| BackgroundGut flora plays a vital role in forming the immune system,preventing colonization by pathogenic microorganisms,maintaining intestinal homeostasis,and improving infant health and development.Microbiomes reshape the immune microenvironment by altering the structure and function of the immune system.The imbalance of the gut microbiota may disturb the microbiota-host interactions,leading to the occurrence and development of some specific diseases,including food-allergic diseases(atopic and asthma)and gastrointestinal diseases(diarrhea,inflammatory bowel disease,and necrotizing enterocolitis).Clostridium perfringens is an anaerobic,gram-positive bacillus,widely distributed in soil,sewage,feces,food and other environments,and is also a component of human and animal intestinal flora.CPF secretes a variety of toxins and enzymes,which are associated with some systemic and digestive diseases,such as gas gangrene,food poisoning,non-foodborne diarrhea and neonatal necrotizing enterocolitis.However,Studies on the association between intestinal C.perfringens colonization and food allergy or cow’s milk protein allergy(CMPA)have not been searched.Our previous clinical study found that the positive rate of Clostridium perfringens in stool culture was as high as 30% in infants with cow’s milk protein allergy with gastrointestinal symptoms,and was as early as 2 days after birth.Therefore,we speculate that premature colonization of C.perfringens is related to the development of food allergy.Food protein-induced proctocolitis(FPIP)is a type of non-Ig Emediated food allergy,but its risk factors and pathogenesis are not fully understood.Our previous study also suggested that CPF may play a role in the development of FPIP.Therefore,we conducted an intervention study to explore the effect of metronidazole combined with probiotics on the outcome of FPIP in infants.MethodsThis study was conducted in two parts.The first part was a Real World Evidence study(RWE).Congenital inherited metabolic diseases,immunodeficiency diseases and digestive tract malformations were excluded.The clinical data of the children were collected,including demographic data,clinical manifestations,feeding patterns,laboratory tests,CPF fecal culture and toxin test.One-way analysis of variance,chisquare test,and correlation coefficient matrix were used to compare the gender,age,clinical characteristics,routine blood test,and stool test between the children with positive CPF and those negative ones.After 12 months of follow-up,two groups were compared in terms of the incidence of food allergy,and the correlation between food allergy and CPF was analyzed by correlation coefficient matrix.The second part confirmed the difference in the diversity of intestinal flora between FPIP infants and healthy controls by 16 srRNA sequencing.The next step was to study the clinical intervention of metronidazole combined with probiotics on FPIP infants.Infants aged1-12 months with clinical manifestations of nausea,vomiting,abdominal pain,bloating,diarrhea,hematopoietin,anemia,hypoproteinemia,and growth retardation were diagnosed as FPIP by food avoidance challenge.Those who were allergic to metronidazole,unstable vital signs,organ failure,or other unfit for inclusion were excluded.The enrolled children were divided into two groups: group A(metronidazole+ probiotics),B(Metronidazole + probiotic placebo),C(probiotics)and D(probiotic placebo).Clinical score and fecal 16 S rRNA sequencing were performed at baseline,2,4,12 and 24 weeks after treatment.Results1.A total of 358 infants carried out fecal cultured for C.perfringens,and 270 infants met the inclusion criteria and were followed up by telephone after 12 months,including 151 males(55.93%)and 119 females(44.07%),with an average age of2.78±2.84 months.There was no significant difference in gender and age between the positive and negative groups of C.perfringens,while the positive rate of C.perfringens in the breastfeeding group(36/50,72.0%)was higher than that in the artificial feeding group(24/126,19.04%)(P=0.0002).2.Food allergy in infants was significantly associated with fecal occult blood(r=0.28),fecal white blood cell(r=0.25),antibiotic use(r=0.29),increased platelet count(r=0.48),and decreased hemoglobin(r=-0.26).3.The diagnostic rates of NEC,food allergy,CMPA and FPIP in the positive group were significantly higher than those in the negative group(7.14% vs 1.27%,48.21%vs 7.59%,37.5% vs 5.06%,32.14% vs 8.23%).The difference was statistically significant.4.A total of 85 stool samples were tested for toxins as follows: α-toxin 97.6%(83/85),β-2 toxin 82.3%(70/85),β-toxin 65.9%(56/85),and no other toxins were detected.83 samples were classified by toxin type: type C 67.5%(56/83),type A 32.5%(27/83).5.Clostridium perfringens was significantly associated with cow’s milk protein allergy in infants aged 0-6 months(r=0.51,P < 0.01).6.16 S rRNA sequencing showed that the gut microbiota composition before FPIP treatment was significantly different from that of the healthy control group(P=0.012,R~2=0.068).7.At the species level,the relative abundance of Enterococcus in the FPIP group was the highest,which was significantly higher than that in the control group,while the relative abundance of Bifidobacterium in the healthy control group was the highest,which was significantly higher than that in the FPIP group.8.The random forest score showed that Enterococcus,Bifidobacterium,Erysipelothrix,Streptococcus,Prevotella,Halomonas,Cononomonas,Lactobacillus,Veillonella differentia were the most effective in distinguishing FPIP from healthy control group.9.Compared with the placebo group,the metronidazole combined with probiotics treatment group had a significant difference in the clinical score at week 4(P=0.013).There were significant differences in the total stool score between the two groups at weeks 4 and 12(P=0.004;P=0.042).Conclusions1.Different feeding patterns affect the colonization of intestinal Clostridium perfringens.The positive rate of intestinal Clostridium perfringens colonization in breast feeding infants is higher than that in artificial feeding infants.2.Food allergy in infants was significantly associated with laboratory tests such as fecal occult blood,fecal white blood cells,antibiotic use,increased platelet count and decreased hemoglobin.3.Intestinal colonization of Clostridium perfringens at 0-6 months of age was significantly associated with food allergy in infants.4.The intestinal flora of infants with FPIP is disordered,and the structure of intestinal flora is significantly different from that of the healthy control group.5.The intestinal flora of infants with FPIP was unbalanced,with significantly increased enrichment of potential pathogens and significantly decreased enrichment of probiotics.6.Metronidazole + probiotics intervention may prolong the time of intestinal flora balance in infants with FPIP,which needs to be further studied with more samples. |
PDF Full Text Request