| In recent decades,antibiotic contamination has become an urgent problem.In aquatic environment,long-term antibiotic stress may also disrupt the structural balance of the fish's gut flora.The intestinal tract of aquatic animals were usually regarded as a significant niche for dissemination of ARGs and direct response of living conditions.Tetracycline is the second most frequently detected antibiotic in the environmental matrix.However,a comprehensive study that the effects of tetracycline on fish intestinal bacterial antibiotic tolerance and intestinal flora structure is still lacking.In this study,goldfish were used as the test subject and treated with different concentrations of tetracycline?0.285,2.85?g/L?for 21 days.Gut samples were obtained on the 7th and 21st days,respectively.The platelet coating method was used to calculate the percent of cultivable intestinal antibiotic resistance bacteria.The gyrB primers were used to identify Aeromonas separated from the plates,meanwhile,the physiological and biochemical tests and 16S rRNA gene sequence were further used.The broth microdilution method was used to measure minimum inhibitory concentrations?MICs?.The fifteen ARGs encoding tetracycline resistance?tet A,tetB,tetC,tetG,tetK,tetL,tetM,tetO,tetQ,tetW,tetZ and tetX?,sulfonamide resistance?sul1,sul2and sul3?and integrons?intI1 and intI2?were quantified by real-time fluorescent quantitative PCR technique?qRT-PCR?.Finally,the intestinal flora structure were characterized by high-throughput sequencing.The main findings are as follows:1.The proportion of resistant bacteria in the low concentration group?L21?and the high concentration group?H7 and H21?group increased significantly after 21 days,increasing to 23.0%;38.43%,62.05%,respectively.This indicates that the treatment of goldfish by tetracycline can significantly increase the resistance rate of intestinal culturable bacteria.2.In this study,21,15,47,26,36 and 19 strains of Aeromonas were isolated in the C7,L7,H7,C21,L21 and H21 groups,respectively.A total of six Aeromonas bacteria were identified,including A.salmonida,A.hydrophila,A.caviae,A.media,V.schubertii and V.sobria.A.schubertii and A.salmonicida in the tetracycline treatment group gradually grew into dominant species.With the increase of tetracycline concentration and treatment time,the dominant species changed to A.Schubertii.3.Among the 47 strains isolated from the control group,38 strains had MIC values of32?g·mL-1,and the remaining 9 resistant strains had MIC of 64?g·mL-1.However,the MIC values of the resistant strains in the L7 and H7 groups increased to 128?g·mL-1.The MIC values of the 1/4 resistant strains in the H21 group were as high as 256?g·mL-1,indicating that sustained tetracycline stress can significantly increase the bacteria tolerance.4.Except for tetB,all target genes were detected in the intestine of goldfish.The relative abundance of most genes increased significantly after tetracycline treatment.Among them,the relative abundance of tetA,tetC,tetG,tetZ and tetO was the highest(higher than 1.0×10-2),indicating that antibiotic pressure is the main cause of increased abundance of resistance genes.In addition,the average relative abundance of tetracycline efflux RGs(1.0×10-2)was significantly higher than that of the encoded ribosome protective protein(average level 1.0×10-3)and encoding tetracycline inactivating enzyme(average level 1.0×10-5)genes.This indicated that the tetracycline efflux pump mechanism is the primary mechanism respond to environmental tetracycline stress.5.Fusobacteria,Firmicutes and Proteobacteria are the three dominant species in goldfish intestinal microbes,accounting for 85.27%to 96.79%of the total abundance.Under the treatment of tetracycline,their diversity were increased,decreased and balanced,respectively.The genus also showed a similar trend.The LEfSe results showed that Fusobacterium,Kaistia,cetobacterium,Meganema,Aminobacter and Bosea were the dominant genus of the goldfish gut in the treatment group.In addition,there is a strong correlation between ARGs and genus Cetobacterium,Rosemonas,Rhizobiales and Bosea,indicating that these genus are potential hosts for ARGs.This study explored the effects of environmental concentrations of tetracycline on gut microbiota resistance and intestinal flora structure.This results are useful for understanding the interaction among the three factors,and will contribute to the formulation of strategies to control antibiotic resistance and the assessment of human health risks. |
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