Diversity And Genetic Environment Of Antibiotic Resistance Genes In Resistant Bacteria From Soil Fertilized With Pig Manure

Animal manure is a hot spot of antibiotic resistance genes(ARGs)due to the extensive use of antibiotics in livestock and poultry industry.In China,a large volume of manures without pretreatment or treated only partly are directly applied on farmlands,which increases the risk of soil contamination caused by antibiotics and ARGs.The application of manure will introduce antibiotics and ARGs into the soil,increasing the abundance and diversity of ARGs in the soil.Soil is a natural reservoir for antibiotics and ARGs.However,with a great introduction of antibiotics and ARGs by manure-based fertilizers,more and more microorganisms in the soil show resistance to different antibiotics.This study mainly focused on the diversity of soil ARGs,the bacterial host of ARGs and the compositions and genetic environments of ARGs in different resistant bacteria isolated from a pig-manured soil.Real-time quantitative PCR(RT-qPCR)was used to compare the abundances of ARGs in soils long-term fertilized with pig manure(M),chemical fertilizer(NPK)and those with no fertilization(Control)in Qiyang Red Soil Experimental Station.The absolute abundance of 16S rRNA genes,five ARGs(tetG,tetW,aadA,sul2,and qacEdeltal)and three MGEs(Mobile gene elements)indicator genes(intI1,tnpA04,and IncW)were quantified.In addition,correlation analysis with gene abundances and the bacterial community composition was employed to predict the potential bacterial hosts of the determined ARGs.The results showed that compared with the control soil,application of NPK had no significant effects on the absolute and relative abundances of the tested genes(p>0.05),while the application of pig manure significantly increased the absolute and relative abundances of these genes(p<0.05).The potential bacterial hosts of aadA,su12,qacEdelta1 and tnpA-04 mainly distributed in Alphaproteobacteria,Deltaproteobacteria and Actinobacteria.In the above experiment,the potential hosts of ARGs and MGEs were speculated without considering the possibility that ARGs and MGEs could be distributed on plasmids and other mobile elements that can be transferred between different bacteria.Therefore,the identification of bacterial hosts of ARGs and MGEs needs further studies.Accordingly,this study further analyzed the composition and genetic environment of ARGs in different resistant bacteria isolated from soil with long-term application of pig manure at the Qiyang Red Soil Experimental Station.Thirty-six strains of resistant bacteria(9 strains of cephalexin,3 strains of chlortetracycline,9 strains of streptomycin,5 strains of ofloxacin,4 strains of sulfadimethoxine and 6 strains of chloramphenicol resistant bacteria)were isolated which belong to Proteobacteria,Firmicutes,Actinobacteria and Bacteroidetes.Resistant bacteria which belong to Proteobacteria and Firmicutes were dominantly isolated.Eighteen resistant bacteria were selected for genome sequencing.The results showed that the general patterns of ARG classes and mechanisms in resistant bacteria were host-specific.Among them,the resistant bacteria selected by cephalexin,chlortetracycline and chloramphenicol all harbored obligative resistance genes with high sequence similarity to known resistance genes.One strain selected by sulfadimethoxine contained an obligative sulfonamide resistance gene sul2 which located on a plasmid,and the other sulfadimethoxine resistant strain SUL3 contained only a point mutated folP which could lead to sulfonamide resistance.Three strains selected by streptomycin all had point mutated rpsL which could cause resistance to streptomycin.The strain screened by ofloxacin(OFC5)did not contain any specific resistance genes or point mutation for quinolone resistance.The resistance of this strain to ofloxacin may be caused by a broad-spectrum antibiotic efflux mechanism.The resistance strains all contained resistance genes that were not related to the corresponding selecting antibiotics.Some of these resistance genes which were not related to the selecting antibiotics showed high similarity to known ARGs from CARD.β-lactam resistance genes were the most widely distributed among all resistant bacteria,which followed by tetracycline and chloramphenicol resistance genes.The results indicated that the resistant bacteria in the pig-manured soil may have a history of multiple antibiotic exposure.There were 7 strains of resistant bacteria carrying ARGs that were prone to horizontal transfer,namely:chloramphenicol resistance genes cat7 were adjacent to transposase genes in C17(Pseudomonas sp.).tetracycline resistance genes tet(Y)were carried by plasmid,sulfadimethoxine resistant gene sul2 were adjacent to transposase genes,which were carried by plasmid likes tetracycline resistance genes(tet(Y),tet(3),tetR)and chloramphenicol resistance genes floR in SUL1(Acinetobacter sp.).β-lactamase resistance genes blaCMY-108 were carried by plasmid,and also were adjacent to integrase genes in SUL3(Klebsiella sp.).β-lactamase resistance genes rm3 were adjacent to integrase genes.β-lactamase resistance genes bla1(1)、blal(4)were carried by plasmid,and also were adjacent to integrase genes,and located in different gene island in Cep5(Bacillus sp.)and ant(2")-Ia were carried by plasmid in Cep5(Bacillus sp.).two tet(L)were carried by plasmid in CTC1(Bacillus sp.).βlactamase resistance genes blaCGB in Cep4(Chryseobacterium sp.).These resistance genes had high potential for horizontal gene transfer and are worthy of further study.This study quantitatively analyzed ARGs in soils with different fertilizers and predicted their potential bacterial hosts based on RT-qPCR and 16S rRNA gene sequencing.,More importantly,the diversity and genetic environments of ARGs in antibiotic resistant bacteria isolated from pig-manured soil were studied by genome sequencing.The results showed that application of pig manure significantly increased the abundance and diversity of soil ARGs.There were significant differences in terms of diversity and genetic environment of ARGs in different resistant bacteria.The overall resistance mechanism of all ARGs showed a certain host-specific difference.Different distributions of the obligative ARGs conferring resistance to different antibiotics were observed in the selected resistant bacteria.β-lactam resistance genes were the most widely distributed,which followed by tetracycline and chloramphenicol resistance genes.The results of our study can provide an important theoretical basis for preventing and mitigating soil ARG pollution.