Formation And Estimation Of Biofilm Of Aeromonas Hydrophila

Aeromonas hydrophila (Ah) is an aquatic organism that could produce cytotoxins and enterotoxins associated with acute gastroenteritis and wound infections in human and hemorrhagic septicaemia of fish, reptiles and amphibians. Ah can form biofilms which are ubiquitous and play a multitude of important roles in different environments. In this work, a method for estimatiing Ah biofilm was established. The factors influent Ah BF formation were discused. And an Ah BF oral vaccine was evaluated.1 A modified method for estimating Ah BF was developed according to O'Toole and Kolter. Overnight culture of Ah J-1 was enumerated. Ah J-1 and diluted 16 dilutions by PBS. 200μL of each dilution was centrifuged for five minutes at 12000 r/min, 4℃. The precipitation was suspended by PBS and washed as above. After the last centrifugation, the precipitation was suspended by 25μL 1% crystal violet (CV) solution and stained 20 minutes. The bacteria stained by CV were precipitated and washed by water three times by centrifugation. Then 200μL of 90% ethanol was added and transferred to ELISA plate. The absorbance at 590 nm was measured. The standard curve of the relationship between the bacteria number and the absorbance at 590 nm was set up. An indirect method for estimating the biofilm formation of Ah by the cell number was developed.2 Kinds of different nutrient media and two kinds of carriers were studied for analying the influent factors of BF formation of Ah. The results showed that MM9 and Mg + LB media increased the biofilm formation of Ah J-1 strain compared to LB medium, and Mg²⁺ could only increase the biofilm formation of Ah within a certain concentration; MOP+LB and 3LB+G, however, inhibited it; Coarse chitin favored it than polystytrene.3 An oral BF vaccine of Ah was prepared by adding heat-killed biofilm bateria as adjuvant to an inactivated Ah vaccine which was developed according to commercialized procedure. Sixty-four mice were divided into four groups (16 mice for each group). GroupI and II for oral BF vaccine [10⁸ CFU/ (100g· d^-1) ] and inactivated Ah vaccine [10⁹ CFLV (100g · d^-1) ] for 15 days respectively. Group III for I.P (10⁷ CFU for each mouse) was boosted by I.P after two weeks. Group IV for control (no vaccined). The serum anti- Ah antibodies were monitored by ELISA every week after immunizing till 60 days post-vaccination. All mice were challenged with 50 LD50 (10⁷ CFU) Ah J-l after 60 days...