| β-Lactam antibiotics are commonly used in livestock and poultry breeding.There are many residues in livestock and poultry feces.When they flow into the environment through feces,it is easy to increase the drug resistance of environmental bacteria and pose a potential threat to public health.At present,the emerging treatment technology of Hermetia illucens shows the advantage of high efficiency and rapid degradation in the early research of tetracycline degradation.Therefore,in this paper,the degradation characteristics,intestinal microbial function,screening of efficient degrading bacteria,antibiotic metabolites and degradation pathways of amoxicillin and penicillin sodium inβ-lactam antibiotics were studied by using black soldier fly larvae(BSFL)transformation system.The main results are as follows:(1)Characteristics of efficient degradation of amoxicillin and penicillin sodium by BSFLThe results showed that after 12 days of transformation,the total degradation rates of amoxicillin and penicillin sodium were 71.00% and 80.89% respectively,which were 4.1times and 2.2 times higher than that of the natural fermentation control group,indicating that the BSFL significantly promoted the biodegradation rate of the two antibiotics.At the same time,the relative expression of β-lactamase gene in the feed was detected by q PCR technology.The results showed that compared with the control group,the expression of related β-lactamase genes amp C/bla DHA,bla OXA10-01,bla-L1 and Cep A in amoxicillin group and penicillin sodium group treated with BSFL were significantly up-regulated,indicating that the transformation of BSFL improved the biodegradation activity of related microbes to β-lactam antibiotics.(2)Community succession and functional analysis of intestinal flora of BSFLIn this study,16 S rDNA high-throughput sequencing technology was used to study the succession of intestinal bacterial community in the process of transforming amoxicillin,penicillin sodium and non-antibiotic feed.The results showed that compared with the non-antibiotic addition group,the intestinal flora of BSFL increased significantlyα diversity increased significantly in the first 8 days of treatment,but decreased significantly in the 12 th day.At the same time,29 unique OTUs of Faecalis,Zymomonas and Micrococcus were found in the intestinal microbes of BSFL in the amoxicillin experimental group,while 33 unique OTUs of Enterobacter,Romboutz and Pseudopallidobacterium were found in the sodium penicillin experimental group.There were 72 core OTUs in the three groups,mainly Proteus,Paenochrobactrum and erysipelas,etc.The results showed that 19 strains and 9 strains of bacteria of different genera showed significant positive correlation with the concentrations of amoxicillin and penicillin sodium respectively,indicating that they may be potential amoxicillin and penicillin sodium degrading bacteria.(3)Screening of efficient degrading bacteria of amoxicillin and penicillin sodium in the intestinal tract of BSFLFive β-lactam antibiotic degrading bacteria were isolated from the intestine of BSFL by selective culture technique.It was identified by 16 S rDNA as BSFL-LAC1 is Morganella morganii,BSFL-LAC2 is Bacillus velezensis,BSFL-LAC3 is Mesobacillus subterraneu,BSFL-LAC4 is Trichophyton mentagrophyte,BSFL-LAC5 is Lichtheimia corymbifera.Through the degradation test in vitro,the results showed that the degradation rates of amoxicillin and penicillin sodium were 16.48%-95.87%.Among them,Morganella morganii BSFL-LAC1 had the best degradation effect,with the degradation rates of 95.87% and 95.65% respectively.Further,the five strains were inoculated into the sterile BSFL transformation system as functional agents.The results showed that Morganella morganii BSFL-LAC1 and Trichophyton mentagrophyte BSFLLAC4 had the best degradation effect,and the degradation rate could reach more than68%,which was 2-3 times higher than that of sterile BSFL,which had better potential application value.(4)Speculation on the degradation pathway of amoxicillin and penicillin sodium by intestinal microbes of BSFLIn this study,LC-MS analysis technology was used to preliminarily analyze the metabolites and degradation pathways of amoxicillin and penicillin sodium degraded by intestinal microbes of BSFL.A total of 7 amoxicillin degradation intermediates and 5penicillin sodium degradation intermediates were identified.It is speculated that the degradation pathway mainly includes hydrolysis reaction,cyclization reaction,deamination reaction,ring opening reaction,oxidation reaction,decarboxylation reaction and electrophilic reaction,which destroys the cyclic structure of β-lactam,which further shows that the intestinal microbes of BSFL can effectively destroy the drug structure of β-lactam antibiotics,and its main role in promoting the degradation of β-lactam antibiotics by BSFL is analyzed from the perspective of material metabolism.To sum up,this subject proves that BSFL are sensitive to typical diseases β-lactam antibiotics have good degradation effect;According to the analysis of dynamic structure succession of intestinal microbes of BSFL,it is speculated that intestinal microbes of BSFL play an important role in the degradation of antibiotics;At the same time,five strains were screened from their intestines β-lactam antibiotics degrading microbes,among which Morganella morganii BSFL-LAC1 and Trichophyton mentagrophyte BSFL-LAC4 can be used as potential degradation promoting bacteria;At the same time,the intestinal microbes of BSFL.The main degradation pathways of β-lactam antibiotics include ring opening,hydrolysis and oxidation.The circular structure of β-lactam preliminarily speculated the metabolic pathway of amoxicillin and penicillin sodium.The results of this study provide a basis for the further use of BSFL and their intestinal microbes to efficiently degrade the pollutants in livestock and poultry feces β-lactam antibiotics provide technical and theoretical support and help China’s Rural Revitalization Strategy and the healthy development of green agriculture. |
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