Deep sea vents home to human pathogenic Vibrios

Vibrio spp. are ubiquitous in the aquatic environment. Technological advances have allowed for the characterisation of many genomic sequences, shedding light on how this genus can survive in a range of habitats. Vibrios can grow in sediments, the water column, and even around hydrothermal vents.

This study conducted by Hasan et al. (2015) has described how a novel species of Vibrio was isolated and characterised, with reference to phylogeny, from deep sea hydrothermal vents. The genomic, functional and phylogenetic analyses carried on this new species, Vibrio antiquarius, revealed the presence of genes associated with symbiosis of a host, adaptation and virulence factors commonly seen in Vibrio species pathogenic to humans.

In 1999 during dives in submersibles along the East Pacific Rise, four samples of water were collected from the surrounding hydrothermal vents and sulphide chimneys. Four mesophilic bacterial isolates were cultured; three of which were Vibrios, one being Shewanella algae. One isolate, Vibrio EX25, was shown to be genetically similar to V. parahaemolyticus and V. cholerae, both of which are human pathogens. However, this strain was phylogenetically distant enough to be described as a separate species, V antiquarius.

The entire genome was sequenced, which showed that V. antiquarius contains >70 genomic islands (in particular, pathogenic islands) and many insertion sequences within the genome. These factors are suggested to give this species, along with other Vibrios, the ability to adapt faster or have quicker responses to changing environmental conditions.

 V. antiquarius has a number of genes allowing for survival in the deep sea, however some were sequenced that are not present in other species: one gene encoding for alkyl hydroperoxide reductase (the most abundant enzyme in the endosymbiont of Riftia pachyptila), multicopper oxidase (an essential gene for manganese oxidation), and delta-9 fatty acid desaturase (essential for growth under immense pressure).

This species of Vibrio has a type III secretion system (T3SS), which enables injection of effector proteins directly into eukaryotic cells of the host. This system allows V. antiquarius to utilise virulence factors; T3SS genes causing diarrhoea as in a cholera infection, RTX toxin and type IV pilin pilA which encodes for proteins expressed during human infection. Along with these genes and those that are ubiquitous, V. antiquarius is able to thrive in the deep sea hydrothermal vent habitat, likely in close association with the inhabiting animals.

A question is raised however, as to the wider use of these pathogenicity genes – could they provide some unknown ecological function? Vibrios can be involved in providing ecological pathways for other organisms (V. fischeri provides bioluminescence in bobtail squid, for example), and so can we write off Vibrios as only the cause of disease, or as a means of ensuring stability in the environment? This paper provides a new perspective on how Vibrios fit into the ecology of the deep sea, and may open up new research options into determining their wider role in the aquatic environment, as a pathogen and ecological stabiliser.

Hasan, N.A., Grim, C.J., Lipp, E.K., Rivera, I.N., Chun, J., Haley, B.J., Taviani, E., Choi, S.Y., Hoq, M., Munk, A.C. and Brettin, T.S., 2015. Deep-sea hydrothermal vent bacteria related to human pathogenic Vibrio species.Proceedings of the National Academy of Sciences, 112(21), pp.E2813-E2819.

http://www.pnas.org/content/112/21/E2813.short 

Not another review of probiotics..

Inspired by todays lecture I am going to introduce one last paper about the impact of probiotics on fish performance. Probiotics feed as diet additives can benefit treated organisms in several ways. Previous studies have shown greater growth performance by improving digestive tract conditions and intestinal activity. It has been also found that probiotics can reduce the presence of pathogens. Further, probiotics have the potential to improve disease resistance by enhancing immunological responses. 

The study by Gioacchini et al. (2014) looked into the impact of probiotics on the immune response of the zebrafish (Danio rerio). Effects of a diet containing the probiotic Lactobacillus rhamnosus IMC 501 on innate immune responses and hepatic stress were investigated. Two experimental groups were analysed. The control group were fed with a usual diet, while the target group were fed a probiotic containing diet. Expression of certain target genes in liver and intestine were investigated by RNA isolation and cDNA synthesis following a real-time PCR.

Expression of three genes (il lb, tnfa, becn1) in liver in intestine which contribute to the innate immune system of zebrafish was measured. Il lb and tnfa are important cytokines in the innate immune response, while becn1 is a novel candidate gene of the innate immunity system.

In the intestine these genes of fish fed with the probiotic diet were upregulated. Gene expression in the liver showed no difference, except of becn1 which was downregulated. This is suggested to be caused by modulated lipid metabolism by inducing a decrease of the autophagic process in zebrafish. Additionally, expression of genes related to stress response on physiological and cellular levels, such as the expression of stress induced genes hsp70l and nr3cl were measured. Probiotic fed fish showed a decrease in the expression of these genes. Further, expression of genes (sod1, gpxl a, nos2a) which are related to oxidative stress response were evaluated. All genes showed a decrease in expression in treatments fed with the probiotic diet. Genes (tp53,casp3a) which are involved in apoptotic processes were also downregulated in the liver. 

All in all the study has shown that the probiotic L. rhamnosus has a positive impact on the innate fish immune system. Cytokines which play a key role in the immune response of zebrafish were upregulated and oxidative stress was reduced. The intestinal microbiota has been changed most likely by the effect of the probiotic.

This paper contributes promising results to the potential application of probiotics in e.g. aquaculture. Benefits of probiotics have been discussed extensively already. More research is needed in terms of technological difficulties, appropriate dosage and evaluation of the efficacy at the farm level. 

Gioacchini, Giorgia; Giorgini, Elisabetta; Olivotto, Ike u. a. (2014): „The Influence of Probiotics on Zebrafish Danio Rerio Innate Immunity and Hepatic Stress“. In: Zebrafish. 11 (2), S. 98-106, DOI: 10.1089/zeb.2013.0932.

Sea breams got guts!

Aquaculture is the most sustainable source of protein based on the feed conversion ratio. Global demand for food is increasing, increasing aquacultures already high economic significance. Mediating disease and developing techniques to reduce cost and increase productivity, e.g. the use of probiotics, are important. So far, many fish gastro-intestinal (GIT) microbiota studies focus on disease and probiotics without fully understanding natural GIT microbiota. Gut microbiota have an influence on the host nutrition and immunity through symbiotic interactions; some genes have importance for virulence, antibiotic resistance and xenobiotic metabolism. Fish GIT’s remain largely understudied with the majority of previous studies based on bacterial cultivation techniques and archaea often completely missed.

Sea bream (Sparus aurata) is an economically important species from the Mediterranean which is fished and produced in aquaculture conventionally and organically. Organic sea bream production requires international standards and certification. Although organic production is significantly less than conventional, 1300 tons of sea bass and sea bream production compared total 275,000 tons in 2012, production is thought to increase as expectations for food sustainability and quality increase.

This study used tag pyrosequencing of 16s rRNA to compare the intestinal structural diversity of bacteria and archaea on wild (fed on a variety of prey e.g. molluscs, crustaceans, and fish), organic (fed sustainable certified fish meal and fish oil: 45% protein and 14% fat) and conventionally- reared sea bream (fed a commercial diet of 46% protein and 17% fat). Gut tissue was removed from 3 fish in each group and washed in sterile, particulate free seawater before DNA extractions.

This study is the first using next generation sequencing (NGS) techniques for the study of the gut microbiome of sea bream. Analysis of communities showed wild and organic sea bream to have higher bacterial diversity compared to conventionally- reared sea bream suggesting organic rearing simulates natural conditions better than conventional methods. Proteobacteria were prominent (above 50% of abundance), a finding that coincides with high abundance at phylum level previously reported in culture experiments. Four operational taxonomic units (OTUs) were found across all individuals. Dominant species were most closely related to genus: β-Proteobacteria Diaphorobacter, a denitrifier known to degrade polyaromatic hydrocarbons; γ-Proteobacteria Acinetobacter; known to metabolise amino acids, aromatic compounds and short-chain fatty acids and; Flavobacterium Cloacibacterium, commonly found in waste water. Only one sample within each group held enough Archaea for amplification, showing very low numbers across all treatments. Those identified were mainly Euryarchaeaota of the methanomicrobia.

This study is difficult to compare to earlier work which uses conventional techniques rather than NGS due to the much higher taxonomic resolution of NGS. Further study is needed to increase our knowledge of the microbiome to make it more comparable and identify differences within the gut e.g. difference between gut tissue, contents and faeces. I have difficulty fully accepting this paper due to its small sample size and confused discussion, with few high resolution studies this study is a start but more in depth analysis is required to increase statistical significance and investigate mechanism of transfer and microbial function within the gut.

Kormas, K. A., Meziti, A., Mente, E., Frentzos, A. (2014) Dietry differences are reflected on the gut prokaryotic community structure of wild and commercially reared seabream (Sparus aurata). Microbiology Open. 3(5): 718-728. 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4234263/

Coral transplantation not as clear cut

While coral transplantation is thought to be restorative to a reef previously decimated, further works have indicated that this may not be the case. A study conducted by Casey et al. (2015), looking into the impacts of grazer damselfish (Stegastes spp.) on the survival of transplanted Acropora muricata, discovered that regardless of the presence of Stegastes, transplantation of corals shifted the coral microbial community in favour of coral disease associated pathogens.

Previous studies have debated the benefit of Stegastes presence on coral reefs; some point towards damselfish promoting a thick growth of an epilithic algal matrix (EAM) and so inhibiting coral growth, and others suggesting that the fish promote recruitment/survival. Thus, the scientists investigated the microbial composition and survival of corals outside and inside the Stegastes territory, using transplanted coral branches.

The work was completed over a year, on a reef 1-3m deep off Lizard Island, Australia. Ten baseline coral samples were collected from territories of Stegastes, and controls collected from outside these areas. The transplanted fragments were directly placed in/outside the territories, and at six months mortality was estimated. Ten fragments from each treatment were sampled for microbial analyses, with the same process being repeated after the one year of transplantation.

Akaike’s Information Criterion (AIC) was used to compare generalised linear models of the data, defining the mortality as low (0-20%), partial (20-80%) and high (80-99%). It was found that after 6 months and a year, the corals transplanted inside the territories suffered a higher loss of tissue and mortality than those transplanted outside.

16S rRNA gene amplicon sequencing was undertaken to retrieve the microbial sequences collected from baseline, six months, and ten months. The OTUs were assigned to categories: autotrophs, heterotrophs and potential pathogens, with the abundances of less than two percent excluded. GLMs were used to analyse differences in relative abundances of the data, taking into account time and treatment type.

The results showed that the microbial communities had shifted in composition. After one year, there was significant increase in abundance of autotrophs in damselfish territories and so a more EAM population; a significant decrease of heterotrophs, and surprisingly there were significantly higher abundances in potential pathogens in all coral fragments regardless of fish presence/absence.

Of these pathogens sequenced, four out of the 21 genera are linked to coral disease, three of these four being associated with black band disease (BBD). This change in coral microbial community could massively affect the ecology of the reef, as loss of coral diversity could lead to decreased resistance to coral disease pathogens.

This paper shows how microbial communities can provide invaluable information about the state of corals, as well as how different trophic interactions impact on the reef as a whole. It provides us with food for thought as to how we can further protect reefs, most likely involving the adaptation of known methods in order to optimise coral health and minimise the risk of disease to already struggling systems.

Casey, J.M., Connolly, S.R. and Ainsworth, T.D., 2015. Coral transplantation triggers shift in microbiome and promotion of coral disease associated potential pathogens. Scientific reports, 5.

http://www.nature.com/articles/srep11903

Calling for backup

Antibiotic resistance is possibly one of the biggest crises we face in the 21^st century, as we are fast running out of effective cures and treatments for bacterial diseases. This is true not just for medicinal uses, but it is also a huge problem faced by the aquaculture and farming industries. Consequently, scientists are desperately looking for new effective ways to treat, or even better, prevent diseases. One of the major fields that has stemmed from this is phage therapy, which involves using various types of phages (often in combination – a phage ‘cocktail’) to reduce infectious bacterial populations.

Phage therapy has had some promising results so far, with many phages detected that are able to successfully reduce the populations of harmful bacteria, however efficiency varies, and there is still the worry that the bacteria will develop a resistance. For both of these reasons, this study looked at the effects of phage therapy with the addition of lysozymes. Using Vibrio parahaemolyticus as the target bacteria, the team tested the efficiency of 3 different Vibrio phages (VP-1, VP-2 and VP-3) both alone, and alongside lytic enzymes isolated from chicken eggs, of all places. The results were surprisingly positive, finding that in all 3 cases, adding these lytic enzymes increased the efficiency of bacterial reduction by the phages.

As can maybe be expected, each phage reacted slightly differently to the addition of these lytic enzymes, though all reacted positively. VP-1 in the presence of lytic enzymes showed bacterial inactivation at a rate almost 7-fold higher than the inactivation achieved when using the phage alone, and VP-2 + lysozyme showed a 2 times increase to the phage only treatment. The lysozymes seemed to have a lesser effect on VP-3, and actually worked better when a lower concentration of the lysozyme was added. However, VP-3 showed the highest bacterial inactivation of all 3 phages when they were being used alone, so it is likely that the limited effects of the lysozymes is because this phage is already highly efficient.

When looking into the effect of lytic enzymes on bacterial populations, and their antibacterial properties, it seems that there is already quite a lot of work being done in this area. While this experiment showed that using lysozymes on their own had no significant effect on bacterial populations, many other scientists seem to think that the exogenous addition of lysozymes could have key antibacterial results. This paper suggests that one of the positives of using lysozymes alongside phages is the slowing of bacterial resistance, as this combination leads to faster bacterial inactivation. I think this is hugely important considering our current position and dilemma with antibiotic resistance. I think an interesting next step would be to combine tried and tested ‘phage cocktails’, possibly with cocktails of lysozymes, to see if the effects can be heightened even further.

L. Mateus, L. C. (2014). Effect of lysozyme addition on the activity of phages against Vibrio parahaemolyticus. Aquaculture, 125-129.

Hydrothermal vents provide Yeti (crabs) with food

Hydrothermal vents are known to be highly productive ecosystems with primary production occurring through chemoautotrophic microbial production. Hydrothermal vents host a range of metazoan communities, which usually have epi and endosymbiont relationships with these microbes. These microbes include chemolithoautrophic bacteria, which can fix carbon in through various pathways, depending on their phylogenetic group. For example, autotrophic Epsilonproteobacteria fix carbon via the reductive tricarboxylic acid (rTCA) cycle whereas autotrophic Gammaproteobacteria do this via the Calvin-Benson-Bassham cycle (CBB). These differences in carbon fixation pathways are measured through the stable carbon isotope fixtures (δ¹³C) of the microbial primary producers. Many physical and chemical properties near the vents can cause differences in these symbiont communities and differences in trophic ecology of the host.

This study by Zwirglmaier et al. investigates the interactions between a new species of Yeti crab (Kiwaidae sp.) and their microbial episymbionts along the Scotia ridge hydrothermal vents. Using 454 pyrosequencing, sangar sequencing and stable isotope analysis coupled with environmental variables that were measured at the two sampling sites, the study aimed to investigate epibiont diversity and community structure which could then be examined and compared to other decapods occupying similar niches. 

Results showed that the sites were different in chemical and physical properties, with site E2 being 353̈°C and E9 being 383°C. Both sites also had different chemical properties. Majority of the Epsilonproteobacteria found were from the genus Sulfurovum, and majority of the Gammaproteobacteria found were from Leucothrix. There was a clear difference in dominating bacteria, with Epsilonproteobacteria being found mainly on E9 and Gammaproteobacteria being found on E2. The study found also that both sites had different stable carbon isotopic fixtures which meant the sites had different food sources (bacteria). The different levels of stable isotopic fixtures stand for different carbon fixation pathways taking place, which match that of Epsilonproteobacteria or Gammaproteobacteria respectively. 

This study shows the importance at looking at regional differences in microbial community structure on hydrothermal vents, as they provide a unique environment for bacteria to thrive in. The use of sequencing techniques and stable carbon isotope analysis proves that the combination of advanced methods can lead to more in-depth views of metabolic pathways. Further study could be done on the metagenome and chemical properties of the vent to provide insights into the relationship between host (Yeti Crab) and epibiont, involving the pathways. Genetic techniques (e.g. qPCR) could also provide information of the level of gene expression coding for metabolic pathways, and compared between different sites.  

Reference:

Zwirglmaier, K., Reid, W., Heywood, J., Sweeting, C., Wigham, B., Polunin, N., Hawkes, J., Connelly, D., Pearce, D. and Linse, K. (2014). Linking regional variation of epibiotic bacterial diversity and trophic ecology in a new species of Kiwaidae (Decapoda, Anomura) from East Scotia Ridge (Antarctica) hydrothermal vents. MicrobiologyOpen, 4(1), pp.136-150.

http://onlinelibrary.wiley.com/doi/10.1002/mbo3.227/epdf

Getting to the core of Yellow Band Disease

The coral holobiont is a complex, diverse community of organisms including symbiotic zooxanthelle and a diverse range of bacteria. Many organisms within the holobiont have positive impacts on the community such as sun protection, nitrogen fixation and photosynthesis. Increased anthropogenic activities and ecological stressors including pollution, fishing and diving, temperature increases have increased the incidence of coral disease significantly. Yellow band disease (YBD) is a slow growing coral disease in reef building corals. YBD is the slow growth of single or yellow/ white radiating bands on the coral surface causing the exposure of bare skeleton. Four species of Vibrio spp. have previously been identified as the cause of YBD.

This study used a combination of molecular tools (bacterial taxonomic profiling, Symbiodinum genotyping and, host transcriptome response) to further understand the mechanisms by which YBD acts on the coral holobiont and investigate how the holobiont responds to the disease. Skeletal tissue cores from colonies of healthy coral (HH), diseased coral (DD) and healthy tissue adjacent to diseased tissue (HD) of O. faveolata were collected from the Puerto Morelos coast, Mexico in September 2008.

PhyloChip hybridisation showed bacterial coral community structure changed dramatically between healthy and diseased colonies with the highest bacterial richness observed in HD which may represent a change in equilibrium or a transitional community phase between healthy and diseased. The most abundant operational taxonomic units (OTU’s) were from sequences previously identified from coral, mammal guts and sediments. Vibrionaceae were more abundant in healthy samples which contradicts, but does not negate previous studies suggesting Vibrio’s pathogenicity to this species. Here, Vibrio is reported at family level, not the species level which have been identified as the cause of many diseases. Anaerobic Firmicutes were abundant in diseased corals suggesting that disease is linked with oxygen limitation; Peptostreptococcaceae, which has been observed in other coral diseases including black band and white plague, was most abundant family in diseased samples. Population shifts between HH and DD corals showed a shift from predominantly gram-negative bacteria in HH corals to gram-positive in DD corals, HD showed a combination of the two.

Diverse Symbiodinium assemblages were observed in diseased corals, this may be due to the detection of lysed cells, or represent a changing community in response to microbial interactions. Host gene transcriptomic responses showed a distinct down regulation of mitochondrial- associated genes and up-regulation of immune and inflammatory responses in diseased corals which could be resultant of nutrient limitation and competition for resources further suggesting nutrient fluxes may impact coral disease.

This study shows that the entire holobiont is affected by coral disease; specifically, the area around the diseased state represents a distinct health state which warrants further study. The abundant bacterial species observed in diseased regions in this study contradict a number of other studies on YBD; this is likely to be resultant of their very small sample size (4 cores per group) however could also depict that the disease is not due to a specific bacterial species and is symptomatic of a response from the whole coral holobiont.

Closek, C. J., Sunagawa, S., DeSalvo, M. K., Piceno, Y. M., De Santis, T. Z., Brodie, E. L., Weber, M. X., Voolstra, C. R., Andersen, G. L., Medina, M. (2014) Coral transcriptome and bacterial community profiles reveal distinct Yellow Band Disease states in Orbicella faveolata. ISME. 8: 2411- 2422. doi:10.1038/ismej.2014.85