Alterations Of Intestinal Microbiota In Severe Acute Pancreatitis And Its Association With Clinical Outcome

Background And Purpose:Acute pancreatitis(AP)is a common disease of digestive system.Severe acute pancreatitis(SAP)is characterized by high infection rate,multiple complications and high mortality.Intestinal infection is the main cause of death in patients with SAP.In recent years,more and more studies have shown that intestinal flora plays an important role in the progression of AP.The balance of intestinal microecology can form intestinal mucosal biological barrier by regulating the immune system and metabolic pathways,thereby reducing bacterial overproliferation and intestinal infection.Studies have shown that changes in certain specific bacteria in the early stages of the disease may affect the progress of the disease,so certain specific bacteria are expected to become markers of disease prognosis and guide early clinical intervention.At present,there is no relevant study on the dynamic changes of intestinal flora in the course of SAP.In this study,the feces within 24 h after admission and weekly fecal follow-up were collected to explore the changes of intestinal flora structure in SAP patients with the prolongation of the course of disease and its relationship with clinical prognosis(infection,death)and intestinal function.Methods:From May 2019 to December 2020,patients with SAP who met the inclusion criteria in the digestive ICU were enrolled.Fecal samples were collected from patients with SAP and healthy people who underwent physical examination in the outpatient department of gastroenterology at the same time.Fecal DNA was extracted,and 16 S rDNA V3-V4 segment was selected for polymerase chain reaction(PCR)amplification.The composition of intestinal flora was identified by Illumina Miseq high-throughput sequencing platform,and the expression peak was detected.Finally,the clinical data of patients were collected for statistical analysis : 1.The difference of fecal flora between SAP patients and healthy people for the first time after admission was compared.2.SAP patients were divided into death group and improvement group according to whether they died,and the dynamic changes of intestinal flora in the course of disease were observed.3.SAP patients were divided into infection group and non-infection group according to whether there were infection complications(pancreatic infection and extrapancreatic infection).The differences in intestinal flora at admission between the two groups were compared,and further divided into groups according to different etiological results.The differences in intestinal flora at admission between the two groups were compared,and the dynamic changes in bacterial abundance were observed.4.According to AGI(Acute Gastrointestinal Injury,AGI)classification to assess whether the intestinal function of SAP patients returned to normal at 1 week,divided into early recovery group(ER group)and non-recovery group(NER group),to study the relationship between intestinal function and intestinal flora changes.Results:1.A total of 100 subjects were included in this study,including 72 cases of SAP patients and 28 cases of healthy control group(HC group).There were 48 males and34 females in SAP group,with an age of 48 ± 13.8 years old and an average onset time of 1.8 ± 0.8 days.There were 13 males and 15 females in HC group,with an age of 48.7 ± 10.9 years old.The baseline data of SAP group and HC group were balanced.2.The difference in the main results of intestinal flora between SAP group and HC group : α diversity showed that the species diversity indexes of intestinal flora such as Sobs and Shannon in SAP group were significantly lower than those in HC group(P < 0.05).PCo A analysis based on bray-curtis distance showed that the structure of intestinal flora in SAP group was significantly different from that in HC group.Further species analysis showed that at phylum level,the relative abundance of Firmicutes and Actinobacteria in SAP group was significantly lower than that in HC group(P < 0.05),and the relative abundance of Proteobacteria was significantly higher than that in HC group(P < 0.05).At the genus level,the relative abundance of Enterococcus and Klebsiella in SAP group was significantly higher than that in HC group(P < 0.05).The relative abundance of Blautia、Bifidobacterium、Romboulsia、Eubacterium＿hallii＿group and Clostridium＿sensu＿stricto＿1 in SAP group was significantly lower than that in HC group(P < 0.05).3.The relationship between the dynamic changes of intestinal flora and mortality : The results of baseline data analysis showed that elderly patients with SAP were more likely to die,and the remaining baseline data were balanced between groups.The results of α diversity showed that with the extension of the course of disease,the Sobs index and Shannon index of the death group showed a continuous downward trend.The Sobs index and Shannon index of the improved group gradually decreased after admission,and the species diversity(Shannon index)showed a recovery trend before discharge.PCo A analysis based on bray-curtis distance showed that there was no significant difference in intestinal flora between the death group at each time point(ANOSIM analysis,P = 0.551),and there was significant difference in intestinal flora between the improvement group at each time point(ANOSIM analysis,P = 0.007),especially between S-T4 and S-T0,which further indicated that there might be a trend of species diversity recovery in the improvement group at discharge.Species analysis showed that the relative abundance of Klebsiella increased gradually with the extension of the course of disease in the death group.The relative abundance of Klebsiella,Escherichia-Shigella and Streptococcus decreased gradually in the improvement group,and Escherichia-Shigella and Streptococcus decreased to the level of healthy people at discharge.The relative abundance of Ruminococcus＿gnavus＿group and Veillonella increased gradually.4.The relationship between the dynamic changes of intestinal flora and infection : the clinical baseline data of the two groups were balanced.The results of αdiversity showed that there was no significant difference in the species diversity of intestinal flora(Sobs,Shannon index)between the infected group and the non-infected group at admission.The results of PCo A analysis based on bray-curtis distance showed that P = 0.23,and further PLS-DA analysis was performed.After ignoring the intra-group difference,it showed that the two groups of samples were distributed in different quadrants,and the intestinal flora was different.Further analysis of LEf Se multi-level species at T0 showed that the dominant bacteria at admission were g＿Klebsiella and g＿Faecalibacterium in the infected group and f＿Lachnospiraceae、o＿Lachnospiraceae、f＿Streptococcaceae、g＿Streptococcus and g＿Ruminococcus＿gnavus＿group in the non-infected group.The patients were further divided into Staphylococcus group,Acinetobacter group,Klebsiella group,Enterococcus group,Enterobacter group and Pseudomonas group according to the results of different etiology.The dynamic change of bacterial abundance histogram was drawn.It was found that the abundance of homologous bacteria in SAP patients with secondary infection was significantly higher than that of HC at admission,suggesting that the excessive proliferation of bacteria at admission may be related to the late occurrence of homologous bacterial infection.5.The relationship between the dynamic changes of intestinal flora and intestinal dysfunction : the clinical baseline data were balanced between groups,and the late infection rate and mortality in the NER group were significantly higher than those in the ER group.α Diversity results showed that with the extension of hospital stay,the species diversity of ER group continued to recover after 1 week,while that of NER group was slowly recovered after 2 weeks,but the difference was not statistically significant.The results of PCo A analysis based on bray-curtis distance showed that there were significant differences in intestinal flora between each time point in ER group(ANOSIM analysis,P = 0.01).However,the ANOSIM analysis of NER group,P = 0.194,was further performed for PLS-DA analysis.After ignoring the intra-group differences,it can be seen that NER＿T0 and NER＿T4 were significantly distributed in different quadrants,and the intestinal flora was different.Species analysis showed that the relative abundance of Enterococcus in the ER group gradually decreased(ER＿T1 VS ER＿T2)with the prolongation of the course of disease,and the relative abundance of Faecallibacterium and Lactobacillus increased at the early stage of the course of disease(ER＿T0 VS ER＿T2).The relative abundance of Klebsiella in the NER group continued to increase after admission,while the relative abundance of Blautia and Faecalibacterium in the early stage of disease continued to decrease,and remained at a low level during hospitalization.Conclusion:1.The intestinal flora diversity of SAP patients was significantly lower than that of healthy people,which was mainly reflected in the decrease of beneficial bacteria and the increase of conditional pathogenic bacteria.When SAP patients were discharged from hospital,the species diversity had a trend of recovery,but it had not recovered to the level of healthy people.2.There were significant differences in intestinal flora between patients with SAP secondary infection and those without SAP secondary infection at admission.Excessive proliferation of the same bacteria occurs in the intestinal tract of SAP patients with secondary infection at admission,suggesting that the abundance and changes of intestinal microorganisms at admission may play a role in the risk stratification and early diagnosis of secondary infection of SAP.3.The recovery of intestinal function in SAP patients may be related to the correction of intestinal flora imbalance.