Screening Of Florfenicol Resistance Genes In Soil From Pigsties By Using Metagenomic Library

Veterinary antibiotics have been increasingly used in livestock farming in recent years. Most antibiotics uptaken by animals couldn’t be metabolized, and they will be discharged with feces in the form of parent compounds, then be put into soil as manure. As a result, more and more antibiotics will accumulate in the soil, and induce the product of antibiotic resistance genes and antibiotic resistant bacterium, which will threaten the health of human and animal. In this study, a swine feedlot with a long term of florfenicol usage was selected as a sampling point, and florfenicol resistance genes in soil were screened by using metagenomic library. The results are as follows:1. Soil genomic DNA was extracted and digested by restrictive endonuclese XhoI and PstI, then fragments length of4-7kb and2.5-4kb were extracted respectively. A soil metagenomic library containing approximately25000clones which has insertion length of4-7kb and15000clones which has insertion length of2.5-4kb was constructed using pUC18as vector and E.coli BL21as the host strain.2. Metagenomic library was screened with florfenicol for florfenicol resistance strains, and about300positive clones were acquired. Two active clones were selected to sequence the insert fragments. We found that one of them contains SdiA gene, which encodes a transcriptional activator that is present in many strains of E. coli. SdiA can promote expression of the AcrAB-TolC efflux pump, and survive E.coli in multiple antibiotics environment. The other one contains a unknown ORF and three genes were already identified in present studies, which encodes a response regulator protein in Methylobacterium, a drug transporter protein, Bcr/CflA family in Oligotropha carboxidovorans OM5, and a short-chain dehydrogenase/reductase SDR in Xanthobacter autotrophicus Py2respectively. In those genes, MFS gene has been proved to work as drug-efflux pumps to confer antibiotic resistance.In this study, we screened florfenicol resistance genes by constructing metagenomic library of pigsty soil, this is the first time for screening antibiotic resistance genes by using metagenomic library in our country. Screening of the ARGs can help us understanding the generation and dissemination in the environment deeply, and evaluating ecologic risk of antibiotics in the environment objectively.