The Molecular Biological And Morphologic Evidence Of Pathogen To The New Disease Fatal Bacterial Granuloma After Trauma

IntroductionIn 1996, a group of patients who suffered from a kind of granuloma disease on face after the recovery of some kind truma and can accelerate to brain necrosis at advanced stage were summarized by our group. The main performance includes increasing dark red pathes on face after truma with different degree, and the brain tissue can be affected by encephalitis mainly. And all patients were dead with 4 years. According to the truma history, pathological findings indicating granuloma, bacterial found with electron microscope, and patients'death, this disease is given the name of fatal bacterial granuloma after trauma (FBGT). We have reached the following achievements after a decade exploration on the pathogenesis of FBGT. Firstly, Propionibacterium acnes (P. acnes) was cultivated successfully in both the skin lesions of such kind of patients on the medium improved by rabbit brain. And the whole 16S rRNA sequences of P.acnes were successfully amplified by the use of polymerase chain reaction (PCR) from the affection area including the necrosis brain. The evidence above ensured P. acnes as the pathogen of the new disease and the death cause of FBGT is P. acnes-induced brain infection. Secondly, based on the clinical features of FBGT, the IAM gene polymorphism causing defective function of the corresponding molecular was thought to be one of the FBGT pathogenesis hypothesises, which was confirmed in one case. Thirdly, through the sub-type identification, the pathgen of FBGT and acne vulgaris belongs to the subtype IA, which is relatively low pathogenicity. Fourthly, when studying P.acnes biofilm (BF), the resistance to antibiotic significantly increased after the formation of BF, which may explain the inconsistent between antibiotic sensitive test and clinical efficacy. Then, the formation mechanism of P. acnes BF in vivo was investigated, which gave solid foundation for FBGT research in vivo. However, the pathogen has not been successfully isolated and cultured in patients'cerebrospinal fluid and brain tissue lesions. And immunohistochemical methods failed to confirm the existence of bacteria in brain. Although the 16S rRNA of P. acnes was amplified with PCR method, a convincing evidence of brain infection is still a problem. To achieve the direct evidence of brain infection is the crucial step for researching the disease further.ObjectivesTo prove the pathogen infecting brain tissue is the same one inducing inflammation on face with molecular biology method.diagnostic system with less tissue from patients, high sensitivity and good repeatability for the early diagnosis and treatment in FBGT patients. Through the combination of the molecular biology thchnique and morphology reaserch methods, the bacterial in brain damage area of FBGT patients is localized. So as to confirm the infection of the brain in FBGT patients is secondary to the face lesions,which induced by the infection of P.acnes. Methods1. Two set of specific primers targeting on P.acnes 16S rRNA and lipase encoding sequence seperately were designed for nested polymerase chain reaction (nPCR) with the use of Oligo6.0 and Primer- BLAST online. Meanwhile, the process of human tissue DNA extraction and the nPCR system was optimized in order to amplify the P.acnes 16SrRNA and lipase coding sequence from both skin lesions DNA and necrosis brain tissue DNA of FBGT patients.2. Make the product of the second pair primers for P.acnes 16S rRNA as the template for digoxigenin-labeled cRNA probe preparation, and locating the P.acnes mRNA in brain lesions of FBGT patients.Results1. Both skin and brain lesions DNA of 9 FBGT patients were amplified with the two set of primers. In the group of 16S rRNA (PAS) primers, it can be found that the result of the first pair primers was negative except the positive control. Then diluting the product of the first round by 1:1000, which is the template for the second round. After the amplification by using the second pair of primers for 16S rRNA of P.acnes, 7 samples can be viewed as positive but with poor band. Correspondly, the same DNA template were amplified by lipase (LIP) premiers, after the first round of nPCR, 8 templates were posivite but with lighter band compareing with positive control. The product was diluted the same way as above. When the second round is done, 9 samples were positive with strong band. Blank control for the same experiment is negative. And the BLAST result of the product defined them as P.acnes.2. With the use of ISH and FISH, positive signal of P.acnes mRNA can be observed widely in brain liquefaction necrosis area of the FBGT patients, most of which is intracellular. After stained with DAPI, further confirmation on the intracellular signal can be made, and the distribution of some signal can also be observed in the same cell with more than one nucleus. No positive signal is seen in sense probe section and normal brain tissue section.ConclusionThe pathogen DNA is amplified by nPCR successfully both in skin lesions and brain lesions of FBGT patients, which proved the infective cause of skin is same one of brain in FBGT patients. On the basis of above evidence, the pathogen P.acnes was successfully positioned in brain tissue of FBGT patient by the combination of ISH and FISH methods. The infection of the brain in FBGT patients was confirmed as secondary to the face lesions, all of which induced by the infection of P.acnes.