| China is the largest country in aquaculture and shrimp farming is a pillar industry for aquaculture.Antibiotics are widely used to prevent and treat bacterial diseases susceptible to shrimp.Due to the abuse of antibiotics,the antibiotic contamination in shrimp and its culture environment,and a large number of antibiotics resistance bacterium(ARB)and antibiotics resistance genes(ARGs)are induced.ARGs is ubiquitous in aquaculture environment and aquaculture products.The food safety problem caused by ARGs pollution in shrimps seriously threatens human health.In this study,the ARGs diversity of shrimps intestinal tract was studied by traditional molecular ecology method,traditional culture method and high throughput sequencing analysis of plasmidome.The total genomic DNA of intestinal microorganism was extracted from 4 vannamei products sold in Guangzhou,and the species and abundance of ARGs were determined by PCR and Q-PCR.We screened and purified 4 kinds of ARB and identified their species,and determined the antibiotic resistance phenotype and genotype of ARB.With mature commercial shrimp,immature healthy shrimp and immature sick shrimp as experimental materials,the method of extracting the plasmidome DNA of intestinal microorganism was established,and the high throughput sequencing and data analysis of the plasmidome DNA of 3 samples were carried out.By PCR,20 of the 30 ARGs of the tetracycline,sulfonamides,quinolones,macrolides,β-lactam and vancomycin were detected.Eight ARGs(tetA,tetB,tetC,tetE,sulⅠ,sulⅡ,qnrS,ermB)were detected in 4 samples,and there was no significant difference in gene copy numbers between groups.The copy number is sorted as tetA≈tet B>sulⅠ>tetE>sulⅡ>qnrS.ARB was screened with tetracycline,sulfadiazine,ciprofloxacin,and erythromycin.The selection rate was tet ARB> qnr ARB> erm ARB> sul ARB under aerobic conditions,and tet ARB> qnr ARB> erm ARB> sul ARB under aerobic culture.57 ARB strains were screened and classified into 10 genera by colony morphology observation and 16 SrDNA homology analysis.Aeromonas was the dominant genus.The resistance phenotype and genotype of 10 strains of ARB were basically consistent,there were ARB with genotype only or genotype only although.After plasmidome sequencing analysis,high diversity and abundance of ARGs were identified in the plasmidomes.The microbial diversity was ranked as mature commercial shrimp> immature sick shrimp > immature healthy shrimp.The diversity of ARGs was ranked as immature sick shrimp > mature commercial shrimp> immature healthy shrimp.The top ten resistant types were quinolone,elfamycin,chlorampheicol,multidrug,tetracycline,aminocoumarin,peptide antibiotic,triclosan,macrolide and nitroimidazole.The Vibrio would be the bacterial pathogen resulting in shrimp death,because this bacterial group held high abundance of ARGs,especial the multidrug resistances. |
