| In humans and livestock breeding,the abuse of antibiotics has led to a wide spread of drug-resistant bacteria in environment.Therefore,so as to avoid an impact on public health, this study analyses diversity of tetracycline-resistant lactose-fermentation Enterobacteriaceae (TR-LFE) which comes from wastewater treatment plant activated sludge pool and lake water biofilm.Last of all,this study does tetracycline resistance genetic testing in activated sludge to find out the proportion of drug-resistant and the distribution of tetracycline-resistance gene in the wastewater treatment plant.After selective cultivation from the extract water samples of the wastewater treatment plant,the results show that on the plate resistance,vast majority of lactose-fermenting Enterobacteriaceae(LFE) is tetracycline-resistant lactose-fermentation Enterobacteriaceae (TR-LFE),accounting for 70%to 90%.However,after a series of post-test showed that the sewage treatment process of wastewater treatment plant cannot decrease large quantity of pathogens.At the same time,excess activated sludge will be shipped to the outside after the treatment of a concentrated pool of gravity concentration and there are no specific treatment methods.So the vast majority of pathogens will be directly discharged into the environment.The tet genotype frequency of tetracycline-resistant lactose-fermentation Enterobacteriaceae(TR-LFE) is between 0%to 69.23%in the influent,activated sludge and effluent samples of wastewater treatment plant.Detected eight-resistant genotypes,such as tet(A),tet(B),tet(C),tet(D),tet(E),tet(M),tet(G),tet(X),not detected ribosomal protection protein encoded tet(O) and tet(S) gene;the frequency of lactose-fermenting Enterobacteriaceae(LFE) is between 3.7%to 68.18%,not detected ribosomal protection protein encoded tet(M),tet(0) and tet(S) gene.After the classification of PCR-RFLP technology,total 328 strains of tetracycline-resistant lactose-fermentation Enterobacteriaceae(TR-LFE) and lactose-fermenting Enterobacteriaceae have been sequenced.The sequencing results are 5 different strains of E.coli,Shigella,and Klebsiella,Aeromonas,and Acinetobacter baumannii. The homologies are 100%～99%.After testing tetracycline-resistant genes of active sludge in different seasons and conditions,we find tet genes are widely distributed in the activated sludge.But wastewater treatment plant cannot be effective in reducing the different types of tet genes in the course of a series of processing.At the same time,detecting drug-resistant genes in Dishui lake biofilm samples proves drug-resistant genes in the wastewater treatment plant are much higher than the general contents of surface runoff in environment.Through real-time fluorescence quantitative PCR technology(qRT-PCR)to quantify the target genes,we find the concentration of tet(A) gene is higher than tet(C) gene in Minhang wastewater treatment plant and the concentration of these two genes is much higher than that of natural lake.Perhaps the use of antibiotic drug and its emission have not been effective controlled in Shanghai,even in China so that the content of antibiotics genes in this region is much higher than other areas.In addition,strains with tet gene to extract plasmid revealed that the majority of strains contain the different size and number of bands.The percentage of obtaining plasmid is over 93.4%.After sequencing tet(A),tet(B),tet(C) and tet(D) genes of the plasmid resistance gene,the results have highly homology with pathogenic Enterobacteriaceae.But only in accordance with the depth of this experimental study,we are unable to observe a significant relationship between the strain with the plasmid and its drug-resistant genes,which requires follow-up study in the further. |
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