Marine fungi – not so fun for pathogens! Marine fungi show antibacterial activity for pathogens in fish aquaculture.

Aquaculture is the fastest growing industry for food production in the world. Much research has focused on how to manage issues in aquaculture both economically and sustainably – one of these problems is disease control. Heavy use of antibiotics has caused the worldwide issues of: antibiotic resistance, environmental toxicity, aquatic pollution, and high-cost implications. Accordingly, there has been recent investigation into safer and more natural options of biological control – here enter the fungi! In previous studies, both marine fungi and sponges have been highlighted for their antimicrobial activity against fish pathogenic bacteria. Sponges hold a rich diversity of bioactive fungi in their tissues. Sponge-associated fungi produce unique bioactive secondary metabolites and many host-specific toxins. The study by Özkaya et al. (2017) investigated antibacterial activities of fungal isolates, that are associated with sponges, against some commonly occurring pathogens in aquaculture. 

The study focused on four of the most well-known fish pathogenic bacteria that cause disease in aquaculture: Vibrio anguillarum, Yersinia ruckeri, Lactococcus garvieae, and Vagococcus salmonarum. V. anguillarum is the most common cause of disease outbreaks for fish and shellfish, causing vibriosis. Y. ruckeri is the agent responsible for the disease yersinoisis – it has caused major economic loss to the salmonid farming industry. L. garvieae is a major problem for a wide range of wild and cultured fish species – both marine and freshwater. Finally, V. salmoninarum is the cause of coldwater streptococcosis; this is an emerging disease for rainbow trout in the European Union which can cause up to 50% broodstock mortality. 

The study investigated 70 strains of marine fungi, isolated from sponges, and used ethyl acetate extracts to screen against the four previously named pathogenic bacteria. Antibacterial assays were performed using the agar diffusion method. Authors also used the co-culture technique to test the production rates of novel bioactive (antibacterial) metabolites – they tested each fungal strain combined with a mixture of all target bacteria.  

Results showed that a total of sixteen fungal isolates had antibacterial activity against at least one pathogen – all these isolates showed antimicrobial activity against V. salmoninarium. Additionally, three isolates had a strong inhibition of growth against all the tested pathogens, including Penicillum canescens and Aspergillus iizukae. P. canescens was the most active against all bacteria collectively, with an inhibition zone of >13mm. Whereas fungi A. iizukae had the strongest activity in the screening of mono-culture against L. garviae and V. Salmoninarum, with an inhibition zone of 30 – 35mm, respectively. The co-culture experiments found an induction of bioactivity in two isolates. A. iizukae gained broad-spectrum activity with high efficiency against test pathogens when fermented in co-culture conditions. 

The study shows that fungi as a use of antibiotics is extremely promising. The antimicrobial activity of fungi varies greatly between differing properties such as: fungal strains, species of targeted pathogen, sponge host species, habitat salinity, seasonal variations, and an array of chemical and bio-physical parameters (there’s quite a lot of varying factors! But this is expected). The study also found that competition between fungi species was also an important factor – the production of secondary metabolites may initiate a defence or attack reaction by stimulating toxin synthesis. Further research is still required to find out the most effective conditions for the use of fungi for antibacterial control, but the benefits appear worth-while. 

Overall, marine fungal metabolites can be a great alternative to commercially banned antibiotics. Marine fungi appear to be a potential source of eco-friendly and non-costly disease control against fish pathogenic bacteria in aquaculture. 

Study of reference: 

Özkaya, F.C., Peker, Z., Camas, M., Sazak Camas, A. and Altunok, M., 2017. Marine Fungi Against Aquaculture Pathogens and Induction of the Activity via Co‐Culture. CLEAN–Soil, Air, Water, 45(8), p.1700238.

Deep-Water Horizon oil spill impairs immune function and increases susceptibility to pathogenic bacteria.

The explosion of the Deepwater Horizon oil platform on April 20, 2010 resulted in the largest release of oil in history, entering the northern Gulf of Mexico causing massive amounts of ecological and economical damage to the ecosystem. Up to 1773 km of coastline from Louisiana to Florida experienced some form of oil exposure. The application of dispersant via aircraft, was intended to remove much of the oil from the ocean surface which resulted in a large portion of sinking to sediments. Exposure to crude oil and its individual constituents can have detrimental impacts on fish species, including impairment of the immune response.

In this study, they examined the effects of exposure to high concentrations of hydrocarbons in crude oil on immune function and increased susceptibility to pathogen infection, in the southern flounder (Paralichthys lethostigm), based on the increased incidents of external lesions/ sores on fish species that are indicative of bacterial infections such as vibriosis.

The experiment consisted of a fully factorial exposure design with contaminated sediment and pathogen exposure as main factors.

Juvenile flounder were exposed to oil-contaminate sediment for 7 days, then challenged with a known fish pathogen for 1 hour, Vibrio anguillarum, that is prevalent in the Gulf of Mexico. V. anguillarum is the causative agent of vibriosis, a haemorrhagic disease responsible for severe economic losses, especially in aquaculture fisheries. The juvenile fish were sampled at 24 hours post bacterial challenge with two fish then being sacrificed from each tank, in which the liver, spleen, kidney, intestine and gill samples were removed and preserved for further processing.

DNA was extracted from samples using PowerSoil DNA Isolation Kit and the relationship between microbial communities in intestine and gill tissues of oil, pathogen and co-exposed fish was determined by amplification and sequencing of the gene encoding 16S rRNA. After denoising and chimera checking, sequence data were separated into operational taxonomic units (OTUs) and predictive metagenomic analysis was performed.

Fish in several oil exposed treatment tanks were observed swimming to the surface and gulping for air, despite oxygen levels being monitored and adequate at those times in comparison to fish from the control tanks, which did not exhibit these behaviours. A lowered haemoglobin concentration has been linked to oil exposure in which down-regulation of β- haemoglobin gene expression, a gene important for erythrocyte production and oxygen transport, occurred suggesting a highly negative impact of hydrocarbons on fish health.

Oil exposure also resulted in reduced expression of IgM mRNA, the primary systemic fish antibody and typically one of the first to respond to bacterial infection. In several fish species, a reduced ability to defend against pathogen infection occurred after exposure to oil or its components. The results suggest that the lesions observed in nGOM fish after Deep-Water Horizon oil spill are the result of an immunotoxic response to oil exposure, resulting in an increased prevalence of pathogenic infections. 

Oil exposure resulted in consistent and clear shifts in overall taxonomically distinct bacterial community composition in gills and intestines of fish, with Alcanivorax being found in increased quantities in marine sediments and impacted by Deepwater Horizon oil where it has been demonstrated that, when supplied with adequate nutrients (nitrogen and phosphorus), it will become the predominant bacterial species in seawater containing petroleum.  Imbalances in these communities can have detrimental physiological effects, important for organismal homeostasis.

This study provides evidence that an imbalance or shift in gut microbial communities can result in the successful establishment of lethal infections by pathogenic organisms, leading to possible disastrous impacts on the aquaculture industry and  the overall diversity of the Gulf of Mexico.

Bayha, K. M., Ortell, N., Ryan, C.N., Griffitt, K.J., Krasnec, M., Sena, J., Ramaraj, T., Takeshita, R., Mayer, G.D., Schilkey, F. & Griffitt, R.J (2017) Crude oil impairs immune function and increases susceptibility to pathogenic bacteria in southern flounder. PLoS ONE 12(5): e0176559. https://doi.org/10.1371/journal.pone.0176559

• Thiruvarangan Ramaraj,
• Ryan Takeshita,
• Gregory D. Mayer,
• Faye Schilkey,
• Robert J. Griffitt 

Continued Bioremediation of Deepwater Horizon Oil Pollution Almost a Decade On

Oil spills can be catastrophic for affected marine ecosystems. Amongst the most devastating of these events was the Deepwater Horizon disaster which began on April 20^th, 2010. Damage to the Macondo Well led to the discharge of an estimated 779 million litres of oil and gas from the sea floor into the Gulf of Mexico. Much of the oil initially deposited onto the nearby sandy coastline was manually cleaned up soon, however, oiled-sand patties continue to be washed ashore to this day. Almost a decade on from the spill, these sand patties provide the most accessible samples of residual Macondo Well oil.   

 There is a natural attenuation of oil in the environment over time, largely through microbial degradation. Researchers found that the Macondo Well oil within deposited sand patties became heavily degraded only two years after the spill, with many residues dominated by oxygenated hydrocarbon degradation products. These oxygenated hydrocarbon products have previously been hypothesised as resistant to decomposition. If so, remaining Macondo Well oil pollution could persist for a long time to come. Consequently, one recent study attempted to identify whether or not microbes were continuing to degrade the remaining, highly weathered Macondo Well oil deposits on affected beaches.

 Bostic et al. collected both oiled and non-oiled sand patties for analysis from intertidal and supratidal zones of three previously studied beaches on the coasts of Mississippi, Alabama and Florida. Initially, samples were solvent extracted and tests were carried out to ensure that acquired oil residues had originated from the Macondo Well. Moreover, the samples were characterised by gas chromatography coupled with a mass spectrometer and a flame ionisation detector. Results confirmed that samples were both Macondo Well-derived and heavily weathered in concordance with previous studies. Furthermore, analysis via thin layer chromatography coupled with a flame ionisation detector revealed that most of the solvent extractable material within the oiled-sand patties comprised oxygenated hydrocarbons (53-69%).     

 Subsequently, microbial phospholipid fatty acids (PLFA) were extracted from both oiled and non-oiled sand patties for analysis of natural abundance radiocarbon (¹⁴C) content. PLFA degrade rapidly following cell death so offer valuable insight into the viable microbial community and their C sources at the time of sampling. By measuring ¹⁴C content within the PLFA, it is possible to identify microbial C sources as either petroleum or as modern, photosynthesis-derived organic matter; petroleum contains significantly depleted ¹⁴C compared with modern organic matter.

 ¹⁴C content measurements revealed that microbial PLFA extracted from oiled-sand patties primarily contained carbon which was petroleum-derived whereas microbial PLFA extracted from non-oiled-sand patties contained carbon consistent with modern organic matter. Accordingly, it is clear that microbial communities within the oiled-patties are utilising different carbon sources to the communities residing within the non-oiled-patties. Additionally, there was no significant difference in ¹⁴C measurements between intertidal and supratidal oiled-patties, suggesting that proximity to fresh carbon sources and moisture had no influence on the ¹⁴C content of microbial PLFA. Therefore, Bostic et al. were able to conclude that petroleum carbon is the primary source of carbon for the microbes inhabiting all studied oiled-sand patties.  

 The results of this study are promising, highlighting the potential for further bioremediation of Deepwater Horizon residual oil pollution, contrary to previous suggestions that such heavily weathered oil could be resistant to degradation. Nevertheless, this study delivers only a snapshot of weathered oil degradation, providing no insight into the degradation rates of the remaining oil. As such, further research should utilise incubation techniques in an attempt to quantify these degradation rates, permitting improved estimates to be made of when affected ecosystems will return to their pre-Deepwater Horizon states. Additionally, employing metagenomics could identify key OTUs in the degradation process of weathered oil, highlighting shifts in oil degrading bacterial communities as oil becomes more heavily degraded over time.

Reviewed Paper:

Bostic, J. T., Aeppli, C., Swarthout, R. F., Reddy, C. M. & Ziolkowski, L. A. (2018). Ongoing biodegradation of Deepwater Horizon oil in beach sands: Insights from tracing petroleum carbon into microbial biomass. Marine Pollution Bulletin, 126: 130-136

Interactions between probiotic, pathogen and protein sources

Probiotics are an integral part of aquaculture practices to increase yields of fish by limiting loss through disease. Modulation of the immune system is a commonly reported benefit, with high potency to stimulate immunity seen under in vitro and in vivo conditions. There are an assortment of common bacteria used including Carnobacterium. The probiotic is naturally occuring in the gut microbiota of salmonids and has displayed antimicrobial abilities, but to be a favourable probiotic it must be able to adhere and grow in the mucus or on the enterocyte surface of the digestive tract.

Hartviksen et al 2015 studied the adherence of a species of this genus, Carnobacterium divergens, and whether it could exclude or displace the common fish pathogen Aeromonas salmonicida using ex vivo methods. This has been utilised for it combines the best of both in vitro and in vivo methods. The use of the intestine environment means results are more applicable to reality, and the removal of it from the organism allowing for flushing etc. enables greater control over variables. There are limitations, for the tissue viability will decrease over time, leading to the use of only one hour incubation as any longer may result in its degradation. Intestines were exposed to either saline (the control) the probiotic, the pathogen, first the probiotic then pathogen or the reverse. The effects of diet, which the fish were given for nine weeks, were also investigated the four variants being: fishmeal (FM), pea protein concentrate (PPC), extracted sunflower (ESF) and feather meal (FeM). Diet was considered as it is known to influence intestinal microbiota and structural integrity.

General exposure to C. divergens showed improvement of the intestinal structure with lower frequencies of intraepithelial leukocytes and debris in the lumen, as well as higher frequency of healthy mitochondria. Intestines exposed to A. salmonicida had greater debris in the lumen with damaged microvilli and enterocytes across all feed types, with FeM having the greatest changes observed up to 7 micrographs per individual showing damage. Having the probiotic before the pathogen showed similar intestinal structuring to the control but the reverse showed in all dietary groups an increase in tissue oedema. Fish fed FeM had an apparent decrease in the prevalence of healthy mitochondria. In the control, diet seemed to have very little effect on the levels of both probiotic and pathogen.

Concerning adherence to the intestine in all treatments where C. divergens was present it increased compared to the saline exposed control; diet had no influence nor was there an interaction between diet and treatment. Levels of the probiotic would lower on exposure to the pathogen in comparison to when solitary, illustrating that A. salmonicida is an opportunistic bacterium that can displace the endogenous probiotic. However pre and post exposures of C. divergens would cause lower levels of the pathogen compared to its singular treatment, so the probiotic had some desired effect.

Alternative protein sources in the form of the varying diets can be seen to not significantly affect the uptake and adherence of both bacteria species. Nor was there any dramatic alterations to intestinal structure following the control or probiotic exposure. However the combination of feed especially FeM with A. salmonicida enhanced structural changes and damage. The mechanism is unknown however as with the control FeM caused no alterations to structure. It is therefore important to consider feed type not only in its appropriateness to the animal but also on whether it increases susceptibility to disease. However the use of C. divergens as a probiotic does seem promising, especially in that the protein source had no effect on its ability to establish so could possibly be supplemented alongside any feed type.

Paper reviewed:

Jose L Gonzalez Vecino, M. (2015). Probiotic and Pathogen Ex-vivo Exposure of Atlantic Salmon (Salmo Salar L.) Intestine from Fish Fed Four Different Protein Sources. Journal of Aquaculture Research & Development, 06(05).

DNA containing extracellular vesicles leading to confusion

The possibilities of cells to communicate and interact with each other and their surrounding are vast. Biofilms are a great example – communication appears on different levels. Cells interchange biomolecules via direct physical contact, release chemical molecules or regulate the expression of genes as a response to the surrounding cell density, known as quorum sensing (Miller and Bassler, 2001). Another way to communicate for microorganisms is the excretion of extracellular vesicles (EVs). Cells of all domains of life produce EVs. These tiny structures, defined by a lipid-membrane can contain lipids, proteins, nucleic acids such as DNA fragments or other biomolecules. The significance of these small “bubbles” regarding biological processes is impressive. Apart from enabling intercellular communication, EVs play a role in pathogenesis, the acquisition of nutrients, in biofilms or can even be part in the cellular defence mechanisms.

Due to the appearance and other characteristics EVs are hard to distinguish from viruses. When those biological particles were first discovered concerns arose in the world of viral research regarding a widely used method for the measurement of viral abundance in seawater. The basis of this standard approach is a fluorescent DNA-binding dye, often SYBR dye. DNA-containing EVs could have been detected via this method unknowingly leading to overestimated numbers of viral abundance.

In the presented study, the main aim was to find out more about DNA containing extracellular vesicles by measuring the size and frequency of fragments and to evaluate the standard method of DNA-binding epifluorescence for the assessment of viral abundance.

The organisms of interest were four different gram-negative heterotrophic bacteria species (Prochloroccocus, Salinicola, Alteromonas and Thalassospira), cultivated as axenic cultures.

Interestingly the DNA content varied significantly among the four marine bacteria. Each of the released DNA fragments possessed unique and species-specific size distribution, between 35bp and 10kb. With the use of SYBR-staining the researches observed a heterogeneous DNA distribution, meaning the fragments were not uniformly scattered among individual vesicles. Therefore only <0.1 vesicles were identified with that method. Exclusively vesicles with large DNA-fragments could be visualized.

The investigated differences in size and amount of DNA fragments on a species-level indicate that vesicles may have a high potential to act as agent of horizontal gene transfer. The researchers underline the importance to look deeper into this subject, because DNA-containing EVs may have a significant effect in the marine environment by acting as defence agents or “vehicles” of transport between targeted host cells of viruses leading to a change of sensitivity to those pathogens.

To assess the questioned method for viral counting and the applicability in the field, seawater samples were collected. Half of the samples were treated with chloroform, which disrupted the lipid-membrane structure of EVs, to enable a first separation of vesicles and viruses. The other half was kept as a control. Eventually the chloroform did not have a significant impact on counts of particles. In sum, the epifluorescence method only led to a fraction of visualized vesicles in the laboratory. Combined with the field-based work where a relatively small decrease in SYBR-bound particles appeared upon chloroform treatment, EVs do not impact the estimates of viral abundance in marine waters notably.

Conclusively, this study is a great example demonstrating that a high quality data collection and analysis needs suitable methods. With new findings previous methods should be questioned, examined, evaluated for they furthermore applicability. Regarding EVs more research is needed in order to understand the exact mechanisms of fragment and vesicle production and to work out the role of EVs on an ecological level to a greater extend.

Article reviewed:

Biller, S. J., McDaniel, L. D., Breitbart, M., Rogers, E., Paul, J. H., & Chisholm, S. W. (2017). Membrane vesicles in sea water: heterogeneous DNA content and implications for viral abundance estimates. The ISME journal, 11(2), 394.

References:
 

Miller, M. B., & Bassler, B. L. (2001). Quorum sensing in bacteria. Annual Reviews in Microbiology, 55(1), 165-199.

Schatz, D., & Vardi, A. (2018). Extracellular vesicles—new players in cell–cell communication in aquatic environments. Current opinion in microbiology, 43, 148-154.