(zhì)粒有P10、P11、P12、P13、P21、P25、P26、P27;具備銅-藥共抗性基因的質(zhì)粒有P1、P3、P10、P12、P13、P27。本論文研究結(jié)果表明:重金屬銅鋅對(duì)病原菌、河水環(huán)境細(xì)菌及豬糞環(huán)境細(xì)菌耐藥性均具有一定的協(xié)同選擇作用。飼料中重金屬銅、鋅添加劑的使用可通過(guò)協(xié)同選擇作用提高豬糞耐藥細(xì)菌的比例,以及耐藥基因的多樣性和表達(dá)豐度,同時(shí)還發(fā)現(xiàn)廣泛使用重金屬添加劑飼料的規(guī)�；B(yǎng)殖場(chǎng)糞便細(xì)菌的抗性菌分離率較高,并分離到攜帶多種抗性基因的耐藥質(zhì)粒,從而導(dǎo)致該環(huán)境的豬糞細(xì)菌具有較高的耐藥基因轉(zhuǎn)移與擴(kuò)散的風(fēng)險(xiǎn)。本課題的研究結(jié)果將為全面認(rèn)識(shí)重金屬在環(huán)境和養(yǎng)殖業(yè)的亂排和濫用對(duì)細(xì)菌耐藥的影響機(jī)制,進(jìn)一步控制耐藥基因在環(huán)境中的轉(zhuǎn)移與擴(kuò)散所導(dǎo)致的超級(jí)細(xì)菌蔓延具有重要意義。
[Abstract]:The prevalence of superbacteria is a major survival problem facing humans. The prevalence of superbacteria is closely related to the transmission and diffusion of antibiotic resistance genes (ARGs). The spread and diffusion of MGEs between environmental bacteria and human pathogenic bacteria pose a great threat to human health and ecological environment. However, the harm of MGEs to environmental drug-resistant bacteria is seriously underestimated and little is known. Among them, the co-distribution of ARGs and heavy metal resistance genes in plasmids can lead to the synergistic effect of heavy metals on ARGs. Nowadays, the selectivity of antibiotic abuse on drug-resistant bacteria has become a hotspot in microbiology. However, the use of heavy metals has less impact on environmental drug-resistant bacteria and ARGs-carrying bacteria. Emissions and widespread use of animal husbandry have led to serious heavy metal pollution in water and pig manure environments, which are closely related to human life. Therefore, it is necessary to analyze the effects and mechanisms of heavy metals on the selection and migration of drug-resistant bacteria in these environments and the ARGs-carrying bacteria in order to effectively control the transformation of bacterial resistance. In order to study the effect of heavy metals on bacterial resistance, this study analyzed the pathogenic bacteria, the groups of bacteria in river water and pig manure environment, the proportion of drug resistance and the expression of drug resistance genes under heavy metal stress. The specific results are as follows: 1. The highest tolerance concentration to tetracycline hydrochloride of Escherichia coli mutant strain increased by 133%; the highest tolerance concentration to tetracycline hydrochloride of Pseudomonas aeruginosa mutant strain increased by 52%; the highest tolerance concentration to tetracycline hydrochloride of Staphylococcus aureus mutant domesticated with heavy metal zinc (Zn) increased by 25%; the highest tolerance concentration to tetracycline hydrochloride of Pseudomonas The maximum tolerance concentration to tetracycline hydrochloride increased by 52.2%. The number and isolation rate of heavy metal and antibiotic resistant bacteria in river water samples cultured under heavy metal Zn stress were significantly increased (p0.05). Compared with the control group, the isolation rate of Cu-resistant bacteria increased by 0.9% and that of Zn-resistant bacteria increased by 11.1%. The isolation rate of resistant bacteria increased by 0.4%, the isolation rate of chloramphenicol resistant bacteria increased by 4.6%, the isolation rate of ampicillin resistant bacteria increased by 2.3%, the isolation rate of streptomycin resistant bacteria increased by 6.1%.3, and the control group (basal feed) and heavy metal treatment group (* 400mg/kg Cu and 1000 mg/kg Zn) were added to the basal diet by high throughput sequencing analysis. The bacterial diversity and species richness of fecal samples were higher in the heavy metal treatment group than in the control group. The bacterial communities of the first ten phyla, class, order, family and genus were analyzed in this study. The dominant phyla in the control group were Firmicutes and Bacteroidetes, accounting for 55.7% and 37.3% respectively, while the dominant phylum in the control group was Bacteroidetes. The dominant phylum in the treatment group was Firmicutes, accounting for 92.12%. Six genera including Oscillospira, Clostridium, SMB53, Ruminococcus, PrevoteLLa, Coprococcus were found in the control group and heavy metal treatment group. Streptococcus (6.78%), Treponema (2.78%), YRC22 (1.94%) and Megasphaera (0.37%) were only distributed in the control group. 4. The number of resistant bacteria and the isolation rate of resistant bacteria in the heavy metal treatment group were significantly higher than those in the control group (P 0.05). The isolation rate of heavy metal resistant bacteria in the heavy metal treatment group was C: U-resistant bacteria 29.58%, Zn-resistant bacteria 41.26%; in the control group, the proportion was: 1.34%, 1.52%. The isolation rate of antibiotic-resistant bacteria in the heavy metal treatment group was: Tet-resistant bacteria 33.00%, Chl-resistant bacteria 21.37%, Amp-resistant bacteria 24.37%, Str-resistant bacteria 25.37%; in the control group, the proportion was: 2.84%, 1.25%, 8.79%, 17.50% respectively. The isolation rate was 15.11% in the treatment group and 0.82% in the control group. The isolation rate of zinc-resistant bacteria was 27.21% in the heavy metal treatment group and 1.02% in the control group. 5. The relative expression abundance of resistance genes (24 resistance genes) on the DNA of pig feces nucleus genome of two different breeding methods was detected by qPCR. The results showed that the resistance of the heavy metal treatment group was 27.21%. The diversity and abundance of gene expression were significantly higher than that of the control group: 19 resistance genes were expressed in the samples of heavy metal treatment group, while 13 resistance genes were successfully amplified in the control group. Only 6 genes (str A, ampC, ermB, ermA, sul2 and pcoA) were expressed in the heavy metal treatment group. The expression abundance of four resistance genes (str B, mefA, sul1, tetX, tetQ, tetW, tetO, tetM, tetG, and zntA) in the heavy metal treatment group was significantly higher than that in the control group (P 0.05), while only three antibiotic resistance genes (ereA, ere B and tetA) in the control group were higher than that in the heavy metal treatment group (P 0.05). In this study, 27 strains of copper, zinc and antibiotic resistant bacteria were isolated and identified. The results showed that 7 strains were Enterococcus faecalis, 7 were Enterococcus sp., 4 were Proteus sp., 2 were Morganella sp., and 2 were Enterococcus faecalis. Enterococcus durans, 2 Enterococcus faecium, 2 Klebsiella sp. and 1 Enterococcus hirae. 7. Ten drug-resistant plasmids carrying multiple resistance genes were isolated from CO-resistant strains isolated from large-scale farms. The electrophoresis showed that plasmid P1 carried eight resistance genes (tetL, tetG, tetM, str A, strB, cmr, ERM B, ere B), plasmid P10, P21, P26 carried six resistance genes; plasmid P11, P12, P25, P27, P13 carried four resistance genes, while P3 carried three resistance genes (tetL, tetM, ERM B); plasmid P10, P11, P12, P21, P25, P26, P27 had zinc-drug co-resistance genes; Copper-drug co-resistance gene plasmids were P1, P3, P10, P12, P13, P27. The results of this study showed that heavy copper and zinc had synergistic effects on the resistance of pathogenic bacteria, river environment bacteria and pig manure environment bacteria. In addition, the diversity and expression abundance of drug-resistant genes were also found. Resistant bacteria isolated from feces of large-scale farms where heavy metal additives were widely used were found to have higher isolation rates, and drug-resistant plasmids carrying multiple resistance genes were isolated, resulting in higher drug-resistant gene transfer and diffusion of bacteria from swine feces in the environment. The results of this study will be of great significance to understand the mechanism of heavy metals'disorder and abuse in environment and aquaculture, and to further control the spread of superbacteria caused by the transfer and diffusion of drug-resistant genes in the environment.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R378

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