Isolation of edwardsiella-specific bacteriophages from fish

Aquaculture, the culture of all types of aquatic animals and plants in fresh, back rush, and marine water environments, is one of the fastest-growing food-producing sectors. This industry frequently suffers heavy losses due to diseases caused by pathogens such as multi-drug resistant bacteria, which are easily transmitted through water and thus capable of infecting a variety of animal and plant species. Edwardsiella species, gram-negative facultative aerobic rod-shaped Enterobacteriaceae, are among the infectious aquaculture pathogens that attack fish. Edwardsiella tarda and Edwardsiella ictulari have been linked to the development of Edwardsiella septicemia (also known as ES, edwardsiellosis, emphysematous putrefactive disease of catfish, fish gangrene, and red disease) in channel catfish, eels, and flounder, as well as Enteric septicemia of catfish.

Edwardsiella tarda has also been linked to zoo infections in a variety of animals. It is a zoonotic disease that can infect a wide range of animals beyond fish, including amphibians, reptiles, and mammals. This bacteria is found in pond water, mud, and the intestines of fish and other marine animals, as well as freshwater species. On the other hand, Edwardsiella ictaluri, the causative agent of enteric septicemia in catfish (ESC), is one of the most common fish pathogens affecting farm-raised channel catfish.

Antibiotics have traditionally been used to control bacterial disease in aquaculture farms and hatcheries. However, the indiscriminate use of antibiotics in aquaculture has resulted in the emergence and spread of antibiotic-resistant bacteria, rendering the culture system ineffective. Another major source of concern is the presence of antibiotic residues in aquatic products, which has resulted in decreased consumption. Aquaculture farmers have also been slow to adopt control and preventive measures due to the high cost and marginal effectiveness of mediated feeds.

Bacteriophages, for example, provide an alternative mechanism for controlling bacterial diseases in aquaculture. Phage therapy typically entails isolating a variety of bacteriophages specific to a bacterial pathogen that can then be combined as a bacteriophage “cocktail.” Because phages can exhibit strong host specificity, express efficient systems for host cell lysis, and spread rapidly in an aquatic medium, there is growing interest in their use to control fish pathogens in the aquaculture industry. Phage therapy is also unrelated to residues found in aquatic products, which are abundant in nature.

Centrifuge, Falcon tubes (15ml and 50ml), Incubator, Orbital Shaker, Refrigerator, wire loops, 10ml syringes, 0.45nm membrane filters, micropipettes

Cotton wool, TSA, TSB, PBS, SM Buffer, Flame, LB Broth, Petri dishes, Gloves, pipette tips, surgical knife, forceps, pair of scissors

1. The fish GIT samples are obtained and chopped into smaller pieces to increase surface area for exposure to their microbial content.
2. The processed samples are then placed in SM Buffer, shaken thoroughly, and transferred into the refrigerator overnight (in case not worked on immediately). Meanwhile, the bacteriophage host Edwardsiella sp from 24-hour pure plate culture is inoculated in TSB and incubated overnight.
3. A portion of the sample in the SM buffer is obtained into 15ml falcon tubes and then centrifuged at 4000rpm for 15 minutes to remove the coarse particles. The supernatant is transferred into sterile 50ml falcon tubes while the residues are discarded.
4. After 24 hours of incubation, the 10mls of the 24-hour TSB bacterial culture is transferred into the 50ml falcon tube mixed with an equal volume of the supernatant obtained from the previous step.
5. The 24-hour bacterial-bacteriophage mixture is vortexed and then centrifuged at 4000rpm for 15 minutes.
6. The supernatant is then filtered using 0.45nm syringe filters and used for further steps, the residue is discarded by autoclaving.

Spot assay is done for the detection of Edwardsiella-specific bacteriophages in the filtered samples.

1. 100µl of the 24-hour TSB host bacterial culture is pipetted into 4mls of melted overlay agar and mixed well.
2. The mixture is then immediately poured over a well-labeled TSA plate and swirled to ensure uniform distribution before solidification.
3. This is followed by pipetting 10µl of the filtrate suspected of bacteriophages and spotting on the corresponding label on the TSA plate.
4. The above setup is allowed to stand for about 15 minutes to allow diffusion and then transferred into the incubator overnight.
5. Plates are then checked for zones of clearance (plaques) and proceeded to the plaque assay.

Bacteriophages are natural enemies of several bacterial pathogens; therefore, they will provide an alternatively safe, non-toxic, abundant in nature, eco-friendly, and scientifically demonstrable method of controlling and preventing infections in aquaculture.

This guest post was written by

Ceaser Aluma Anyanzo, Undergraduate student, School biosecurity and laboratory sciences, Makerere university.