Distribution And Spread Of Antibiotic Resistance Genes In Bacteriophage In Pig Farms Environment

With the increase use and abuse of antibiotics in the process of livestock and poultry breeding,the occurrance of pathogens is getting more and more serious.Also,with the continuously discovered and widely spread of newly antibiotic resistance genes?ARGs?and antibiotic resistant bacteria?ARB?among different hosts,which seriously threatens human health.Evidences have shown that bacteriophage can serve as an important reservior for ARGs and promote the horizontal transmission in the environment,suggesting a more frequent contaction between bacteriophage and bacteria than expected.Therefore,it will be of great significance for further study of transmission mechanism before exploring the distribution and spread of ARGs in bacteriophage in the farm environment.In this study,we screened pig feces,arable soil and wasterwater samples from a commercial farms in China for 32 clinically relevant ARG types(sulfonamide resistance genes?sul1,sul2,sul3?,macrolide resistance genes?ermA,ermB,ermF,mefA,vatB,mphA,ereB?,colistin resistance gene mcr-1,quinolone-resistant genes?qnrA,qnrB,qnrS,oqxA,oqxB,aac?6'?-Ib-cr,qepA?,?-lactam resistance gene(bla_TEM,bla_SHV,bla_CTX),linezolid resistance gene optrA,chloramphenicol/florfenicol resistant genes?cmLA,floR,fexA,cfr,fexB?and tetracycline resistance genes?tetM,tetA,tetB,tetT,tetW?)to assess the presence of the ARG in bacteria and bacteriophage and further to estimate the contribution of bacteriophages to the dissemination of antibiotic resistance according to the transduction ability value?TA?.The results of detection rate of 32 resistant genes showed that ARGs in feces,wastewater,and soil phage DNA was only 34.38%?11/32?,37.50%?12/32?,and 15.63%?5/32?.However,the low detection rate still cannot underestimate the spread characteristics of some important drug resistance genes,especially for the genes sul1,bla_TEM,ermB,floR and tetM that were detected in all sample phage genomes?only detection rate in soil samples is low?.These results suggest that phage may play an important role in the horizontal transmission of these 5 resistant genes,especially from the feces to the sewage.In order to more accurately reflect the real sample environment,the detection rate of single ARGs in all samples was higher than 50%were further conducted the quantitative analysis.Further quantitative results showed that the abundance of ermB gene in feces and floR in wastewater was the highest in the bacterial DNA,whereas in the phage DNA the ermB in feces and bla_TEMEM in the wasterwater occupied the highest.To further explore and assess the transduction ability of bacteriophage in spreading resistance,the introduction of TA values can provide a preliminary assessment of gene exchange capabilities between bacteriophage and bacteria.Therefore,the ratio index of the abundance of ARGs in bacteriophage and bacteria was introduced in this study as an estimator of bacteriophage ability to transmit ARGs.The results of TA value showed that qnrA is the highest,reaching a magnitude of 10^-1?meant that for every 10 copies in bacteria,there would be one copy transferred to a phage?.However,we found that its abundance and detection rate did not exhibit a relatively high level.These results suggest that though some ARGs are low in abundance and detection rate,they still have a strong ability to transmit and we should not underestimate the horizontal transfer ability mediated by bacteriophage transduction.On the other hand,these results also illustrate that ARGs abundance and detection rates used alone probably be not suitable for comprehensively judging the contribution of bacteriophage to the dissemination of antibiotic resistance.A more suitable model is the application of three indices;occurrence rate,absolute abundance in bacteriophage and the TA value as warning and monitoring tools for environmental ARG assessments in bacteriophages.