| Antibiotic resistance genes?ARGs?have become a new environmental pollutant endangering public health,which is induced by the abuse of antibiotics in the medical treatment and cultivation.Nowadays,there are few reports revealed drug-resistant bacteria and ARGs pollutions in aquatic products.The results of studies show that ARGs pollution is serious and various in aquaculture water and sediments.As a result,it is necessary to carry out more researches on the distribution of drug-resistant bacteria and the pollution status of ARGs of aquatic products,to set up the method for ARGs detection.In this study,we focused on six major aquatic products in Zhejiang province including Larimichthys crocea,Carassius auratus,Pelteobagrus fulvidraco,Mylopharyngodon piceus,Misgurnus anguillicaudatus,Macrobrachium rosenbergii.The drug-resistant bacteria were picked up from aquatic products using agar plates containing antibiotics?sulfadiazine,gentamycin sulfate,enrofloxacin,cefalexin,oxytetracycline,chloramphenicol?,then isolates were identified based on 16S rDNA gene sequencing.Bacterial strains were tested for their susceptibility to compound sulfamethoxazole,gentamycin,enrofloxacin,cefalexin,tetracycline and chloramphenicol.ARGs including sul2,aac?6'?-Ib,qnrS,blaPSE,tet A,cmlA,floR,were detected by PCR amplification.The coincidences of resistance phenotype and genotype for the isolates were analyzed.Finally,three multiplex PCR systems were established for aac?6'?-Ib,cmlA,tet A,floR,sul2,int?1,and quantitative real-time PCR systems were developed to detect and quantify tetA,cmlA,aac?6'?-Ib,sul2,int?1,qnrS,blaPSE.The main results were generalized as follows:1.136 culturable drug-resistant bacteria were picked up by agar screen plates and were classified into 22 genera,49 species according to the 16S rDNA sequencing data.Most of which belonged to genus of Aeromonas and Shewanella,which accounted for 30.1%and14.0%.Among them,29.3%Aeromonas were oxytetracycline-resistant strains,and most of Shewanella were chloramphenicol-resistant or sulfadiazine-resistant.The dominant drug-resistant bacteria in different samples were different.In the Larimichthys crocea,Shewanella were dominant.In the Carassius auratus and Mylopharyngodon piceus,Aeromonas were the dominant bacteria.Most of strains belonged to Pseudomonas in the Peltebagrus fulvidraco,and Aeromonas and Myroides marinus were the dominant bacteria in the Misgurnus anguilicaudatus.In the case of Macrobrachium rosenbergii,Citrobacter were more common.2.The resistance of isolates to 6 antibiotics was different.The resistance rates of the isolates to gentamycin were less than 30%,and the resistance to compound sulfamethoxazole and cefalexin were serious.The resistance rates of the isolates to cephalexin were between 4%and 60%,and resistance rates of the isolates to compound sulfamethoxazole were between10%and 59%.Some of the isolates were resistant to 5 antibiotics.In the Peltebagrus fulvidraco and Misgurnus anguilicaudatus,multi-drug resistance strains were not detected,and the multi-drug resistance rates of the strains from Larimichthys crocea,Carassius auratus,Mylopharyngodon piceus,Macrobrachium rosenbergii were no less than 25%.The coincidence rates between resistant phenotypes and genotypes of enrofloxacin,tetracycline and chloramphenicol were high,ranging from 67%to 79%.3.Three multiplex PCR detection systems for ARGs were established.System 1 could detect aac?6'?-Ib,cmlA,sul2,int?1 simutaneously.System 2 was established for aac?6'?-Ib,cmlA,floR,int?1 and system 3 was established for aac?6'?-Ib,tet A,int?1.These multiplex PCR systems had good specificity and high sensitivity.According to the established multiplex PCR systems,the commercial Macrobrachium,Carassius auratus,Pelteobagrus fulvidraco and Larimichthys crocea were detected.The results showed that all samples habored sul2,int?1,tetA,floR,and the detection rates of aac?6'?-Ib and cmlA were low.4.Quantitative real-time polymerase chain reaction used to quantify tetA,cmlA,aac?6'?-Ib,sul2,int?1,qnr S,blaPSE was established.The detection limit of blaPSE was6.6×104 copies/?L,and the detection limits of other ARGs were between 11.5 and 202copies/?L.Among the four kinds of aquatic products,the relative abundances of blaPSE were high,ranging from 1.17×10-44 to 5.96×10-3,and the relative abundances of aac?6'?-Ib were low,ranging from 2.38×10-88 to 6.71×10-7.In this study,only the correlation between the relative abundance of ARGs and int?1was analyzed,and the correlation between the amount of antibiotic residues and the content of related drug resistance genes in the samples can be further analyzed.We can continue to optimize the qPCR reaction system,establish a multiplex real-time PCR method to detect and quantify ARGs at the same time. |
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