Group post: Tracing the carbon flow from diatoms to bacteria in a benthic biofilm

The microphytobenthic community provides a vital carbon source for higher trophic levels in coastal ecosystems. In addition, through the formation of a biofilm the microphytobenthos –typically dominated by benthic diatoms- stabilises soft sediments and provides settlement cues for intertidal organisms. Low molecular weight exudates released by diatoms may be directly utilised by bacteria, whereas high molecular weight compounds such as extracellular polymeric substances (EPS) may need to be hydrolysed by extracellular enzymes.

Miyatake et al. (2014), traced the in situ carbon flow from benthic diatoms through heterotrophic bacteria in an intertidal sediment over 5 days using a pulse-chase method, advantageous over laboratory based techniques by preserving environmental variables such as wave, sediment, and pore water dynamics. The study was conducted on an intertidal flat covered by a diatom mat in The Netherlands, 0.15 m below the mean tidal level. Two 50 x 50 cm frames were inserted into the sediment to a depth of 8 cm to contain the carbon flow. ¹³C Sodium bicarbonate was sprayed onto the sediment to a final concentration of 1 g [¹³C] sodium bicarbonate m². The first sampling occurred after 4 hours (pulse period) and subsequent sampling at 12 h, 1 d, 2 d, 3 d, and 5 d (chase period). 16S rRNA (Mag-SIP) and phospholipid derived fatty acid (PLFA) biomarkers were used for the identification of the major active microbial groups. A wide range of oligonucleotide probes was used to generate clone libraries for Mag-SIP bacterial analysis derived from total RNA and captured 16S rRNA. Water extractable carbohydrates were extracted to identify the intermediate compounds produced by the diatoms and those bacterial groups which assimilated them.

Out of the primary producers; diatoms were predominant with Gammaproteobacteria, Bacteroidetes, and Deltaproteobacteria being the main heterotrophic bacterial groups. Data from both ¹³C-PLFA and ¹³C-rRNA suggest a fast transfer of label from diatoms (60 nmol ¹³C g^-1 dry weight [dry wt]) to bacteria (7 nmol ¹³C g^-1 [dry wt]) during the first twenty-four hours, which suggests an exudation of low-molecular-weight organic compounds by diatoms that could be directly utilised by bacteria. Following this initial fast transfer, labelling of bacteria continued at a slower rate (13 nmol ¹³C g^-1 [dry wt]), which coincided with the degradation of carbohydrates in water-extractable extracellular polymeric substances (EPS) initially produced by the diatoms.

Unexpectedly, secondary labelling was also discovered for the diatoms. They may have used the EPS as external storage of carbon since the DIC was low.  On the other hand it is possible that the diatoms gradually reincorporated a variety of EPS and although this wasn’t the focus of the study, there are papers supporting this theory such as Smith and Underwood 2000 which showed that reserve compounds were used in the dark.  It was expected that there would be some specialized bacterial groups associated with the mat fulfilling different nutrient niches. However, the results suggest that the heterotrophic bacterial community equally shared the diatom organic matter. This study demonstrates the close nutrient coupling between benthic diatoms and heterotrophic bacteria. This study didn’t consider the action of macrofauna on the nutrient pathway concerned. Considering the open nature of this in vivo experiment. Burrowing organisms may have caused bioturbation within the sediment, or molluscan grazing upon the biofilm may have occurred. Considering the importance of benthic biofilms as a carbon source for higher trophic levels it is important to consider these processes.

Reference:
Miyatake, T., Moerdijk-Poortvliet, T. C., Stal, L. J., & Boschker, H. T. (2014). Tracing carbon flow from microphytobenthos to major bacterial groups in an intertidal marine sediment by using an in situ 13C pulse‐chase method.Limnology and Oceanography, 59(4), 1275-1287.

Protozoan parasites threaten tropical bathing waters

Pollution in the form of sewage is a major contributor to the fouling of shallow tropical coastal waters. These warm coastal water can harbour various pathogenic microbes, capable of causing harm to human health. This study looks specifically at two protozoan intestinal parasites, Giardia duodenalis and Cryptosporidium parvum, both environmentally robust in their cyst stages allowing prevalence in sewage waste waters entering the marine environment. In recreational coastal areas, the likelihood of these pathogens causing harm is increased by activities such as swimming and water sport activities. However, in the tropics the levels of this harm have not yet been estimated. Tropical areas are of particular interest in terms of harbouring pathogens as temperature and sunlight irradiation may increase their survival. This combined with the posing threat of climate change and increasing population densities in coastal areas is cause of significant concern for human health in bathing waters.

In this study, the potential risks were assessed in Venezuela’s central coast, an area of high intensity recreation usage. They used SYBR green I real time PCR to look for genetic markers of Cryptosporidium and Giardia cysts specifically and traditional culture methods to assess for faecal indicator bacteria FIB) and water quality deterioration. Risk of each species was determined by the level of cysts found in the areas using a dose response model, showing that Cryptosporidium has a risk value of 0.026 and Giardia has a a risk value of 0.00572, equating to a 39 and 50% chance of rate becoming ill once infected, respectively. Both of these were below the U.S. EPA upper bound on recreational risk of 0.036 however, 95th percentile estimates for Giardiasis for children exceeded the 0.036 level, suggesting levels of contamination may not be completely safe. A Monte Carlo uncertainty analysis was performed in order to determine the probability distribution of risks. 35% of the sites were shown to harbour Giardia cysts while Cryptosporidium were only detected in 14%. This level increased with the extent of sewage pollution and bather density during collection based on FIB levels. Giardia is the most common protozoan parasite found in human faecal samples in clinical laboratories for parasitic examination, explaining its widespread occurrence in the tested bating waters.

This study highlights the importance of pathogen specific detection for detection of disease risk in specific geographical areas. It shows how the risks of parasitic diseases acquired whilst undertaking recreation activity in tropical waters can be estimated using quantitative microbial risk assessment. Future research is needed into understanding fully the relationship between sewage pathogens and surrogate indicators in tropical waters, however this research highlights the advantages of using microbial tracking to identify sewage pollution. I think that issues such as sewage pollution are increasingly needed with the changing climate, as research into more tropical parasitic microbes will need to be applied to more temperate regions as temperatures increase and species distributions change with the climate. It is important that reliable detection methods are implemented soon.

Betancourt, W., Duarte, D., Vásquez, R. and Gurian, P. (2014). Cryptosporidium and Giardia in tropical recreational marine waters contaminated with domestic sewage: Estimation of bathing-associated disease risks. Marine Pollution Bulletin, 85(1), pp.268-273.

Papers for revision

As previously discussed in the final workshop for the module, I have included below the web addresses for the Ocean Acidification and Biofilm papers which Anita and I read for our global change project. I hope they are of use.

Intertidal epilithic bacteria diversity changes along a naturally occurring carbon dioxide and pH gradient. 

http://onlinelibrary.wiley.com.plymouth.idm.oclc.org/doi/10.1111/1574-6941.12368/epdf

Microbial biofilms in intertidal systems: an overview.

http://www.sciencedirect.com.plymouth.idm.oclc.org/science/article/pii/S0278434300000224

This paper addresses microbial biofilms in general, however, this does include some information on pH reduction effects on biofilm structure and associated microbes.  

My final blog post-I'll sign off with some gastrointestinal bacteria from turbot, lovely.

The gastrointestinal (GI) tract of fish is very complex, containing up to 107-108 colony-forming units (A unit used to measure the number of viable bacteria). The GI tract composition for farmed adult turbot Scophthalmus maximus hasn’t been previously studied using metagenomics (Xing et al., 2013). Xing et al., (2013) therefore looked into this, using metagenomics alongside 16S rRNA analysis, aiming to characterize taxonomic distribution and metabolic potential of the S.maximus microbiome, assess the bacterial diversity and see how the microbiome is related to the environment.

Xing et al., (2013) used 10 adult S.maximus alongside water samples from China. The GI tract of each fish was removed, and the contents squeezed out. The mucus was also collected, along with the bacteria present in the seawater. The genomic DNA was extracted and metagenomics and 16S rRNA were carried out to assess the taxonomic composition and functional diversity of the microbiome.

They found that both the GI tract and mucus samples of the fish were largely made up of Protebacteria (a major bacterial group) and Firmicutes. It was also found that the GI tract might possess bacteria that are initially associated with the seaewater. Both quorum sensing and biofilm formation were found to be overabundant when compared to other metagenomes. The genes associated with these were found to be mainly in species within the group Vibrio. The species also showed an overrepresentation of the systems associated with protein folding and stress responses. Alongside this, the genes related to human activity, such as antibiotic and heavy metal resistance were also detected. Therefore, it can be inferred that humans are affecting the GI microbiome in marine aquaculture species. (Xing et al., 2013)

Being one of the very early pieces of research using metagenomics on a farmed species, I feel this study is clearly of huge importance both in the present and for the future of aquaculture. It gives a clear idea of the bacteria that are associated with the fish, in this case, a high level of Vibrio. Also showing a detection of the genes associated with antibiotic resistance shows that the fish microbiome in aquaculture is being compromised by humans. Xing et al., (2013) suggest that aquaculture may effect the microbiome of the fish. Finding this out has huge implications in terms of being able to manage aquaculture to give the healthiest fish and the best yield. I feel using metagenomic profiling in the GI tract of fish holds potential for giving an insight into the bacteria associated with it and so may be of great use in the future of aquaculture. However, the number of samples and species that this is conducted on needs to be hugely increased in order to give broader, more applicable results.

Reference:

Xing, M., Hou, Z., Yuan, J., Liu, Y., Qu, Y., Liu, B. (2013). Taxonomic and functional metagenomic profiling of gastrointestinal tract microbiome of the farmed adult turbot (Scophthalmus maximus). FEMS Microbiology Ecology. 86, 432-443.

Who wants salty fish???

The importance of microflora is well documented both in the literature and in this blog. Briefly, symbiotic bacteria present in the GI tract of organisms confer a number of benefits from vitamin production to increased immunity. Within the field of microbiomes, examination of the symbionts and how they respond to biotic and abiotic influence is not numerous. This lack of understanding may be particularly important in light of climate change, where changes in the microbial community could affect host fitness. Some studies have purported that a microbiome has contributed vastly to the evolutionary success of man. However, how these assemblages assemble is poorly understood, as such much of the literature is filled with debate. A multitude of hypotheses aim to explain this, two of the main ones are niche-appropriation, a competition driven process and neutral theory, where stochastic processes determine colonisation. With this in mind, Schmidt et al. (2015) investigated the role of salinity acclimation on the microbial consortia present in/on the euryhaline fish, Poecilla sphenops (Mexican Molly).

After acclimation to eliminate the effects of previous environmental history, fish were acclimated between 0 and 2ppt. Following acclimation fish were exposed to four salinities for 12 days; 0, 5, 18 and 30ppt. Fish were then homogenised, tank water was filtered and DNA analysis undertaken for both sample types.

Results showed that the Gammaproteobacteria were the main constituents of the microbiome in all fish, across all salinity treatments. Generally, the same 2-5 OTUs were present in all individuals; however the frequency of these changed in accordance with the prevailing salinity. Vibrio and Enterobacteriaceae spp. replaced Aeromonas and Cetobacterium spp. at higher salinities. Furthermore, significant overlap was observed between the microbiomes of fish at 0 and 5ppt, and 18 and 30ppt. There were however, some outliers to this general rule, where a particular OTU was far more abundant in an individual despite being subject to the same conditions. Significant differences were observed between the microbial communities present in tissues and the surrounding water. Further, microbial communities in the water did not affect those in the tissues of fish.

Researchers concluded that microbial community assembly is a competition driven process and not a stochastic one owing to the lack of effect external microbial communities had on host colonisation. Additionally, changes in salinity caused changes in the microfloral composition to be observed; some species would not have the capacity to function optimally at varying salinities, and would thus be outcompeted. As such the neutral theory of microbiome assembly seems to be apparent. Despite this, there are some potential flaws in this investigation, many of which are detailed by authors themselves. The potential role of disease (some fish died before experimentation), replication cycles and diet were not considered; likewise analysis of the initial microbiome was not undertaken. Whilst this investigation does provide some interesting insights in to microbiome dynamics in response to the changing environment, it is evident that a more informed experimental design would be beneficial and thus, make the results more convincing. To expand upon this investigation, a multi-stressor approach may be useful. In addition, with the use of axenic fish, could the role of the microbiome in fitness be examined? Again as with most studies, the Fungi, Archaea and eukaryotes were ignored, despite evidence suggesting their significance in microbiome dynamics.  

Jack 

References

Schmidt, V. T., Smith, K. F., Melvin, D. W., & Amaral‐Zettler, L. A. (2015). Community assembly of a euryhaline fish microbiome during salinity acclimation. Molecular ecology

Ocean acidification and host–pathogen interactions

Ocean acidification (OA), caused by anthropogenic CO₂ emissions is considered one of the major threats to marine ecosystems and seawater pH is predicted to decrease by 0.4 units by the end of the 21^st century. Many studies have shown the strong negative impacts of future OA, with calcifying organisms being particularly at risk due to their dependence on calcium carbonate. However, there is little evidence of the impact of OA on potentially associated heterotrophic bacteria. OA appears to influence organisms differently and as such the interactive processes between organisms, such as pathogens and hosts, are likely to vary. Asplund et al., (2014) set out to investigate the relationship between Mytilus edulis and Vibrio tubiashii in terms of pathogen growth, viability and virulence, the host growth, immune defence and stress response and the interaction between these organisms under OA conditions.

Adult bivalves are able to accumulate high numbers of pathogenic bacteria without being infected as the generally posses a number of defence mechanisms against these invasive microorganisms. In the arms-race between hosts and pathogens, the balance may however shift if the host is exposed to other stressors, such as OA, as the host may have to concentrate more resources on processes like shell formation or growth. OA appeared to have no distinct effect on the virulence of V. Tubiashii. M. Edulis immune responses, such as maeocyte numbers and phagocytotic capacity, appeared to be unaffected by OA either. However, OA had a negative effect on the average shell growth and impaired crystalline shell structures as expected. Despite no evident impact on host immunity or growth and virulence of the pathogen, V. tubiashii was more successful in infecting mussels exposed to long-term OA compared to those maintained under ambient conditions. The viability of OA exposed V. tubiashii decreased when exposed to haemocytes extracted from control mussels but interestingly the viability of OA exposed V. tubiashii increased when encountering haemocytes from OA exposed mussels.

This study highlights the importance of studying the impact of OA on the interaction between different organisms and that results from studies focusing on each organism’s response separately could be very misleading. As climate change does not only affect OA, but other factors such as temperature as well, it would be interesting to test the combined effects of OA and increased temperature on pathogen-hosts interactions. OA is also likely to change the bioavailability of metals ions and as this paper also mentioned, there are many metal ion dependant enzymes that affect virulence in vibrios. This means that it is possible that the virulence of the vibrios will increase putting further stress on the host increasing the chance of infection. Furthermore, this study used adults injected with an inoculated solution. This is of very little ecological significance as it is an unlikely scenario in any ecosystem. The authors also mention that adults are normally resistant and it is the juvenile stage that is the most susceptible to infection. The next step in the study of M.edulis -V. tubiashii should focus more on the juvenile stage as it is more sensitive to pathogens.

Asplund, M. E., Baden, S. P., Russ, S., Ellis, R. P., Gong, N., & Hernroth, B. E. (2014). Ocean acidification and host–pathogen interactions: blue mussels, Mytilus edulis, encountering Vibrio tubiashii. Environmental microbiology, 16(4), 1029-1039.

http://onlinelibrary.wiley.com.plymouth.idm.oclc.org/doi/10.1111/1462-2920.12307/epdf

Where do mollusc murderers hide during winter?

Mollusc farming has become a major portion of global aquaculture, with oysters considered second only to cyprinids in importance of aquaculture products. Recently however, cultivated European populations have been suffering from mass mortality episodes.  Mortality in the summer in sheltered areas, where these outbreaks generally occur, can reach 80/100%. The causes behind these outbreaks are still poorly understood and are thought to be attributed to complex interactions among molluscs, pathogens and their environment. Two pathogens associated with the summer mortalities are Vibrio splendidus and Vibrio aestaurianus. Although some of their biology and toxicity within host organisms is now understood, little is known about the ecology of these pathogens outside of their bivalve hosts. The lack of information on the aquatic ecology and lifestyle limits the understanding of their role in the occurrence of mortality outbreaks. Vezzulli et. al. (2015) extensively investigated the ecology of these species in an aquatic brackish environment.

Using the strains LGP32 and 01/32 from V. splendidus and V. aestuarianus respectively, both related to diseases and mortality in Crassostrea gigas, they conducted laboratory microcosm experiments to assess the persistence of these strains in both seawater and sediment in 5°C and 25°C temperature and 20‰ and 35‰ salinity. The capability of LGP32 and 01/32 to interact (adhere) with both chitin particles and plankton crustaceans in vitro was analysed as well as the biofilm formation of both strains on PVC surfaces. Finally, in order to validate results obtained, the occurrence and temporal variations of V. splendidus and V. aestuarianus-clade bacteria were investigated in the Goro lagoon.

LGP32 generally lost culturability in all experiments in seaweater after a short incubation time (less than 5 days) and entered a viable but not culturable (VBNC) state. Whereas, 01/32 lost both viability and culturability in seawater within 5 days in almost all experimental settings suggesting 01/32 may be more demanding in its living requirements than LGP32. The short culture time for both strains also suggests the laboratory conditions of the experiment were not suitable for either strain. Both culturability and viability were higher in sediment experiments for the two Vibrio species, which may be due to sediment providing surfaces for biofilm development and the concentration of organic matter being higher than surrounding water. The presence and activity of two ligands (MSHA and GlcNAc) mediating the attachment of V. cholerae to chitin surfaces was also analysed. 01/32 was found to have both ligands and inhibition experiments supported their role in attachment of this strain to chitin-containing surfaces. However, although LGP32 contained the mshA gene, inhibition experiments pointed to a non-significant contribution of either ligand. Biofilm formations constitutes a successful survival strategy, however, although LGP32 showed a greater capability to form biofilms on PVC surfaces and possibly why LGP32 had a higher persistence in lab experiments, both strains showed lower capability than V. cholerae. When exposed to prolonged low temperatures, both strains entered a VBNC state. Interestingly though, VBNC cells of both strains retained virulence related factors and were smaller than 0.2 μm. This suggests they may remain undetected in some experiments and can reactivate under favourable conditions and infect hosts. The lagoon had an absence of a clear temporal trend in Vibrio spp. as well as the presence of culturable vibrios in seawater during cold periods. This could be due to a high nutrient content in the Goro lagoon. Similar to lab experiments, sediment samples also contained the highest proportion of viable and culturable vibrios.

The multitude of tests performed in this study allows us to understand more of the ecology and biology of these bivalve pathogens, however, some tests may be limited in their depth. Binding experiments only tested binding on chitin and copepods. The use of other substrates associated with bivalve morphology may have been beneficial to this study. Similarly in biofilm experiments, although PVC may allow tests to be easily repeatable and comparable, using a plastic may have less real world relevance. Therefore, biofilm formation of these strains may be different on organic surfaces in the natural environment. With VBNC bacteria found to retain virulence factors means monitoring these populations in locations of bivalve aquaculture could be important in determining disease risk in summer months. Monitoring will also have to account for the smaller than 0.2 μm size. As found in Goro lagoon though, culturable populations can survive through winter probably due to increased nutrients. The presence of aquaculture in habitats will probably also increase the amount of nutrients present in the habitat. Therefore, a significant proportion of culturable pathogens may remain in the environment. This could result in summer months having a large population of pathogenic bacteria and help cause the mass mortality of bivalves.

Vezzulli, L., Pezzati, E., Stauder, M., Stagnaro, L., Venier, P., & Pruzzo, C. (2014). Aquatic ecology of the oyster pathogens Vibrio splendidus and Vibrio aestuarianus. Environmental microbiology.

http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12484/abstract;jsessionid=AAD534EA8D57255E79EB5828EBFAD99B.f02t03?deniedAccessCustomisedMessage=&userIsAuthenticated=false

A symbiosis or body hijack?

A symbiosis between a eukaryote and bacteria conveys a mutualistic relationship whereby an enhancement of fitness is evident in both partners. But at what stage does a symbiosis become a hijacking?

The worm Olavius algarvensis resides in shallow water sediment, migrating between the oxic and anoxic. Whether voluntarily or not, this worm has undergone an extreme body makeover; whereby it is now exists in a gutless form, without a mouth or an excretion system. O. algarvensis relies solely on it’s four bacterial symbionts for all its nutritional demands. The community of bacteria is made up of two aerobic denitrifying gammaproteobacteria, and two anaerobic sulfate reducing deltaproteobacteria. Sulfide is scare in the shallow sediment, so the sulfate reduction provides energy for the deltaproteobacteria, whilst simultaneously providing sulfide for the gammaproteobacteria, which can be used for autotrophic CO₂ fixation. 

The balance seems too perfect, and all organisms need to acquire nutrients from external sources. Kleiner et al (2012) shows that the ideal energy source for this migrating worm and its suite of microbes is carbon monoxide (CO). CO has a large redox potential and can be transferred to a number of electron acceptors, and thus provides a ubiquitous energy source for both anaerobic and aerobic bacteria. The use of CO as an energy source was supported by genes for CO dehydrogenase in all bacterial symbionts, and additionally by the unexpectedly high concentration of CO in the sediment.

As the worm did not hold on to its excretory system in its bodily cutbacks, an efficient waste management system is in place, which conserves energy by recycling waste products. The fermentative waste products produced by the worm in anaerobic conditions, is utilised by the delta-proteobacteria. Additionally the author hypothesises that one of the gammaproteobacteria may also contribute to waste management, as it produces an enzyme (3-HPB) that would allow it to fix CO₂, but also assimilate waste products. Furthermore, the gene for the 3-HPB-enzyme has been acquired through horizontal gene transfer, and may have been key in the early establishment of the symbiosis.

Consistent with the frugal nature of this symbiosis another mechanism for conserving energy is proposed.  In the first step of sulfate reduction pyrophosphate is produced, and must be removed from the cell in order to reduce sulfide. Yet the removal is energy costly and essentially wasteful. Here the bacteria are thought to store the pyrophosphate from sulfate reduction using a pyrophosphate enzyme (pryophosphatease), which is bound to the cell membrane. The pyrophosphatease acts as a proton pump creating a proton motive force, by hydrolysing pyrophosphate instead of ATP. The author infers that this might be a common feature in all sulfate reducers.

This intricate balance between the bacterial symbionts functioning together, like a well-oiled machine, leaves me questioning when does a eukaryotic organism just become a house for a bacterial community? This worm would not survive alone in this environment, so the bacteria are essential, but would the worm have dispersed elsewhere had the symbionts not moved in? Or would it have come to the end of it’s evolutionary line?

Reference:

Kleiner, M., Wentrup, C., Lott, C., Teeling, H., Wetzel, S., Young, J., ... & Dubilier, N. (2012). Metaproteomics of a gutless marine worm and its symbiotic microbial community reveal unusual pathways for carbon and energy use. Proceedings of the National Academy of Sciences, 109(19), E1173-E1182.

Virus-like particles used to control HABs

Harmful algal blooms (HABs) can cause mass mortality of fish and other aquatic organisms through the depletion of oxygen from the water, the contamination of the surrounding air, blockage of gills and production of toxins and reactive oxygen species. HABs are becoming a global issue with recurrent cases along many coastal areas. Many of the commonly used techniques to control HABs (e.g. ozonation, chlorination, ultrasonic treatment, etc.) have limited success and also have the capacity to disrupt marine ecosystems. Safer biological strategies for mitigating HABs have been proposed that utilise marine bacteria, algal viruses, protozoa and macrophytes. Kang et al., (2015) set out to investigate the potential use of virus-like particles (VLPs) to deliver algicidal compounds directly to harmful algae.

The toxic dinoflagellate Heterocapsa circularisquama is widely spread along the Chinese coast from Japan to Hong Kong and causes severe die-off of cultured clams, mussels, razor shells and oysters. The capsid protein of HcRNAV34, a single stranded RNA virus that infects and lyses H. circularisquama, produced from Escherichia coli  was self-assembled into VLPs  in vitro and employed as a carrier for the potent algicidal compound thiazolidinedione 49 (TD49). The self-assembled VLPs were significantly smaller than the authentic HcRNAV34 capsid reflecting the various morphological forms a virion may adopt during maturation. However, despite this difference in size, the TD49-HcRNAV34 VLP showed the same host specificity as the native particles. The TD49-encapsidated VLPs showed a more potent target-specific algicidal effect than TD49 alone by increasing TD49 local concentrations near the target cells. In contrast, encapsulated TD49 protected non-host cells at low concentrations (0.5μM) even after 72h. Interestingly, at 5.0μM the VLP lost their protective effect for non-host cells by 72h and resulted in higher growth inhibition rates that free TD49. One of the possible explanations for this is the protection of TD49 by VLP from degradation, hence prolonging its otherwise short half-life.

VLPs have some major advantages for the delivery of algicidal compounds to harmful algae such as high target specificity and thus protection of non-target species, the use of a naturally occurring biodegradable protein and stabilisation of the algicidal compound from degradation in sea water. However, the stabilisation of the algicidal compounds could also be considered a disadvantage as it can increase the effect on non-target species once the VLPs have degraded as it makes the algicidal compounds linger around for longer. More work needs to be done on the production and stability of VLPs as HABs can be comprised of more than one strain and different types of VLPs would need to be deployed. It would also be interesting to test the efficiency of other algicidal compounds while encapsulated in VLPs and see whether the findings of this paper apply to other compounds as well.

Kang, B. S., Eom, C. Y., Kim, W., Ju, S. Y., Ryu, J., Han, G. H., ... & Kim, S. W. (2015). Construction of target‐specific virus‐like particles for the delivery of algicidal compounds to harmful algae. Environmental microbiology, 17(4), 1463-1474

http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12650/abstract?deniedAccessCustomisedMessage=&userIsAuthenticated=false

Intestinal microbial species composition in Atlantic cod

The intestinal microbial community provides a variety of crucial functions for their vertebrate hosts. However, the microbiota in individual is influenced by host-specific selection and it has been shown that a selective group of microbiota is common across different host of the same species. This concept of core microbiota, although it is widely studied in mammals, few studies have been done on fishes. The core microbiota is said to be the key which provides a minimal functionality in the healthy gut and the colonisation factors appear to be conserved among wide range of vertebrates, including fish. The diversity of fish lineages provides the opportunities to investigate the factors that influence the composition of the vertebrate intestinal microbiota. In this study, Star et al. (2013) describe microbial intestinal communities of eleven individual Atlantic cod (Gadus morhus) caught at a single location and quantify a core microbiota based on an extensively 454 sequenced 16S rRNA library of the V3 region.

Using high throughput sequencing, they identified 573 operational taxonomic units (OTUs) from a total of 280447 sequences and found that 10 OTUs are shared at 97% sequence similarity among the 11 individual Atlantic cod. They found that the shared OTUs is dominated by the orders of Vibrionales (50%), which is prevalent in the Atlantic cod intestinal microbiota, followed by variable numbers of Bacteroidales (17%), Erysipelotrichales, Clostridiales, Altermonadales and Deferribacterales. Furthermore, they found that the intestinal microbiota is dominated by a few abundant OTUs, while the majority of OTUs is present at rare frequency. This pattern is similar to intestinal composition in zebrafish or humans.    

However, the utility of the core microbiota concept at a taxonomic level has raised questions due to limited evidence of universally abundant species in humans. Additionally, the authors stated that the number of shared OTUs identified in the paper may be an overestimation. This is because the fishes were kept in the same tank, and thus experience the same environmental conditions before sampling. They assumed that similar environment is more likely to homogenize microbial communities, rather than promote individual differences. Therefore, a different experiment would be required to investigate is the shared OTUs are due to environmental factors or are species specific.

In conclusion, individual variations in the OTUs in wild-caught Atlantic cod suggest that a complex combination of factors influences the gut microbiota composition. However, such variation has gone unobserved in previous studies of natural populations of teleost, which may affect estimates of the number of shared OTUs among hosts.   

Star, B., Haverkamp, T. H., Jentoft, S. and Jakobsen, K. S. (2013) Next generation sequencing shows high variation of the intestinal microbial species composition in Atlantic cod caught at a single location. BMC Microbiology. 13: 248.

the 'resuscitation' of dormant Vibrio cholerae

The bacterium Vibrio cholerae has been very nicely described on numerous occasions during these last few weeks throughout the blogs. Therefore, I won’t dwell on describing it again and crack on with this study by Nayeembul Bari et al., (2013). Detecting Vibrio cholerae is no easy task due to the fact that the bacteria have a dormant state in which it isn’t active but still remains viable. In mammals, growth of the intestine has been reported to trigger these dormant cells and ‘resuscitate’ them into an active state in which they can cause cholera. However, the signals behind this process remain unknown. The aim here was to bring these signals to light.

Strains of dormant Vibrio cholerae were isolated and plated with the appropriate antibiotics. The strains came from water samples from Dhaka that have previously shown evidence of ‘conditionally viable environmental cells’ (CVEC), this meaning they have to potential to be cholera causing. Only samples that didn’t contain live, active bacteria were used. They then prepared 2 different autoinducers, which are signalling chemicals associated with quorum sensing in bacteria, thought to play a role in the ‘resuscitation’ process. These were then introduced to the plated strains and it was then observed as to whether these autoinducers had any resuscitating effects on the strains (Nayeembul Bari et al., 2013).

They found that the dormant form of the bacteria could easily be converted into culturable cells by exposing them to both of the autoinducers used. This is a hugely important finding, as the autoinducers used here can be produced by many bacterial species at any time. This clearly makes detection of Vibrio cholerae a hard task.

The data suggests that this can happen both within and outside the host in the form of intestinal or environmental bacteria. This may go some way to explaining the seasonality of cholera, with different temperatures/salinities etc activating the dormant bacteria to become active under different conditions. The environmental conditions may work to increase survival of human intestinal bacteria and so may increase the likelihood of cholera causing blooms (Nayeembul Bari et al., 2013).

The authors state that this study sets up the potential for future research. I agree that this is most definitely the case. It shows that cholera seasonality is ultimately dependant on communication between bacterial species. These communications could be much further looked into to uncover which bacteria are interacting and how they are doing so. Any information found here would hugely help in the overall effort to help reduce the number of outbreaks. They also suggest that other bacterial species may have a dormant state, so this opens up even more possibilities for future research; will the autoinducers resuscitate these too? Overall I feel this study provides a large amount of interesting aspects for future research, and has the potential to aid in the tackling of cholera prevention. It is still a long way from being able to accurately predict cholera outbreaks but the research gives strong evidence for how the dormant cells become active and so have to potential to cause outbreaks.

Nayeemul Bari, S., Roky, M., Mohiuddin, M., M., Mekalanos, J., Faruque, S. (2013). Quorum-sensing autoinducers resuscitate dormant Vibrio cholerae in environmental water samples. Proceedings of the National Academy of Science. 110, 9926-9931.

Genomic island variability facilitates Prochlorococcus-virus coexistence

Both Prochlorococcus and the viruses that infect them are ubiquitous in the marine environment. The underlying mechanisms that allows the coexistence of Prochlorococcus and viruses in nature remains unclear. However, one such hypotheses that have been put forward to explain this phenomena is the continuous arms race between Prochlorococcus and strain-specific viruses. In this paper, they combined whole-genome sequencing and PCR techniques to investigate the effect of resistance on genome evolution and the genomic mechanisms behind the long-term coexistence.

The experiment studied the genome of 77 substrains that is selected for resistance to 10 podoviruses. The substrains used are from two different ecotype which differs in their geographic distribution. These substrains have shown majority of mutations in the hypervariable genomic island, with 71% localised to a single genomic island, ISL4, indicating that this is a virus susceptible region. The mutations affects viral attachment to the cell surface and imposed a fitness cost by decreasing growth rate to the host. This identification of resistance-conferring mutations in the genes suggest that the genomic islands are dynamic and genomic mutation occurs not by chance but rather are directly associated with viral selection pressure.  

Mutation confer resistance to specific phages might be seen as favourable to the survival of Prochlorococcus, but the authors have shown that it allow more rapid infection by other phages. Which is termed as “enhanced infection dynamics” fitness cost. In this study, they have shown that at least 16 out of the 23 mutant substrains studied have showed adaptive cost resistance- which is either reduced growth rate of more rapid infection by other viruses.

To test for the coexistence of cyanobacteria-virus, their findings suggest that resistant Prochlorococcus population probably exist in the environment and is highly diverse. The experiments have shown high rate of mutation which leads to resistance in the substrain MED4. This spontaneous mutation is 10-1000 fold higher than other bacteria.

It is well accepted that gene acquisition is facilitated by horizontal gene transfer mediated both by phages and by other mobile genetic elements, and that the genomic islands are a repository for such horizontally acquired genes. Horizontal gene transferred in genomic islands is beneficial to microbes as it facilitates constant gene flow and generates numerous polymorphisms which effectively reduce the population size for infection by any particular phage.   

The authors proposed that the “arms race” between bacteria and their viruses leads to the emergence of resistant bacteria in both a sequential and accumulative process, resulting in a continuum of cyanobacteria with different but overlapping ranges of viral susceptibility. This also further brings in the “numerical refuge hypothesis”. This study have thus evidence that viral-attachment genes are preferentially located in genomic islands and that viruses are a selective pressure enhancing the diversity of both island genes and island gene content. This diversity emerges as a genomic mechanism that reduces the effective host population size for infection by a given virus, thus facilitating long-term coexistence between viruses and their hosts in nature. 

Avrani, S., Wurtzel, O., Sharon, I., Sorek, R. and Lindell, D. (2011) Genomic island variability facilitates Prochlorococcus–virus coexistence. Nature. 474, 606-608.

I’ve caught something fishy! : Application of probiotic, prebiotic and synbiotic for the control of streptococcosis in Tilapia Oreochromis niloticus

Disease within aquaculture can impact fish health and decimate stock numbers, the study of Widanarni and Tanbiyaskur (2015) addresses the use of probiotics, prebiotics and synbiotics (a combination of probiotics and prebiotics) through Tilapia (Oroechromis niloticus) feed to regulate the bacteria Streptococcosis. Infection of Streptococcosis agalactiae in O.niloticus can cause lesions and lethargy, and in extreme cases presumed water loss from the digestive tract leads to death. Considering the reduction in health and possible lethal effects of S.agalactiae, its impact on aquaculture and economy could be great.

Three treatments were added to the Tilapia feed and tested to assess their effects on the management of S.agalactiae. Additionally two controls were used; one consisting of a feed with no treatment and exposure of the fish to S.agalactiae (positive control) and a second with no feed treatment and no exposure to S.agalactiae (negative control). Bacillus sp. NP5 was used as the probiotic and oligosaccharides (from sweet potato) were used as the prebiotic, the treatments were sprayed onto artificial feed. Artificial feed was given to the fish three times a day, with the treated being administered once a day for 14 days. On the 15^th day the fish were injected with a dose of S.agalactiae equivalent to LD50. The parameters measured were daily growth rate (DGR), feed conversion ratio (FCR) and survival rate.

After injection of S.agalactiae, those Tilapia which were treated with the probiotic, prebiotic or synbiotic possessed an extremely low count of S.agalactiae, the controls however possessed a substantially high count of S.agalactiae. Target organs of the liver, kidney, brain and eyes were examined on the 3^rd and 4^th week after infection by S.agalactiae, during the 3^rd week there were S.agalactiae counts for all the treatments. By the 4^th week the synbiotic treated fish possessed no traces of the S.agalactiae. The probiotic and prebiotic treated fish displayed presence of S.agalactiae in the brain, the positive control displayed S.agalactiae in all the target organs.

Of all the treatments the most successful was the synbiotic, this provided the probiotic bacteria with enough resource to increase their number well and increase enzyme activity. The synbiotic increased the growth of the Tilapia due to a healthy digestive bacterial community and activity. Due to the number of probiotic bacteria in the fish which received the three treatments the Tilapia could efficiently digest the food it received. This study by Widanarni and Tanbiyaskur (2015) provides clear definitive results, although the in depth detail of the positive control biological results described in the discussion appear to be unnecessary and detract from the hypothesis of the study.                  

       

Widanarni and Tanbiyaskur. (2015) Application of probiotic, prebiotic and synbiotic for the control of streptococcosis in Tilapia Oreochromis niloticus. Pakistan Journal of Biological Sciences. 18 pp.59-66

http://scialert.net/qredirect.php?doi=pjbs.2015.59.66&linkid=pdf

Phytoplankton Promote V. parahaemolyticus Persistence

Vibrio parahaemolyticus, is another malignant Vibrio, causing acute gastroenteritis (and occasionally death) if shellfish in which it has accumulated is under cooked or improperly handled. Ubiquitous in marine waters and distributed worldwide it causes an estimated 4,500 cases in the USA annually. The bacterium has a highly seasonal life-style in brackish coastal waters where it is most common. Increasing in abundance during warm summer months but effectively vanishing in winter by entering a viable but non-culturable (VBNC) state. Like other Vibrio species, it possess a particular affinity for chitin and attaches to chitin-based surfaces such as zooplankton exoskeletons. Chitin is also produced by phytoplankton such as diatoms and is a structural component of their cell walls. Given that increases in diatom abundance have been associated with increased in Vibrio spp. abundances it is possible that adherence to chitin compounds is a mechanisms for Vibrio parahaemolyticus persistence along with VBNC. Frischkorn et al. examined this, firstly by assessing the role that type IV pili pay in attachment to chitin and then examining the association with a real diatom species.

The role in attachment of two type IV pilins was assessed, mannose-sensitive haemagglutinin (MsHA) and chiten-regulated (PilA) pilus. These mediate biofilm formation and cell-cell binding respectively. Additionally the role of GlcNAc binding protein A (GbpA) which facilitates adherence to chitin in V. cholera was examined. Knockouts were created for each gene and biofilm formation on polystyrene and attachment to chitin beads was assayed. Knockouts showed both reduced biofilm formation and attachment compared to wild type bacteria, with double knockouts for both pilins doing worst. Whereas GbpA knockouts were not as affected. This indicated that the pilins act synergistically to facilitate attachment and are more essential for attachment than GbpA. Adherence to the diatom Thalassiosira weissflogii, which produces chitin containing fibrils, was then tested. Attachment to the diatom was assayed throughout the growth curve and increased from the early stationary phase though to death phase. Bacteria were also observed attached to free floating fibrils shed by the diatom. T. weissflogii is known to increase its chitin production during these phases as structural nutrients become starved. Attachment to non-chitinous products was ruled out and knockouts showed that the two type IV pili are important for binding to the diatoms chitin. These results highlight the ability of V. parahaemolyticus to associate with an abundant diatom species during period of the growth phase analogous to a bloom collapse. Potentially providing a method of outbreak prediction by monitoring the dynamics of diatom species, which are relatively easy to identify. This is particularly important in the Pacific Northwest as remote sensing data is currently not good enough to deal with the intricate coastlines and high cloud cover.

I thought this study was excellent, being both highly applied and extremely interesting. A number of future avenues of research spring to mind. Is direct attachment of the bacterium observed in field samples and is the association consistent over other chitin producing diatoms? Overall, I think the study shows that numerous different chitin producing taxa are important in the ecology of Vibrios. Given this, I would not be surprised if associations with other chitin producers e.g. fungi are present. More broadly, the study highlights the link between science for the sake of it and that of an applied ilk. This study is about a public health issue, but none of it would have been possible without an excellent of both the pathogen and the diatom species. This clearly shows that you cannot have one without the other, both are interconnected and to ignore blue skies would be of serious detriment to real world applications. 



Reference: Frischkorn, K.R., Stohanovshi, A. and Paranjpye, R. (2013). Vibrio parahaemolyticus type IV pili mediate interactions with diatom-derived chitin and point to an unexplored mechanism of environmental persistence. Environmental Microbiology, 15, 1416-1427.