Three Dimensional Model Construction Propionibacterium Acnes (the Pathogen Of Fatal Bacterial Granuloma After Trauma) Biofilm And The Interaction Between The Biofilm And Microenvironment PH

Fatal bacterial granuloma after trauma (FBGT), a chronic and fatal infective disease most following slight wound of facial skin, was found and denominated by Professor Tianwen Gao in 1996. With the support of the National Natural Science Foundation (30271196), the pathogen of FBGT has been identified as Propionibacterium acnes (P. acnes), and the intracellular infection is the cause of death of FBGT patients.The patients of FBGT received a long-term, combinating, alternative antibiotic therapy according to the vitro drug sensitivity test, but most of them still deceased. The incompatibility of drug sensitivity test and clinical curative effect puzzled us in determining the pathogenesis of FBGT and treating the patients effectively. Biofilm (BF) is an important living model of bacterium, significantly increasing the antibiotic resistance of bacterium. The theory of BF may explain the contradiction between vitro test and vivo therapy reasonablely. Dr. Xianlong Qi successfully established the vitro model of P. acnes BF, and preliminarily analyzed the character of P. acnes BF with scanning electron microscope, and proved that BF could significantly increased the resistance of P. acnes to antibiotics such as lincomycin (National Natural Science Foundation 30670117). The result partially explains the contradiction between drug sensitivity test and clinical curative effect. But the study about the three-dimensional (3D) structure of P. acnes BF is still deficient.The mononuclear macrophages of innate immunity play the dominant role in the early stage of immune reaction against P. acnes, and the Th1 cells of adaptive immunity play the dominant roll in the late stage. Usually, P. acnes is phagocytosed and phagocytized by mononuclear macrophages without chronic granulomatous infections. But P. acnes can survive the damage of the acid lysosome, existing in the tissue of FBGT. In our previous study, we found that in the conditions of defective mononuclear macrophage and Th1 cells, P. acnes may formulate BF to protect themselves again the acid microenvironment of lysosome, which might partly clarify the pathogenesis of FBGT.Therefore, to gain more evidence of pathogenesis and antibiotic resistance of FBGT, the author focused on the further research about 3D reconstraction of P. acnes BF and the interreaction between the P. acnes BF and pH of microenvironment, which was expected to push the research about P. acnes. Objects1. Observed the live bacterium, dead bacterium and extracellular polymeric substances (EPS) in P. acnes BF with multi-fluorescein stain, confocal scanning laser microscopy (CSLM), and software of 3D reconstruction. After different incubating period, analyzed the pattern of P. acnes BF and inner characters of BF.2. Established the vitro model of P. acnes BF in different pH microenvironment. Analyzed the effect of pH and incubating period on the BF formulation and P. acnes proliferation, and the effect of P. acnes BF on the microenvironment. Then study the ability of P. acnes toleration and BF formulation in different microenvironment pH.Methods1 Three-dimensional reconstruction of P. acnes BF.1.1 Cultivated P. acnes on cover slips for 5d, 10d, 15d and 20d to form vitro BF model.1.2 Marked the live P. acnes, polysaccharides on dead P. acnes and in EPS with fluorescein diacetate (FDA) and rhodamine-ConA, respectively.1.3 After different incubating period, observed the change of live P. acnes, dead P. acnes, EPS in BF, and then analysed the superficial and inner characters of P. acnes BF. 2 Interaction between P. acnes BF and microenvironment pH.2.1 Established vitro P. acnes BF model in 96-well plate in different pHIncluded P. acnes in pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0. The incubating period was 24h, 48h, 72h and 96h. Sterile media of pH7.0 was set as negative control and blank well was set as blank control.2.2 The effect of different pH and incubating period on P. acnes BF formulation.Stained the biofilm in the well with crystal violet (CV), and analyzed the effect of different pH and incubating period on P. acnes BF formulation.2.3 The effect of different pH and incubating period on P. acnes proliferating in BF.Compared the effect of different pH and incubating period on P. acnes proliferating in BF with sodium XTT- phenazine methosulfate (PMS) chromatometry. Analyzed the ability of P. acnes proliferation and BF forming in different pH microenvironment.2.4 Effect of P. acnes BF on the pH of media.Compared the pH of media after different incubating period, and analyzed the regulation of P. acnes to the pH of microenvironment during P. acnes forming BF. Results and discussionThe vitro model of P. acnes BF was established and the characters of P. acnes and BF were observed in a natural condition.1 Observation of inner structure of P. acnes BF after long-term incubation.A series of characters were found after scanning the inner P. acnes BF after the incubation from 24h to 96h.(1) With P. acnes BF maturating, live P. acnes decreased in the inner BF. When incubating for 20d, the live P. acnes gathered as colony style.(2) The aggregation of EPS was earlier than live P. acnes. EPS of P. acnes BF increased with time, aggregating as scattered granula, and formed high-dense ridge-like structure at last.(3) With the development of mature P. acnes BF, live bacterium aggregated in the thick part of EPS gradually. At 20d, the distribution of live P. acnes was compatible with the EPS. Some live P. acnes were not covered with EPS, they might be the P. acnes released at the late stage of BF development.1.2 Three-dimensional reconstruction of P. acnes BF.After incubating for 24h, EPS of different thickness covered all the BF. The wide tunnel and excurrent part to the base was presented below the typical mushroom-like structure. Most of live P. acnes aggregated as small colonies in the mushroom-like nodule and covered with thick EPS. In the thinner part, the structure that small live P. acnes intercrossing with EPS was presented. With the BF forming, live P. acnes becreased obviously but EPS increased generally, perhaps because increased EPS reduced the transport of nutrient. Live P. acnes aggregating as small colonies in the thick part of EPS might be relative to the quorum sensing system (QSS). P. acnes formed BF on smooth glass after incubating in vitro experiment and still survived in thick EPS after 20d. It proved that P. acnes had strong survival ability, could induce chronic infections and resisted antibiotics. It suggested that full dosage sensitive antibiotics therapy should be administered at early stage to prevent BF formulation.2 Interreaction between P. acnes BF and microenvironment pH.2.1 The effect of different pH and incubating period on P. acnes BF formulation.The quantities of P. acnes BF in different pH and different period were obviously various. From 24h, BF of pH3.5-9.0 began increasing, but only pH 4.0-8.5 were significantly higher than negative control and other pH groups(P<0.05). Groups of pH5.5-7.0 were obviously higher than negative control and other pH groups(P<0.05) all the time, pH6.5 was the peak. P. acnes formed BF in pH4.5-7.0 faster than other groups.2.2 The effect of different pH and incubating period on P. acnes proliferating in BF.The effect of pH and incubating period on P. acnes proliferating in BF was similar to BF forming. P. acne proliferated significantly higher in pH5.5-7.0 groups than in other groups during all the periods (P<0.05), the crest value was at pH6.0 and pH6.5. At 96h, pH 4.0-8.5 were significantly higher than negative control and other pH groups(P<0.05). P. acnes proliferated in pH4.5-7.0 faster than other groups.2.3 Effect of P. acnes BF on the pH of media.After incubating for 96h, the pH value of groups pH3.0-5.5 increased and the pH value of groups pH7.5-8.5 decreased. But there were no statistical differences (P>0.05).P. acnes could formed BF and proliferated in microenvironment of pH 4.0-8.5, especially in pH4.5-7.0, and the formulation and proliferation was faster in pH 5.5-7.0. It suggested that neutral and metaacid microenvironment benefited the P. acnes surviving and forming BF. Series disease have been proved associating with P. acnes and BF, such as FBGT, acne, sarcoidosis, infective endocarditis, intracranial infeion, postoperative infection of bone and joint, primary biliary cirrhosis, hyperplasia of prostate gland, and endophthalmitis, involving many organs and tissues including skin, blood, brain, bone, liver and eyes. The normal pH of most human tissues and humor is within 4.0-8.5, and it is profitable to the survival and proliferation of P. acnes. The formulation of BF induces that P. acnes adhere tightly to the tissue, increase the antibiotic resistance of P. acnes, and lead to chronic infections.Bacterium is adapted well to microenvironment, including strongly acid and alkaline pH. The research suggested that the drift to pH6.0-7.0 after incubating for 96h was not statistically different, but it suggested that P.acnes perhaps had the capability of adjusting the microenvironment.The erzymes in lysosome are acidic hydrolase, and the better pH for the activity of them is about 5.0. It proved that P. acnes could proliferate and form BF at pH4.0 suggested that P. acnes could survive in the acidic microenvironment of lysosome. Our research partly proved the interaction between P.acnes BF and microenvironment pH, and presented some theoretical supplement for the pathogenesis and anti-infective therapy of P.acnes.Conclusion1 The reconstruction and analysis of P.acnes BF suggested the characters of P.acnes BF inner structure and development.2 The research confirmed the ability of P.acnes surviving and forming BF in pH4.0-8.5 microenvironment,partly explained the reason of P.acnes causing infections and inducing antibiotic resistance in many human organs and tissues.The research partly proved the interaction between P.acnes BF and microenvironment pH, supported that P.acnes BF played a dominant role in the clinical antibiotic resisitance of FBGT, and presented some important theoretical evidence for the pathogenesis and anti-infective therapy of P.acnes.