Intervention Of Konjac Glucomannan And Its Derivatives On Antibiotic-Induced Gut Microbiota Disorders And Resilience

The inappropriate use and abuse of antibiotic caused increasing clinical bacteria antibiotic resistance and the flourish of multidrug-resistance bacteria,leading to a dramatic rise in global mortality rates and a huge social and economic burden.However,most current studies are more inclined to the distribution and spread of Antibiotic Resistance Genes（ARGs）in the natural environment,the distribution characteristics of gut ARGs and the changes of intestinal flora and ARGs under antibiotic stress are less concerned.Dietary fibers have been widely used in gut microbiota regulation,further involved in the modulation of host immunity and metabolic pathways to treat or prevent diseases.Nevertheless,the underlying mechanisms of dietary fibers in the alleviation of antibiotic induced dysbiosis are unclear,let alone their effects on ARGs.Antibiotics can not only kill pathogenic bacteria and also affect other symbiotic bacteria.Intestinal microorganisms,mainly obligate anaerobic bacteria,colonize the gut mucosal epithelium surface to form a biofilm,which prevent the adhesion and colonization of opportunistic pathogens with "colonization resistance",any force that changes the intestinal micro-ecological environment to disrupt the normal intestinal flora structure,such as long-term and/or large-term use of antibiotics,can cause the passage of pathogenic microorganisms from the gastrointestinal（GI）tract to extraintestinal sites,such as the mesenteric lymph node complex（MLN）,liver and spleen through the damaged mucosal barrier,thus bacterial translocation（BT）occurs,and further triggers inflammatory responses,posing a potential risk to host health.Deacetylated Konjac Glucomannan（DKGM）was produced from native Konjac Glucomannan（KGM）under the treatment of alkali and heating.Konjac Oligo-glucomannan（KOGM）could be obtained by enzyme,acid,irradiation and/or mechanochemical degradation from KGM.Nowadays,KGM,DKGM and KOGM have been three main application forms of konjac in food industry,with large market demand and great development potential.Preliminary studies have shown that they all have certain probiotic effects,but no report focused on their role in the regulation and recovery of antibiotic induced gut microbiota and ARGs disorders.While polysaccharides have been proven to repair intestinal mucosa,no studies have reported the effect of KGM and its derivatives on BT.Based on the extensive research background of prebiotics in regulating gut microbiota,in the treatments of clinical postpartum,postoperative and intestinal diseases,doctors may recommend prebiotic supplements（such as inulin and fructo-oligosaccharides）to help restore the disordered microbiota while taking antibiotics.However,there are few studies on whether dietary fiber may affect the pharmacokinetics of antibiotics in vivo.Based on the above research background,in this study,firstly,DKGM and KOGM were prepared,and their structures and physicochemical properties were characterized and compared with native KGM.An in vitro fermentation was then carried out to explore the link between the structure and properties of KGM and its derivatives and their role in regulating gut microbiota.pH values,gas production,short-chain fatty acids（SCFAs）content,and gut microbial diversity were analyzed.Next,amoxicillin,neomycin sulfate,vancomycin and metronidazole were selected to prepare an antibiotic cocktail（Abx）.Metagenomics and metabolomics were applied to study the effect of KGM and its derivatives on the regulation and recovery of Abx-induced disturbance of gut microbiota and ARGs in mice.Besides,the impact of KGM and its derivatives on BT and inflammation caused by antibiotics were also emphasized.Finally,the effect of KGM and its derivatives on pharmacokinetics of antibiotics was investigated.This study will gain a comprehensive understanding of the intervention of KGM and its derivatives on gut microbial dysbiosis and recovery,lay a foundation for mitigating the long-term negative effects of antibiotics on gut microbiota,and provide theoretical support for the better use of antibiotic and disease control.The main findings were as follows:（1）Structural alterations of KGM and its derivatives changed their molecular chain aggregation and water binding ability,thus affected the susceptibility to enzymatic degradation,leading to the distinct microbial composition and outcomes profile in fermentation.KOGM with relatively simple structures was rapidly utilized by gut microbes,followed by structurally disordered KGM and more compact,ordered DKGM.The fermentability of KGM was higher than that of KOGM,followed by DKGM,and the deacetylation reaction weakened the fermentability of KGM.（2）Antibiotic treatment caused gut microbial disturbances,characterized with significantly reduced microbial diversity.Proteobacteria became the dominant phylum instead of Bacteroidetes,while the proportions of Bacteroidetes and Firmicutes were greatly reduced,so that a variety of ARGs,mainly multidrug-resistant ARGs,were enriched.KGM and its derivatives did not significantly increase gut microbial diversity during antibiotic administration,but altered the composition of gut microbiota,thereby driving ARGs to more normal ratios.By inhibiting antibiotic-induced flourish of Proteus_f<sub>Morganellaceae,Proteus_mirabilis,Escherichia_coli,unclassified_g<sub>Escherichia,unclassified_o<sub>Enterobacterales,unclassified_g<sub>Klebsiella,Klebsiella_michiganensis,Morganella_morganii,unclassified_f<sub>Enterobacteriaceae and other species in phylum Proteobacteriaceae,KGM and its derivatives reduced antibiotic-induced multidrug resistant ARGs,such as msbA、adeL、arlR、evgS、cpxA、farA genes,which develop drug resistance with increased efflux pump expression and vmlR gene,which alters antibiotic target.In addition,the intervention of KGM and its derivatives could retain a certain structure of normal intestinal microorganisms,and selectively control the ARGs host of phylum Proteobacteria,thus reduced the gene abundance of vanHO（by changing the target of antibiotic resistance）.bcrA（by increasing efflux expression）,and kdpE（by increasing the efflux pump expression）,and decreased the resistance of gut microbes to glycopeptide,peptide and aminoglycoside antibiotics.（3）Antibiotic treatment resulted in the disorder of multiple metabolic pathways.The intervention of KGM and its derivatives maintained the metabolism of primary bile acids to secondary bile acids.Besides,KGM and its derivatives significantly promoted the metabolism of flavonoids,and significantly increased the level of propionic acid,concurrently they urged the disordered metabolism of the glycerophospholipids caused by antibiotics to a more normal status.（4）When the antibiotic administration was stopped,the microorganisms in each group were recovered to a certain extent after a 14-day recovery period,but the diversity was not completely recovered.Natural recovery has led to the abnormal proliferation of genus Bacteroides,while other genera did not recover well,this resulted in the higher abundance of multidrug-resistant ARGs than that in normal condition.Akkermansia_muciniphila,from phylum Verrucomicrobia,overgrew too in natural recovery（AN group）.But the species in phylum Firmicutes and unclassified_dBacteria recovered poorly.The intervention of KGM and its derivatives decreased the proportion of Bacteroidetes and Verrucomicrobia,increased the proportion of Firmicutes,and promoted the recovery of species with less resilience,such as Muribaculum_sp._NM65_B17,Prevotella_sp._CAG:873,Muribaculaceae_bacterium,Muribaculaceae_bacterium_Isolate104_HZI,and Porphyromonadaceae_bacterium,pushed the microbial composition ratio to a more normal level.By controlling the reproduction of the above ARGs host,the intervention of KGM and its derivatives significantly reduced the abundance of Multidrug-resistant ARGs,such as baeS（resistance by increasing the expression of efflux pumps）,optrA（resistance by protecting the target of antibiotic action）,evgS（resistance by increasing efflux pump expression resistance）and tetracycline ARGs,such as tetA（58）（resistance by increasing efflux pump expression）.They also promoted the restoration of genes such as oleC（resistance by increasing efflux pump expression）of MLS ARGs,TaeA（resistance by increasing the expression of efflux pumps）of pleuromutilins ARGs,ugd（resistance by changing the target of antibiotic action）of peptides ARGs,Streptomyces rishiriensis parY mutant conferring resistance of aminocoumarins（resistance by changing the target of antibiotic action）of aminocoumarin ARGs.Among them,the KK group,DD group,and KOKO group,in which KGM and its derivatives were continuously administrated during and after antibiotics,had better reconstruction effects than the KN group,DN group,and KON group,in which KGM and its derivatives were only administrated during antibiotics treatment.（5）KGM and its derivatives could mitigate the destruction of colonic tissue caused by antibiotics through increasing the production of SCFAs,alleviating gut microbiota disturbance,and promoting the recovery of gut microbiota.Thereby KGM and its derivatives reduced BT incidence and the following inflammatory responses.（6）The effects of KGM and its derivatives on the absorption,distribution and elimination of oral administrated antibiotics varied with the property of dietary fiber and the types of antibiotics.The ingestion of KGM and its derivatives improved the absorption of metronidazole,but did not affect the absorption of neomycin sulfate and vancomycin,thereinto,KGM hindered the absorption of amoxicillin.KGM and its derivatives had no effect on the in vivo distribution of amoxicillin and metronidazole,but DKGM and KGM hindered the distribution of neomycin sulfate and vancomycin,respectively.KGM and its derivatives promoted the elimination of amoxicillin.KGM and KOGM promoted the elimination of neomycin sulfate and vancomycin,but DKGM had the opposite effect.KGM and its derivatives prolonged the elimination half life（t1/2β）of metronidazole to varying degrees,the order from short to long is DKGM<KOGM<KGM.