3 Phages Walk Into a Vibrio

Apostichopus japonicas, commonly known as the Japanese sea cucumber is a temperate shallow water species found along the coasts of south east Asia. Its common use is as an edible consumable and as such is considerably over fished. Sea cucumbers aquaculture is a rapidly growing, extremely profitable industry. The industry has the largest economic output for any single marine species in the whole of China. As with any aquaculture, disease has a serious negative effect on both yield and profit. Skin ulceration syndrome is one of the more serious diseases that affects cucumbers and is commonly caused by Vibrio species, in particular Vibrio splendidus.

At present antibiotics are used to control bacterial diseases, however, phage therapy is an alternative method which does not result in environmental pollution or antibiotic resistance. Li et al., sought to isolate and test the efficiency of phages that protect cucumbers against V.splendidus infection. Being the first study to assess the potential of a phage cocktail lytic to V.splendidus. 3 phages were isolated from raw sewage obtained from cucumber hatcheries. The sewage samples were introduced to pellets of V.splendidus which were then filtered through a 0.22 μm filter after a 24 hour incubation period. The presence of phages was then confirmed using the Double-Layered Agar Method. The phages were inspected using a Transmission Electron Microscope and their morphological features including head, tail and tail fibers were recorded. Cultures of V.splendidus were incubated in a 96 well plate for 6 hours with the turbidity of plates being visually examined to calculate the lytic capability of the 3 phages. The inhibition of bacterial growth was then tested using either a cocktail of all 3 phages in a ratio of 1:1:1 or each phage separately. A 96 well plate was inoculated and each phage dilution was added with a control for plate sterility, phage suspension sterility and bacterial culture included. All plates were incubated for 16 hours before the optical density being measured at 600 nm.

360 cucumbers were divided into 6 groups. Each group was divided into 3 tanks each housing 20 cucumbers. The cucumbers were fed for 60 days with each treatment being fed a separate diet of either an ‘unsupplemented’ control, antibiotic, all 3 phages or each phage separately. At days 20, 40 and 60 the levels of acid phosphatase and nitric oxide synthase were measured. 90 fresh cucumbers from the same source were selected and separated into 3 treatments; a blank control, an untreated group and a treated group. The treatment was phage injection by coelomic injection and was done to test the survival rate and enzyme activity in the coelomic fluid.

The electron microscopy of the phages revealed that all 3 belong to the order Caudovirales, with two belonging to the family Myoviridae and one belonging to the family Siphoviridae. The effects of each of the 3 phages on V.splendidus showed a significant decrease in the optical density at 600 nm compared to the control culture. For the ‘unsupplemented’ diet the survival rate was 18% whereas both antibiotic and phage cocktail treatments had 82% survival rates. The 3 phages individually showed far higher survival rates than the ‘unsupplemented’ treatment but not as high as the 3 combined. There was a significant difference detected between phage PVS-1 and PVS-3 but not between PVS-2 and the other phages. After the 60 days, there was no significant difference in weight gain or ingestion rate across any of the treatments. The levels of enzymes did significantly increase in cucumbers fed phages over the ‘unsupplemented’ treatment. There was however, no significant difference between antibiotic and ‘unsupplemented’ cucumber enzyme levels.

This study is very interesting and very useful for combatting current global problems. It follows on from a lot of previous studies and provides further evidence to support the move away from antibiotic use. Whilst showing that phage therapy is just as effective as antibiotics the authors also discovered 3 new bacteriophages lytic to V.splendidus and imaged them. The study uses proven microbiological techniques which are very robust and provide good, usable results in my opinion. My main qualm with this study is the size of it. I feel whilst done very well the authors attempt to do too much in one study. If each experiment was done separately or less was done, then more time and resources could have potentially been spent on individual elements. Whilst this study serves its purpose well, allowing for the potential disease reduction of aquaculture species and the reduction of antibiotics in the environment, the potential for broader future study is low. 

Li, Z., Li, X., Zhang, J., Wang, X., Wang, L., Cao, Z. and Xu, Y. (2016). Use of phages to control Vibrio splendidus infection in the juvenile sea cucumber Apostichopus japonicus. Fish & Shellfish Immunology, 54, pp.302-311.

http://www.sciencedirect.com/science/article/pii/S1050464816301772