| The prevalence of superbugs is a major problem for human beings.The prevalence of superbugs is closely related to the spread and spread of antibiotic resistance genes(ARGs).At present the study on ARGs mainly concentrated in the field of Medicine(environmental pathogens and hospital),and the environment of resistant bacteria by ARGs(antibiotic resistance genes)plasmid MGEs(Mobile genetic elements)between bacteria and human pathogenic environment spread and spread,causing a great threat to human health and ecological environment,but the harm to the environment of drug-resistant bacteria seriously underestimated,and knows little.In addition to ARGs,most of the heavy metal resistance genes were mainly located in the plasmid,and the co distribution of ARGs and heavy metal resistance genes could lead to the synergistic effect of heavy metals on ARGs.At present,the selection of antibiotic resistant bacteria has become a hot topic in the field of microbiology.However,the impact of heavy metals on the environment resistant bacteria and their carrying ARGs is less.The discharge of industrial wastewater and widely used in anima L husbandry and water environment in pig manure led to heavy metal pollution is serious,and the environment is c Losely related to human life,so it is necessary to analyze the influence of selection and transfer of heavy metals on drug-resistant bacteria in these environments is carrying ARGs and mechanism for the transfer of effective control of bacterial resistance and super bacteria to provide a theoretical basis to spread.In order to study the effect of heavy metals on bacterial resistance,this study analyzed under heavy metal stress,pathogenic bacteria,and the bacteria species and drug resistance of pig manure and river water environment and the proportion of resistant gene expression,the main results are as follows: 1,The heavy metal Cu domestication of Escherichia coli mutations obtained the maximum tolerance concentration of tetracycline hydrochloride strain increased 133%;mutant of Pseudomonas aeruginosa strains of tetracycline hydrochloride maximum tolerated concentration increased by 52%.The maximum tolerated concentration of tetracycline resistant strain of Staphylococcus aureus was 25% higher than that of the starting strain,and the maximum tolerated concentration of tetracycline resistant mutant of Pseudomonas aeruginosa increased by by Zn.2,After heavy metal stress of Zn cultured in water samples of heavy metal and antibiotic resistant bacteria number and separation rate were significantly improved(p<0.05),compared with the control group,Zn treatment group Cu resistant bacteria isolation rate increased by 0.9%;Zn resistant bacteria iso Lation rate increased by 11.1%;hydrochloride tetracycline resistant bacteria isolation rate increased by 0.4% chloramphenicol resistant bacteria;separation rate increased by 4.6%;ampicillin resistant bacteria isolation rate increased by 2.3%;the separation of streptomycin resistant bacteria was increased by 6.1%.3,Bacterial diversity and species richness of pig manure samples were analyzed by high throughput sequencing in the control group(basal feed)and heavy metal treatment group(400mg/kg Cu and 1000 mg/kg Zn were added to the basal diet),The diversity of heavy metal group was higher than that of control group.This study analyzed the abundance of the phylum、class、order、family and genus of each classification level of bacterial communities:Firmicutes and Bacteroidetes were dominant bacterias phylum in the control group,accounted for 55.7% and 37.3%,while the dominant phylum of heavy metal treatment group was Firmicutes,accounting for 92.12%.The abundance of the analysis was found in ten of the genera: Oscillospira,Clostridium,SMB53,Ruminococcus,Prevotella,Coprococcus were distributed s in the control group and the heavy metals treatment group.Streptococcus(6.78%),Treponema(2.78%),YRC22(1.94%),Megasphaera(0.37% only in the control group.4,The number of resistant bacteria and iso Lation rate of resistant bacteria in heavy metal treatment group were significantly higher than those in control group(P<0.05).The percentage of heavy metal resistant bacteria in the heavy metal treatment group was: Cu resistant strain 29.58%,Zn resistant strain 41.26%;The proportions in the control group were 1.34%,1.52%.The isolation of antibiotic resistant bacteria in the heavy metal treatment group was as follows: tet resistant strain 33%,Chl resistant strain 21.37%,amp resistant strain 24.37%,str resistant strain 25.37%;The proportions in the control group were: 2.84%,1.25%,8.79%,17.50%.The separation rate of copper drugs resistant bacteria in the heavy metal treatment group was 15.11%;in the control group,it was about 0.82%.The separation rate of zinc drugs resistant bacteria in the heavy metal treatment group was 27.21%,which was higher than that in the control group,which was about 1.02%.5,The relative expression of the resistance genes(24 resistance genes)in the of the pig feces genomic DNA was detected by qPCR,The results showed that the number and abundance of resistance genes in the treatment group were significantly higher than those in the control group: Among them,19 resistance genes were expressed in the heavy metal treatment group,Among them,19 resistance genes were expressed in the heavy metal treatment group,In the control group,13 resistance genes were amplified successfully,There were 6 genes only expressed in the heavy metal treatment group(str A,amp C,erm B,erm A,suL 2 and pco A).The 13 resistance genes were expressed in all the two groups,The expression of the 10 genes(str B,mef A,suL 1,tet X,tet Q,tet W,tet O,tet M,tet G,and znt A)in the heavy metal treatment group was significantly higher than that in the control group(P< 0.05).However,only 3 antibiotic resistance genes(pcoA,pcoD,copA)were higher in the control group(P < 0.05).6,The resistance rate of pig manure samples in the large-scale pig farms was higher.In this study,27 strains of copper,zinc and antibiotic resistant bacteria were isolated,Molecular identification was carried out,The results showed that 7 strains of Enterococcus faecalis,7 strains of Enterococcus sp.,4 strains of Proteus sp.,2 strains of Morganella sp.,2 strains of Enterococcus durans,2 strains of Enterococcus faecium,2 strains of Klebsiella sp.,1 strains of Enterococcus hirae.7,A total of 10 resistant plasmids carrying a variety of resistance genes were isolated from a total of resistant strains isolated from large-scale farms.Amplification of the resistance gene by PCR: plasmid P1 carrying 8 resistance genes(tetL、tetG、tetM、strA、strB、cmr、ermB、ereB),plasmid P10,P21,P26 carrying 6 resistant genes;P11,P12,P25,P27,P13 carrying 4 resistance gene,and P3 carriying 3 resistance genes(tetL、tetM、erm B);The plasmid with zinc drug resistance gene are P10,P11,P12,P13,P21,P25,P26,P27 and so on,with the exception of and;The p Lasmid with copper drug resistance gene are P1,P3,P10,P12,P13,P27.The results of this study showed that the heavy metal copper and zinc had a synergistic effect on the pathogenic bacteria,the river water environment bacteria and the resistance to environmental bacteria.The use of copper and zinc in feed additives by pig manure resistant bacteria enhance proportional selection,and the expression of multidrug resistance gene number and abundance,it was also found that the resistant bacteria of fecal bacteria in scalefarming feed additives widely used heavy metal separation rate is higher,and separation of resistant plasmids carry multiple resistance genes,risk which leads to the resistance gene environment of swine manure bacterium has a high transfer and diffusion.The results of this study will provide a comprehensive understanding of the mechanism of heavy metals in the environment and breeding industry,the impact of abuse and abuse,It is of great significance to further control the spread of super bacteria caused by the transfer and diffusion of drug resistance genes in the environmentthe. |
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