| Commensal bacteria reside in the gastrointestinal tract and affect host health both locally and systemically.The relationship between these symbiotic bacteria and their host is far more complex than simple coexistence with nutritional benefits.Recently,the more obscure contributions of symbiotic bacteria to host health have been uncovered and the "hygiene hypothesis",which states that exposure to commensal bacteria is necessary to protect the host from unrelated immune diseases,has been proposed and debated.Firmicutes(50%-70%)and Bacteroidetes(10%-30%)comprise of more than 98%of the human intestinal flora.In recent years,many diseases have been discovered a close relationship with intestinal flora disorder and probiotics become a new treatment for many diseases.Probiotics are live microorganisms confer health effect when administered in adequate amounts.At present,the most developed probiotic are Lactobacillus and Bifidobacterium,from Firmicutes and Actinobacteria,respectively.Surprisingly,though Bacteroidetes is the second largest group of intestinal flora it is still hasn't the representative available and it should be a gap of history for development of probiotics.As the member of Bacteroidetes,Bacteroides fragilis has arisen the public attention recently thanks to some publications revealing its huge health effects.B.fragilis is a ubiquitous Gram-negative obligate anaerobe that colonises the lower gut of mammals,and Bacteroides species constitute a numerically prominent part of the gut flora.Despite comprising 1%or less of Bacteroides sp.,B.fragilis accounts for more than 50%of anaerobic infections in humans,which predominantly lead to intra-abdominal abscesses.B.fragilis was initially considered to be an opportunistic pathogen;however,it is now recognised to exhibit both pathogenic and beneficial characteristics in the host.B.fragilis has been shown to exert powerful immunoregulatory benefits predominantly mediated by polysaccharide A(PSA),a zwitterionic polysaccharide,shown to protect animals against various inflammatory diseases such as trinitro-benzene-sulfonic-sulfonic acid and Helicobacter hepaticus-induced colitis.PSA also exert protection against central nervous system(CNS)demyelination in experimental autoimmune encephalomyelitis,and protection from Bartonella henselae-induced damage.Another report demonstrated that application of B.fragilis relieved ASD symptoms in mice,exhibiting considerable probiotic potential.Previously we isolated a new B.fragilis from feces of a health infant.Trying to develop this strain as a microorganismal agent,we designed experiments mainly based on China Pharmacopeia 2015 edition.Accordingly,microorganismal agent is characterized as microorganism located in human or nontoxic bacteria promoting normal gut microbiome growing and active,being applied in preventing or curing diseases caused by dysbacteriosis.As a commensal bacterial,B.fragilis displays impressing probiotic characteristics,such as curing effects on immune deficiency,inflammatory bowel disease or ASD.It makes confidence that B.fragilis be a potential probiotic in the future.In this study we aim to test it whether safe or not.Furthermore,some fundamental characteristics will be confirmed in this research,providing useful information for researches of this microorganism.Accordingly,accurate identification not only helps understand the biological and scientific characteristics of a strain,but also makes it easy to track and monitor its safe use in human.Correct identification analysis includes gene analysis,morphological and cultural characteristics as well as some biochemical features.Risk of transferable antibiotic resistance is one of the most important parts of assessment of safety since it might lead to serious infection.Therefore,only those proven without transferable antibiotic resistance can be addressed as the safe.In addition,resistance to extreme environment in upper gastrointestinal tract,adhesion to colon cells,resistance to air in combination with nontoxicity to animals and cells are also included in this assessment.Purpose:1.Build phylogenetic tree based on 16s DNA and whole gene sequencing to identify its identity,and find potential toxicity related gene and antibiotic resistance gene trough comparison with known database.2.Understand its morphological and cultural characteristics,resistance to air and adherence to colon cells in vitro.3.Analyze its biochemical features and main products.4.Confirm its resistance to antibiotic.5.Scan its tolerance to gastric and intestine fluid,ox bile.6.Identify whether this strain is safe to mice and colon cells.Material and method:1.Identification of gene DNATotal DNA was extracted from this B.fragilis,then amplified,sequenced and analysed by blast analysis to figure out the nearest strain to it.Build phylogenetic tree based on whole gene sequencing and compare with standard strain to confirm its source.Annotate virulence related gene and antibiotic resistance gene through comparison with know database.2.Morphological and fundamental characteristicsCultured this strain anaerobically for 48h in TSA agar plated and observed its structure by gram staining and by scanning electrochemical microscopy and transmission electron microscopy.This strain was grew by 1:100 in new culture medium,and cultivated anaerobically for 24 hours.Value of OD600 was monitored every two hours.1×109CFu/ml of this strain was cultured under exposure to air for 96 hours,37?,viability would be confirmed at 0,4,8,24,36,48,72,96h;LoVo cells were seeded at(5-10)×104 cells/well in 24-well plates overnight.Bacterial cells were harvested and adjusted to(5-10)×106 CFU/ml(multiplicity of infection,MOI=100)with cell culture medium,co-incubated with LoVo cells at 37? for 30,60 or 90 min.The adherent bacteria were serially diluted and spread on TSA agar plates.3.Biochemical features and main products analysisCultured the bacteria into a Biolog biochemical identification plate for 16-24 h and then read by micro-plate reader,standard strain as control.Dripped few 3%hydrogen peroxide into bacteria precipitation to test catalase.Inoculated the bacteria into gelatin medium with a needle,cultured at 37?for 48 h and placed in 4? for 3-4 h.Cultured the bacteria with solid medium containing blood to determine hemolysis.Cultured the bacteria with semi-solid medium to determine motility.Collected and analysed the supernatant.4.Antibiotic resistance experimentVerified the result of the potential antibiotic resistance gene found by comparison with known database.Collected and diluted bacteria to 107cf/ml,cultured with antibiotic or with saline in 96 microplate for 48h.Then read by microplate reader and determine the minimal inhibitory concentration(MIC).5.Tolerance to gastric and intestine fluid,ox bileGastric fluid with pH value equal to 2.0,3.0 4.0 containing pepsin and intestine fluid with trypsin were prepared.Bacteria were cultured under above liquid for 0,1,2,4 h at 1×109CFU/ml.104 CFU/ml,106 CFU/ml,108 CFU/ml of this strain were cultured in 0%,1%,2%,4%ox bile.Viability was determined by plate counting.6.Toxicity to animal and colon cellsAnimal safety experiment:Three groups were divided:middle dosage group(5×1010 CFU/day),high dosage group(5×1010 CFU/day)and supernatant group(0.5ml/day).SPF level Balb/C mice ranging from 6-8 week old were infected by oral gavage per day for 5 days,observed for 12 days after gavage.The state and body weight were recorded every day.Blood routine index,hepatorenal function and pathological condition were detected in high dose group.Cell safety experiment:In this study we screen in vitro safety of this B.fragilis with a newly emerged non-invasive and label-free approach that monitors cellular proliferation and viability in real time.This real-time cellular analysis(RTCA)approach continuously monitors cells condition trough impedance detection by iCELLigence apparatus.1)Optimal seeding density of cells:Preplanned seeding density of LoVo and HT-29 cells were prepared by serial two-fold dilution.A volume of 300ul cell suspension was added to each well which contains 12×104,6×104,3×104,1.5×104,0.75×104,0.375×104,0.1875×104 cells,respectively.Culture media was designed as background control in this assay.Optimal seeding density and compromising time would be determined based on growth characteristics provided by software.Live bacteria(MOI=200),bacteria cell lysate(MOI=200),culture supernatant(100ul/450ul)cytotoxicity:B.fragilis,L.casei(probiotic control)and EHEC(pathogenic group)infected LoVo or HT-29 cells(6 × 104 cells)for 80-85h.Medium group was performed as control.Result:1.This is a strain of Non-toxic B.fragilisThe results of 16S rDNA gene sequencing demonstrated that the nucleotide sequence of this strain was up to 99%identical to that of type stain B.fragilis ATCC 28285(GenBank database),and showed high similarity to JCM11019.According to the phylogenetic tree built based on whole gene sequencing,this strain was originated from the same branch with ATCC 25285,confirming that the isolate was a strain of B.fragilis.24 virulence related genes and 11 antibiotic resistance genes were discovered through comparison with ARDB,VFDB database.Most above virulence genes were found to encode capsule,flagella,surface antigen and so on.Therefore we believe that there isn't any gene related to toxicity found in this strain.Remarkably,this strain was classified as non-toxic B.fragilis because bft gene was absent in it.Furthermore,all of the drug resistance gene were located in chromosome rather than in the only plasmid,confirming its safety without transferable drug resistance.2.Morphological and fundamental characteristicsBy Gram staining and direct observation by scanning electrochemical microscopy and transmission electron microscopy,this strain was found to be Gram-negative,and rod shaped with rounded ends.The physiological and biochemical features of this strain were also in accordance with B.fragilis as described in Bergey's manual.This train entered exponential phase after 8 h,and progressed to stationary phase at around 16 h.Through our study,we selected late logarithmic phase(12-16 h)cells because of the peak in the number and viability of bacteria.As most assays are performed aerobically,we tested the viability of this strain after exposure to air ant it showed resistance to air for at least three days.Survival rates gradually decreased with few surviving cells at 72 h.This demonstrated that this B.fragilis is not a strictly anaerobic bacterium,and sufficiently air resistant for the in vitro assays performed in this study.The results of an adhesion assay to assess B.fragilis(MOI=100)adhesion to LoVo cells are performed.Differences in the adhesion rate between each time point were not significant(p>0.05),but the adhesion rate showed a steady increase over time reaching 4.18 ± 1.18,5.25 ± 1.03 and 5.51 ± 1.60%of the adhesion activity to Lo Vo cells at 30,60 and 90 min,respectively.This result demonstrated the adherent ability of B.fragilis to colon cells in vitro.3.Biochemical features and products analysisAnalyse the biochemical features and product of this strain for further identification.There some differences between this strain and standard strain in metabolising N-acetyl D-glucosamine,alanine,salicin and so on.But both of them showed positive in catalase test,weak positive in gelatin liquefaction test,non-hemolysi and non-motile.Lactic acid,acetic acid,succinic acid,propionic acid and phenylacetic acid were the main product of them.4.Drug resistanceThis strain was resistant to cefepime,kanamycin,streptomycin but sensitive to ceftriaxone,trimethoprim,clarithromycin,chloramphenicol,tetracycline and levofloxacin.5.Tolerance to stimulated digestive fluidAn in vitro test was adopted to test the survival rate after exposure to SGF,SIG and bile under different conditions(pH or concentrations)and time points.In this study,this strain was unable to survive on exposure to SGF of pH 2.0(data not shown),but survived well with SGF at pH 3.0 or pH 4.0,and with SIF at pH 6.8.It showed high resistance to bile,which was consistent with a previous study that reported that B.fragilis showed good tolerance to bile salt.6.This B.fragilis is safe to animal and colon cellsThere was no death during animal experiment,and the difference of change of body weight per day was non-significant(p=0.46)between middle dosage group and control group.In high dosage and supernatant experiment,similar results were obtained,differences(p=0.83 and p=0.9598)were non significant as well.The difference of blood routine index,hepatorenal function and pathological condition were not significant between high dose group and control group.Compared with HT-29 cells,LoVo cells are much more bigger in size and more flexible in morphology.Considering needs for following experiments,density of 6×104cells per well was selected thanks to its suitable growing profile and stable proliferating tendency.Furthermore,we would compromise cells at 12-14h because during this time period HT-29 cells were logarithimic growing while LoVo cells had already reached plateau.Log phase and plateau compose two vital parts of cell growth and how added substances affect these parts represents cytotoxicity in different aspects.Intact bacteria(MOI=200),cell lysate(MOI=200)as well as supernatant(100ul/450ul)of B.fragilis,L.casei and EHEC infected LoVo and HT-29 cells,data of NCI were monitored for 80-85h.According to the results,none of live bacteria,cell lysate or culture supernatant of B.fragilis impairs HT-29 cells and LoVo cells,since NCI didn't fall in whole experiment in comparison with probiotic control(L.casei)or medium control.Conclusions:1.Strain applied in this research is identified as a strain of non-toxic B.fragilis,originating from the same branch with ATCC25285.2.Show similar morphological and culturing characteristics in accordance with B.fragilis,with desirable tolerance to air and adhesion to colon cells.3.Display similar biochemical features with ATCC 25285,and mainly produce lactic acid,acetic acid,succinic acid,propionic acid and phenylacetic acid.4.This strain was resistant to cefepime,kanamycin and streptomycin,but all the drug resistance genes only located in chromosome,without risk of transferable drug resistance.5.This strain survives well in simulated gastric fluid(pH 3.0),intestine fluid and ox bile.6.This strain is safe to animal.No death or weight loss happened by mice after oral infected with 5×1010,5×1011CFU or supernatant of it.This strain is safe to colon cells.Normalized cell index is stable after infection by live bacteria,cell lysate or supernatant of it. |
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