Dynamic Alteration Of The Gut Microbiota In A Rat Model Of Intra-abdominal Infection And The Early Diagnosis Of Sepsis By DGGE

Intra-abdominal infections(IAIs)is a diverse group of diseases that are encountered commonly in surgical practice.Most IAIs could be controlled effectively and with low associated morbidity through removal or repair of the infected focus,treatment with pathogen-specific antimicrobial therapy,and restoration of anatomy if resection is performed for definitive source control.However,in some instances,overwhelming infection leads to gut barrier dysfunction,intestinal dysbiosis and excessive activation of the immune response,generalized inflammation,systemic inflammatory response syndrome(SIRS),sepsis and multiple organ dysfunction syndrome(MODS).So the study of gut microbiota and early diagnosis of bacterial translocation(BT)is very important.The gut luminal microbiota accommodates about 400 different species of bacteria,comprising 10 3 to 10 14 microorganisms.Their genome is over 100 times as large as human beings' genome.Most of gut microbes have a profound influence on human physiology,such as energy harvest,immune system development,homoeostasis and prevention of noncommensal organisms from expanding.Meanwhile,more and more scientists have recognized the importance of gut microbiota.Since most of microbial community members are unculturable,it is meaningful to define the composition of the gut microbiota and reveal the role of the microbiota in the pathogenesis of IAIs.Furthermore,the novel molecular techniques such as denaturing gradient gel electrophoresis(DGGE)make it easier now.Therefore,we undertook the study(1)to characterize the changes of gut microbiota in the pathogenesis of IAIs using DGGE method and(2)to observing association between epitheliall barriers dysfuction and intestinal dysbiosis in the same model.Meanwhile,we used DGGE(3)to detect the rate of bacterial infection in peritoneum,liver and blood and assessed whether DGGE could make diagnosis of bacterial infection earlier.Part ? Dynamic alteration of the gut microbiota in a rat model of intra-abdominal infectionStudy ? The rat model of IAIs and ileal microbiota alteration in IAIsObjectives:To set up the rat model of IAIs and to illustrate ileal flora shifting patterns according to CASP time course and to define meaningful bacterial species.Methods:Rats were randomized into six groups.A group of rats were sacrificed just after anesthesia(control),while other five groups underwent CASP and were euthanized at 3,6,12,24,48 hours following surgery respectively.At each point,survival rate was observed.Six survival rats were selected and detected the obdominal infection.Luminal bacterial DNA was extracted from ileal contents at 0,3,12,24,48 hours following CASP respectively.The V3 region of the 16S ribosomal RNA gene was amplified by polymerase chain reaction(PCR)and examined by DGGE.After image digitization,microbiota similarity and diversity index were elevated and principal component analysis(PCA)was performed.Samples from different surgery time points were compared with the control group.Bands of interest from DGGE gels were cut,sequenced and matched.Results:All rats survived in control group and 3h group.The abdominal is normal at 3 hours post CASP.The survival rate was 87%and we detected few ascites in 6 hous group.The survival rate was 65%and the intestines were edematous slightly at 12 hours post CASP.After 24 hours,The survival rate was 43%.The abdominal infection was observed and the intestines were edematous and adhesive.In 48 hours group,only 12%of rats survived and the severe abdominal infection was detected.DGGE of ileal microbiota showed that gut flora pattern shifted at 6 hours after CASP.The 48 hours infection ileal flora were most significantly different from normal groups ileal microbiota.The specific bacterial species that contributed to the dysbiosis were identified by sequencing.Helicobacter cetorum,Pasteurella sp.,Escherichia sp,Uncultured bacterium and Shigella flexneri proliferated continuously after IAIs.However,Lactobacillus amylovorus,Escherichia coli,Uncultured Firmicutes bacterium and Lactobacillus sp diminished continuously.Conclusions:CASP was a good model to imitate IAIs.Ileal microbiota changed in IAIs injury longitudinally.Proteobacteria overgrowth and Firmicutes reduction characterized the dysbiosis.Study ? Colonic microbiota alteration in IAIsObjectives:To illustrate colonic flora shifting patterns according to CASP time course and to define meaningful bacterial species.Methods:Rats were randomized into six groups.Luminal bacterial DNA was extracted from colonic contents at 0,3,12,24,48 hours following CASP respectively.The V3 region of the 16S ribosomal RNA gene was amplified by PCR and examined by DGGE.After image digitization,microbiota similarity and diversity index were elevated and PCA was performed.Samples from different surgery time points were compared with the control group.Bands of interest from DGGE gels were cut,sequenced and matched.Results:DGGE of colonic microbiota showed that gut flora pattern shifted at 6 hours after CASP.The 24 and 48 hours infection colonic flora were most significantly different from normal groups colonic microbiota.The specific bacterial species that contributed to the dysbiosis were identified by sequencing.Escherichia sp,Bacteroides coprosuis,Escherichia coli proliferated continuously after IAIs.Lactobacillus gasseri,Lactobacillus gallinarum and Uncultured Firmicutes bacterium diminished continuously.Conclusions:Colonic microbiota changed in IAIs injury longitudinally.Proteobacteria,Bacteroidetes overgrowth and Firmicutes reduction characterized the dysbiosis.Study ? Gut barrier dysfunction in IAIs rat modelObjectives:To observe ileal and colonic epithelium changes and compare with time course of gut microbiota shifts in the IAIs model.Methods:Rats were randomized into six groups.The ileal and colonic epithelial barriers were checked by microscopy at 0,3,12,24,48 hours following CASP respectively.Ileal and colonic epithelium tight junctions(TJ),ZO-1 were examined by immunofluorescent staining and electron microscope.The level of serum TNF-?,IL-? and IL-10 were assessed by ELISA.Results:Three hours post CASP showed slightly ileal villus damage.The highest injury scores were observed at 48 hours after CASP(p<0.001 vs control group);Whereas colonic mucous was normal at 3,6,12 hours after surgery.Twenty-four hours post CASP showed slightly colonic mucous damage.At 48 hours,the damage of colon was the severest.Immunofluorescence assay also demonstrated that,during infection time course,from 3 hour to 48 hours,ZO-1 delocalized from apical cellular border.The damage of TJ in ileum was detected in 3 hours group and the most severely damage happened at 48 hours post surgery by electron microscope.The serum IL-1 and TNF-a concentration elevated as early as 3 hour after CASP and reach the highest level at 24 hours(p<0.001 vs control group).However,IL-10 level increased at 12 hours and peaked at 24 hours post IAIs.Conclusions:The damage of ileal epithelium was earlier than ileal dysbiosis.While the damage of colonic epithelium was later than colonic dysbiosis.Part ? The early diagnosis of sepsis by DGGE Study ? The early diagnosis of infection in peritoneum after CASPObjectives:To compare the infection rates of peritoneum after CASP by conventional culture and DGGE techniquesMethods:At given time points(3,6,12,24 and 48 h after CASP surgery),animals were sacrificed.The skin of the abdomen was cut open in the midline after thorough disinfection and without injury of the muscle.5 ml of Sterile saline solution was injected into and aspirated out of the peritoneal cavity twice to rinse out bacteria from the peritoneal cavity.Aliquots of a serial log dilution of this peritoneal lavage fluid were plated on Columbia blood agar and MacConkey plates;CFU per entire peritoneal lavage were detected after overnight incubation at 37?.Another 200 ?l of solution was tested:the V3 region of the 16S ribosomal RNA gene was amplified by PCR and examined by DGGE.Results:Peritoneum infection was observed by culture in 16.7%of rats at 3 hours and it was 50%by DGGE.At 12 hours,peritoneum infection rates were 50%by culture,100%by DGGE,they have significantly different.Peritoneum infection was detected in all rats by culture and DGGE at 48 hours;The polymicrobial infection rates of peritoneum were 33%by DGGE and 16%by culture.At 12 hours,the infection was detected in all rats by DGGE while the rates were only 50%by culture(p=0.046).The polymicrobial infection rate of peritoneum was observed in all rats by culture or DGGE at 24,38 hours post CASP.Conclusions:Peritoneum infection was found at 3 hours post surgery.DGGE is more sensitive to the diagnosis of peritoneum infection.Study ? The early diagnosis of infection in liver after CASPObjectives:To compare the infection rates of liver after CASP by conventional culture and DGGE techniquesMethods:At given time points(3,6,12,24 and 48 h after CASP surgery),animals were sacrificed.200mg of liver were homogenized in 4ml of sterile phosphate buffered saline(PBS)buffer.Ten microliters of 10-fold dilutions of liver suspensions were plated on Colombia blood agar and Mac-Conkey plates.CFU per entire peritoneal lavage were detected after overnight incubation at 37?.Another 200 mg of solution was tested:the V3 region of the 16S ribosomal RNA gene was amplified by PCR and examined by DGGE.Results:No Liver infection was observed by culture at 3 hours and it was 16.7%by DGGE.At 12 hours,liver infection rates were 33%by culture,50%by DGGE,they had no significantly different.At 48 hours,the infection was detected in all rats by DGGE while the rates were only 50%by culture(p=0.046).No polymicrobial infection of liver was observed by culture or DGGE at 3,6 hours.The polymicrobial infection rates of liver were 33%by DGGE and 0%by culture at 12 hours.The polymicrobial infection was detected in all rats by DGGE while the rates were only 33%by culture at 48 hours(p<0.05).Conclusions:Liver infection was found at 3 hours post surgery.DGGE is more sensitive to the diagnosis of liver infection.Study ? The early diagnosis of bloodstream infections after CASPObjectives:To compare the rates of bloodstream infection after CASP by conventional culture and DGGE techniques.Methods:At given time points(3,6,12,24 and 48 h after CASP surgery),animals were sacrificed.100?l of blood were plated on Columbia blood agar and MacConkey plates;CFU per entire peritoneal lavage were detected after overnight incubation at 37?.Another 200 ?l blood was tested:the V3 region of the 16S ribosomal RNA gene was amplified by PCR and examined by DGGE.Results:No bloodstream infection was observed by culture of at 6 hours and it was 16%by DGGE.At 24 hours,bloodstream infection rates were 16%by culture,50%by DGGE,they had no significantly different.Bloodstream infection was detected in all rats by DGGE at 48 hours;No polymicrobial bloodstream infection was observed by culture or DGGE at 3,6,12 hours.At 24 hours,the infection was detected in 50%of rats by DGGE while the rates were 0%by culture(p<0.05).The polymicrobial infection rates of bloodstream were 50%by DGGE and 16%by culture at 48 hours.Conclusions:Bloodstream infection was found at 6 hours post surgery.DGGE is clearly superior to the diagnosis of bloodstream infection.