The Effect Of Lactic Acid Bacteria With Wide-spectrum Antimicrobial Activity On The Gut Microbial System Of Infants With Diarrhea

This article analyzes the diversity of lactic acid bacteria(LAB)in diarrhea and healthy infants'feces,and conducts a preliminary study on the probiotic properties and antibacterial activity of isolated LAB.One strain with wide-spectrum inhibition on common food-borne pathogens was selected,and the effect of the strain selected on the intestinal microorganisms of diarrhea infants were investigated in vitro.1.800 LAB were isolated from 300 diarrhea samples and 85 healthy samples collected from hospital,and 135 strains were randomly selected for identifying to species or subspecies by 16S r RNA gene combined with morphological characteristics and physio-biochemical characteristics.The results showed that Enterococcus accounted for the largest proportion in infant feces,with health and diarrhea of 82.93%and 84.04%,mainly were Enterococcus faecium,Enterococcus faecalis,and Enterococcus avium.Besides,members of the genera Lactobacillus was also detected,with health and diarrhea of 17.07%and 15.96%,of which Lactobacillus casei,Lactobacillus paracasei,Lactobacillus fermentum and Lactobacillus plantarum were mainly in healthy infants'feces,and Lactobacillus casei,Lactobacillus fermentum,Lactobacillus paracasei and Lactobacillus rhamnosus were most commonly in diarrhea feces.2.LAB isolated from infant feces were screened for salt-tolerance,acid-alkali tolerance,temperature tolerance,antibiotic sensitivity,hydrophobicity and aggregation,simulated gastrointestinal survival test,and antibacterial activity test,and L.casei ZX633 were selected with wide-spectrum and high efficient antimicrobial activity.This strain can survive in the low temperature environment of 5°C and 10°C,grow well in the salt concentration of 3.0%and 6.5%,and can grow in the acid and alkali environment of p H 3.5-p H 10.In addition,this strain showed resistance to colistin sulfate,gentamicin,penicillin,vancomycin,ciprofloxacin,and sensitive to tetracycline,erythromycin,chloramphenicol,rifampin,ampicillin;with high hydrophobicity(10%),auto-aggregation of 40%,co-aggregation with pathogens ranging from 5.14%to27.03%.Moreover,the cell-free supernatant of L.casei ZX633 has varying degrees antibacterial activity of 10 pathogens such as Micrococcus luteus,Escherichia coli,Staphylococcus aureus,Salmonella enterica,Listeria monocytogenes,Pseudomonas aeruginosa and Bacillus subtilis.The antibacterial effects were not observed after the CFS treated with protease,the bacteriostatic activity of LAB did not disappear after catalase treatment,and when the p H was adjusted to 5.5,it still had an inhibitory effect on Micrococcus luteus ATCC 4698 and Bacillus subtilis ATCC 6633.3.The feces of infants with diarrhea were treated with a concentration of 10~5CFU/g L.casei ZX633,and analyzed the effect on intestinal microorganisms in infants with diarrhea in vitro.The viable counts of main microorganisms and the concentration of short chain fatty acid(SCFA)in infant feces were respectively measured by microbial dilution plate method and high performance liquid chromatography(HPLC).The results showed that diarrhea samples after treated with L.casei ZX633,the viable counts of yeast,E.coli,LAB and aerobic-bacteria increased which tended to the healthy group,and the formic acid and propionic acid contents were significantly reduced which had no significant difference with healthy group.The V3-V4 region of the 16S r DNA gene was sequenced by Illumina high-throughput sequencing technology.Alpha diversity analysis and cluster analysis indicated that the abundance for diarrhea group was higher than healthy group and the diversity was lower,which both decreased after treatment.The gene sequence classification showed that the healthy,diarrhea group and treatment group were mainly composed of four phylums:Actinobacteria,Proteobacteria,Firmicutes and Bacteroidetes.At the genus level,in diarrhea group,the abundance of Streptococcus and Bacteroides(14.21%and 10.05%,respectively)was higher than that the healthy group(3.26%and 0.05%,respectively),the abundance of Escherichia-Shigella(7.56%)was lower than the healthy group(27.63%),which all(10.86%,7.29%and 11.29%,respectively)developed to the healthy group.This study shows that L.casei ZX633 isolated from the feces of healthy infant has a broad-spectrum bacteriostatic effect on pathogens,and can improve the intestinal microbiota of infant with diarrhea.Therefore,it can be used as a new natural probiotic bacteria to reduce bacterial diarrhea for further research.