Preliminary Study On Degradation Characteristics And Drug Resistance Mechanism Of Oxytetracycline-degrading Strain OTC-16

Oxytetracycline,one of the tetracycline antibiotics,is often used as feed additive in animal husbandry to prevent animal diseases and promote animal growth because of its low cost and easy to use.However,the extensive application and incomplete degradation of oxytetracycline in animal husbandry have caused widespread environmental residues and the spread of resistance genes,which seriously threatened human survival and safety.Microbial degradation is the main method to remove antibiotic residues in natural environment.Based on the previous screening of a high efficient oxytetracycline-degrading strain OTC-16,the strain was identified by traditional combined molecular methods in the study.The environmental adaptability of the strain to degrade oxytetracycline was studied with the inoculation amount,initial pH and temperature as influence factors.The actual effect of the strain to degrade oxytetracycline in soil under different pH conditions was studied.At last,bioinformatics was used to predict the resistance genes of the strain and the actual resistance of the strain was also determined.The results are as follows:?1?The strain was identified as Arthrobacter nicotianae using conventional combinding with molecular means.In addition to oxytetracycline,strain OTC-16 could also degrade tetracycline and chlortetracycline to some extent.A preliminary study on the mechanism of the strain showed that the strain OTC-16 mainly reduced the oxytetracycline concentration through the biochemical degradation pathway instead of the simple cell surface adsorption,and the degradation-related active substances acted mainly after being secreted extracellularly.The environmental adaptability of strain OTC-16 in oxytetracycline degradation was studied.The results showed that OTC-16 strain grew well at a pH range of 6.0-10.0,and had strong adaptability to alkaline environment;The strain OTC-16 can grow under a 0-500 mg·L^-1 oxytetracycline content,and the strain still grow well when the concentration of oxytetracycline is as high as 500 mg·L^-1.The strain OTC-16 exhibited the best degrading effect on oxytetracycline when the inoculum amount,initial pH,temperature,and initial concentration of oxytetracycline were 3%,pH 9.0,30?and 150 mg·L^-1,respectively.?2?The degradation effect of strain OTC-16 in different soils was evaluated by soil simulation test in the laboratory.The experimental results showed that the degrading bacteria OTC-16 obtained in this study had unique advantages in removing oxytetracycline residues in alkaline soil environment.After 10 days of culture,the degradation rate in acid,neutral and basic soil was increased by 16.0%,6.3%and 40.7%,respectively.Moreover,the appearance of the strain won^/t deteriorated physical and chemical properties of the soils.The addition of manure and straw could increase the organic matter content in the soils,but inhibited the degradation of oxytetracycline.To solve this problem we suggest a separate agricultural measures of improving soil and removing antibiotic residues.The analysis of 16S rRNA and 18S rRNA gene abundance in soil indicated that,the addition of degrading bacteria OTC-16,manure and straw can significantly increase the number of bacteria in the three soils,and the effect of manure is more obvious than the others;the straw itself could not increase the number of bacteria,however the number of bacteria in the alkaline soil increased sharply when the straw was mixed with the degrading bacteria;the addition of the degrading strain OTC-16 had little effect on the amount of fungi in the three soils,while manure and straw significantly increased the number of fungi in acidic and neutral soils.In alkaline soils,only straw can increase the abundance of fungi.?3?The whole genome sequencing of oxytetracycline-degrading strain OTC-16 showed that the total length of the genome was 3 797 724 bp;it contained a circular chromosome of 3 643 989 bp and two plasmids?a 123 894 bp linear and a 29 841 bp circular?.A total of 3 561 genes were obtained by gene annotation,of which 23 were resistant genes,most of which were distributed on plasmids.Through the annotation of the drug resistance gene?ARDB?database,we found that there are multiple drug resistance genes TetV,Tet33,sul1,ant2ia,vanre,vanrc,cml_e6,cml_e8,catb3,cara in the OTC-16genome.TetV and Tet33 belong to the efflux pump gene,and few papers about gene Tet33 has been reported so far.The drug resistance spectrum test showed that the OTC-16 strain can tolerate 15 kinds of antibiotics such as oxytetracycline,tetracycline,sulfamethoxazole,sulfamethoxazole and the like.The results were basically consistent with the drug resistance genes predicted by bioinformatics,which basically revealed the antibiotic resistance mechanism of the strain.