Effects Of Difloxacin On Rumen Microbial Structure And CAZy For Yak And The Effects Of Three Stimulating Factors On Their ARGs

As a primary consumer,ruminants play a vital role in the energy flow and material circulation of the ecosystem,and their rumen microorganisms play a key role in these functions.Ruminants,especially domestic ruminants,have their rumen microbes directly affected by the use of antibiotics,and inevitably come into contact with environmental antibiotic residues and pollutants such as mycotoxins,which may harm the health of the organism and the stability of the ecosystem.In order to reveal the effects of difloxacin（DIF）on the rumen microbial structure and carbohydrate enzymes（CAZy）,and to study the characteristics of DIF,cefquinome（CEF）and aflatoxin B1（AFB1）on the resistance types and resistance mechanism types of antibiotic resistance genes（ARGs）in ruminants,fifteen 42-month-old,healthy,well-developed male yaks were selected and randomly divided into 5 groups.According to the recommended dosage of the instructions,the Cef group and the Dif group were orally administered with CEF 1 1mg·kg^-1and DIF 1 ml·kg^-1,respectively;E1 groups and E2 groups were fed AFB₁20μg·kg^-1and 60μg·kg^-1,respectively;Group C was the control group.The rumen fluid samples of yaks were collected and DNA was extracted.Genomic sequencing and bioinformatics analysis of rumen microorganisms was performed.The results showed that:1)The original data was 185.3G by sequencing,and the data after quality control was 74.2G.The average data quality value has little fluctuation and the base quality is stable.A total of 909634554 reads were obtained,9,171,462 contigs were assembled,and 11,933,585 ORFs were predicted.2)At the level of phylum,DIF decreased the relative abundance of Bacteroides,Proteobacteria,etc.,while the abundance of Firmicutes,Euryarchaeota etc.increased.At the level of genus,DIF decreased the relative abundance of Prevotella,Bacteroides,while the abundance of Chryseobacterium,Streptococcus,etc.increased.3)Among the CAZy,the abundance of CBM17,CBM25 etc.was up-regulated （P<0.01 or P<0.05）;The abundance of GH14,GH53,CE8,CE13,CBM21,CBM26and dockerin etc.were all down-regulated（P<0.01 or P<0.05）.4)In ARGs species,CEF increased tet W gene abundance（P<0.05）,and DIF increased tet Q abundance（P<0.05）;5)In ARGs resistance types,CEF increased the abundance of tetracycline antibiotic and cephalosporin antibiotic resistance genes（P<0.05）.DIF increased the abundance of tetracycline antibiotic and aminocoumarin antibiotic resistance genes（P<0.05）.When the dose of AFB₁was 20μg·kg-1,the abundance of aminocoumarin antibiotic and penem antibiotic resistance genes increased（P<0.05）.When the dose of AFB1 was 60μg·kg-1,the abundances of 9 resistance genes such as penems antibiotic and cephalosporins antibiotic increased（P<0.05）.6)In ARGs resistance mechanism types,CEF increased the abundance of tetracycline-resistant ribosomal protection protein（P<0.05）,DIF increased the abundance of Erm 23S ribosomal RNA methyltransferase and tetracycline-resistant ribosomal protection protein（P<0.05）.When the dose of AFB1 was 60μg·kg-1,the abundance of three resistance mechanisms such as ATP-binding cassette（ABC）antibiotic efflux pump increased（P<0.05）.7)All three treatments significantly increased the host species of tetracycline antibiotic resistance genes.In conclusion:Difloxacin（DIF）affected the rumen microbial structure,and changed the relative abundance of the dominant phylum and genus,and inhibited the function of some beneficial bacteria.It also changed the gene abundance of some family in CAZy,which hindered the degradation of starch and pectin by rumen microbes,and reduced the efficiency of microbial degradation of plant cellulose.Not only CEF,DIF increase the abundance of some ARGs species,resistance types and enzymes related to resistance mechanisms,thereby increasing rumen microbial resistance,but also AFB₁has a similar effect.Moreover,the high-dose AFB₁has a greater range of effects on antibiotic resistance than antibiotics.These three factors also cause new types of host microorganisms carrying a tetracycline antibiotic resistance gene,thereby enhancing the horizontal gene transfer,accelerating the spread of ARGs,and enhancing the resistance of microorganisms to tetracycline antibiotics.