The Mechanisms Of Bacterial Translocation During Severe Acute Pancreatitis

Acute pancreatitis is one of the most common gastrointestinal disorders requiring acute hospitalization worldwide,with a reported annual incidence of 13-45 cases per 100,000 persons.It has become apparent that there are two phases of acute pancreatitis:an early phase(usually within the first week of onset)and a subsequent phase occurring after the first week of onset of the disease.The first phase is characterized more by the presence or absence of organ failure.The late phase is characterized by infectious complications following bacterial translocation of intestinal bacteria and late mortality.And the infection of pancreatic necrosis by gut bacteria is a major cause of morbidity and mortality in patients with severe acute pancreatitis.Two important factors which have been proposed as promoting bacterial translocation in patients with severe acute pancreatitis are physical injury of the gastrointestinal epithelium and the gut flora dysbiosis.Vertebrates harbor a densely populated resident microbial community,which consists of bacteria,viruses and fungi,particularly in mucosal organs,such as the oral cavity and the intestine.The intestinal tract is one of the most complex and diverse ecosystems,comprising up to 1014 cells of 300-500 differemt mcrobial species,many of which have never been cultured.In healthy individuals,Gram-negative Proteobacteria and Bacteroidetes,and Gram-positive Firmicutes,such as Clostridiales and Lactobacillales,are the major phyla among intestinal eubacteria,whereas methanogens are the predominant intestinal archaea[2].Although the gut microbiota has previously been studied in the context of inflammatory diseases,it has recently become clear that this microbial community has a beneficial role during normal homeostasis,modulating the host's immune system as well as influencing host development and physiology,including organ development and morphogenesis,and host metabolism.Techniques for the fast and accurate detection of bacterial infection are critical for early diagnosis,prevention and treatment of bacterial translocation in clinical severe acute pancreatitis(SAP).Bacterial DNA is stable,and PCR-based methods are highly sensitive and specific to detect minimal amounts of bacterial DNA in serum of patients with acute pancreatitis.This study is aimed at unraveling intestinal microbiota alteration pattern and identifying specific bacterial species that differ significantly using denaturing gradient gel electrophoresis(DGGE)method as well as observing intestinal epithelium change in rat models of severe acute pancreatitis.PART1 Establish an model of experimental severe acute pancreatitis(AP)in rats by retrograde injection of sodium taurocholate into biliopancreatic duct through duodenal wallObjective:To investigate the relationship between the gut microbita and bacterial translocation in the model of experimental severe acute pancreatitis(AP)in rats induced by retrograde injection of sodium taurocholate into biliopancreatic duct through duodenal wall.Methods:24 cases of rats were randomly divided into a model control group(n=6)and Severe acute pancreatitis(n=18).At 2,6 and 12 hours(h)after operation,pancreatic tissues were performed HE staining to judge whether the models were successful or not.Liver function,renal function and the mortality were evaluated.Results:At 12h after operation,the survival rates in control group(100%)were higher than in the model SAP group(29%).The serum level of Amylase and Lipase were significantly increased in all SAP groups compared with the control group.The liver function,including ALT and AST,were increased significantly in SAP group.However,other measurements,including creatinine levels,Urea nitrogen were no significantly difference between two groups.Conclusions:The model of experimental SAP induced by retrograde injection sodium taurocholate into biliopancreatic was stable,higher mortality,and caused more severe pathological injury and could be used for investigate the pathogenesis of severe acute pancreatitis.PART2 Dynamics of the ileal microbiota and intestinal epithelium in a rat model of severe acute pancreatitisObjective:Infection of pancreatic necrosis with intestinal flora is accepted to be a main predictor of outcome during severe acute pancreatitis.This study aimed to identify ileal flora dysbiosis pattern and bacterial species that changed significantly in a rat model of severe acute pancreatitis,and illustrate time courses of both epithelial alterations and gut flora variations in the same injury.Methods:24 rats were randomized into four groups(n=6/group).A group of rats were sacrificed just after anesthesia(control),while other three groups underwent retrograde injection of sodium taurocholate into biliopancreatic duct and were euthanized at 2,6 and 12 hours following reperfusion respectively.Denaturing gradient gel electrophoresis of ileal microbiota showed that gut flora pattern changed early after SAP models were induced.Results:We observed that the gut flora pattern changed significantly in comparison with the sham control group at 6 hours after the establishment of the SAP model,and then started to recover towards normal pattern.The specific dysbiosis were characterized by Escherichia coli proliferation and Lachnospiraceae and Lactobacilli reduction.And the intestinal barrier function was impaired after the establishment of the SAP model,in comparison with the sham control group.Conclusion:This study shows for the first time that the models of SAP results in colonic flora dysbiosis that follows epithelia damage,and identifies the bacterial species that contribute most.PART3 The colonic microbiota alteration pattern and intestinal epithelium change in rat models of severe acute pancreatitisObjectives:The bacterial contamination of pancreatic necrosis in acute pancreatitis is supposed to occur through translocation of intestinal bacteria.To date,the pathophysiology of the underlying bacterial translocation is poorly understood.Increased gut permeability and gut flora dysbiosis may be the initial phenomenon in this process.The present study investigated the colonic microbiota alteration pattern as well as observed colonic epithelium change in rats with SAP.Method:Denaturing gradient gel electrophoresis(DGGE)was used to monitor the colonic microbiota of control rats and experimental rats that 2,6,and 12 hours following severe acute pancreatitis which was induced by retrograde injection of sodium taurocholate into biliopancreatic duct.Colonic samples and its contents at 1 cm distal to the cecal-colonic junction was harvested for morphological studies,streptavidin-peroxidase immunohistochemistry examination.Results:Denaturing gradient gel electrophoresis of colonic microbiota showed that gut flora pattern changed early after severe acute pancreatitis,differed significantly at 6 hours of model was induced,and then started to recover towards normal pattern.The specific dysbiosis were characterized by Escherichia coli proliferation and Lachnospiraceae and Lactobacilli reduction.As shown by immunohistochemical staining,greatly reduced expressions of ZO-1 protein were detected in rats with SAP.And the structures of junctional complexes were disrupted and the gaps between cell junctions were wider after the SAP induced.Conclusions:Severe acute pancreatitis results in colonic flora dysbiosis.And tight junction in rats with SAP was disrupted in the intestinal epithelium.Both of them may be a key factor contributing to bacterial translocations in rats with SAP.PART4 The bacterial translocation in rat models of severe acute pancreatitisObjectives:Techniques for the fast and accurate detection of bacterial infection are critical for early diagnosis,prevention and treatment of bacterial translocation in clinical severe acute pancreatitis(SAP).In this study,the availability of a Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis,(PCR-DGGE)method in detection of bacterial colonization in SAP rat models was investigated.Method:Samples of peripheral blood and abdominal collections were collected from the rats before and after(2,6 or 12 hours)severe acute pancreatitis model was induced.PCR and DGGE were employed to detect bacterial translocation in blood and abdominal collections.Conventional microbiological culture methods were also applied for all the samples.Results:Bacterial DNA was detected in abdominal collections from 66%after 2hours in rat of severe acute pancreatitis.Translocated bacteria in rats with acute pancreatitis were primarily constituted of opportunistic pathogens derived from the gut,including Escherichia coli,Shigella flexneri,Enterobacteriaceae bacterium,Acinetobacter lwoffii,Bacillus coagulans,and Enterococcus faecium.29%samples were documented positive by culture method,which was far less than that from the DGGE analysis(29%vs 66%,p<0.05)at 6 hours after the establishment of the SAP model.Conclusion:Our study demonstrated that unlike traditional microbiological techniques,PCR can early detect the presence of bacteria in the blood and abdominal collections of rats with severe AP.These results indicated that bacterial DNA-dependent molecular techniques were able to detect nonviable bacteria that could not be grown in culturesConclusions:The model of experimental SAP induced by retrograde injection sodium taurocholate into biliopancreatic was stable,higher mortality,and caused more severe pathological injury and could be used for investigate the pathogenesis of severe acute pancreatitis;This study shows for the first time that the models of SAP results in colonic flora dysbiosis that follows epithelia damage,and identifies the bacterial species that contribute most.Severe acute pancreatitis results in colonic flora dysbiosis.And tight junction in rats with SAP was disrupted in the intestinal epithelium.Both of them may be a key factor contributing to bacterial translocations in rats with SAP.Our study demonstrated that unlike traditional microbiological techniques,PCR can detect the presence of bacteria in the blood and abdominal collections of rats with severe AP.Therefore,this latter method in conjunction with DGGE is potentially an extremely useful tool in predicting septic morbidity and evaluating patients with the disease.