The Characteristics Of Veillonella And Its Inhibition Of Enterohemorrhagic Escherichia Coli Infection

There are a large number of microbial florae in the intestines of human and animals,and they interact with each other to form a complex intestinal microbial environment.Veillonella is an anaerobic symbiotic bacterium in the intestines of human and animals,which can utilize organic acids such as lactic acid to metabolize and produce short chain fatty acids such as acetic acid.As the core lactate-utilizing bacterium in the intestine,Veillonella regulates the dynamic balance of lactic acid and has physiological functions such as maintaining intestinal health and inhibiting pathogen infections.In this study,we isolated and identified multiple Veillonella,and explored their growth characteristics and individual morphology.And we Studied the antibiotic sensitivity and biofilm formation ability of the strains of Veillonella.In order to explore the potential probiotic function and mechanism of Veillonella and lay a foundation for its development as a potential probiotic,we studied in vitro and in vivo interactions between Veillonella and EHEC O157:H7.The main research content and results are as follows:(1)Twelve strains of Veillonella were isolated and identified from samples such as human oral cavity,infant feces,and dairy goat feces by using a selective culture medium.Based on the housekeeping gene rop B gene and 16 S r RNA gene of Veillonella,PCR technology was used to identify the strains at the species level.The study publicly stored four potential development value strains of Veillonella at the China Industrial Microbial Strain Conservation and Management Center(CICC),and obtained the strain preservation number V.rogosae CICC 25158,V.nakazawae CICC 25189,V.nakazawae CICC 25170 and V.ratti CICC25149.The DNA sequence of the strains were submitted on NCBI and the sequence number was obtained: 16 S r RNA genes: V.rogosae OM267728.1,V.nakazawae OM267781.1,V.parvula OM838404.1,V.ratti OL597939.1;rop B gene: V rogosae OM830707,V.nakazawae OM830708,V.parvula OM809111,V.ratti OM830709.(2)Use scanning electron microscope to observe the individual morphology of the four isolated species of Veillonella,and observe that Veillonella is spherical or ellipsoidal,with a diameter of about 0.3-0.5 μm.Arranged in a single or short chain.According to the standards of the American Clinical and Laboratory Standards Institute,the MIC values of penicillin,piperacillin-tazobactam,cefoxitin,tetracycline,moxifloxacin,clindamycin,metronidazole,and vancomycin against four strains of Veillonella were determined by Agar dilution method.The result showed that V.rogosae and V Nakazawae were resistance to penicillin,V.ratti was resistance to piperacillin-tazobactam,and the four strains were sensitivity to other antibiotics.The bacterial biofilm forming ability of four strains of Veillonella was measured by using crystal violet staining method.V.parvula has the strongest biofilm forming ability among the four bacteria,6-13 times that of the other three species of Veillonella.(3)Studied the growth rule of co-culture in vitro between V.ratti and EHEC O157:H7.When V.ratti and EHEC O157: H7 co-cultured in vitro,compared to EHEC O157:H7 pure culture,the time for V.ratti and EHEC O157: H7 co-cultured system to enter the stable phase was delayed by 3 hours.In the co-culture system of V.ratti and EHEC O157:H7,the number of viable bacteria decreased significantly after 24 hours of cultivation compared to 9 hours of cultivation.The p H value in the system gradually decreased,which inhibited the growth of EHEC O157:H7.(4)The metabolism of short chain fatty acids of V.ratti and EHEC O157: H7 co-cultured in vitro was studied using gas chromatography.After 48 hours of co-cultivation between V.ratti and EHEC O157: H7,the main metabolites were acetic acid(201.44 mg/L)and propionic acid(236.24 mg/L).The yield is 2-3 times that of EHEC O157: H7 pure culture.The total amount of short chain fatty acids metabolized in co-culture was 439.64 mg/L,which was 5.4times that of EHEC O157: H7 pure culture(82.27 mg/L).When EHEC O157: H7 co-cultured with V.ratti,the expression levels of QS regulatory genes(lux S,qse A,qse B,qse C,qse E,qse F),LEE virulence island related regulatory genes(eae,esp D,esp A,ler,grl A,grl R),adhesion factor genes(efa1-a,efa1-b),and toxin genes(pag C,exh A,stc E,vt2 A,vt2B)were downregulated(p < 0.05).This indicates that during the interaction process of co-culture,the expression of EHEC O157: H7 virulence genes were to some extent inhibited.(5)To study the inhibitory effect and mechanism of V.ratti on EHEC O157: H7 in vivo infection,we used piglet intestinal ligation animal experiments.Compared with the EHEC O157: H7 positive group,the EHEC O157: H7 infected piglets with V.ratti intervention increased the abundance and diversity of gut microbiota,arranged the intestinal villi more neatly and orderly,with more complete brush like edges,significantly improving the degree of damage to the small intestine structure and tissue of piglets.After V.ratti intervention in piglets,the expression of QS regulatory genes(lux S,qse A,qse B,qse C,qse E,qse F),LEE virulence island related regulatory genes(eae,esp D,esp A,ler,grl A,grl R)),adhesion factor genes(efa1-a,efa1-b),and toxin genes(pag C,exh A,stc E,vt2 A,vt2B)werw downregulated(p < 0.05).Which revealed that V.ratti intervention in the gut of piglets suppressed the expression of EHEC O157: H7 related pathogenic factor genes,alleviated the infection of EHEC O157: H7 and its damage to the gut.The research results indicated that V.ratti is a functional intestinal probiotic with development potential,which has potential probiotic functions such as inhibiting pathogen infection,increasing short chain fatty acid production,and regulating stable intestinal microbiota.