| Quorum sensing (QS) means a process of cell signaling transmission between bacteria, which activated bacterial genes expression, bioluminescence, violence factors, biofilm formation by the communication of autoinducers (AIs) secreted by themselves. In the natural environment, bacteria don’t exist in the way of a single colony or single bacterial biofilm, but in the form of multiple biofilm. Then, in the mixed biofilm multidimensional communication must be proceeded between bacteria. Aeromonas hydrophila, widely found in fresh water, sewage and sludge, soil and human feces, is a kind of typical zoonotic pathogens of people, animals and fish. It can cause aquatic a variety of diseases and even diarrhea. Quorum sensing signal AI-2 can be identified by A. hydrophila, which played an important role in its pathogenicity and biofilm formation. This experiment was studied exogenous signal molecule on A. hydrophila isolate biofilm formation, including the adhesion ability of biofilm, minimum inhibitory concentration (MIC) of ampicillin, cefotaxime, streptomycin sulfate, biofilm morphology by scanning electron microscope(SEM).METHOD A. hydrophila strain was selected by screening culture medium and identified with 16SrDNA sequencing analysis. A. hydrophila luxS fragment was detected by PCR. The presence of AI-2 in the culture supernatant and its luminescence intensity was examinated by AI-2 indicator Vibrio harveyi BB170 strain. By using crystal violet staining for detection of A.hydrophila biofilm adhesion when added into a exogenous culture supernatant; by broth microdilution method detection of A.hydrophila drug sensitivity to ampicillin, cefotaxime, streptomycin sulfate; SEM observation of A.hydrophila biofilm morphology under the existence of exogenous culture supernatant.RESULT The samples collected from fish markets, were screened and identified by 16SrDNA sequencing as A.hydrophila, which only contained enterotoxin alt gene. luxS fragment and up-/down-stream luxS sequences was amplified by PCR. Meanwhile AI-2 activity results showed that A.hydrophila supernatant could induce BB170 luminescence. It’s demonstrated that A. hydrophila harbored luxS gene and synthesize AI-2. AI-2 activity results also showed that exogenous supernatant can promote BB170 indicator luminescent, in which the luminescence activity of Salmonella supernatant was the largest, and the Achromobacter supernatant was the lowest, only reached to 60%. The results of drug sensitivity test showed that, when adding Salmonella supernatant, MIC of A. hydrophila strain to ampicillin and streptomycin sulfate, was 8 times and 4 times respectively compared to control group, and was sensitive to cephalosporin, which MIC value was less than 0.13μg/ml; when adding Achromobacter supernatant, MIC value to cefotaxime and streptomycin sulfate was the same as the control group, and value of ampicillin was 4 times higher than that of control group. SEM results displayed that different exogenous supernatant differed from formation of A.hydrophila biofilm. High relative AI-2 activity of Salmonella supernatant can promote A.hydrophila strain aggregation to form a colonization. It’s found that single cell around had a trend of aggregation. On the contrary, when adding low relative AI-2 activity of Achromobacter supernatant, A.hydrophila biofilm pictures displayed that there was no colony forming, but was observed a "silk" between the cells, and that this kind of "silk" always linked like a bridge during biofilm formation process.CONCLUSION The exogenous signal molecule plays an important role in the formation of bacterial biofilm. The culture supernatant contained AI-2. The high relatively AI-2 activity can much more promote the bacterial biofilm formation, increase antibiotic resistance, strengthen the structure of biofilm. But the resistance ability was closely related with different types of antibiotics. |
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