Experimental Research On Separation, Identification And Immunological Effect Of A Drug-resistant Bacillus Proteus

BackgroundThe birth of vaccine brought revolutionary changes to the disease prevention of mankind. Practices have proved that the immunity prevention is the most economical, convenient and effective ways of preventing and controlling diseases. Following with the annihilation of smallpox in 1980s, and with the rapid development of immunology, microbiology and molecular biology, the development of vaccines has advanced greatly. Reviewing the developmental history of vaccines, we can divide the vaccines into the classic vaccines and the novel vaccines according to different production technologies. The principle of classic vaccines directly inoculates nontoxic or attenuated pathogenies into the bodies of human beings or animals as the vaccines, to stimulate the body immunity system to engender the specific immune response, so as to prevent the occurrence of diseases, while the novel vaccines are the ones produced by modem biological technologies, such as genetic recombination, genetic engineering, protein engineering, etc., and therefore have unexampled advantages than the classic vaccines. But since the existence of some problems, the novel vaccines still cannot instead the classic vaccines, which are widely used nowadays, in a short period. As to the bacteria, virus infectious diseases and so on, the bacterial vaccines are still an important direction of research. Especially, with the extensive application and the unreasonable employment of antibiotics, the drug-resistant and multi drug-resistant bacteria have been seriously threatening the health of human beings and animals. It is an important research direction on the treatment of some epidemic and multi drug-resistant infectious diseases in recent years that induces the bacteria's molecular mechanism of engendering the drug resistance, overcomes the bacteria drug resistance of cells, strictly controls the use of antibiotics, and develops some natural antibacterial peptides and some specific bacteria vaccines to control the infection from genetics, such as bacterial chromosome, plasmid, transposon, etc., and biochemistry, such as occurrence of inactivating enzyme, active effiux, permeability barrier, metabolism, change of target position, formation of biofilm, etc. There are about 15 kinds of bacterial vaccines currently used in the world, of which 12 kinds have been included in relevant regulations of our country, namely the cholera vaccine, typhoid vaccine, dysentery vaccine, chincough vaccine, polysaccharide vaccine of epidemic encephalitis, vaccine of b-type Haemophilus influenzae, polysaccharide vaccine of pneumonia, leptospira vaccine, Lyme disease vaccine, beg vaccine, brucellin, plague vaccine and anthrax vaccine. All these vaccines have acquired certain curative effect in the clinic application. At present, there are some other kinds of bacterial vaccines reported successively.This research adopted the Bacillus Proteus as the immunogen. The Bacillus Proteus is a group of bacteria with different sizes and shapes. They have obvious pleiomorphy, sometimes round, while sometimes threadlike. They have flagellums all over the body. They are moveable and move actively. They have no gemma and capsule, have the aerobic characteristic or the facultative anaerobic characteristic and belong to the Gram-negative bacteria. The Bacillus Proteus widely exists in the water, the corrupted organic substance among earth and intestinal canal of human beings and animals. It belongs to the conditioned pathogen and often causes the secondary infection, e.g. otitis media chronica, wound infection, etc. In addition, it can cause cystitis, infantile diarrhea, food poisoning, and so on. The Bacillus Proteus includes the proteus vulgaris, Balcillus proteus mirabilis, proteus morganil, proteus rettgeri and proteus inconstants. Among all these types, the proteus vulgaris and the Balcillus proteus mirabilis have relative close relations with the clinic. Especially the Balcillus proteus mirabilis can cause the blood poisoning, which results in a very high death rate. So, the control of diseases caused by Bacillus Proteus is very important to the treatment of the clinic infectious diseases. The main mechanism of disease control by bacterial vaccines lies in stimulating the body immune system to induce the body fluid immune response and the cell immune response, hence to release cytokines and engender antibodies, so as to destroy or dissolve the bacteria or viruses. With the increasing deepening of the signal and approach research activating the lymphocytes, the quite important function of the dendritic cells in the courses of antigen transfer and immunoreaction appears gradually. The incidence of hepatitis B is high in our country and is an important pathogeny of the liver cancer and the terminal hepatic diseases. By research, the immune signal expression of dendritic cells, the cell immunity and the body fluid immunity of the hepatitis B patients are obviously abnormal compared with those of healthy ones. Therefore, this research chose the in vivo/in-vitro tests, such as the signal expression of dendritic cells in the peripheral blood and the cytokine secretion of hepatitis B patients, the multiplication of splenic cells and the cytokine secretion of splenic cells of mice, etc., to study the influence of Bacillus Proteus to the signal expression of dendritic cells, the cell immunity and the body fluid immunity, so as to establish the experimental foundation for the application of the Bacillus Proteus as the bacterial vaccine.ObjectiveThis research adopted the intestinal bacteria to choose and distinguish the growth mediums and separate the Bacillus Proteus in the urine specimens of patients suffered form the repeating infections of urinary system, and then infect the mice with the separated Bacillus Proteus after the further identification and the subculture tenuation and analyze the multiplication ability of splenic cells outside the body and the splenic cell's secretion level of IL-2 and IFN-γ, as well as the change of antibodies. Meanwhile, we analyzed the signal expression of dendritic cells in the peripheral blood and the change of the IFN-γlevel of hepatitis B patients, and further explore the influence of Bacillus Proteus to the cell immunity and the body fluid immunity through the flow cytometry and the elisa spot test, so as to establish the experimental foundation for the research of Bacillus Proteus vaccine.Method1. Take the urine specimens of patients suffered form the repeating infections of urinary system, inoculate the specimens in the plate of intestinal bacteria selective medium (S, S agar) and the plate of differential medium (emb agar) respectively, place the plates in the 37℃incubator for 18～24h cultivation, observe the colony characteristics, find the colony, of which the characteristics accord with those of the Bacillus Proteus, from the cultivated plate, and make a further identification of the selected colony.2. Make the preliminary identification of the elected colony by ways of the observation after the Blendon silver staining, the trisaccharide iron agar culture test, the dynamic indole urease test and the phenylalanine deaminase test.3. Make special treatments on the Bacillus Proteus separated from the urine specimens of patients suffered form the repeating infections of urinary system, so as to abate the former toxicity, make the toxicity test to prove the reduction of toxicity, and then perform the final identification of the bacteria treated specially by ways of the trisaccharide iron agar culture test, the dynamic indole urease test, the phenylalanine deaminase test, migration and growth test, the sulfureted hydrogen generation test, the gelatin liquefaction test, the cirtrate utilization test, the indole test, the amino acid decarboxylases test, the glucide (alcohols, glucoside) fermentation test (maltose, xylose) and the chloromycetin sensitivity test. After they are identified and assured, inoculate, cultivate, gather, sterilize, centrifugate and purify the specially treated bacteria and dilute them into the 2×10¹¹/mL stock solution, and then perform the sterility test and the assay of the stock solution.4. Dilute the Bacillus Proteus stock solution into the liquid of 2×10¹⁰/mL with the RPMI-1640 culture fluid. Collect the haparin anticoagulant peripheral blood of the chronic hepatitis B patients and the healthy persons, separate the single karyocyte of the peripheral blood, and cultivate and induce the differentiation of dendritic cells in a directed way out of the body. The control group is 500μL of RPMI-1640 culture medium (containing 10% of Bovine calf serum)+the cultivated dendritic cells+the Bacillus Proteus diluent, and the expression levels of CD80 and CD86 are detected by the flow cytometry.5. Dilute the Bacillus Proteus stock solution into the liquid of 2×10⁹/mL with the distilled water, and inoculate the diluent into the abdominal cavity of the BALB/C mouse. Check the change of the mouse splenic cell's multiplication ability after the in vitro culture of PHA, Bacillus Proteus and ConA by the way of ³H-TdR uptake method; detect the serum antibody level of the mouse by the ELISA method; and, using the ELISA detect reagent kit, to detect the splenic cells' concentrations of IFN-γand IL-2 after they have been cultivated with the Bacillus Proteus.6. Take the haparin anticoagulant peripheral blood to prepare the peripheral blood lymphocyte of human beings. The experiment is divided into 5 groups, i.e. adding the culture medium, the HBcAg, the HBcAg+Bacillus Proteus culture fluid, the HBsAg, the HBsAg+Bacillus Proteus culture fluid respectively, and then respectively detect the cell factor IFN-γsecretion of the peripheral blood lymphocyte of the five groups with the ELISPOT.Result1. Separation, identification and generation of the Bacillus Proteus(1)The separated bacteria accorded with the characteristics of the Bacillus Proteus colony.(2)The preliminary identification proved that the bacteria might be the Bacillus Proteus.The trisaccharide iron agar medium was black and released the H₂S gas; the result of the dynamic indole urease test positive in dynamics, indole and urease; the phenylalanine deaminase test generated the green color and the result was positive too.(3)The final identification has proved that the bacteria separated are the Bacillus Proteus.The result of the toxicity test proved the toxicity of the Bacillus Proteus decreased obviously after the subculture. In addition, the results of the trisaccharide iron agar culture test, the dynamic indole urease test, the phenylalanine deaminase test, migration and growth test, the sulfureted hydrogen generation test, the gelatin liquefaction test, the lipoidase (corn oil) test, the indole test, the maltose test and the xylose test were positive in toto, the result of the amino acid decarboxylase test is negative, the result of the cirtrate utilization test was d, the sensitivity to the chloromycetin was R, which proved that the original bacteria were the Bacillus Proteus. Then the original bacteria were made into the 2×10¹¹/mL Bacillus Proteus stoke solution.2. The Bacillus Proteus can remarkably increase the CD80 and CD86 positive cell proportion of the in vitro cultivated dendritic cells of the chronic hepatitis B patients.By separating the peripheral blood dendritic cells of the HBV infected patients and the healthy persons, it was found that both the CD80 and CD86 positive proportion of healthy persons were larger than those of the chronic hepatitis B patients(P=0.000, P=0.000); after 48h cultivation of the Bacillus Proteus, the expression rate of CDS0 and CD86 of chronic hepatitis B patients had increased remarkably (P=0.000, P=0.000), while the change of positive expressing percentage of CD80 and CD86 of healthy persons was inapparent (P=0.185, P=0.118).3. The Bacillus Proteus has certain influence to the immunity of mice.(1)The Bacillus Proteus can enhance the multiplication ability of the splenic lymphocyte of mice.After the Bacillus Proteus inoculation in the mouse's abdominal cavity (two weeks with one time per week), it was detected by the ³H-TdR uptake method that, after the PHA or Bacillus Proteus culture, the treated lymphocytes appeared a remarkable multiplication reaction compared with those of he control group (the PHA group t=-27.860, P=0.000; the Bacillus Proteus group t=-14.630, P=0.000).By cultivating the splenic cells together with the Bacillus Proteus and the ConA, all the multiplication reactions of the splenic cells stimulated by three different dosages of Bacillus Proteus had remarkable differences comparing with the control group (the 0.5 mL experiment group, the 1 mL experiment group and the 2mL experiment group are all P=0.000).(2)The influence of the Bacillus Proteus to the peripheral blood antibody IgG2a of miceWe detected the IgG2a, a subtype of the serum antibody IgG, of the inoculated mice, and the result proved that the mouse Bacillus Proteus of all the three experimental groups could induce the generation of IgG2a antibody on the mouse body; which had a remarkable difference compared with that of the: control group (0.25mL experimental group: P=0.015, 0.5mL experimental group and thelmL experimental group: P=0.000).(3)The Bacillus Proteus can accelerate the cell factors IFN-γand IL-2 secretion of the mouse splenic cell.After the mice were inoculated with different dosages ((0.1mL,0.3mL,0.9mL) of Bacillus Proteus, we cultivated the splenic cells with the Bacillus Proteus for 48 hours and found the cell factors IFN-γ, and IL-2 secretion of the experimental groups had a remarkable increase compared with those of the control group (with injection of physiological saline into the abdominal cavities of the mice) ( Three different dose experiment groups are all P=0.000), and the increase of the 0.9mL experimental group was the most remarkable(compared with 0.1mL experiment group and 0.3mL experiment group are all P=0.000).4. The Bacillus Proteus can accelerate the IFN-γsecretion of peripheral blood lymphocyte of the chronic hepatitis B patients.We separated the peripheral blood lymphocytes of the hepatitis B patients and detected the cell factor IFN-γsecretion of peripheral blood lymphocyte of the HBV infected patients. It proved by the result that the number of spots appeared in the HBcAg + Bacillus Proteus experimental group was remarkably higher than that of the HBcAg experimental group (P=0.031,χ²=0.797), and the number of spots appeared in the HBsAg + Bacillus Proteus experimental group was also remarkable higher than that of the HBsAg experimental group (P=0.048,χ²=1.033).Conclusions1. It is reliable for the method of separating the Bacillus Proteus among the urine specimens of patients suffered form the repeating infections of urinary system by the intestinal bacteria selective medium and differential medium, and the toxicity has decreased a lot after the further identification and the subculture tenuation.2. The Bacillus Proteus can remarkably increase the CD80 and CD86 positive cell percentage of the peripheral blood dendritic cells of the HBV infected patients, while it will not increase the CD80 and CD86 positive cell percentage of the peripheral blood dendritic cells of the healthy persons.3. After the Bacillus Proteus infect the mice, they can promote the in vitro multiplication ability and the secretion levels of IL-2and IFNγof splenic cells.4. The Bacillus Proteus can accelerate the IFN-γsecretion of peripheral blood lymphocyte of the chronic hepatitis B patients...