Combined Contamination Of Tetracycline And 3,4-benzo(A) Pyrene Cause High Resistant Mutation In Teta(C) Gene

In recent years,the concentration of antibiotics in the environment has been increasing,and the resistance of microorganisms to antibiotics has also improved.Numerous studies have shown that abnormal increase in abundance of Antibiotic Resistance Genes(ARGs),can lead to high-resistant in microorganisms.However,several studies have shown that microorganisms can acquire high-resistant through the mutation of ARGs.There are various genetic mutagenic substances in the environment,for instance,3,4-benzene(Bap),a type of polycyclic aromatic hydrocarbons(PAHs).In this study,we choose Bap and tetracycline(TC)as pollutants,and choose tetA(C)gene as test subject to explore the impact of combined pollution of TC and Bap on ARGs.After tetA(C)mutant gene library was first established,we screened out high-resistant tetA(C)mutant strains from it and then the tetA(C)gene mutation site-high resistance relationship code table was obtained by the use of DNA-seq.Afterwards,we cultivated strains in some treatments with different concentration of Bap and TC for 14 days to analyze the link between combined pollution and probabilities of occurrence of high-resistant tetA(C)mutant strains through the help of code table.After sufficient verifying tests,we analyzed the contamination level of Bap,TC and mutants of tetA(C)and the potential relationships between them in soil samples gathered from realistic environment.In this study,we provided scientific basis for ARGs environmental health warning and ecological risk assessment as well as theoretical support to alleviation of ARGs pollution in soil environment by degrading organic pollutants and antibiotics.The major research results are as follows:1.We created a testing strain carrying pACYC 184 plasmid and optimized the error-prone PCR,based on which established the tetA(C)mutant gene library.2.We tested the minimum inhibitory concentration(MIC)of TC to testing strains was 100 mg/L,,based on which we screened out 20 tetA(C)mutant strains with high resistance from the tetA(C)mutant gene library to establish high-resistant tetA(C)mutant gene library.After established the tetA(C)gene mutation site-high resistance relation code table by the use of DNA-seq,we found that mutation sites which enhanced resistance were concentrated in the latter part of tetA(C)gene.3.We spread the testing strains on LB plates with Bap(0,2,5,10,20 and 30mg/L)and TC(0 and 10mg/L)of various concentrations for 14 days.We screened and counted highresistant strains in both the treatment group,and calculated the multiple relation between the high-resistant strains of the treatment group and the control group(without Bap and TC).The results showed that when the concentration of TC was 10mg/L,the multiple was positively correlated with the concentration of Bap;while without adding TC,Bap concentration has no obvious relation with the multiple.Then,we selected 10 stains from each group of highresistant strains for tetA(C)gene sequencing.The sequencing results were compared with tetA(C)gene mutation site-high resistance relation code table and the gene mutation was analyzed.After the comparison,we calculated the proportion of high-resistant mutant strains generated due to gene mutation to all the high-resistant mutant strains,based on which we analyzed the influences of TC/Bap combined pollution on high-resistant mutation of tetA(C)genes.The results show that when the concentration of TC was 10mg/L,the proportion was positively correlated with the concentration of Bap;while without adding TC,the proportion has no obvious relation with Bap concentration..4.We gathered soil samples in Nanjing and the peripheral areas to test the contents of Bap and TC and extract soil DNA.Co-existence of Bap,TC and tet(A)was found in the soil sample of Hengliang dairy and it was also found that the similarity between tet(A)gene and the original tet(A)gene was as high as 98%-100%,that is,some tet(A)genes had mutated.It was tested that the resistance of the mutated tet(A)strains was higher than that of the unmutated tet(A)strains.In the sample soils collected from the vegetable fields,there were only TC and tet(A)genes,among which the similarity between tet(A)gene and the original tet(A)gene was as high as 100%,that is,tet(A)gene did not mutate.Comparing the test results of these two soil samples,it could be found that it is more possible to have high-resistant mutant genes in the environment with Bap,which accords with the results of the experiments cultivation testing strains treated with Bap and TC.