| Antibiotic growth promoters(AGPs)are widely used in livestock and poultry diets because of their low price and good growth-promoting effect.Pork is the main source of animal protein for humans,and the amount of antibiotics used in pig production each year is huge.However,in recent years,infections caused by resistant bacteria have seriously threatened the safety of human life and property.Antibiotics can promote the acquisition and maintenance of bacterial resistance,so reducing the use of antibiotics can effectively alleviate bacterial resistance.China began to ban the addition of AGPs in feed in July 2020.According to the experience of AGPs restriction in Europe and some studies done in our country,after the prohibition of AGPs,livestock and poultry become more prone to pathogen infection,diarrhea and even death,especially for weaned piglets whose digestive and immune systems are immature.Banning AGPs will cause serious economic losses to farmers.Therefore,the animal husbandry industry urgently needs to find effective AGP alternative to alleviate the pressure caused by the prohibition of the AGPs.The mixture of lactulose and Bacillus coagulans as a synbiotic has the potential to become an AGPs substitute.However,there is a lack of systematic evaluation on the probiotic function of this synbiotic.So,in this study,we added this synbiotic to the feed of weaned piglets,while using chlortetracycline as a positive control,to comprehensively evaluate the probiotic effects of this synbiotic on microorganism composition,fecal metabolism,and bacterial resistance through metagenomics,metabolomics,and other methods.We hoped this study would provide a theoretical basis for the research of AGPs substitutes in the animal husbandry industry.This study was composed of three following parts:1 Effects of the synbiotic containing lactulose and Bacillus coagulans on fecal microflora composition,function,and fecal metabolism of weaned pigletsThe first experiment was aimed to explore the effects of the synbiotic containing lactulose and Bacillus coagulans on fecal microflora composition,function,and fecal metabolism of weaned piglets.Eighteen[Duroc×Landrace×Yorkshire]weaned castrated boars(at 27 or 28 days of age)with similar body weights were selected and randomly divided into 3 groups with 6 replicates in each group.The control group(CTR)was fed with a basal diet(antibiotic free),the antibiotic group(ANT)was fed a basal diet supplemented with 75mg/kg chlortetracycline,and the synbiotic group(SYN)was fed a basal diet supplemented with lactulose at 10 g/kg and Bacillus coagulans at 2×10~9 CFU/kg.The test lasted 29 days,and the fecal samples were collected on d29.Metagenomics,metabolomics and gas chromatography were employed to detect changes in microbiota and metabolites.The results showed that,in microbiology,the content of Bacillus coagulans in the SYN group was higher than that in the ANT and CTR groups(P<0.05),while no difference was detected between the ANT group and the CTR group(P>0.05).In addition,the results of microbial KEGG function analysis showed that,compared with the CTR group,the SYN group had a significant impact on seven microbial functional pathways,including glycine serine and threonine metabolism(ko00260),phenazine biosynthesis(ko00405)and biotin metabolism(ko00780)(P<0.05),while no difference was detected between the ANT and CTR groups(P>0.05).Compared with the ANT group,the biosynthesis of neomycin,kanamycin and gentamicin(ko00524),pantothenic acid and coenzyme A biosynthesis(ko00770)pathway were significantly enriched in the SYN group(P<0.05).Compared with the CTR group,both the ANT group and the SYN group had increased fecal contents of L-pipecolic acid and pyridoxamine(P<0.05).Compared with the ANT group,the SYN group had improved the contents of three organic acids(such as linoleic acid),D-lactose,cyclohexylamine,nicotinate,adenine,and N6,N6,N6-trimethyl-L-lysine(P<0.05).In addition,5-aminovaleric acid,D-galacturonic acid,and 2-oxoadipate in the SYN group were lower than those in the ANT group(P<0.05).The KEGG pathway enrichment analysis showed that compared with the CTR group,the lysine degradation pathway was enriched in both the SYN and ANT groups(P<0.05).Three additional pathways were enriched in in the ANT group,including phenylalanine metabolism,vitamin digestion,and arginine and proline metabolism(P<0.05);Six pathways were enriched in the SYN group,including galactose metabolism,taste transduction,carbohydrate digestion and absorption,ABC transporters,tryptophan metabolism,and starch and sucrose metabolism(P<0.05).Compared with the ANT group,four pathways were enriched in the SYN group including lysine degradation,linoleic acid metabolism,ABC transporters and unsaturated fatty acid biosynthesis(P<0.05).There was no significant difference in the concentration of SCFAs among the three groups(P>0.05),except that the proportion of propionic acid in the SYN group was lower than that in the ANT group(P<0.05).Based on the correlation analysis,Bacillus coagulans is significantly related to a variety of metabolites involved in nutrient transport,carbohydrate metabolism,amino acid metabolism and appetite regulation.In addition,Bacillus coagulans is also significantly associated with microbial glycine,serine and threonine metabolism(ko00260),neomycin,kanamycin and gentamicin biosynthesis(ko00524),biotin metabolism(ko00780).In summary,dietary supplementation of the synbiotic containing lactulose and Bacillus coagulans increased the content of Bacillus coagulans,enhanced the functions of amino acid metabolism and carbohydrate metabolism of the flora,and increased the content of various sugar and organic in the fecal samples of weaned piglets.2 Effects of the synbiotic containing lactulose and Bcaillus coagulans on antibiotic resistant genes in feces of weaned pigletsThe second experiment was aimed to explore the effects of the synbiotic containing lactulose and Bacillus coagulans on antibiotic resistant genes in feces of weaned piglets.The experiment design was the same as part one.The fecal samples on d29 of the experiment were collected,and the changes of antibiotic resistant genes were detected by the metagenomic sequencing.Compared to the CTR group,the abundance of genes conferring tetracycline resistance were significantly increased in the SYN and ANT groups(P<0.05).Compared to the CTR group,the composition of the plasmid and insertion sequences(ISs)were different in the SYN and ANT groups(P<0.05).The abundance of plasmid NZ＿LR536659.1,which carried a variety of resistance genes,was increased in the SYN and ANT groups(P<0.05).The abundance of thirteen ISs was different in the SYN and ANT groups(P<0.05),and ISs such as ISBco1,ISBco2,ISBco3,ISBco5 and ISBco6 were only detected in the SYN group.In conclusion,the synbiotic containing lactulose and Bacillus coagulans exerted high risks in ARGs enrichment and increased the burden on ARGs elimination.3 Effects of the synbiotic containing lactulose and Bacillus coagulans on Escherichia coli resistance in the feces of weaned pigletsThe third experiment was aimed to explore effects of the synbiotic containing lactulose and Bacillus coagulans on E.coli resistance in the feces of weaned piglets.The experiment design was the same as part one.Fresh feces samples of weaned piglets were collected on d1,d8,d15,d22,and d29 during the test period,and the chlortetracycline resistance rate of E.coli was tested.And after that,the antibiotic sensitivity of E.coli strains in the feces were detected.The resistance rate of fecal E.coli to chlortetracycline increased in the ANT group compared with the CTR group(P<0.05),while there was no differenence between the SYN and the CTR or the ANT group(P>0.05).The resistance rate of E.coli isolates remained at a high level,and the phenomenon of multi-drug resistance was prominent.The resistance rate of E.coli isolates to gentamicin and ceftiofur decreased over time(P<0.05).In summary,the replacement of antibiotics with the synbiotic containing lactulose and Bacillus coagulans in the diet alleviate the resistance of fecal E.coli of weaned piglets. |
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