Study On The Source Of Infection In Infected Pancreatic Necrosis Based On Intestinal Flora Imbalance

Background And Aim:Acute pancreatitis(AP)is a common disease of digestive system,of which about20% develop into severe acute pancreatitis(SAP).Patients with SAP are prone to organ dysfunction in the early stage of the disease,and the mortality rate can reach15-20%,and if infectious complications occur in the later course of the disease,the mortality rate is as high as 30%.Intestinal-derived infection is considered to be the main cause of infectious complications in patients with AP,and relevant studies have detected intestinal conditional pathogenic bacteria in pancreatic necrotic drainage fluid and peripheral blood of AP patients,suggesting that they may enter the circulatory system and abdominal tissues and organs through the damaged intestinal mucosal barrier,causing intestinal-derived infection.However,no study has been conducted to directly determine the correlation between the flora of different sites and intestinal flora in patients with AP by retaining samples of pancreatic necrotic drainage fluid,peripheral blood and deep sputum.Based on this,the aim of this study was to analyze the flora of pancreatic necrotic drainage fluid,blood and deep sputum from patients with AP undergoing the first minimally invasive intervention for pancreatic necrosis,to analyze their homology with intestinal flora,and to further validate the theory of intestinal-derived infection in AP.Methods:This study prospectively included AP patients were hospitalized in the Department of Gastroenterology,the First Affiliated Hospital of Nanchang University,from October 2021 to March 2022.During the first minimally invasive intervention of pancreatic necrosis,samples of pancreatic necrotic drainage fluid,blood,fecal and deep sputum were collected at the same time.Based on Pac Bio SMRT sequencing technology,full-length 16 S r DNA sequencing was performed to analyze the structural characteristics of the pancreas,blood,intestinal and lungs of the patients.The clinical data of patients were collected.According to the results of laboratory culture of pancreatic necrotic drainage fluid during minimally invasive intervention of pancreatic necrosis,the patients were divided into infected pancreatic necrosis(IPN)group and sterile pancreatic necrosis(SPN)group.The difference of intestinal flora between the two groups and the correlation between pancreatic,blood,pulmonary flora and intestinal flora were compared between the two groups.Results:1.A total of 22 patients were included in this study,including 16 patients in the IPN group and 6 patients in the SPN group.There were no statistical differences in baseline data between the two groups in terms of gender,age,cause of AP,mode of first minimally invasive intervention,time from onset to admission and time from onset to first minimally invasive intervention.2.Composition characteristics and comparison of intestinal flora between IPN group and SPN group: α diversity showed that there was a trend of lower intestinal flora species diversity indices such as Chao1 index and Shannon index in IPN group than SPN group,but the difference was not statistically significant(P > 0.05).PCo A analysis based on bray-curtis distance showed that there was a significant difference in the structure of intestinal flora between the IPN and SPN groups(Adonis test,P <0.05).Further species composition analysis was performed,and the results showed that at the phylum level,the relative abundance of the beneficial phylum Verrucomicrobia was significantly reduced in the IPN group compared to the SPN group(0.9% vs.12.6%;P < 0.05);at the species level,the conditionally pathogenic bacteria Escherichia coli,Ralstonia sp,Sphingomonas leidyi and Caulobacter sp.were significantly more abundant in relative abundance in the IPN group compared to the SPN group(P < 0.05).The expression abundance of Escherichia coli in the fecal samples of both groups was further verified by q PCR,and the results showed that the relative abundance of Escherichia coli in the fecal of IPN group was significantly higher than that in the SPN group(P < 0.05).3.Intestinal-pancreatic flora correlation between IPN and SPN groups: Species composition analysis of the pancreatic flora of IPN and SPN groups,at the phylum level,the pancreatic flora of both groups was predominantly Proteobacteria.At the species level,the dominant pancreatic flora in the IPN group were Serratia marcescens、Ralstonia sp.、Escherichia coli、Delftia tsuruhatensis and Staphylococcus epidermidis,among them,Escherichia coli was the common conditionally pathogenic bacteria in the intestine,while the dominant flora in the pancreas of the SPN group were Serratia marcescens、Ralstonia sp.、Delftia tsuruhatensis、Sphingomonas leidyi、and Caulobacter sp..There were 105 ASVs in the pancreatic and intestinal flora in the IPN group,accounting for 29.2% of the number of ASVs in the pancreas,and 10 ASVs in the pancreatic and intestinal flora in the SPN group,accounting for 10.3% of the number of ASVs in the pancreas.The intestinal and pancreatic flora were more similar in the IPN group compared to the SPN group(P<0.001).Comparison of beta diversity distances based on bray-curtis distances likewise showed that the distances between intestinal and pancreatic flora were closer in the IPN group than in the SPN group(P<0.01).4.Intestinal-blood flora correlation between IPN and SPN groups: Species composition analysis of blood flora in IPN and SPN groups showed that at the phylum level,blood flora in both groups were predominantly Proteobacteria.At the species level,the dominant blood flora of the IPN group were Ralstonia sp.、Delftia tsuruhatensis 、 Sphingomonas leidyi 、 Caulobacter sp.、 Escherichia coli and Enterococcus faecium,where Escherichia coli、Enterococcus faecium were common conditionally pathogenic bacteria in the intestine,while the dominant groups of blood in the SPN group were Ralstonia sp.、Delftia tsuruhatensis、Sphingomonas leidyi、Caulobacter sp.and Bacteroides vulgatus,of which Bacteroides vulgatus was common in the intestine.The blood and intestinal flora in the IPN group had a total of145 ASVs,accounting for 21.7% of the blood ASVs,and the blood and intestinal flora in the SPN group had a total of 42 ASVs,accounting for 10.1% of the blood ASVs.The intestinal and blood flora were more similar in the IPN group compared with the SPN group(P<0.001).However,there was no statistical difference in the distance of beta diversity between the intestinal and blood flora of the IPN and SPN groups(P > 0.05).5.Intestinal-pulmonary flora correlation between IPN and SPN groups: Species composition analysis of pulmonary flora in IPN and SPN groups,at the phylum level,the pulmonary flora of both groups was dominated by Proteobacteria、Firmicutes、Bacteroidetes.At the species level,the dominant pulmonary flora in the IPN group were Prevotella＿＿unclassified 、 Streptococcus＿＿unclassified 、 Pseudomonas aeruginosa、Serratia marcescens and Acinetobacter baumannii,the dominant flora of the pulmonary flora of the SPN group were Serratia marcescens、Fusobacterium nucleatum、Streptococcus＿＿unclassified、Prevotella＿＿unclassified and Streptococcus mitis,and the dominant pulmonary flora of both groups were significantly different from the dominant flora of the intestine.However,the pulmonary and intestinal flora in the IPN group had a total of 70 ASVs,accounting for 4.3% of the pulmonary ASVs,whereas the pulmonary and intestinal flora in the SPN group had a total of 13 ASVs,accounting for 1.3% of the pulmonary ASVs.The intestinal and pulmonary flora were more similar in the IPN group compared to the SPN group(P<0.001).Comparison of beta diversity distances based on bray-curtis distances likewise showed that the distances between intestinal and pulmonary flora were closer in the IPN group than in the SPN group(P<0.05).6.The correlation difference between the flora of each part of the IPN group and the intestinal flora: Comparing the number of ASVs shared by each part of the patients in the IPN group with the intestinal flora,the similarity between the pancreatic flora and the intestinal flora in the IPN group was the highest(29.2%),followed by the similarity between the blood flora and the intestinal flora(21.7%),and the similarity between the pulmonary flora and the intestinal flora was the lowest(4.3%).The differences between the two comparisons were statistically significant(P< 0.01).Conclusions:(1)There are significant differences in the structure of intestinal flora between IPN patients and SPN patients,mainly in the imbalance of intestinal flora(decrease of beneficial bacteria and increase of pathogenic bacteria)in IPN patients,indicating that it may have an important role in the development of pancreatic infection in AP patients.(2)The homology of pancreatic,blood and pulmonary flora with intestinal flora is higher in IPN patients than in SPN patients,and the pancreatic flora of IPN patients is most similar to the intestinal flora,followed by the blood flora,and the pulmonary flora have the lowest similarity to the intestinal flora.This suggests that infections at various sites in IPN patients may be partially of intestinal origin,especially pancreatic infection.