Effects Of Empirical Antibiotic Therapy On The Early Development Of Gut Microbiota And Metabolites In Preterm Infants And Study On The Role Of Vitamin A In Neonatal Necrotizing Enterocolitis

PART Ⅰ EFFECTS OF EMPIRICAL ANTIBIOTIC THERAPY ON THE EARLY DEVELOPMENT OF GUT MICROBIOTA AND METABOLITES IN PRETERM INFANTSObjective: The early postnatal period is the most important stage in the assembly of intestinal microbiota.Antibiotics are commonly prescribed to newborn preterm babies with high risk of infection in order to avoid early onset sepsis.However antibiotics have a significant effect on the composition of intestinal microbiota.Our study aims to investigate the effects of one-week empirical antibiotic therapy on the early development of intestinal microbiota and their metabolites,which may provide theoretical basis for the rational use of antibiotics.Methods: The study subjects were preterm infants who were sent to the Children’s Hospital of Chongqing Medical University immediately after birth.According to the type of antibiotics,the antibiotic groups were divided into two groups,one group was treated with Piperacillin/ tazobactam（Pt group）,another was treated with Penicillin combined with Latamoxef（PL group）,and the control group was free of antibiotics（AF group）.The fecal samples of each baby in the three groups on Day3 and Day7 were collected.High-throughput sequencing that based on the 16 S r DNA was used to analyze the composition of intestinal microbiota in the three groups,and liquid chromatography-mass spectrometry（LC-MS）platform was used to analyze the metabolites in the three groups.Results: Both PL and Pt groups showed significant lower Shannon index on Day7 compared to those on Day3（P<0.01 for PL group,P<0.05 for Pt group）,but the Shannon index in AF group showed no statistical difference on Day7 compared to that on Day3（P>0.05）;Partial least squares-discriminate analysis（PLS-DA）showed that the differences in bacterial compositions in all the samples of antibiotic groups on Day3 and Day7 were more obvious than AF group;The microbial community analysis results showed that Bacteroidetes and Actinobacteria were significantly different among the three groups on Day3 at phylum（P<0.001 and P<0.05,respectively）,but Proteobacteria,Firmicutes,Bacteroidetes and Actinobacteria all have statistical differences among the three groups on Day7（P<0.05 for Proteobacteria and Firmicutes,P<0.001 for Bacteroidetes,P<0.01 for Actinobacteria）;Comparing the proportion of bacteria on Day3 with that on Day7,we found only the decrease in Actinobacteria in Pt group and increase in Bacteroidetes in PL group have statistical differences（both P<0.01）;At genus level,there were significantly differences in Pseudomonas（the proportion in Pt group was 23.73%,which is the highest among the three groups,P<0.05）,Enterococcus（Pt group containing 23.26% was highest among the three groups,P<0.01）,Lactobacillus（PL group containing 28.04% was highest among the three groups,P<0.001）,and Sphingomonas（PL group containing 6.64% was highest among the three groups,P<0.001）among the three groups on Day3,and the proportion of Klebsiella（AF group containing 64.39% was highest among the three groups,P<0.05）,Enterococcus（Pt group containing 48.12% was highest among the three groups,P<0.001）,Escherichia-Shigella（PL group containing 17.25% was highest among the three groups,P<0.05）,Clostridium_sensu_stricto₁（AF group containing 5.62% was highest among the three groups,P<0.01）,Peptoclostridium（AF group containing 5.60% was highest among the three groups,P<0.001）,Pseudomonas（PL group containing 2.688% among the three groups,P<0.01）were significantly different on Day7;Comparing the proportion of bacteria in each group on Day3 to that on Day7,Enterococcus was increased（P<0.05）and Escherichia-Shigella（P<0.05）,Pseudomonas and Acinetobacter（both P<0.01）were decreased in Pt group on Day7,the decrease of Lactobacillus,Acinetobacter and Sphingomonas in PL group had significant differences（P<0.01 for Lactobacillus,P<0.05 for Acinetobacter and Sphingomonas）,the increase of Klebsiella and decrease of Escherichia-Shigella and Acinetobacter had statistical significances in the AF group（P<0.05 for Klebsiella and Escherichia-Shigella,P<0.001 for Acinetobacter）.According to the fecal metabolomic analysis,We found Gluconic acid,L-valine,L-serine,Glycerol（both P<0.05）and L-Proline（P<0.01）were significantly different among the three group on Day3,and six kinds of metabolites including L-valine and citric acid（both P<0.001）,linoleic acid and L-glutamate（both P<0.01）,L-tyrosine and pantothenic acid（both P<0.05）had statistically significant differences on Day7 among the three group;Besides,there were only seven metabolic pathways had statistically significant differences on Day3（both P<0.05）,but 22 metabolic pathways exhibited significant differences among the three groups（both P<0.05）and there were almost opposite metabolism activities representation of the 22 pathways between the PL and AF group on Day7.Based on PLS-DA,the differences in metabolite compositions between the antibiotic groups and AF group were more remarkable on Day7 compared to that on Day3,and different type of antibiotic have different effects on the types of metabolites;The correlation analysis of fecal metabolites and bacterial compositions showed that the essential metabolites including linoleic acid,L-glutamate,L-valine and pantothenic acid were significantly increased in the AF group,and showed a positive correlation with Veillonella,Citrobacter and Peptoclostridium in the AF group,while this four kinds of metabolits had negative correlation with the bacteria that had high abundance in antibiotic groups,especially with the bacteria in PL group.Conclusion: Significant changes were observed in the composition of gut microbiota as well as the metabolites and metabolic pathways after one-week antibiotic treatment.The pathogenic bacteria were increased after prolonged use of antibiotic,such as Escherichia-Shigella and Enterococcus.Besides,regardless of the type of antibiotic,the community diversity of the antibiotic group on Day7 was significantly lower than that on Day3.And the change trends of metabolites and related metabolic pathways were consistent with the results analyzed by high-throughput sequencing.The differences in metabolites and metabolic pathways between the antibiotic groups and AF group were more remarkable with the time of antibiotic use increasing,and most of the high content metabolites in each group were positively correlated with high abundance bacteria in its own group.The production of normal metabolites and the activity of key metabolic pathways were decreased in the antibiotics groups,and the usage of Penicillin combined with Latamoxef had more powerful influences on intestinal microbiota and metabolites than treated with Piperacillin/tazobactam.PART Ⅱ STUDY ON THE ROLE OF VITAMIN A IN NEONATAL NECROTIZING ENTEROCOLITISObjective: Neonatal necrotizing enterocolitis（NEC）is an acute and serious intestinal disease that affects newborns,especially preterm infants.It is generally accepted that NEC is caused by an invasion of bacteria and exaggerated inflammation due to an immature intestinal barrier and immunity system of newborns.Vitamin A（VA）is an essential nutrient for normal physiological metabolism,and it plays important role in the regulation of epithelial growth and immune function in human body.The first aim of our study is to clarify the correlation in VA,microbiota and NEC through detecting the changes of intestinal microbiota and the level of VA in infants with NEC.By making clear the above changes and verifying the correlation,we would manage to investigate the effects of VA supplementation on the development of NEC in vivo and in vitro,the findings are able to provide a new target to cure or prevent NEC.Methods: Clinical samples collection and analysis: The serum samples and fecal samples from NEC patients and controls were collected.The levels of serum retinol in patients were detected with the use of highperformance liquid chromatography（HPLC）.The microbial communities of NEC and controls were detected by 16 S r DNA high-throughput sequencing.In vivo experiments,the newborn mice received an intragastric administration of VA（VAS group）and PBS（PBS group）for 7 days and then were established as an NEC model by formula feeding and hypoxia/cold stress.The levels of serum retinol in mice of two groups were detected by HPLC.Intestinal tissue slices were stained with hematoxylin and eosin（HE）,and pathological damage in the structure of the ileum were graded by a blinded evaluator in order to compare the severity of NEC in VAS and PBS group;the microbial communities of NEC mice treated with VA or PBS were detected by 16 S r DNA high-throughput sequencing approach;the levels of inflammatory factors were measured by ELISA and q PCR,and the expression of tight junction proteins in the intestinal mucosa of NEC mice in VAS and PBS groups were detected by western blot.In vitro experiments,we used a Caco-2 cell monolayer to established a well trans-well system,and then the Caco-2 cells monolayers were treated with retinoic acid,the integrity of epithelial barrier was analyzed by the measure of transepithelial electrical resistance;after RA treatment,the levels of inflammatory factors in Caco-2 cells monolayers were measured by q PCR and ELISA,and the expression of tight junction proteins were detected by WB.Results: The serum level of VA in the NEC group was significantly lower than that in the control group（P<0.05）.Comparing with controls,the proportion of Proteobacteria was significantly increased and the proportion of Bacteroidetes was significantly decreased in the NEC patients（P<0.05 and P<0.01,respectively）.In vivo experiments,the serum VA level in VAS group was significantly higher than PBS group（P<0.01）.Compared with the PBS group,the abundance of Proteobacteria was lower in the VAS group（P<0.05）,while the proportion of Bacteroidetes was significantly higher（P<0.01）.The results of q PCR and ELISA showed the expression levels of IL-6 and TNF-α were significantly decreased in the intestinal tissues of mice in the VAS group comparing to PBS group（P<0.05 for IL-6 and P<0.01 for TNF-α at m RNA level;P<0.001 for IL-6 and P<0.05 for TNF-α at protein level）.The NEC score of histopathologic evaluation in VAS group was obvious lower than PBS group（P<0.05）,indicating that only mild inflammation exhibited in the intestinal wall of VAS group,while obvious intestinal mucosal injuries were observed in the PBS group.Besides,the expressions of claudin-1,occludin（both P<0.05）and ZO-1（P<0.01）exhibited statistical significant increase compared the VAS group to the PBS group.In vitro experiments,TEER values were significantly increased in the RA treatment groups and RA treatment also could mitigated LPS-induced damage to Caco-2 cells monolayers（both P<0.01）;the results of q PCR and ELISA showed the levels of IL-6 and IL-8 were significantly decreased（At m RNA level: P<0.001 for IL-6 in 5R-10 L group and 2R-10 L group,P<0.0001 for IL-8 in 5R-10 L group,P<0.001 for IL-8 in 2R-10 L group;At protein level,P <0.01 for IL-6 and IL-8 in 5R-10 L group,P <0.05 for IL-6 and IL-8 in 2R-10 L group）;the results of western blot indicated that the expressions of occludin（P<0.05）,claudin-1 and ZO-1（both P <0.05）were increased in 5R-10 L group after RA treatment,while only claudin-1 was increased in 2R-10 L group after RA treatment,the differences in Occludin and ZO-1 between 2R-10 L group and 0R-10 L group were not statistically significant.Conclusion: The low level of VA,immature intestinal barrier function and changes in intestinal flora（the increase in Proteobacteria and decrease in Bacteroidetes）were risk factors that underlie the pathogenesis of NEC,which leading to the pathogens invaded to the intestinal mucosa causing excessive inflammation.VA supplementation could decrease the proportion of Proteobacteria,and increase the proportion of Bacteroidetes.It also could down-regulate the levels of inflammatory factors,such as IL-6,IL-8 and TNF-α,and improve the expressions of tight junction proteins and enhance the integrity of epithelial barrier.VA exerted significant regulating effects on the development of NEC.