重金属铜锌对病原菌及环境细菌耐药性的影响

发布时间：2018-09-17 06:33

【摘要】：超级细菌的流行是当前人类面临的重大生存问题。超级细菌的流行与抗生素抗性基因(ARGs,antibiotic resistance genes)通过的传播与扩散密切相关。目前对ARGs的研究主要集中在医学领域(病原菌和医院环境),而环境耐药细菌的ARGs可通过质粒等可移动遗传元件(MGEs,Mobile genetic elements)在环境细菌和人体病原菌之间传播和扩散,从而对人类健康和生态环境造成极大威胁,但目前对环境耐药细菌的危害严重低估,认识很少。质粒除了携带ARGs外,多数重金属抗性基因也主要定位于其中,ARGs和重金属抗性基因在质粒上的共分布可以导致重金属对ARGs具有协同选择作用。目前抗生素的滥用对耐药细菌的选择作用已成为微生物学研究的热点,但重金属的使用对环境耐药细菌及其携带ARGs的影响涉及较少。工业废水的排放以及畜牧养殖业的广泛使用导致水体和猪粪环境中重金属污染严重,而这些环境系统与人类生活关系密切,因此有必要分析重金属对这些环境中的耐药细菌及其携带ARGs的选择与迁移的影响及机制,为有效控制细菌耐药性的转移及超级细菌的蔓延提供理论基础。为了研究重金属对细菌耐药性的影响,本研究具体分析了在重金属胁迫条件下病原细菌,河水及猪粪环境细菌的类群、耐药比例以及耐药基因表达的情况,具体结果如下:1、经重金属铜(Cu)驯化得到的大肠埃希氏菌突变菌株对盐酸四环素的最高耐受浓度提高了133%;铜绿假单胞菌突变株对盐酸四环素最高耐受浓度提高了52%。经重金属锌(Zn)驯化的金黄色葡萄球菌突变株对盐酸四环素最高耐受浓度比出发菌提高了25%;铜绿假单胞菌突变株对盐酸四环素最高耐受浓度提高了52%。2、通过重金属Zn胁迫培养之后的河水样本重金属和抗生素抗性菌的数量和分离率均有显著提高(p0.05),与对照组相比,Zn处理组Cu抗性菌分离率提高了0.9%;Zn抗性菌分离率提高了11.1%;盐酸四环素抗性菌分离率提高了0.4%;氯霉素抗性菌分离率提高了4.6%;氨苄青霉素抗性菌分离率提高了2.3%;链霉素抗性菌的分离率提高了6.1%。3、通过高通量测序分析对照组(基础饲料喂养)和重金属处理组(在基础饲料中添加400mg/kg Cu和1000 mg/kg Zn)猪粪样本细菌多样性与物种丰度,重金属处理组比对照组的猪粪菌群多样性高。本研究分析了丰度前十的门、纲、目、科、属各分类层级细菌群落:对照组的优势菌门为厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes),分别占了55.7%和37.3%,而重金属处理组的优势菌门为厚壁菌门(Firmicutes),占了92.12%。分析的丰度前十的菌属中发现,颤螺菌属(Oscillospira)、梭菌属(Clostridium)、SMB53、瘤胃球菌属(Ruminococcus)、中间普氏菌(PrevoteLLa)、粪球菌属(Coprococcus)等六个属在对照组和重金属处理组均有分布。而链球菌属(Streptococcus)(6.78%)、密螺旋体属(Treponema)(2.78%)、YRC22(1.94%)、巨球型菌属(Megasphaera)(0.37%)仅在对照组分布。4、重金属处理组的抗性菌数量和抗性菌分离率显著高于对照组(P0.05)。重金属抗性菌的分离率在重金属处理组的比例为:Cu抗性菌29.58%,Zn抗性菌41.26%;在对照组的比例分别是:1.34%,1.52%。抗生素抗性菌在重金属处理组的分离率为:Tet抗性菌33.00%,Chl抗性菌21.37%,Amp抗性菌24.37%,Str抗性菌25.37%;在对照组的比例分别是:2.84%,1.25%,8.79%,17.50%。铜药协同抗性菌在重金属处理组的分离率为15.11%;在对照组是0.82%。锌药协同抗性菌在重金属处理组的分离率27.21%,在对照组是1.02%。5、通过qPCR检测两种不同养殖方式的猪场粪便核基因组DNA上的抗性基因(24个抗性基因)相对表达丰度,结果表明重金属处理组抗性基因的表达多样性和丰度明显高于对照组:其中19个抗性基因在重金属处理组样本有表达,而对照组样本中有13个抗性基因扩增成功,仅在重金属处理组表达的基因有6个(str A、ampC、ermB、ermA、sul2和pcoA)。在两组均表达的13个抗性基因中,有10个抗性基因(str B、mefA、sul1、tetX、tetQ、tetW、tetO、tetM、tetG、和zntA)在重金属处理组的表达丰度明显高于对照组(P0.05),而仅有3个抗生素抗性基因(ereA、ereB和tetA)在对照组的表达丰度高于重金属处理组(P0.05)。6、规模化养猪场的猪粪样本抗性菌具有较高的分离率。本研究从中分离到27株重金属铜、锌和抗生素协同抗性菌,对其进行了分子鉴定,鉴定结果发现有7株为粪肠球菌(Enterococcus faecalis),7株为肠球菌属(Enterococcus sp.),4株为变形菌属(Proteus sp.),2株为摩根氏菌属(Morganella sp.),2株为坚强肠球菌(Enterococcus durans),2株为屎肠球菌(Enterococcus faecium),2株为克雷伯氏菌属(KLebsiella sp.),1株为海氏肠球菌(Enterococcus hirae)。7、从规模化养殖场分离的共抗性菌株中提取到10个携带多种抗性基因的耐药质粒。携带的抗性基因PCR扩增电泳图显示:质粒P1携带8个耐药基因(tetL、tetG、tetM、str A、strB、cmr、erm B、ere B),质粒P10、P21、P26携带6个耐药基因;P11、P12、P25、P27,P13则携带4个耐药基因,而P3携带3个耐药基因(tet L、tetM、erm B);具备锌-药共抗性基因的质粒有P10、P11、P12、P13、P21、P25、P26、P27;具备铜-药共抗性基因的质粒有P1、P3、P10、P12、P13、P27。本论文研究结果表明:重金属铜锌对病原菌、河水环境细菌及猪粪环境细菌耐药性均具有一定的协同选择作用。饲料中重金属铜、锌添加剂的使用可通过协同选择作用提高猪粪耐药细菌的比例,以及耐药基因的多样性和表达丰度,同时还发现广泛使用重金属添加剂饲料的规模化养殖场粪便细菌的抗性菌分离率较高,并分离到携带多种抗性基因的耐药质粒,从而导致该环境的猪粪细菌具有较高的耐药基因转移与扩散的风险。本课题的研究结果将为全面认识重金属在环境和养殖业的乱排和滥用对细菌耐药的影响机制,进一步控制耐药基因在环境中的转移与扩散所导致的超级细菌蔓延具有重要意义。
[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.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378

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