Effects Of Antibiotics On Intestinal Flora

BackgroundThe discovery and production and use of antibiotics is a big step forward of human medical history,the United States food and drug administration（FDA）for the first time in 1950 approved the antibiotics can be use as animal feed additives,and as a result,antibiotics in improving feed conversion rate,prevention and treatment of animal infectious diseases has played a very important role[1].Antibiotics can well prevent and treat infectious diseases and save countless lives,but if overused,they can also bring great harm,such as intestinal flora disorder,reducing the stability and diversity of intestinal flora.Of course,certain antibiotics may also have beneficial effects on intestinal flora.For example,the antibiotic Rifaximin is not absorbable in the intestinal tract,and it produces a symbiotic effect on intestinal microbial flora by increasing the bacterial species abundance of several beneficial bacteria.Different results showed that rifaximin increased the abundance of F.prausnitzii and Lactobacillus[2-3].Different antibiotics have different effects on intestinal flora.High-throughput sequencing methods for evaluating community studies have shown that in healthy adults treated with the broad-spectrum antibiotic ciprofloxacin,nearly 30%of the affected intestinal flora,including the number and type of bacteria,but it returned to their previous state after one month[4].Two other early studies looked at the short-term effects of clindamycin and amoxicillin on intestinal flora and found that short-term damage to intestinal flora diversity did not affect overall intestinal function[5-6].Also have a clinical trial,66 healthy adults accept amoxicillin,clindamycin,ciprofloxacin,minocycline or a placebo for standard courses of treatment,and then follow-up,the results show that the drug 1 months later,the subjects returned to normal intestinal flora,only clindamycin bigger influence on the intestinal flora,more than four months.But after one year,all the antibiotics had no significant effect on the intestinal flora compared with the control group[7].At present,the abuse of antibiotics has become a common phenomenon in the world,especially in China.The intestinal microbiome in the neonatal period is responsible for immunology education to a certain extent[8].The use of antibiotics may have long-term consequences,and early use of antibiotics is associated with an increased risk of obesity,asthma,and Crohn’s disease[9-12].In addition,the use of antibiotics was also directly related to recurrent Clostridium difficile infection（CDI）[13],the formation of vancomycin resistant enterococcus（VRE）[14]and the emergence of antibiotic resistance[15].It is worth noting that the height of the response and recovery of the human intestinal flora after antibiotic treatment may be different among individuals,such as the antibiotics ciprofloxacin[16],clarithromycin and metronidazole[17].Human intestinal flora is an aggregation of all microorganisms living in the gastrointestinal tract,a complex microecosystem,and a complex,extensive and diverse collection of microorganisms including bacteria,viruses,archaea and single-celled eukaryotes[18].About 10 trillion bacteria live in the human gut,where they maintain a dynamic balance that allows them to resist the invasion and colonization of pathogens.Intestinal flora refers to the microorganisms distributed in the human intestinal tract and constitute a microecosystem together with the human intestinal system,which can well maintain the normal intestinal function of the human body[19].It is known that the biodiversity between different healthy individuals is basically greater than expected,and can be changed rapidly through changes in diet,and it also differs greatly from age and geography[20].In healthy human intestinal bacterial phyla,there are 29 species is known and two kinds of bacteria is dominant,respectively is mainly composed of Gram-negative bacteria genera Bacteroidetes and mainly composed of Gram-positive clostridia genera Firmicutes,other bacterial phyla,such as Actinobacteria,Proteobacteria（including Escherichia coli）and Fusobacteria are found less[21].The intestinal microbiota can well adapt to the host and coexist symbiotically with the host,which can contribute to the development of the intestinal tract,the improvement of intestinal barrier function and the maintenance of immune and metabolic homeostasis,which are key factors for health[18].There is also growing evidence that early communication between host and microbiome is essential for establishing and maintaining the health of the individual.Moreover,the early microbiota seems to provide the necessary stimulation for the full development of intestinal and immune functions,and to have an impact on the distal organs and the whole body[22-31].Under physiological conditions,intestinal flora maintains a dynamic balance with the body,playing an important role in maintaining health.Once this balance is broken,it will bring a series of diseases to the body,such as obesity,non-alcoholic fatty liver etc.Therefore,intestinal flora has become a hot research object in recent years.Although our understanding of the composition of the human gut microbiota has improved significantly over the past decade,the complex interactions between the gut microecosystem and its host are still poorly understood.The diversity in different parts of the human gut is very different,such as the surface adherent flora and the enteric lumen flora,as well as the small intestine and colon microflora[20].Recent advances in this field are mainly the result of the comprehensive development of complex intestinal microecosystem research.At the end of last century,the main research methods of intestinal microbial composition were culture.In recent years,with the development of non-culture technology and the improvement of large-scale DNA sequencing platform,these tools have become the current standards for microflora research[32,33]The new understanding of the gut microbiota has revolutionized many concepts of modern medicine in recent years.The ecological imbalance of microbiota is related to the emergence and development of diseases.Next-generation sequencing technology can fully analyze the composition of gut microbes without the limitations of traditional culture methods.In addition,the introduction of functional techniques such as metabolomics and proteomics has contributed to comprehensive analysis,resulting in stronger insights into the function of microbiomes in individual health and disease.These tools can alter the factors that affect the microbiome,and the data can help explain the pathophysiological aspects of certain diseases,thereby identifying new potential therapeutic targets.Among the many factors that affect the flora,the widespread use of antibiotics has great influence.In addition to their benefits in treating infectious diseases,some drugs have harmful effects on intestinal flora,which in turn may have long-term effects on the host.Therefore,therapeutic regulation of intestinal flora by selection of probiotic-acting antibiotics,probiotics,and fecal bacteria transplantation seems likely to prevent or even reverse antibiotic-induced dysregulation[34].We aim to study the effect of antibiotics on intestinal flora of healthy individuals in Central China,and adopt metagomogenomics method to conduct statistical analysis on intestinal flora of individuals taking antibiotics and those not taking antibiotics,so as to obtain different results of intestinal flora in different aspects,which will lay a foundation for further research.ObjectiveThe purpose of this study is to study the differences of intestinal flora at species level and functional level between healthy individuals receiving antibiotics and healthy individuals not receiving antibiotics in central China,and to conduct statistical analysis,so as to provide evidence for further studies on the physiological pathology and diseases related to intestinal flora or oral flora.MethodsWe prospectively collected blood,urine,and stool samples from 202 participants,including those taking antibiotics（n=65）and those not taking antibiotics（n=137）.Metagenomic sequencing was performed on all blood,urine,and stool samples.We performed a series of microecological data analyses,including species composition analysis,α diversity analysis and β diversity analysis.The specific steps are as follows:（1）screening participants;（2）Sample collection and DNA extraction;（3）Sequence data processing;（4）Bacterial diversity and taxonomic analysis;（5）Statistical analysis.Results1 For species level diversity analysis,there was no significant difference of the shannon index and Simpson index between the antibiotic group and the non-antibiotic group.Comparing the distance of Jenon-Shannon Divergence（JSD）from two samples in the antibiotic group and the non-antibiotic group,there were significant differences.Compared the Bray-Curtis（Bray）distance between two samples in the antibiotic group and the non-antibiotic group,there were significant differences.PCoA analysis based on JSD distance showed that the first principal component was significantly different between the antibiotic group and the non-antibiotic group.The second principal component showed no significant difference between the antibiotic group and the non-antibiotic group.The PCoA analysis based on Bray distance showed that the first principal component was significantly different between the antibiotic group and the non-antibiotic group.The second principal component showed no significant difference between the antibiotic group and the non-antibiotic group.Significance tests were performed for the species whose abundance was not 0 in each sample and was greater than 20 in the antibiotic group and the non-antibiotic group,and 19 species were found to have significant differences between the antibiotic group and the non-antibiotic group（P-value<0.05,FDR=0.35）.The phenotypic age was found to be significantly different between the antibiotic group and the non-antibiotic group in t-test（P-value<0.05 in T-test）.In chi-square test,there were significant differences in phenotypic antibiotic type and sex between the antibiotic group and the non-antibiotic group（Chi-square test,P-value<0.05）.Permanova analysis of phenotypes was performed based on the Bray distance at the species level.Three phenotypes,namely gender,Body Mass Index（BMI）and antibiotic grouping,had significant effects on intestinal microorganisms（P-value<0.05）,while the other phenotypes had no effects.CCA analysis of phenotypes based on species markers showed that gender and BMI had significant effects on intestinal flora species.Heat map analysis shows that the three related species are all concentrated in the non-antibiotic group,that is,Coprococcus_sp_ART55₁,Prevotella_copriand Bacteroides_compiled Losilyticus,indicating that the significant difference of the three species is related to the phenotype.2 In functional level diversity analysis,the Shannon index and Simpson index showed no significant difference between the antibiotic group and the non-antibiotic group.Diversity analysis compared the JSD distance between two samples in the antibiotic group and the JSD distance between two samples in the non-antibiotic group,showing significant differences.There was no significant difference between the Bray distance between two samples in the antibiotic group and in the non-antibiotic group.PCoA analysis based on JSD distance showed no significant difference in the first principal component between the antibiotic group and the non-antibiotic group.There was no significant difference in the second principal component between the antibiotic group and the non-antibiotic group.The PCoA analysis based on Bray distance showed no significant difference in the first principal component between the antibiotic group and the non-antibiotic group.The second principal component showed no significant difference between the antibiotic group and the non-antibiotic group.Significance tests were performed on the pathways whose abundance was not 0 and whose frequency of occurrence was greater than 5 in each sample.It was found that 23 pathways showed significant differences between the antibiotic group and the non-antibiotic group（P-value<0.05,FDR=0.52）.The phenotypic age was found to be significantly different between the antibiotic group and the non-antibiotic group in t-test（P-value<0.05 in T-test）.In chi-square test,there were significant differences in phenotypic antibiotic type and sex between the antibiotic group and the non-antibiotic group（Chi-square test,P-value<0.05）.Permanova analysis of phenotypes was performed based on Bray distance based on the pathway level.BMI and gender phenotypes had significant influence on intestinal microorganisms（P-value<0.05）,while other phenotypes had no influence.Heat map analysis showed that there were 8 related pathways in the antibiotic group and 15 related pathways in the non-antibiotic group.Conclusion1.At the species level,the β diversity analysis compared the JSD and Bray distances between two samples in the antibiotic group and the non-antibiotic group had statistically significant.PCoA analysis showed that the first principal component was significantly different between the antibiotic group and the non-antibiotic group.Significance test was conducted for each species whose abundance was not 0 and whose occurrence frequency was greater than 20,and it was found that there were significant differences between the antibiotic group and the non-antibiotic group for 19 species.Statistical analysis showed that gender and BMI had significant effects on intestinal flora species.Heat map analysis shows that the three related species are all concentrated in the non-antibiotic group,that is,Coprococcus_sp_ART5 5_,Prevotella_copri and Bacteroides_compiled Losilyticus.2.At the functional level,the β diversity analysis compared the JSD distance between two samples in the antibiotic group and the non-antibiotic group had statistically significant.In each sample,the pathways with the occurrence times of non-0 abundance greater than 5 were tested for significance in the antibiotic group and the non-antibiotic group,and significant differences were found in 23 pathways.Permanova analysis showed that BMI and sex had a significant effect on gut microbes.Heat map analysis showed that there were 8 related pathways in the antibiotic group and 15 related pathways in the non-antibiotic group.3.The purpose of this study is to analyze the influence of antibiotics on intestinal flora,which can provide a certain basis for future studies on oral flora and antibiotic resistance prescription,and lay a certain foundation for us to further study human microecology in the future.