Category: STUDENT’S PERSPECTIVE

Fish plays a vital role in the human diet, and consumption per capita is increasing; however, the expansion of industry and agriculture causes contamination of the natural and man-made aquatic environment, which affects the health of organisms, raising safety concerns about its use for human consumption. Consumption of fish products, particularly raw or undercooked fish, is frequently linked to human diseases. As a result, it is critical to research these pathogens in order to ensure the safety of fish. The presence of various bacterial species in fish, including those pathogenic to humans, has been linked to direct contact with contaminated water or feed. Pseudomonas species are bacterial pathogens that infect fish.

Pseudomonas species are Gram-negative, aerobic bacilli measuring 0.5 to 0.8, μm by 1.5 to 3.0 μm. Motility is by a single polar flagellum. Species are distinguished by biochemical and DNA hybridization tests and they belong to the family Pseudomonadaceae and which includes four genera: Frateuria, Pseudomonas, Xanthomonas, and Zoogloea. Bacteria colonization can be observed on fish skin and gills.

Misuse of antibiotics as prophylactic agents in disease prevention is common and contributes to the development of antibiotic resistance Alternatives to antibiotics such as antibacterial vaccines, bacteriophage, and their lysins, have been applied to curtail the increasing emergence of antibiotic-resistant bacteria due to the imprudent application of antibiotics in aquaculture

Bacteriophages (phages), viruses infecting bacterial species, demonstrate clear antimicrobial activity against an array of resistant species, with high levels of specificity and potency. These are viral species that specifically infect bacteria by injecting their genetic material into the host bacterial cell. They are ubiquitous in aquatic and terrestrial ecosystems and in the microbiome of animal species. In fish, phages are present on skin surfaces and intestines, where they act as a natural predator to the bacterial microbiome.

Centrifuge, Incubator, Incubator shaker, Refrigerator, 10ml Syringes, Syringe filters (0.22nm), Micropipettes, Autoclave

Falcon tubes (15ml and 50ml ), Gloves, TSA, TSB, SM buffer/ PBS buffer, LB overlay, Flame, Petri dishes, Pipette tips, Scalpel blades, Forceps, Pair of scissors, Cotton wool.

1. The fish GIT are obtained and processed into smaller pieces 
2. The processed samples are then placed into SM buffer, shaken thoroughly, and transferred into the refrigerator at 4°C overnight
3.  Simultaneously Pseudomonas bacteria (bacteriophage host) pure culture is inoculated in TSB and incubated at 30°C overnight.
4. 10ml of the sample in SM buffer is picked and placed in a 15ml falcon tube and centrifuged at 3500 rpm for 10 minutes.  The supernatant(assumed to contain phages) is transferred into 50ml falcon tubes and then mixed with the pure culture of pseudomonas bacteria from TSB(10ml) followed by incubation In the incubator shaker for 24 hours. Pure Pseudomonas bacteria is inoculated in TSB overnight.
5. After 24 hours if incubation the sample is removed from the incubator shaker and then distributed into falcon tubes (15ml) followed by centrifugation at 3500 rpm for 10 minutes 
6. The supernatant is placed into the syringe connected to a syringe filter (0.22 nm) and the filtration product will be collected into a clean and sterile falcon tube 
7. Spot assay will be performed to check for the presence of the Pseudomonas-specific bacteriophages 
8. 4mls of the LB overlay will be measured and transferred into a clean and sterile falcon tube followed by pipetting 100 μl of the overnight bacterial culture in TSB, the mixture will be mixed well and then immediately poured into the TSA plate forming a Double layer Agar (DLA)
9. 10 μl of the sample assumed to contain phages will be measured and spotted into the Double layer agar 
10. The plate is then placed into the incubator overnight; the residue is autoclaved for proper discarding 
11. The presence of plaques (zone of clearance) indicates the presence of phages.

Bacteriophages are viruses that infect and utilize bacteria as their host. They can reside in the bacterial genome as a prophage, or enter the lytic phase, take over the bacterial gene expression machinery, synthesize new phage particles, lyse the host, and release up to hundreds of phage progeny. Lysis occurs during the late phase of the lytic cycle when the phage endolysin and a holin molecule are produced. The holin creates holes in the cell’s lipid bilayer allowing the phage endolysin (peptidoglycan hydrolase) to escape and degrade the structural portion of the cell wall. These (and other phage-encoded proteins) have been shown to inhibit bacterial growth. The ability to inhibit growth or kill bacteria makes both the bacteriophage and their gene products a rich source of potential antimicrobials.

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.