Character Of Proteus Vulgaris Biofilm Of Chinese Alligator And Study On Its Forming Gene

Proteus vulgaris is one of the opportunistic pathogen, widely distributed in nature and intestinal of reptiles, birds, mammals and other animals. The pathogen has great potential harm to human health and the development of breeding industry. It can cause the host disease and even death under certain conditions. Proteus vulgaris is one of the important pathogens of proteus sp., which can casue septicemia, urinary tract stones, gout, chronic otitis media, trauma, infection and it also can be the pathogen of cystitis, infant diarrhea, and food poisoning. It has become the most important pathogen of hospital cross-infection. In recent years, reports of Proteus diseases gradually increased.In the development of breeding industry, antibiotics have played an important role in the control of bacterial diseases, and promote the development of the breeding industry. But now, the attendant problems have gradually highlights. The resistance of other opportunistic pathogen in the host to antibiotics becomes increased when we use antibiotics to control bacterial diseases. Now, bacterial resistance phenomenon has become the focus, catching people’s attention. Furthermore, the pathogen must colonize and evade the body’s defense system, and then they can cause infection. Phagocytic cells as the most important defensive barrier of the body can swallow the pathogen; play an important role in its removal. Bacterial biofilm (BF) as one of the important mechanisms of drug resistance of bacteria, widely exists in the nature, has the very strong resistance and immune escape, and is one of the main causes of chronic infection.The proteus vulgaris used in this study was isolated from a sick Chinese alligator at the Anhui Research Center of Chinese Alligator Reproduction. Biofilm of the 18 P. vulgaris were analyzed and the results showed that 14 of them have a strong ability to form biofilms; 3 of them have a middle ability to form biofilms; Only 1 of them has a weak ability to form biofilms, account for 77.8%,16.7% and 5.5% respectively.A kanamycin-sensitive strain (Proteus vulgaris XC 2) of the strong biofilms formation ability strains was selected as receptor bacteria of the random transposon insertion mutant. For the convenience of the follow-up tests, the biofilm formation conditions were optimized. Results showed that the biofilm formation in optimum conditions for containing 0.2% glucose or sucrose as carbon source in the LB medium, cultured at 37℃ for 24 h. Before transposon mutagenesis, we also tests the phagocytic ability of RAW264.7 cells to biofilm and planktonic of Proteus vulgaris XC 2 which revealed that the phagocytosis of biofilm and planktonic by RAW264.7 cells are similar.A kanamycin-resistant strain of E.coli with PUT mini Tn5 was selected as the donor in triparental conjugation. Transconjugants were passed by three times to get 500 mutants.The biofilm biomass of each mutant strain was determined using crystal violet method,3 biofilm-deficient strains were obtained at last. One of them was dead in the next subculture. For the remaining 2 mutant, TAIL-PCR was used to amplify the flanking sequence of the insertion fragment. After amplification, sequencing, comparison, the inactivated gene glpC was determined in one of the two strains. Unfortunately, we failed to determine the inactivated gene of the other mutant.In the analysis of biological characteristics to the mutant strain, the tolerance of mutant strain to pH, sensitive to the tested antibiotic reduced obviously, the organic solvent tolerance is related to the tested organic solvent, the phagocytosis of RAW264.7 phagocytic cells to proteus vulgaris is related to the inactivation or not of glpC gene, and has no direct relationship to the existence state of bacteria (biofilms or plantonic).