Phage cocktails: combatting Vibriosis in aquaculture

Aquaculture is one of the fastest growing industries in the world. However, bacterial infections limit the development of production (Oliveira et al., 2012). Vibriosis is responsible for most of the outbreaks, resulting in huge economic costs. The use of antibiotics to combat this has led to resistant strains. Phage therapy can be an eco-friendly alternative to antibiotic treatment in aquaculture systems to prevent and control pathogens. Phage cocktails, containing two or more phages, have the potential to overcome the problem of phage bacterial resistance. The aim of the study by Mateus et al. (2014) was to evaluate the efficiency of cocktails to control Vibrio spp. infections in aquaculture. V. parahaemolyticus was chosen to be used as the bacterial host in this study as it is a human bacterial pathogen which can lead to gastroenteritis contracted from contaminated.

Three phages (VP-1, VP-2 and VP-3) were isolated from the Cortedas Freiras aquaculture system in Portugal. The phages were tested either by themselves, as a combination of two, or all three combined. The occurrence of lysis zones were used to assess bacterial susceptibility to the bacteriophage. Phage survival was determined by calculating the phage titer. The study found that phage therapy using cocktails was significantly more efficient at inactivating V. parahaemolyticus and had higher burst sizes and shorter lytic cycles than single phage treatment. This could be due to the phages exploiting different bacterial receptors to be absorbed by the host. It was also determined that all three phages had a long period of viability in marine water, between five to seven months.

This report is believed to be the first known study on Vibriosis and phage cocktail control. The authors have proved that these cocktails are a viable alternative to antibiotics in aquaculture and the problem of phage resistant bacteria can be circumvented by the use of a combination of phages (Chan et al., 2013). All phage cocktails were found to be more efficient at controlling bacteria growth than single phages. Phage cocktails have an increased efficiency with a higher and faster rate of inactivation and a delay in the development of bacterial resistance. This could lead to a more effective and environmentally friendly method of controlling pathogens in aquaculture systems. However, they did not prevent bacterial regrowth and would likely have to be reintroduced into the systems periodically. There is a high potential for expansion of the use of phage cocktails in aquaculture which will require further research and the identification of crucial pathogens.

In conclusion, the important findings of this paper show that phage cocktails with two or three phages increase the efficiency of phage therapy against Vibrio, delay the development of resistance by the bacterial host and high burst sizes and short latent periods increase the efficiency of bacterial inactivation. Also, phages have a long period of survival in the environment are more efficient. The identification of these characteristics will help recognise the most effective phages in the future. This knowledge is greatly beneficial to the continual development of the aquaculture industry.

Reviewed paper:

Mateus, L., Costa, L., Silva, Y., Pereira, C., Cunha, A., & Almeida, A. (2014). Efficiency of phage cocktails in the inactivation of Vibrio in aquaculture. Aquaculture, 424-425, 167-173. http://dx.doi.org/10.1016/j.aquaculture.2014.01.001

Other mentioned papers:

Chan, B.K., Abedon, S.T., Loc-Carrillo, C., 2013. Phage cocktails and the future of phage therapy. Future Microbiol 8, 769–783.

Oliveira, J., Castilho, F., Cunha, A., Pereira, M., 2012. Bacteriophage therapy as a bacterial control strategy in aquaculture. Aquac. Int. 20, 879–910.

Role of viruses in a changing climate

As professor William Wilson highlighted in his lecture, the role of viruses within marine systems is commonly overlooked. Phytoplankton are known key drivers of global processes such as primary production and biogeochemical cycling. Marine coccolithophores, such as Emiliania huxleyi, are enclosed by CaCO₃ plates called coccoliths. The sinking and dissolution of these plates results in the sequestration of dissolved inorganic carbon, known as the carbonate pump. Coccolithophores form mesoscale blooms which are characterized by an exponential increase in abundance followed by a crash, initiated by lytic viruses. Despite being well studied, there are considerable discrepancies within the literature regarding the effect of ocean acidification on algal bloom dynamics. Some studies have found that reduced pH levels result in increased calcite production whereas others have reported reduced calcification, and some have even suggested no effect. The effects of decreased pH on the viruses of these algae and their interaction with their host are yet not known and may indeed contribute to this varied response of coccolithophores to ocean acidification. Accordingly, Highfield et al. (2017) investigated the impacts of elevated pCO₂ levels (760 pmv) on the structure and diversity of both the algal host, E. huxleyi and its viruses (EhVs), through a mesocosm experiment conducted in a Norwegian fjord.

The progression of the bloom was monitored through chlorophyll a concentration (flurorometrically determined), pigment analysis (High Pressure Liquid Chromatography) and cell counts of E. huxleyi and EhVs (flow cytometry). Carbon fixation was used as a proxy for primary production and was determined using ¹⁴C measurements of surface water samples. To determine genetic diversity, Polymerase Chain Reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) analyses were carried out using primers specific to the calcium binding protein gene (gpa) for E. huxleyi and the major capsid protein gene (mcp) for EhV.

The results of the study suggest a negative effect of ocean acidification in this instance as a lower pH was found to significantly reduce phytoplankton biomass and primary production of an E. huxleyi dominated bloom. Within the elevated treatment, they found high variability between replicates, with significant differences in the abundance of coccolithophores. Despite this, the genetic diversity of the E. huxleyi remained stable across the replicates, treatments and the duration of the experiment. The authors therefore posit that viral infection by EhV may be driving these differences in abundances. Notably, they found that elevated pCO₂ levels can affect both the composition and diversity of EhV as in contrast to their algal host they did not stabilise through the succession of the bloom and were not consistent across replicates. Environmental stress may affect EhVs host ranges and infection characteristics (burst size and latent period) and may have caused a change in virus genotype resulting in a change in viral community composition.

This study really demonstrates the importance of including viruses when studying marine systems, particularly in the context of climate change. Indeed, the authors provide evidence that suggests a potential knock-down effect of increased pCO2 on E. huxleyi driven by viral responses to acidified conditions. Despite this, it is still not clear whether the observed response is due to the environmental conditions affecting viruses directly or their interaction with the host. Future experiments should aim to better dissect the host and viral responses, in order to accurately predict their ecological interactions in future ocean acidification scenarios.

Referenced paper:

Highfield, A., Joint, I., Gilbert, J., Crawfurd, K., & Schroeder, D. (2017). Change in Emiliania huxleyi Virus Assemblage Diversity but Not in Host Genetic Composition during an Ocean Acidification Mesocosm Experiment. Viruses, 9(3), 41. http://dx.doi.org/10.3390/v9030041

Neuston, we have an ‘in-situ’ation in the SML!

Wang et al (2014) describes phytoneuston as phytoplankton communities that dominate the sea-surface microlayer (SML). The SML differs from sub surface water (SSW) and sub bottom water (SBW) as they are comprised of different community compositions and biomass, which is the focus of this study. The SML is a 1000 micron biogeochemical boundary between the atmosphere and the sea that has distinct roles in processes like gas and heat exchange, particle cycling, microbial loops (as larvae feed on microalgae), etc. The SML phytoneuston also provides an insight to increase in temperature, nutrients and UV rays, decrease in oxygen concentrations, etc (Wang et al 2014). Dapeng Cove in the Daya Bay in the South China Sea was used as the study area for this experiment, which experiences reduced water flux and elevated nutrient levels (40-fold increase in the nitrogen: phosphorus ratio).

A volume of 0.5L of the SML sample was collected using a glass plate sampler with the plate having been submerged vertically and withdrawn gently. The SSW and the SBW samples were collected with a Niskin bottle sampler at 0.5m below the surface and equally above the bottom, respectively. The subsamples for chlorophyll A and dissolved nutrients (like total nitrogen and total phosphorus) were filtered in the field and analysed. Water temperature, salinity, and dissolved oxygen were determined in situ.

Based on microscopic observations of phytoplankton communities, species in the phytoneuston were different to other phytoplankton groups in the underlying column. This suggests that the SML harbour favourable micro-niches for these organisms to thrive in, which eliminates factors such as physical transportation via mixing; and that there is almost a shift from phytoplankton dominance to cyanobacteria dominance.

Although the total diversity in the SML was significantly high (with abundant phosphorous), cyanobacteria were highly enriched in the SML with Lyngbya, Oscillatoria and Synechococcus as the principal contributors (Wang et al 2014). Their increased abundance in the SML (especially in temperate regions during the summer) is suggested to be due to buoyancy strategies (photo-protective accessory pigments), high growth rates, high temperature and UV radiation resilience (despite natural warming as well as receiving large volumes of discharged warm water), lowered grazing pressure, production of antioxidants, etc. Cyanobacteria correlates positively with nutrients, water temperature and salinity and inversely correlate with dissolved oxygen, diatoms and phytoplankton. This is because cyanobacteria are able to exploit remineralized organic compounds produced as a result of diatom (and other phytoplankton) mortality (Wang et al 2014).

The results of this study also showed that diatoms dominated the SSW and the SBW and only 10% of the SSW and the SBW were in the SML as they have a low tolerance to high temperature and UV rays. They suggested that the small fraction of diatom densities that were measured was due to mixing. Dinoflagellates (Alexandrium tamarense) and chlorophyll A also showed the same trend, due to actively motile cells in flagellates and high temperatures and UV radiation damaging chlorophyll A as well as grazing by protists.

This study highlights how environmental parameters can be established by studying the composition of the phytoneuston community of a certain area. This can be used to determine the fate of aquaculture and the communities inhabiting these waters due to aquaculture as well as increased human population. This paper serves as a good base as it provides evidence for positive correlation between phytoplankton and diatoms, and an inverse relationship between the two and cyanobacteria. However, the microscopic methods used should be associated with size-fractionated pigment analysis and other molecular methods to further gain an understanding of the phytoneuston community, as small flagellates and picoplankton could have been ignored under the microscope. Another potential future study could be to understand the extent and type of protist grazing and to also understand how phytoneuston overcome this pressure (if at all).

Reviewed Paper:

Zhao-Hui Wang, Shu-Hua Song, Yu-Zao Qi (2014). A comparative study of phytoneuston and the phytoplankton community structure in Daya Bay, South China Sea. Journal of Sea Research 85, 474-482.

No Use Crying Over Spilt Oil

Microbial bioremediation plays an important role in the removal of pollutants as a result of oil spills in the marine environment. A study by Bovio et al. (2017) focussed on the fungal community of a marine site in the Mediterranean Sea which had been contaminated by an oil spill, as little research has been conducted regarding mycobiota. Different techniques are used for removing oil after spills but these often have economic, ecological and technical drawbacks so it is necessary to find bio-based alternative systems (Zhang et al., 2011). Bacteria and fungi could potentially be used to treat pollutants as they use crude oil as their main carbon source (McGenity et al., 2012). Much like bacteria, fungi have also been found to produce biosurfactants, which reduce surface tension and increase uptake of crude oil (Das & Chandran, 2011). This study aims to better understand the role of fungi in bioremediation.

The samples were collected on the 4^th June 2013 at Gela, Sicily where there had been a recent and persistent oil spill. Several areas of testing were conducted.

Fungal isolation and identification:

Seawater was filtered and strains were cultured for taxonomic identification. Soil dilution plate technique was used for the sediment samples and fungi were classified according to macroscopic and microscopic features and molecular analysis. The water samples had high fungal biodiversity with 67 taxa found and 12 new species identified that were previously discovered in seawater and the sediment samples had 17 taxa and 14 new species. Both sample types were dominated by 94% Ascomyota.

Fungal growth on crude oil:

Fungi were tested for their capability to grow on crude oil (this experiment used Arabian Light). The percentage stimulation was calculated and compared with the controls. All 142 fungi were able to grow but with different efficiencies. In the water samples, 24% were stimulated by crude oil presence; 49% insensitive; 27% inhibited. In the sediment samples, 22% were stimulated; 45% insensitive and 33% inhibited.

Crude oil degradation assay:
4 strains were selected that had been stimulated by crude oil. The capability to degrade oil in liquid cultures was tested using DCPIP colorimetric assay. Fungal development was determined by biomass dry weight. A. terreus was the fastest to degrade. There was a correlation between DCPIP disappearance and crude oil degradation. In this experiment, the fungi expressed enzymes only as a response to crude oil and used it was a source of nourishment because the biomass was found to be significantly higher with the presence of oil. 

This study concluded that fungi have the potential to restore marine environments after being contaminated by oil spills and highlighted the existence of an important living fungi community straight after an oil spill.  As this is the first report on mycobiota bioremediation of an oil contaminated site in the Mediterranean Sea it is a very important study which can form the basis for further research as fungi have been neglected in studies up until now. 21% of species isolated have been reported as present for the first time in the marine environment. Bovia et al. (2017) have highlighted the high capacity of some strains of fungi to degrade oil, using it as their sole Carbon source. P. citereonigrum and A. terreus have been identified as having very high bioremediation potential which requires further analysis and could lead to an effective and natural solution for removing pollutants. This report has presented novel knowledge regarding fungi and can be used to develop biological systems to reduce and improve the effects of future oil spills in the marine environment.

Paper discussed:

Bovio, E., Gnavi, G., Prigione, V., Spina, F., Denaro, R., & Yakimov, M. et al. (2017). The culturable mycobiota of a Mediterranean marine site after an oil spill: isolation, identification and potential application in bioremediation. Science Of The Total Environment, 576, 310-318. http://dx.doi.org/10.1016/j.scitotenv.2016.10.064

Other papers referenced:

Das, N., Chandran, P. (2011). Microbial degradation of petroleum hydrocarbon contaminants: an overview. Biotechnol. Res. Int. 2011, 1–13.

McGenity, T.J., Folwell, B.D., McKew, B.A., Sanni, G.O. (2012). Marine crude-oil biodegradation: a central role for interspecies interactions. Aquat. Biosyst 8 (10), 10–1186.

Zhang, Z., Hou, Z., Yang, C., Ma, C., Tao, F., Xu, P. (2011). Degradation of n-alkanes and polycyclic aromatic hydrocarbons in petroleum by a newly isolated Pseudomonas aeruginosa DQ8. Bioresour. Technol. 102 (5), 4111–4116.

An alternative to chemical dispersants in response to marine oil spills?

Biosurfactants are produced by a variety of microorganisms and can be either of low molecular weight type or high-molecular weight type (known as bioemulsifiers). Biosurfactants have a range of applications, including in petroleum and cosmetic industries, in medicine and in bioremediation. When used for bioremediation, biosurfactants can be used to increase the bioavailability of hydrophobic compounds (e.g. hydrocarbons). Despite this, they are not used in response to oil spills occurring in the marine environment, where chemical dispersants are solely used. However, the toxicity and negative effects of chemical dispersants on the activity of natural oil-degrading microorganisms has raised concerns.

As biosurfactants are biodegradable and have low toxicity, they could be used to develop biodispersants and provide an alternative to chemical dispersants. This has led to an increased interest in identifying biosurfactant-producing marine microbes that are able to function in marine ecosystems with low water activity and extreme temperatures. A study by Raddadi et al. (2017) aimed to identify marine bacteria capable of producing biosufactants when grown on soybean oil and/or glucose-based media and then characterise the activity and stability of the biosurfactants produced under the challenging conditions seen in the marine environment.

Bacteria collected in marine sediment samples from harbours in southern Italy were isolated using three successive spreading serial dilutions. Based on colony morphology, 43 isolated strains were then inoculated in modified mineral salt medium (mMSM) with either glucose or soybean oil as a carbon source and once bacterial growth had occurred the cell-free culture supernatants were collected using centrifugation. The supernatant was then filter-sterilised and screened for biosurfactant/bioemulsifiers (BS/BE) production by measuring emulsification activity, drop collapse and interfacial surface tension; 26 isolates were found to produce BS/BE when grown with glucose while 16 of these isolates were found to produce BS/BE when grown on soybean oil (a cheap and readily available source of carbon) with the results suggesting that mainly bioemulsifiers were produced. Identification of the 26 BS/BE-producing isolates using 16S rRNA sequencing showed the isolates belonged to the genera Bacillus, Thalassospira, Halomonas and Marinobacter. The BS/BE produced by the isolates were found to be active and stable at extreme temperatures and low water activity, reflecting the challenging conditions of the marine environment, as well as up to 30 months of incubation. The BS/BE were also shown to have good environmental compatibility as they exhibited low toxicity and were capable of dispersing crude oil in artificial marine water. Based on their findings, the authors conclude that the nonpathogenic Marinobacter sp. are suitable for large-scale BS/BE production.

In summary, the authors identified a range of marine bacteria capable of producing BS/BE which were active and stable under the challenging conditions seen in the marine environment and were also environmentally compatible; the nonpathogenic Marinobacter sp. was suggested to be the most suitable option for large-scale BS/BE production. The results of this study are promising as an alternative to chemical dispersants. However, the statement that the BS/BE produced by the isolates have low toxicity and thus good environmental compatibility is perhaps premature as the only measure of toxicity was the effect on bioluminescence of Vibrio fischeri; only having one measurement of toxicity could be misleading as other measures may produce different results and so presents a possible limitation of this study and an area that requires further research.

Reviewed paper:

Raddadi, N., Giacomucci, L., Totaro, G., & Fava, F. (2017). Marinobacter sp. from marine sediments produce highly stable surface-active agents for combatting marine oil spills. Microbial cell factories, 16(1), 186.

https://pdfs.semanticscholar.org/ad7e/91b6312ef3a5cdf9cb1140ac99669ce2e5b0.pdf