| As one of the substitutes for antibiotics,microecologic agents have been widely used in the culture of Litopenaeus vannamei.Microecologic agents not only solve the potential harm of antibiotic residues to cultured animals and human beings,but also increase the number of probiotics and decrease the number of pathogenic bacteria by regulating the structure of microbial communities.Thus it played a role in the prevention and control of diseases.However,the quality of the microecologic agents is difficult to prove,and the presence of the drug-resistant bacteria in the microecologic agents can also pose a potential threat to the aquatic animal and the human health.Therefore,it is of great significance to study the drug resistance bacterias of the commercial aquatic microecological agents and study the effect of the drug-resistant probiotics to the structure of the microbial communities in the intestinal and the culture water of shrimp.It is of great significance to guide the production of the healthy microecologic agents.The thesis is divided into four parts:1.Isolation and identification of drug-resistant strains in microecological agents.The agar dilution method was used to screen 9 kinds of aquatic microecologic agents(Bacillus,Enterococcus and Lactobacillus)purchased on the market.The results showed that drug-resistant strains were isolated from 9 kinds of microecologic agents,and 100 strains were isolated.Among the 100 isolates,57 strains were Bacillus,and 23strains were Enterococcus.But only 44 strains are listed in the list of feed microbial strains specified by the Ministry of Agriculture in China in 2013.In addition,15pathogenic strains were isolated,and the proportion of pathogenic strains was 15%.This indicates that probiotics in aquatic microecological agents that bought from market may have potential safety risks.2.The resistance phenotype and genotype detection of the drug-resistant microecological agents.Drug sensitivity test was used to detect the drug resistance level of gentamicin,enrofloxacin,norfloxacin,florfenicol,sulfadiazine,sulfamerazine,tetracycline,terramycin,vancomycin,and then detect the resistance genes(fluoroquinolone,amphenicol,sulfonamide,tetracycline,aminoglycoside and glycopeptide)and mobile genetic elements by Polymerase Chain Reaction.The results showed that 100 isolates were resistant to 8 antibiotics except enrofloxacin.Among them,the drug resistance rate of florfenicol was the highest(100%),and the rate of sulfonamide resistance genes was also higher(99%).Inaddition,46%of the strains carried fluoroquinolone,amphenicol,sulfonamide,tetracycline,aminoglycoside and glycopeptide resistant genes.15 isolates carried 4 removable genetic elements simultaneously,and 95%carry more than one removable genetic element.This indicates that the drug-resistant genes in the microecologic agents has the potential to spread and transfer.3.Effects of drug-resistant probiotic to the microbial community in the intestinal of Litopenaeus vannamei.A Bacillus subtilis strain with tetM resistance gene on Tn916 transposon and tetM,tetA,ant(3')-Ia(aadA)resistance gene on plasmid was screened from the isolated strain.The concentration of Bacillus subtilis is 10~8CFU/g and fed to Litopenaeus vannamei.High-throughput sequencing technique was used to detect the changes of microbial community in the intestinal of shrimp at different feeding time and qPCR technique was used to detect the changes of drug-resistant gene abundance.The results showed that the abundance and diversity of Bacillus subtilis changed little in intestinal of Litopenaeus vannamei at 14d,but increased at 21d.In the classification of dominant bacteria,Proteobacteria was the main phylum in the bacteria group and the control group at different sampling time.As two probiotic rhizobium of Proteobacteria,the abundance of Cupriavidus was obviously lower in later feeding stage than that in the early feeding stage,and the relative abundance of Burkholderia in different samples was all lower than the control group.Vibrionaceae was also the main strain,and the relative abundance of Vibrio and Photobacteriumr in the intestinal tract was significantly higher than that of the control group in 42d.In addition,the relative abundance of tetM resistant genes in bacteria group detected was higher in 14d than that in the control group.The relative abundance of tetM resistance gene was positively correlated with the number of Comamonadaceae and Brucellaceae.Feeding with Bacillus subtilis could increase the number of pathogenic bacteria,decrease the number of probiotics and increase the resistance gene in intestinal of Litopenaeus vannamei,which would provide some reference for regulating the production of microecologic agents.4.Effects of drug-resistant probiotic to the microbial community in cultured water of Litopenaeus vannamei.A Bacillus subtilis strain with tetM resistance gene on Tn916 transposon and tetM,tetA,ant(3')-Ia(aadA)resistance gene on plasmid was screened from the isolated strain.The concentration of Bacillus subtilis is 10~8CFU/g and fed to Litopenaeus vannamei.High-throughput sequencing technique was used to detect the changes of microbial community in cultured water of shrimp at different feeding time and qPCR technique was used to detect the changes of drug-resistant gene abundance.The results showed that at 14d,21d and 42d after feeding with Bacillus subtilis,the abundance and diversity of microorganisms in shrimp culture water were significantly increased.At 54d,the diversity of microorganisms was larger than that of control group,but the abundance was a little less than that of control group.The microbiological community structure of14d was similar with 21d after feeding Bacillus subtilis,but 14d and 42d were significantly different from the control group,42d and 54d tended to be similar,but it was different from the early stage,the microbial community structure of bacteria group and control group turned to be similer at 54d.In the classification of dominant bacteria,Alphaproteobacteria was the most abundant group in the bacteria group and the control group at each sampling time,Rhodobacteraceae was the highest family in it,and the number of Rhodobacteraceae increased gradually following the continuous feeding.Flavobacteriaceae and Alteromonadaceae are also two kinds of familys with high relative abundance in water,it was found that the relative abundance of them decreased gradually with the continuous feeding of Bacillus subtilis.The abundance results of drug resistance genes showed that tetM resistance genes in bacteria group was higher than that of the control group on all samples,tetA and ant(3')-Ia(aadA)resistance genes were higher in the bacteria-fed group than in the control group on the 14d and 21d,but lower on 42d and 54d.No positive correlation was found between the drug resistance gene and microbe.The increase of the abundance of drug resistance gene may be related to the transfer of the resistance genes.Feeding with Bacillus subtilis could reduce the relative abundance of some probiotics and increased the abundance of drug-resistant genes,which would provide some reference for some departments to strengthen quality inspection of the microecological agents to avoid potential harm.We detected some drug-resistant genes and removable genetic elements in probiotics in aquatic microecological agents,which may increase the number of drug-resistant genes in breeding environments.At the same time,the presence of pathogenic bacteria in microecological agents may cause diseases in farmed animals as well as in humans.Feeding this probiotics to Litopenaeus vannamei will reduce the richness and diversity of intestinal microbial community structure,improve the richness and diversity of microbial community structure in shrimp aquaculture water.The abundance of drug resistance genes in intestinal tract and aquaculture waters of shrimp may increased due to the existence of probiotics containing drug-resistant genes and removable genetic elements. |
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