| In this paper,tetracycline-resistant bacteria（TRB）isolated from pig manure and soil with pig manure were used as materials.According to the sensitivity characteristics of different strains to midgut fluid of earthworms,20 strains were selected to construct simulated food with sensitive flora and insensitive flora respectively.The two types of microflora simulated food combined with strain inactivation and tetracycline addition treatment were used to carry out earthworm feeding experiments in artificial environment.The characteristics of bacterial community composition,abundance of tetracycline resistance genes（TRGs）and its relationship with the bacterial community structure of earthworm manure were studied.The results provide a scientific basis for analyzing the changes of drug resistance genes from food sources in the process of earthworm overfeeding and accurately evaluating the mechanism of earthworm action on drug resistance genes in the composting process and soil ecosystem.The main results obtained in this paper are as follows:1.The sensitivity analysis of earthworm midgut fluid was performed on 95 TRB strains by plate culture.The results showed that 41 strains of the tested TRB showed different degrees of sensitivity to the midgut fluid of earthworms,among which the extremely sensitive,highly sensitive,moderately sensitive and low sensitive strains accounted for 14.6%,17.1%,39%and 29.3%of the sensitive strains,respectively.It indicated that the sensitivity of the tested strains to the midgut fluid of earthworms were mainly moderate and low sensitivity.Combined with the analysis of the taxonomic information of the tested strains,it was found that the sensitivity of TRB to the midgut fluid of earthworms was Firmicutes>Proteobacteria>Actinobacteria>Bacteroidetes.2.Based on the sensitivity and taxonomic information of the above strains to midgut fluid of earthworms,the simulated diets of sensitive TRB and insensitive TRB were constructed and the earthworms were raised in laboratory.The abundance of vermicompost TRGs in each treatment at the first week and the sixth week after feeding were measured,and the results showed that:（1）The food treatment of drug-resistant bacteria significantly promoted the formation of vermicompost TRGs,and the treatment of insensitive bacteria was more obvious.tetB,tetZ and tetX were the main contributors to the formation of vermicompost TRGs,and the increases ranged from 58.35%to 153.24%,-10.33%to 11.71%and-2.41%to 304.51%compared with the control.Bacterial inactivation treatment obviously inhibited the formation of vermicompost TRGs in the sensitive flora treatment,but significantly promoted the formation of vermicompost TRGs in the insensitive flora treatment.Compared with the active flora treatment,inactivation significantly inhibited the formation of vermicompost tetB,tetZ and tetX in the sensitive flora treatment,and the formation of vermicompost tetX in the insensitive flora treatment,but promoted the formation of tetB and tetZ treated by insensitive flora.（2）Compared with the control,the addition of tetracycline significantly promoted the formation of vermicompost TRGs in the sensitive flora treatment,but inhibited the formation of vermicompost TRGs in the insensitive flora treatment.The main reason was that the abundances of tetZ and tetX in the two types of flora treated vermicompost differed in response to tetracycline treatment.Compared with the treatment without tetracycline,the addition of tetracycline significantly promoted the formation of vermicompost TRGs in the two types of bacteria diets in the early feeding period.This result was closely related to the increase of the abundance of vermicompost tetB and tetX in the sensitive bacteria group and vermicompost tetX in the insensitive bacteria group.In the later stage of feeding,the addition of tetracycline significantly promoted the total TRGs of vermicompost treated with sensitive flora,but inhibited the formation of TRGs treated with insensitive flora.3.Illumina high-throughput sequencing technology was used to determine the bacterial community composition of vermicompost on the 6th week of different bacterial groups.The results show that:（1）Microflora,inactivation and tetracycline treatment all affected the species richness and diversity of vermicompost bacterial community.Among them,the food flora led to a decrease in the species richness and diversity of the vermicompost bacterial community.The inactivation treatment significantly increased the species richness of the insensitive bacterial group treatment.The addition of tetracycline significantly improved the species richness and diversity of the insensitive flora in the food treatment.The two types of drug-resistant bacteria diet treatment significantly changed the vermicompost bacterial community structure.Inactivation treatment weakened the effect of sensitive flora on the bacterial community structure of vermicompost.The addition of tetracycline weakened the effects of the two types of bacterial groups on the bacterial community structure of vermicompost,but promoted the evolution of the vermicompost community structure in the inactivated inactivated bacterial group treatment and led to a significant difference between the two types of inactivated bacterial groups.（2）Food-derived drug-resistant bacteria,tetracycline and inactivation treatments all had different regulatory effects on the composition of vermicompost bacterial community.From the phylum level,the changes in the abundance of Proteobacteria and Actinobacteria were shown.The results of genus-level cluster analysis showed that the insensitive bacterial group treatment had a greater impact on the composition of the vermicompost dominant genus than the sensitive bacterial group treatment.Inactivation treatment did not change the effects of the two types of flora on the composition of dominant genera in vermicompost,while tetracycline treatment enhanced the effect of sensitive flora on the composition of dominant genera in vermicompost.（3）Co-occurrence network analysis was performed on the bacterial community composition of vermicompost and the abundance of TRGs in different treatments.The results showed that the dominant bacterial genera accounted for 66.67%,45.45%,70%,53.33%and 45.45%of the potential hosts of vermicompost tetB,tetC,tetZ,tetL and tetX,respectively.And the changes of the relative abundance of the main drug resistance gene tetB in vermicompost were closely related to the changes of the relative abundance of Streptomyces.Changes in relative abundance of tetZ correlated with changes in relative abundances of Humibacter and Pseudomonas.The relative abundance of tetX was closely related to the relative abundance of Klebsiella et al. |
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