Ecological Function Diversity Of Intestinal Bacterial Community In Earthworm Under Soil Antibiotic Stress

With the increasing application of organic fertilizer recently,a large amount of residual antibiotics has been released into the environment,therein posing great threat against both environment security and public health.Therefore,it is of great significance to investigate the toxicity of antibiotics on the widely-applied bioindicator-earthworm.As bionidicator of soil contamination,earthworm gut also played an important role in the transformation of various contaminants in soil environments.The ingestion of antibiotics,antibiotic resistance bacteria(ARB),and antibiotic resistance genes(ARGs)made the earthworm gut a potential favorable micro-environment for the transmission of ARGs in the soil.However,little attention has been paid to the earthworm's largest organ—the intestine,and the microbiome that colonize it.The main objectives of this study were to 1)the toxicity of soil tetracycline(TC)towards earthworm coelomocytes;2)investigate the diversity of the bacteria community inhabiting the earthworm gut;3)changes in the composition of the bacterial community and its functions in the earthworm gut in response to varying tetracycline stress;4)tetracycline resistance of earthworm gut and soil microbes.The main results showed that:(1)A total of 87 cultivable isolates that resistanted tetracycline(TC)and/or sulfadiazine(SD)were obtained,most of which belonged to phylum Firmicutes,genus Bacillus.Meanwhile,the counts of isolates with TC-SD dual resistance were higher than those with sole SD or TC resistance.Moreover,higher ARB counts and diversity were detected in the earthworm gut by high-throughput sequencing technique than those by the classical plate cultivation.(2)Tetracycline residues were detected in the intestines of earthworms under different experimental treatments.With the increase in tetracycline concentration,the intestinal enzyme activity of earthworm in the filter paper experiment showed a trend of increasing first and then decreasing.However,the intestinal enzyme activity of earthworm increased all the time in the soil culture experiment.In the present study,the physiological state of the earthworm coelomocytes was characterized using a flow cytometer,and the toxicity of TC towards earthworm coelomocytes is dose-and time-dependent.(3)When TC concentration increased from 0 to 35.7 ?gcm-2,the percentage of the Proteobacteria phylum declined significantly,while the proportions of the Firmicutes,Planctomycetes and Atinomycete phyla clearly increased(p<0.05).Meanwhile,the levels of TC resistance genes tetC,and tetW increased with the increasing TC concentration,in contrast to the declined abundance in denitrifying genes nirS and nosZ(p<0.05).The watersoluble TC fraction was the main stimulant for ARG expression in the gut.The genes level was also correlated with the abundance of Firmicutes and Proteobacteria.(4)A total of 129 ARGs were detected in the earthworm gut and adajacent soil.Earthworm gut bacteria increased the abundance of 71 ARGs in soil,of which the Planctomycetes played a major role.Species harboring both ARGs and functional genes were detected in 20 phyla.The functional genes in earthworm gut were mostly positively correlated with ARGs,while the forest soils were mostly negatively correlated.The abundance of antibiotic efflux ARGs in earthworm intestinal was 10.1%lower than that of soil,and the antibiotic target protection ARGs was almost twice that of other treatments.In addition,60 functional genes were detected in earthworm gut and soil,and the functional genes coexisting with ARGs weremainly involved in metabolic pathways with glycolysis,nitrate to nitrite and sulfate to ammonium persulfate.Overall,the combination of conventional cultivable bacteria isolation and highthroughput sequencing methods provided a comprehensive understanding of the ARB composition in the earthworm gut.The results demonstrate that the earthworm gut is a hospitable micro-environment for ARB colonization.The potential role of earthworm intestinal ARB and ARGs proliferation in soil environments warrants further research.Meanwhile,these results provide new ways to investigate the ecotoxicological risks posed by residual tetracycline in environment.