A Study On The Differences In Fecal Bifidobacterium Species Between Patients With Type2Diabetes And Healthy Individuals

[Background]With the improvement of our living standards, the proportion of diabetes especially type2diabetes in the world showed a sharp rise in the prevalence of the trend of diabetes in recent years. There are more than240million people around the world have been diagnosed with diabetes currently, experts predict that in2025, there is estimated to be more than3.5million people suffer from diabetes. The increased prevalence of diabetes has started to threaten the health of us, and has brought heavy economic burden to our society. Diabetes has become a major public health problem that strategies aimed at the prevention and treatment of diabetes are needed.Researches of etiology and pathogenesis of diabetes are the basis of controlling the prevalence of the disease. There has been growing consensus that the increased prevalence of type2diabetes cannot be attributed solely to changes in the human genome, nutritional habits, or the reduction of physical activity in our daily lives. The gut microbiota was recently proposed as an environmental factor responsible for the control of body weight and energy metabolism, which is closely linked to obesity and metabolic disorders such as type2diabetes.This complex gut microbiota contains an enormous number and diversity of microorganisms. It is composed of1013to1014bacterial cells, representing at least10-fold more cells gene than exist in the human body. This microorganism community could now be considered as a "microbial organ" localized within the host in that it plays an important role in maintaining human health. Many studies have demonstrated that the gut microbiota is involved in regulating lipogenesis and associated with the development of obesity and type2diabetes. One report indicates that type2diabetic patients have greater amount of the total amount of Enterobacter and less numbers of Bacteroidetes. One report indicates that the gut microbiota can significantly increase the energy extraction and fat storage. Increased intestinal monosaccharide absorption and hepatic triglycerides production promote insulin resistance,which may lead to the development of type2diabetes. A series of experiments has shown that a microbial dysbiosis caused by high-fat diet can lead to the inflammatory processes which they called’metabolic endotoxemia’.The inflammatory is associated with insulin resistance and type2diabetes.Bifidobacterium is among the most predominant bacteria found in human gut. It is one of probiotics which play beneficial roles in a host’s health. Bifidobacterium plays an important role in maintaining the balance between gut microbiota and host, as well as the host’s normal metabolism. The decline of the amount of Bifidobacterium can lead to a microbial dysbiosis and inbalance,which promote the development of metabolic disorders. Previous studies have showed that Bifidobacterium can improve glucose tolerance and restore glucose-induced insulin secretion, as well as lower the levels of endotoxaemia and proinflammatory cytokines in adipose tissue, improve the metabolic disorders of type2diabetes finally. Another animal study has showed that Bifidobacterium adolescentis can improve the microbial dysbiosis tone and dyslipidoses. In addition to the fact that these species are highly prevalent in the human gut, they can also easily be added or removed from probiotic food preparations, making them therefore ideal candidate for potential clinical interventions in diseases associated with gut microbiota. So it is significant to explore the alteration of Bifidobacterium in type2diabetes patients.Quantitative analysis is the basis of researches on gut microbiota. Currently, traditional plating methods are used for the enumeration of gut microbiota.But these timeconsuming traditional plating methods have some major limitations such as lack of accuracy and stability, and the results can be easily affected by some factors such as environment,culture medium and technique.So it is necessary for us to establish a rapid, precise and stable method to analyse flora. Real-time quantitative PCR with species-specific primers can provide a rapid and sensitive method for more accurate quantification of bacteria, and will be a useful tool for studies on the ecology of gut microbiota.More studies on the differences in fecal Bifidobacterium between patients with type2diabetes and healthy individuals are needed. There are still no studies on the alterations of each species.So we designed16SrRNA-targeted genus-and species-specific PCR primers for a selected group of fecal Bifidobacterium species including total Bifidobacterium, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium infantis and used real-time quantitative PCR to assess their copy numbers in fecal samples of patients with type2diabetes and healthy individuals.At the same time, we analyzed the correlation between these Bifidobacterium species and some metabolic parameters in patients with type2diabetes to provide some data for the analysis of correlations between Bifidobacterium species and type2diabetes. [Objective]1. To assess whether there are changes in the amount of fecal Bifidobacterium species including total Bifidobacterium, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium infantis in patients with type2diabetes and non-diabetic persons as control.2. To analyze the correlation between these Bifidobacterium species and some metabolic parameters in patients with type2diabetes.[Methods]1.This cross-sectional analysis included50patients with type2diabetes. and30control subjects in Mingjing Hospital in Foshan.These patients with type2diabetes were diagnosed by WHO1999criterion. The patients were interviewed following a questionnaire assessing:individual health status (including chronic or acute diseases), body length, weight, and computing the Body Mass Index and Waist/Hip Circumference.2.We took the blood of patients with type2diabetes to test the levels of FPG,2hPG, HbAlc、FCP、2hPCP、TC、TG、HDL-C、LDL-C、CRP.3.Faecal samples collection and processing:we mixed two gram wet faecal samples with20ml PBS and then centrifuged many times, collected the bacteria precipitate.4.Extraction of Total DNA from Stool Samples:DNAs were extracted using the TIANamp Bacteria DNA kit (TIANGEN BIOTECH, Beijing) according to the manufacturer’s instructions. The DNA concentration was determined using a NanoPhotometer TM (Uvikon923, USA).5.The standard lines:We designed16SrRNA-targeted genus-and species-specific PCR primers for the selected group of fecal Bifidobacterium species including total Bifidobacterium, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium infantis. Then PCR amplification was performed for each species. The standard line of each species was established by qPCR using the7500Fast Real-time PCR System (Applied Biosystems, USA) with plasmids.6.Quantitative analysis of bacteria:Bacterial groups in80fecal samples were quantified by qPCR using the7500Fast Real-time PCR System (Applied Biosystems, USA).7.Statistical analysis:Statistical analysis were performed using the SPSS17.0software package.The data which fit the normal distribution were expressed as the mean±SD. Differences between the diabetic and control groups were assessed using the Independent-Samples T Test.The data which did not fit the normal distribution were expressed as the median(P25～P75). Differences between the diabetic and control groups were assessed using the Mann-Whitney U Test. Correlations between these Bifidobacterium species and metabolic parameters in patients with type2diabetes were computed by Pearson Rank correlation when the data fit the normal distribution while Spearman Rank correlation when did not fit the normal distribution. P<0.05considered significant difference.[Results]1.The amouts of Bifidobacterium species:The copy numbers of total Bifidobacterium, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium infantis in patients with type2diabetes were10.97(10.25～11.26),3.49(3.26～3.81),3.94(3.76～4.13),3.44(3.34～3.57),3.80(3.23～4.66) respectively. The copy numbers of these Bifidobacterium species in control group were11.56(10.97～11.92),3.68(3.30～4.48),4.02(3.52～4.12),4.01 (3.72～4.28)、4.23(2.92～4.72) respectively. Type2diabetic patients had less numbers of total Bifidobacterium and Bifidobacterium adolescentis than controls (P=0.000respectively).There are no significant differences in Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis between patients with type2diabetes and healthy individuals (P=0.101,0.676,0.964respectively)2.There were no correlations between the copy numbers of total Bifidobacterium, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium infantis and FPG、2hPG、HbAlc、CRP、FCP、2hPCP、TC、 TG、HDL-C、LDL-C in patients with type2diabetes (P>0.05respectively)[Conclusion]1.Type2diabetic patients had less numbers of total Bifidobacterium and Bifidobacterium adolescentis than controls, and hence it suggests that type2diabetic patients have alterations in the gut total Bifidobacterium and Bifidobacterium adolescentis. Such alterations may play a role in the development of type2diabetes.2.There may be no correlations between the copy numbers of fecal Bifidobacterium species and disorders of glycometabolism and lipometabolism, islet function, inflammatory levels in patients with type2diabetes.