Study On The Intestinal Flora Of Patients With Colorectal Cancer And The Mechanism Of Pks~+ Escherichia Coli Causing Colorectal Cancer

Colorectal cancer(CRC)is one of the most three common cancers in the world,causing considerable harm to the health of people around the world.As the largest and most complex micro ecosystem of human body,intestinal microorganisms have a profound impact on human health.Under normal physiological conditions,the intestinal microorganisms are in a dynamic balance.However,this balance is greatly affected by factors such as diet,lifestyle,disease,and environment.Once this balance is broken,the disorders of intestinal microorganisms will cause diseases such as CRC,but the law of its incidence still needs to be studied.CRC screening can reduce the occurrence and death of CRC by identifying precancerous lesions before malignant transformation or early detection and treatment of CRC.However,the current early screening methods have not made great progress in improving the disease-free survival rate of colorectal cancer patients.Colonoscopy is invasive and not widely accepted;the noninvasive screening test based on stool has a low sensitivity for detecting colorectal adenoma,especially for early adenoma screening.Therefore,the screening rate for early CRC is still very low,so that the incidence and mortality of CRC remain high,so a non-invasive and economical screening method that is sensitive to early and high-risk adenoma of colorectal cancer needs to be explored.In recent years,genotoxic E.coli(pks~+Escherichia coli,pks~+E.coli)containing the pks gene cluster has received much attention because of its potential role in the occurrence of CRC,but the mechanism of CRC caused by pks~+E.coli is still unclear.The purpose of this project was to study the intestinal flora of CRC patients and the changes of flora before and after surgery,to isolate and culture pathogenic bacteria,and to screen pks~+E.coli to act on CRC mice to verify its impact on the occurrence and development of CRC,so as to provide research basis for early diagnosis and surgical prognosis of CRC.The main research contents and experimental results were as follows:Sixteen CRC patients were selected to collect fecal samples before and after the operation,cancerous tissues and cancer adjacent tissues during the operation,and the composition and changes of the flora were analyzed.The number of OTU,Chao1,ACE,Shannon and Simpson were taken into the study.The bacterial diversity varied greatly from patient to patient,and some patients had a very low diversity index.It was showed that the bacterial diversity in the fecal samples of the patients after the operation increased,and the diversity index in the cancer adjacent tissues was higher,but the average index of the diversity index of the samples from the six sources was not significantly different.Comparing the bacterial community structure of all patients before and after surgery,the diversity of fecal samples after surgery was higher than before surgery,and the bacterial abundance of Enterobacteriacea and Ruminococcaceae decreased after surgery.Comparing the bacterial community structure in cancer tissues and cancer adjacent tissues of all patients,it was found that there was no significant difference in the level of phylum;from the level of genus,there were differences in the bacterial community composition of cancer tissues and cancer adjacent tissues.The proportion of pathogens in the cancer adjacent tissues were lower than that in cancer tissues,and the content decreased as the distance from the cancer tissues increased.OTU-based phylogenetic analysis showed that the main bacterial type in the samples was E.coli,and the classification information of more OTUs involved were all recognized pathogenic bacteria or conditional pathogenic bacteria,which could cause multiple organ infections.It showed that the individual intestinal microbes of the patients were quite different,and different patients had their own specific intestinal microbes,the composition of the intestinal bacteria in patients after surgery were quite different from that before the operation,and there were differences in the bacterial composition of cancer tissues and cancer adjacent tissues.The intestinal microorganisms of patients with cancer were mainly pathogenic bacteria and conditionally pathogenic bacteria,which were quite different from those of healthy people.It is still very important to prevent infection of patients with colorectal cancer.The 33 strains of Enterobacteriaceae and Enterococcaceae were isolated and cultured from the cancer tissues of CRC patients by aerobic culture,including 21strains of Escherichia coli,9 strains of Klebsiella pneumoniae,1 strain of Klebsiella quasipneumoniae,and 2 strains of Enterococcus faecalis.Combined use of light-weight air-tight isolation gloves operation box,anaerobic gas-generating bag,sealed culture tank,sealed culture bag and other simple equipment to prepare a anaerobic environment,isolated and cultured 28 strains of Enterobacteriaceae,Enterococcaceae and Streptococcaceae from the tissues of CRC patients,including 12strains of Escherichia coli,10 strains of Klebsiella pneumoniae,1 strain of Klebsiella quasipneumoniae,2 strains of Enterococcus faecalis,and 3 strains of Streptococcus anginosus.The 21 aerobic E.coli and 12 anaerobic E.coli isolated from CRC tissues isolated in this experiment and 19 aerobic E.coli strains isolated from fecal samples of CRC patients in 2017 were amplified by using pks genomic island detection primers clb Q,clb A,clb B.The results showed that anaerobic strain crc2-6 and aerobic strain crc3-6 were pks~+E.coli.The CRC mouse model based on colitis was established by one-off intraperitoneal injection of azoxymethane(AOM)and three cycle feeding with dextran sulfate sodium(DSS).During the modeling process,mice in the experimental group were fed with pks~+E.coli crc2-6 viable bacterial suspension,and mice in the control group were given saline.Compared with the control group,the mice in the experimental group treated with crc2-6 had increased intestinal inflammation,thickened colorectal intestinal wall,obviously shrunk of intestinal mucosa,significantly reduced body weight,reduced survival rate,and aggravated intestinal diseases.The results of histopathological examination showed that the progress of canceration in mice after crc2-6 treatment was accelerated,the number of cancerous cells increased significantly,and the detection rate of Ki-67 and PCNA positive cells increased significantly.It showed that pks~+E.coli crc2-6 strain could promote colorectal inflammation to a certain extent,and then promoted the occurrence and development of CRC.