Phage Cocktail-intestinal Microbiota Transplantation For Experimental Treatment Of Bacterial Colitis And Its Molecular Mechanism

Salmonella is one of the major foodborne enteric pathogens causing food poisoning worldwide and the third most common cause of diarrheal deaths,posing a serious public health threat and causing significant economic losses.Although antibiotics have a therapeutic effect on infections caused by Salmonella,the misuse of antibiotics has led to drug resistance and antibiotic residues in medical clinics and livestock farming,making the prevention and control of Salmonella a major public health challenge worldwide.Therefore,the development of new antimicrobial agents is the focus of attention.Phages,viruses that specifically infect and lyse host bacteria,are an ancient therapy that is gaining renewed attention and promises to be a novel antibiotic alternative for combating bacterial infections,especially drug-resistant bacterial infections.Many studies have now confirmed the efficacy of phage therapy against certain bacterial-induced infections in experimental animals as well as clinical infections in humans.Moreover,phage cocktail therapy is usually better than single phage therapy in terms of therapeutic efficacy and delaying the production of phage-resistant bacteria.However,there are reports that even phage cocktails do not completely eliminate the bacteria,which suggests that monotherapy is hardly a foolproof solution.Combination therapy may be a major trend in future clinical treatment.There are reports of successful application of phage therapy in combination with antibiotics to effectively avoid the emergence of antibiotic-resistant bacteria and effectively control the emergence of phage-resistant bacteria.However,the indiscriminate bactericidal effect of antibiotics may aggravate the disorder of intestinal flora and may aggravate the disease,so the treatment effect is not satisfactory for the infection caused by intestinal pathogenic bacteria.The intestinal flora,as the"forgotten organ",has been shown to be associated with many gastrointestinal diseases.Restoration of its homeostasis can help the body to defend itself against many pathogenic and non-pathogenic diseases.Therefore,the association of phages with intestinal flora may be more effective in controlling infections caused by intestinal pathogens.To address this scientific question,this paper investigates the molecular mechanism of using phage cocktail-intestinal microbiota transplantation for the experimental treatment of bacterial colitis and its effects using S.Typhimurium-induced colitis as an experimental animal model.In this study,two S.Typhimurium phages,named v B?Sen S-En JE1 and v B?Sen S-En JE6,were isolated from different sources of sewage,using S.Typhimurium 01E,a model capable of causing colitis in mice,as the host bacterium,and their general biological properties were further investigated,and their genomes were sequenced and bioinformatically analysis.The results showed that both phages v B?Sen S-En JE1 and v B?Sen S-En JE6 had good temperature(30°C-60°C)and p H(4-10)stability and a broad lysis spectrum.v B?Sen S-En JE1 was able to lyse 41/54 strains of Salmonella serotype O:4,and v B?Sen S-En JE6 was able to lyse 46/54 strains of Salmonella serotype O:4 and O:9 serotypes of Salmonella.Also based on their morphology,genome size,G+C content,number of open reading frames,distribution of functional modules and genomic homology analysis,these two strains were found to be classified as phages of the family Siphoviridae,subfamily Jerseyvirinae,genus Jerseylikevirus,which are all lytic phages.The above results laid the foundation for the subsequent therapeutic studies of v B?Sen S-En JE1 and v B?Sen S-En JE6.We further evaluated the inhibition of phage v B?Sen S-En JE1 and v B?Sen S-En JE6 and their cocktails(Phage cocktail(Pc))in vitro and their therapeutic effects in a mouse colitis model.In vitro results showed that Pc significantly retarded the proliferation of phage-resistant bacteria compared with the phage-alone treatment group,with bacterial load>10~8 CFU/m L in the phage-alone group and approximately10~3 CFU/m L in the Pc-treated group.in vivo results showed that Pc was effective in reducing the bacterial load of mouse colonic tissue(<10~2 g)(P<0.5)compared with the phage-alone treatment group,but still could not completely remove bacteria at 144h after treatment.These results indicate that Pc is more effective than phage alone in bacterial inhibition as well as in vivo treatment.Subsequently,we investigated the in vitro bacterial inhibitory effect of Pc in combination with anaerobically cultured mouse intestinal microbiota(ACMIM).The results showed that the inhibition activity of ACMIM itself against S.Typhimurium 01E in vitro was concentration-dependent,with ACMIM completely clearing the bacteria on the second day of co-culture,while 1:10or 1:100 dilutions of ACMIM failed to completely clear the bacteria even on the seventh day after co-culture.The combination of Pc and ACMIM at 1:10 dilution completely cleared the bacteria by day 5 after infection,indicating that the combination was more effective.The results of 16Sr RNA sequencing showed that ACMIM was similar to normal mouse fecal flora,and the abundance of Lactobacillus spp.increased significantly after 7 days of co-culture with Pc and 1:10 diluted ACMIM or 1:10 diluted ACMIM alone.High levels of short-chain fatty acids(SCFAs)and reuterin were detected in the medium.These results suggest that the synergistic effect of Pc in combination with ACMIM may be related to the increased abundance of Lactobacillus spp.in ACMIM and the production of SCFAs and reuterin by Lactobacillus spp.We continued to explore the therapeutic efficacy of Pc in combination with intestinal bacterial populations.We evaluated the therapeutic efficacy of the combination of Pc and fecal microbial transplantation(FMT)(Pc FMT)on S.Typhimurium 01E-induced colitis in mice.The results showed that Pc FMT completely cleared S.Typhimurium and significantly improved the pathological damage of colonic tissue compared to single Pc or single FMT.In addition,16Sr RNA sequencing showed that Pc FMT effectively restored the balance of Firmicutes/Bacteroides and Pc FMT treatment significantly increased the abundance of Lactobacillus and increased the levels of SCFA and reuterin.It is consistent with the results of the combination of Pc and ACMIM in vitro.In the follow-up study,Pc was combined with L.reuteri(a bacterium that can produce reuterin and acetate)(Pc LR).The results showed that the therapeutic effects of Pc LR and Pc FMT were similar,and compared with the single Pc treatment group or even the healthy control group,Pc LR treatment significantly increased the number of goblet cells and the expression level of Mucin 2(P<0.01).Meanwhile,compared with Pc treatment group,Pc LR significantly increased the expression level of Occludin(P<0.05).The above results show that the combination of Pc and LR is more conducive to the recovery of intestinal barrier.To explore the molecular mechanism of Pc and LR association in depth.We combined Pc with LR supernatant(Pc LR-Sup)and LR inactivator(Pc LR-In)respectively,and the results showed that Pc LR-Sup was similar to Pc LR in terms of complete bacterial clearance and significant increase in the number of goblet cells and transcript levels of Mucin 2.Also high concentrations of SCFA and reuterin were detected in the LR supernatant.The above results suggest that the synergistic effect of Pc in combination with LR may be related to the high levels of SCFA and reuterin produced by LR.Pc was further combined with acetate(Pc Ac)or reuterin(Pc Re)or acetate and reuterin(Pc Re Ac)to evaluate its therapeutic effect on mouse colitis model.The results showed that Pc Re Ac could achieve similar therapeutic effects as Pc LR and Pc LR-Sup,with the same significant increase in the number of goblet cells as well as the transcriptional level of Mucin 2.It was shown that the synergistic effect of Pc in combination with FMT was actually the SCFA and reuterin produced by Lactobacillus spp.in FMT in synergy with Pc to promote rapid recovery of the intestinal barrier and effective resistance to pathogenic bacteria thus achieving better therapeutic effects.In conclusion,we found that the combination of Pc and intestinal microbiota(ACMIM,FMT)can effectively kill S.Typhimurium in vitro and effectively treat severe inflammatory colitis caused by S.Typhimurium.In this combination therapy,Pc play the role of"eliminating pathogens",that is,killing specific pathogens specifically;FMT mainly plays the role of"strengthening vital qi",that is,increasing the concentration of probiotics(reuterin and SCFA)by increasing the abundance of probiotics(LR),thus helping to restore the intestine to help restore the intestinal barrier and assist the phage to clear S.Typhimurium.Due to the complex composition of the intestinal microbiota,which may even contain harmful components,LR can be used directly instead of FMT and bound to phages of specific pathogens to treat intestinal inflammation caused by the respective pathogens.This study provides a new idea for the treatment of inflammatory intestinal diseases caused by specific bacterial infections.