| Background and purposeCronobacter sakazakii (Cronobacter sakazakii, CS), belonging to the Enterobacteriaceae,which is a week flagella, motion and facultative anaerobic leather gram negative bacillus that parasitic in the intestinal tract of humans and animals. The bacteria is the major cause of infants, especially the premature infants were easily infected with meningitis, septicemia and necrotizing enterocolitis. The mortality rates are as high as 40%～80% and the survivors of the infection could easily lead to severe neurological sequelae. Since 1961, it is reported two cases that caused by the meningitis of Cronobacter sakazakii, successively after in the world within the scope of reported incidents of a series of neonatal infection. At Fuyang, Anhui of China, the inferior infant formula milk powder incident has caused the government to attach great importance to it in 2004,for the first time they checked it from 87 copies of infant formula milk powder separation to 11 strains of this bacteria, the detection rate was up to 12.6%. The children had a head enlargement and unresponsive,so the bacteria maybe plays an important role in the pathogenesis of this case. At present, ampicillin and gentamicin or chloramphenicol is the gold standard for the infection of Cronobacter sakazakii.But the widespread use of antibiotics has the inevitable hazards:1) anti infective drugs can easily lead to human microecological imbalance; 2) resistance; 3) the vast majority of anti infective drugs is designed for microorganisms, but the relatively neglect the host’s role in anti infection. Therefore, supplying and studying new drugs and alternative methods of antibiotic therapy has attracted more and more attention.The Cronobacter sakazakii how to pass through the intestinal barrier caused by central nervous system infection remains unclear presently, but in the pathogenesis of neonatal meningitis, Cronobacter sakazakii firstly engraftment in neonatal intestinal, then penetrate in the intestinal barrier and the blood brain barrier,。After that, it can cause bacteremia and meningitis, so giving the prevention of Cronobacter sakazakii penetration of intestinal epithelial cells into the blood, also becomes the key link of the effective prevention which is caused by bacteremia meningitis.On the basis of that, probiotics is often used as a preventive and antibacterial treatment of complementary or alternative, especially it played a great advantage role in the protection and the treatment of various caused by germs, and the conditions of pathogenic bacteria in intestinal adhesion, invasion and growth aspects. Probiotics also was known as micro ecological preparation or live bacteria preparation, according to the definition of the European food and Feed Association in 2003, probiotics refers to something taht can have one or more of the host have been shown to have functional health benefits of living microorganisms when a sufficient number of intake, such as Lactobacillus genera and Bifidobacterium, propionic acid bacteria, bacillus, yeast, and so on. Research has demonstrated that probiotics can prevent and treat various of infectious diseases, the mechanism mainly includes:directly interact with the pathogenic microorganisms, through substitution, exclusion and competition mechanism to inhibit pathogen growth and adhesion of colonization; Adjust the intestinal micro ecology and promote environmental symbiosis steady; they play the anti infection effect through the site and nutrient competition and blocking the on intestinal mucosa to induce destruction and hematogenous metastasis; induce colonic mucin gene expression upregulation and intestinal mucosal surface mucin secretion to antagonize the effect caused by the adhesion of bacteria invasion and transfer across the cell translocation.For example the prevention and treatment of probiotics used in early neonatal meningitis, which can overcome many disadvantages of antibiotics. Recently there has been a study that is the first to demonstrate the probiotic Lactobacillus rhamnosus can inhibit the adhesion and invasion of E. coli K1 strains in neonatal rat intestinal adhesion and invasion, resultied in a significant reduction in the incidence of bacteremia and meningitis. But whether probiotics can inhibit the adhesion and invasion of Cronobacter sakazakii and reduce the incidence of bacteremia and meningitis. it is still not clear now.The selection of Lactobacillus rhamnosus and new probiotics B.fragilis as the research object. LGG is one of one Lactobacillus strains isolated from healthy human intestinal tract, which is the most widely studied probiotics of human. It can live in gastrointestinal tract, high-adhesion-rate, colonization ability, lower cholesterol, promote cell division, regulate and the balance of intestinal flora function, strengthen the natural defenses against infection, it also has a positive effect on improvement of various intestinal diseases. Bacteroides fragilis is a common human anaerobic bacteria, non-spore forming, no power, enterotoxigenic and does not produce enterotoxin. Enterotoxigenic strains was studied more, may cause diarrhea in humans and animals, but the non-enterotoxigenic Bacteroides bacteria gut colonization in the human. For children, promote the growth and development and improve the immunity can enhance the barrier function of intestinal epithelial cells which have obvious therapeutic effect on respiratory and digestive tract of some of the common and frequently occurring disease. It is a new kind of living biological preparation that has a role of eliminating intestinal inflammation, treating diarrhea and autistic disorder and other functions.In summary, the main purpose of this study:assessing the Lactobacillus rhamnose (LGG) and Bacteroides bacteria (BF SK08) which was separated from a healthy full-term newborns fecal, that two kinds of probiotic wheather can inhibit the adhesion and invasion of ATCC 29544 to the epithelial cells of the small intestine (Caco-2), thereby give the prevention and treatment of bacteremia meningitis caused by Cronobacter sakazakii. (1) in vitro, we used Caco-2 cell as a model, detected the adhesion and invasion of Cronobacter sakazakii to Caco-2 cells and the adhesion properties of two kinds of probiotics on Caco-2 cell. (2) With competition exclusion replacement method, discussed the two kinds of probiotics wheather can inhibit the adhesion and invasion of Cronobacter sakazakii on Caco-2 cells and determineed the way of probiotics how to adhere and inhibit Cronobacter sakazakii on Caco-2 cell. (3) We used Western blot to verify the expression regulation of two kinds of probiotics on mucin gene MUC-2,so as to reaserch probiotics wheather can antagonistic Cronobacter sakazakii to induce mucin gene expression and decreased play to antagonize the pathogen adhesion and invasion effects. (4) The neonatal rat model of probiotics inhibit Cronobacter sakazakii intestinal bacillus hematogenous metastasis and the experiment of treatment and prevention of neonatal rat model. Through the research above, observe Lactobacillus rhamnose LGG and new Bacteroides SK08, that two probiotics inhibit bacteremia meningitis caused by Cronobacter sakazakii, also preliminary explore the effects of probiotics inhibit the adhesion and invasion of pathogen, and prevent blood bank transfer into the brain whether is related with the regulation of intestinal mucin gene expression.Research methods1.The adhesion and invasion experiment of Cronobacter sakazakii in vitroIn vitro, intestinal epithelial Caco-2 cells as adhesion and invasion detection object and specific methods:Caco-2 cells before inoculation and infection, (about 105), bacterial inocula used does not contain antibiotic medium weight hanging to be long to a monolayer. To each hole, add 1 x 107 bacteria into the culture of Caco-2 cell monolayers in 24 pore plate (the cell infection ratio was 100), at 37 degrees Celsius, with 5% CO2 culture case were incubated with education for 90 minutes. In order to calculate the number of bacterial cell adhesion cells with medium washed three times, adding 100μl 0.5% Triton X-100 lysis were incubated for 8 minutes and immediately joined the 50μL of distilled water. The cells were lysed before incubation and washing stage, try to keep the integrity of the monolayer cells. Repeated pipetting after aspirate samples, gradient dilution (10-1-10-4) after coated with oxacillin and ampicillin resistance plate count the number of colonies. Adhesion rate= intracellular bacteria number/incubation bacteria number x 100%, repeat three times and get the average.For invasion assay, with each hole in the 1 x 10’cfu bacteria into the cultured Caco-2 cells in 24 pore plate (the cell infection ratio was 100), at 37 degrees celsius, with 5% CO2 culture case were incubated with sterile 90min. Medium for washing 3 times, then the content of medium for 100 mg/mL gentamicin 1 hours incubation, the extracellular bacteria were killed. Medium washed three times, adding 100μL 0.5% Triton X-100 lysis were incubated for 8 min after and immediately joined the 50μL of distilled water and lysed cells. Repeated pipetting after aspirate samples of gradient dilution (10-1-10-4), the resistance plate count the number of colonies. Invasion rate= intracellular bacterial count/incubation bacteria number* 100%, or the relative rate of invasion, repeat three times and get the average.2. Probiotics inhibit the adhesion and invasion experiment of Cronobacter sakazakiiAccording to the reference, the competition exclusion displacement experiment to literature, control group and experimental group and control group:Caco-2+ ATCC 29544 incubation 3 h; The experimental group is divided into three different groups:(1) competition group:Caco-2+ATCC 29544+SK08 and LGG, incubated 3 hours; (2) replacement group:Caco-2+ATCC 29544 incubated 1 h plus probiotics (BF SK08 and LGG)to hatch 2 h (3) rejection group:Caco-2+probiotics (BF SK08 and LGG) incubated 1 h and ATCC 29544 to hatch at 2h. The pre training good 24 hole in Caco-2 cells, the cells grow to 80%(about 105-106 cell volume), caused by bacteria and probiotics according to the above groups were incubated, gentamycin and then incubated sterile 1 h, Triton-X 100 cell lysis, LB coated bacteria incubator culture plate count for 24 h,calculate the adhesion rate and invasion rate.3. Probiotics induce the expression of MUC-2 gene of intestinal epithelial cellsExperiments were divided into four groups, respectively for the probiotic group, probiotics and pathogenic bacteria, pathogenic bacteria group and control group, the probiotic group was divided into Caco-2+BF SK08, Caco-2+LGG, Caco-2+LGG+BF SK08, probiotic and pathogenic bacteria group is divided into Caco-2+BF SK08+ ATCC 29544, Caco-2+LGG+ATCC 29544, Caco-2+LGG+BF SK08+ATCC 29544, pathogenic bacteria group Caco-2+ATCC 29544. Control group is for Caco-2 cells, cells were seeded in the cells in a Petri dish long to a monolayer, by the method of adding ATCC 29544,3h incubation, the cell suspension was collected and protein extraction and Western blot was used to detect the probiotic and pathogenic bacteria induced the expression of MUC-2 in the intestinal epithelial cells.4. The intestinal hematogenous metastasis of Cronobacter sakazakii in the neonatal rat modelAccording to the references, Sprague Dawley rat littermates from 16 litters (about 160),2-3 days of age, with 20 rats in each group, prevention and treatment of experiments, in particular to:(1) prevention trials:fossa in 4 litters, divided into the experimental group and the control group, the experimental group 2 days of age were fed stomach BF SK08, LGG, BF SK08+LGG and dose for 108 CFU, continuous irrigation stomach 2D. In the control group were administered the same dose of PBS. 3D (5 days), all rats were intragastric dose of 108 CFU/of ATCC 29544. After 2D rats were killed, were collected from the intestinal contents, blood, cerebrospinal fluid, the resistance of plate count. (2) treatment experiment:fossa in 4 litters, divided into experimental group and control group, all neonatal rat 2 day old irrigation stomach doses of 108 CFU of ATCC 29544.3D (5 days of age), mice of the experimental group were treated with gastric BF SK08, in the LGG and BF SK08+LGG and 108 CFU per dose, control group were administered the same dose of PBS. After 2D rats were killed, we collected from the intestinal contents, blood, cerebrospinal fluid. Coated plate count.Result1. Cronobacter sakazakii has an adhesion and invasion ability on Caco-2 cell. The E.coli E44 was used as a positive control, DH5a as negative control.They are stick attached for 3 h, the adhesion effect of ATCC 29544 is smaller than positive control group of E44 (relative adhesion rate was 31.3%), it is higher compared with negative control group of DH5a(P<0.05); when the bacterial adhere it for 3h, the invasion experiment testing addition of gentamicin was added the gentamicin, the results showed the adhesion of ATCC 29544 is close to the positive control group of E44, and was higher than that of the negative control group of DH5a (P<0.05). Respectively at 1,3, and 5 h time point, we take the sample in bacteria coated plate culture. The results showed that the adhesion and invasion of ATCC29544 on cells for 3 h after infection was highest, with the passage of time, the adhesion of bacteria is declined (P<0.05).2. Probiotics (LGG and BF SK08) inhibit the adhesion and invasion of Cronobacter sakazakii on Caco-2 cells and it is in a dose-dependent manner. Respectively do adhesion and invasion experiment with different concentrations (106,107,108) BF SK08 and LGG to inhibit ATCC 29544. The results showed that with the concentration of BF SK08 and LGG increasing, the relative invasion rate of Cronobacter sakazakii decreased significantly, that intruded into the pathogenic bacteria in Caco-2 cells was significantly reduced, and the inhibition is significantly, and the results showed BF SK08 inhibited is slightly less than LGG. (P<0.05).The competitive exclusion displacement experiments. The results show that: compared with the control group, each experimental group invasion rate were decreased (P< 0.05), but the competition and rejection group inhibition is higher than replacement group, rejection group of inhibitory effect is higher than in group competition, statistically significant differences (P< 0.05), suggest that BF SK08 and LGG, that two kinds of probiotics has a role in inhibiting the pathogen ATCC29544 on Caco-2 cell invasion, and indirectly guide the preventive effect of probiotics is stronger than the treatment effect.3. We study the adhesion and invasion of the probiotic antagonistic pathogenic bacteria with using the WB method. Each cell total protein extraction, using β-actin as a reference, WB assay for the detection of probiotic and pathogenic bacteria induced intestinal epithelial cell line Caco-2 mucin gene expression. Results showed that adding probiotics, the MUC-2 genes of Caco-2 cell were obviously up-regulated, and not join the probiotic group of MUC-2 gene expression is not significantly reduced, together with probiotics and pathogenic bacteria group is gene expression did not change significantly, indicating that the probiotic induced MUC-2 gene expression up regulation may be one of the protective mechanism of pathogen induced by the antagonist. MUC-2 changed along with the body pathological changes, will appear mucin expression abnormalities include sticky protein gene level and protein level changes, reduced sulfation degree, glycosylation and protein high-grade structure and spatial structure change, thus affecting the play their protection function, lead to disease occurrence. The experiments to elucidate the MUC-2 gene in human node biological functions in the cancer cells and further study the gene and probiotics, cause there is a certain relationship between the bacteria. This suggests that probiotics on induced inhibition of bacteria adhesion attached invasion of intestinal epithelial cells is inseparable from the protective effect of MUC-2 gene carried by the host itself.4. Cronobacter sakazakii intestinal hematogenous metastasis of newborn rats, and set the prevention group and treatment group,48h after from the intestinal content, blood, cerebrospinal fluid (CSF) detection. The results showed that the prevention group and treatment group, the number of probiotics and pathogenic bacteria group of neonatal rat intestinal colonization of ATCC 29544 significantly less than in the control group (P< 0.05), without the occurrence of bacteremia, cerebrospinal fluid was also not detected in the ATCC 29544. Control group was intragastrically administered pathogens, prevention group (n= 20) and neonatal rat in 20 rats appeared bacteremia (>105cfu/mL),14 in cerebrospinal fluid (CSF) appear ATCC 29544 strains. Treatment group (n= 20) in suckling rats 20 appeared bacteremia (>105cfu/mL),15 in cerebrospinal fluid (CSF) appear ATCC 29544 strains. While the probiotic prevention group of 20 rats. LGG group has 12 rats appear bacteremia (>105cfu/mL), group SK08 has 11 rats appear bacteremia (>105cfu/mL), mixed probiotic group has 11 rats appear bacteremia (>105cfu/mL), three groups of cerebrospinal fluid were not rats appeared ATCC 29544 strains; In the probiotic treament group of 20 rats, LGG group has 14 rats appear bacteremia (>105cfu/mL), SK08 group and mixed probiotic group has 12 rats appear bacteremia (>105cfu/mL), SK08 group and probiotic mixed groups of cerebrospinal fluid were not appeared ATCC 29544 strains, and LGG group has only 1 rat appear ATCC 29544 strains in CSF.The results showed that fragile intends to inhibit pathogenic bacteria penetrate the intestinal barrier BF SK08 and probiotics LGG, thereby reducing the quantity of bacteria in blood brain barrier, with BF SK08 and probiotics LGG prevention and treatment of mouse meningitis has certain effect.Conclusion1. Bacteroides bacteria SK08 and probiotics LGG can adhere to intestinal epithelial cells and inhibit significantly the adhesion and invasion of Cronobacter sakazakii;2. Bacteroides bacteria SK08 and probiotics LGG can promote up the expression level of intestinal epithelial cell mucoprotein MUC-2 gene expression and protect intestinal epithelial cells against pathogens of injury, this may be one of the protective mechanism against the adhesion and invasion of pathogen;3. The neonatal rat in vivo experiments showed that Bacteroides bacteria SK08 and probiotics LGG can be used to prevent and treat the meningitis of Cronobacter sakazakii;4. Combined with the experiments in vitro and in vivo, Bacteroides bacteria SK08 and probiotics LGG induced the meningitis caused by Cronobacter sakazakii, the prevention effect is better than the treatment effect. |
PDF Full Text Request