Separation,Purification Of Escherichia Coli Bacteriophage And Its Application In Mariculture

Bacteriophage is a kind of virus inflecting prokaryotes, which is widely distributed in the nature with wide varieties. China, as a large country of aquaculture, has always ranked the first in productivity of aquaculture in the world for many years, however, the deterioration of water quality and emerging of drug resistant strains have brought huge economic loss to aquaculture, so bacteriophage treatment is re-concerned.In order to solve the problem of deteriorated water quality and frequent emerging of drug resistant strains, in this research, direct method was used to take samples in drain outlet of Xiamen Port and bridgehead of Yanwu Bridge, South Ditch of Xiamen University, Furong Lake and Pearl Bay. After enrichment of bacterium suspension, double-layer agar plate method and liquid purification method were used to separate and purify2lytic Escherichia coli bacteriophage EXO1and EX02for host infection test. The titer under cryopreservation and one-step reproduction curve of EX01were determined and EX01was used to treat domestic sewage, pre-treat aquaculture water and treat aquaculture medium-term water and Scylla temporary culture seawater. The result will provide data for the disease prevention and treatment of mariculture with important theoretical significance and application value. The main results are as follows:1. E. coli was taken as the host and2strains of lytic E. coli bacteriophages EX01and EX02were separated from natural water body. Through electron microscope observation, EX01was tadpole-shaped bacteriophage with shrinkable tail. With about70x110nm triacontahedron head, tail length about100-130nm and width18-22nm and structures such as tail sheath and tail fiber, it belongs to Myoviridae. EX02is a short-tail bacteriophage, with about20nm icosahedron head, tail length about2-3nm, belonging to Podoviridae.2. Bacteriophages EX01and EX02were used for host infection test to5different bacteria, the result indicated that as a broad-spectrum bacteriophage, EX01could infect E. coli DH5a and the other2strains of E. coli(E.coli S1and E. coli S2) separated from domestic sewage; while EX02could only infect E. coli S2. Neither EX01and EX02could infect vibrio W(Vibrio sp.) and pseudomonas putida.3. Cryopreservation titer test was conducted against EX01, the bacteriophage was filled separately in the form of suspension under-18℃for preservation and was determined every7days, until no bacteriophage was detected. The result showed that the suspension cryopreservation method could preserve bacteriophage for above30days and EXO1was still infectious in the56days of preservation.4. The one-step reproduction curve of bacteriophage EXOl infectious bacteriophage was determined, the result indicated that the latent period of EXO1was about25min and rise period was25-35min, the absorption rate within15min was85.6%and the average burst size after splitting was135.05.5. Bacteriophage EXO1with titer of1.81x108pfu-mL-1was used to treat the domestic sewage for1hour, the resulted indicated that the total bacterial count reduced by34.6%and E. coli count by89.0%, showing that bacteriophage EXO1has a better elimination effect to Escherichia coli in domestic sewage.6. Bacteriophage EXO1was used to pre-treat the unused fresh aquaculture sea water and no bacteria were detected both before and after treatment, indicating that after filtration pretreatment, the sea water contained extremely few bacteria, appropriate for aquaculture.7. Bacteriophage EXO1with titer of1.47×108pfu-mL-1was used to treat the aquaculture medium-term water collected from oyster fry tank for1hour, the result indicated that the total bacterial count reduced by13.3%and E. coli count by82.4%, showing that EXO1has a better elimination effect to E. coli in the aquaculture medium-term water.8. Bacteriophage EXO1with titer of1.04×108pfu-mL-1was used to treat the Scylla temporary culture sea water for1hour, the result indicated that the total bacterial count increased by7.7%and E. colicount reduced by98.3%, showing that EXO1has good better elimination effect to E. coli in blue crab temporary culture sea water. The increasing of total bacterial count might be the result of flora succession and mass propagation of contaminating microorganism.9. The titer of EXO1was set as7.02×102pfu·mL-1,7.02×103pfu·mL-1,…,7.02×106pfu·mL-1to treat Scylla temporary culture sea water respectively, with sampling time1hour,2hours, and3hours. The result indicated that under the same titer, the total bacterial count increased with the treatment duration, count of E. coli presented a reduction trend with the increasing of treatment duration and maintained stable after reduction of98-99%. Under the condition of the same duration, the total bacterial count first reduced and then increased with the increasing of bacteriophage titer, the titer corresponding to the minimum value was7.02x103pfu-mL-1, the count of E. coli presented a reduction trend with the increasing of bacteriophage titer and maintained stable after reduction of98-99%.The above results shows that EX01has a better elimination effect to E. coli in aquaculture and EX0l has potential for application in Aquaculture.