Vibrios: A Big Family?

Vibrios have undoubtedly been a recurrent theme in our lectures this year: from their importance as symbionts in the Hawaiian squid Euprymna scolopes to their role as potential pathogens, vibrios occupy a wide variety of geographical and ecological niches. Especially the close association of vibrios to zooplankton due to the presence of a specific chitinase may have facilitated their widespread distribution. But what about the genetic relationship between vibrios occupying different niches:  did adaptive radiation lead to genetic segregation or do Vibrio populations have a panmictic population structure?  

Hoffmann et al. (2012) aimed to clarify Vibrio population dynamics within natural marine environments by studying genetic relatedness and phylogenies of both sponge-derived (SD) and shallow, coastal water Vibrio populations. Sponges have a diverse microbiota (e.g. see Robyn’s blog “Shedding light on the function of marine sponge microbiota”), including vibrios, and occupy a range of benthic habitats ranging from shallow waters to 900 metre depth. Vibrio species have been reported to be involved in both secondary metabolism and synthesis of antibacterial compounds in sponges; however it remained unclear how these SD Vibrio species are related to the nearshore counterparts.

Hoffmann et al. used a variety of state-of-the-art techniques to elucidate this relationship between different Vibrio populations. Basically Vibrio strains very isolated from sponge samples and cultured in highly selective agar. PCR, 16 rRNA gene sequencing, 16S-23S rRNA intergenic spacer region (ISR), multi-locus sequence analyses (MLSA), as well as phylogenetic analyses were then used to investigate the degree of relatedness and to construct phylogenies.

In accordance with previous studies, they found that most SD Vibrio isolates (74%) clustered within the Vibrio Harveyi clade. The MLSA approach was applied on these isolates to further distinguish the phylogenetic relatedness of SD and the coastal reference Vibrio strains. Results pointed towards strong evidence for extensive recombination among SD vibrios and their nearshore counterparts strains, isolated from a variety of globally distinct locations, which supported the hypothesis that SD and shallow-water vibrios are not two disparate populations, but members of a larger single panmictic group. Moreover there was evidence for horizontal gene transfer occurring between both groups, suggesting that DNA may be exchange even across these expansive niches.

Overall these findings may help to explain the wide distribution of vibrios, but particularly interesting are the implications of this study on the movement of marine pathogens such as the highly pathogenic V. parahaemolyticus O3:K6 clone, which spread from Calcutta in India to South-East Asia, Atlantic and Gulf coast of the United States, Europe, Africa and South America. In fact, the panmictic population structure could be a critical component of the global distribution of the O3:K6 pandemic strain. Like Vezzulli (see Vicky’s comment on my blog “Early warning systems for Vibrio disease risks”), Hoffmann et al. suggested that the association of vibrios with copepods could provide a means by which vibrios traverse disparate niches, from the near-shore pelagic to the deep benthic habitats, which is supported by the copepod life cycle overlapping these two regions.  

I think this is a very interesting study which links together several themes covered in previous blogs: sponge microbiota (authors briefly mention in their conclusion that sponges may support Vibrio speciation events), horizontal gene transfer, vibrios and chitin, and vibrios as human pathogens. 

Hoffmann, M., Monday, S. R., McCarthy, P. J., Lopez, J. V., Fischer, M. & Brown, E. W. 2012. Genetic and phylogenetic evidence for horizontal gene transfer among ecologically disparate groups of marine Vibrio. Cladistics, 1–19.

Is today’s sewage treatment actually what’s best for our health…..?

Is today’s sewage treatment actually what’s best for our health…..?

 

Relating to our feature lectures looking at sewage and its treatment along with prevalent microbes that are present and cause severe health problems this paper was relevant as it looked at whether sewage microflora could be used as a potential indicator for effective water sewage treatment or whether the methods undertaken at present are not effective enough.

The study by Maruthi et al (2013) aimed to see whether the sewage treatment in Vishakhapatnam is effective as it could and should be, although this does not relate to things closer to home it does provide an insight into whether the treatment in the UK is effective enough and provides a potential method of what could be done here to test the efficiencies.

Environmental surveys are necessary to understand and document the occurrence and distribution of bacteria that could cause negative effects within any population, not just the human one. Numerous studies looking at this microflora and this has only increased since sewage has been classed as an emergent pollutant as they are commonly found in watercourses.  The city in question, is a city situated on the coast of the Bay of Bengal and currently has an unscrupulous amount of sewage daily, these figures average about 330 million litres per day of sewage, and of this only 25mld are treated, hence the need for a study like this and a massive increase in treatment protocols.

The method undertaken involved 10 sites that were previously selected from along the coast of this city, at sewage exit points, these water samples were then transported back to the laboratory for further testing. The microflora that was being looked at were selected prior to this, and compiled in a table within this paper. The key measurements recorded were the total coliform count, the total bacterial count and also the total salmonella count. These were not the only measurements undertaken as bacterial fungal counts were also taken of select species and the same for a select group of Helminthes (Family of parasitic worms).

The results found that even after sewage treatment a large abundance of animal and fecal related coliforms were still present after the treatment which in this case is chlorination, the general range of microflora was quite large and this depended on the area taken from but overall the fact that there is still a significant amount of these organisms present is alarming. So this study has demonstrated the fact that chlorination cannot provide the complete answer to treatment and should be used in accordance with another, If not a few more processes. Yes this study looked just at one city but the general principle and results can be applied globally. So this needs to be addressed and I still found this paper both relateable and interesting.

Any questions please ask.

Ollie.

Available at:

http://theecoscan.in/Journal_PDF/Spl2013_v3-06%20Y.%20Avasn%20Maruthi.pdf

Reference:

Maruthi,Y., Hossain,K., Rao,S. and Kumar,Y. (2013). Sewage Microflora is a potential indicator of effective sewage treatment. The Ecoscan, Volume III, 35-39.

Aquaculture and Vibrios

Vibrios are gram negative, curved and rod shaped, microbes which are most commonly found in coastland and estuarine waters which are natural inhabitants of the marine environment. Many groups are free living though some can form pathogenic or symbiotic interactions with eukaryotic hosts and often alternate between growing within the host or prolonged survival within aquatic habitats. In vibrios such as these it is the ability to form and maintain a biofilm mode of living that is key to their survival and transmission. Growing as a biofilm is the preferred way of growth for alot of microbial life as it enhances the growth of the microbes that form the biofilm as they have better access to nutrients. It also increases the survival rate of the microbes by providing protection against predators and antimicrobial agents. Vibrio biofilms are a particular problem for aquaculture as they can infect the fish stocks relatively quickly and transmit across the stocks quickly.

This study focussed on the effect that the supernatant of marine bacterial culture has upon the biofilm formation of vibrio sp. The authors screened samples of vibrio sp for biofilm inhibitors and then used light microscopy in order to observe and document the biofilms as they developed. After running intial attachment assays and Bacterial Adhesion To Hydrocarbons (BATH) assays upon cultures it was found that bacterial culture supernatants of Bacillus pumilus (S8-07) and Bacillus indicus (S6-01) were able to destroy the architecture of the biofilm and reduced the surface hydrophobicity of the vibrio sp. which is an essential requirement for biofilm formation. The paper concludes with the suggestion that the two strains previously mentioned could also be used to control the proliferation of vibrio biofilms in aquaculture.

Nithya C., Pandian SK., 2010, The in vitro antibiofilm activity of selected marine bacterial culture supernatants against vibrio spp, Archives of microbiology, 192, 843-854

Natural Antifouling compounds from cyanobacterium Lungbya majuscule

The current most widely used method of boifouling control in marine environments involves the use of highly toxic compounds. Use of these chemicals is undesirable due to their highly toxic nature which can affect marine life. It is therefore desirable to find an alternative form of control. Many potential marine-derived antifoulants have been identified from organisms which are naturally exposed to larvae of fouling organisms and produce anti-fouling chemicals. Synthetic analogues of such naturally produced antifoulants can be developed and mass produced for commercial use.

Cyananobacteria are known to produce many useful secondary metabolites but few have been looked at for possible antifouling (AF) properties.  These secondary metabolites have been shown to possess useful antifeed properties against fish, herbivores and marine invertebrates. Some secondary metabolites identified from Lyngbya majuscule have been shown to possess such antifeed properties and the authors hypothesised that these compounds may inhibit invertebrate larval settlement.  In this study twelve secondary metabolites were purified from L. Majuscula and their effect on barnacle larvae settlement was assessed. Some metabolites were found to be highly toxic, killing more than 90% of the barnacle cyprids. Others showed significantly lower toxicity (<15%) and also showed high anti settlement activities ( >80% of the barnacle were unsettled). The best anti settlement molecule of the 12 tested was found to be dolistain 16 which was tested at many different concentrations and was found to have an EC₅₀ at 0.003 µg ml^-1.

Further evaluation of this molecule was carried out in the field at concentrations of 10.0, 1.0, 0.1 and 0.01 µg ml^-1.All four concentrations showed significant AF activities against the settlement of barnacles. Plates containing dolastatin 16 at a concentration of 10.0 µg ml^-1 showed barnacle counts below 25 individual and 0.01 µg ml^-1  showing  less than 100 individuals. When compared to the control with counts of greater than 200 barnacles it can be seen that a significant reduction in settlement occurs, at the lowest concentration. Therefore indicating that dolastain 16 is a highly effect inhibitor of barnacle settlement.

This study shows that cyanobacteria could be used in future as a source an environmentally friendly antifouling compounds and this group should be further explored to find more possible AF compounds. Prior to this study only one cyanobacterial secondary metabolite had been explored for possible AF properties therefore this study provided a valuable contribution to the field, widening the number of molecules tested. Secondary metabolites such as the ones tested in this study could provide an alternative to the current methods of antifouling control. Future work should focus on incorporation of molecules such as dolastin 16 into antifouling paints and further work is needed to assess the AF properties of other secondary metabolites to find the most appropriate.

 Lik Tong Tan , Beverly Pi Lee Goh , Ashootosh Tripathi , Mui Gek Lim , Gary H. Dickinson , Serina

Siew Chen Lee & Serena Lay Ming Teo (2010): Natural antifoulants from the marine cyanobacterium Lyngbya majuscula ,

Biofouling: The Journal of Bioadhesion and Biofilm Research, 26:6, 685-695

This paper can be found at: http://www.tandfonline.com/doi/pdf/10.1080/08927014.2010.508343

What Has Prevented Genetically Engineered Microbes From Cleaning Up The Sea?

Microbial bioremediation is defined as the process by which micro-organisms degrade or transform hazardous organic compounds into non-toxic substances. A variety of microorganisms are capable of efficiently degrading toxic compounds and xenobiotic in the environment have either been isolated or engineered. Since naturally occurring microorganisms are not capable of degrading all toxic chemicals genetically engineered microorganisms have been the essential for bioremediation.

The first genetically engineered microbe was created by Ananda Chakrabarty in 1971; the microbe was an alternative form of the genus Pseudomonas and was capable of breaking down crude oil. These microbes as well as another, Deinococcus radiodurans (the most radiation-resistant organism) were not used for bioremediation because of public concern and regulations. In the 1980s-1990s many biomed companies began and increased research into genetic engineering. Due to high technical costs and difficulties involved in regulation a switch from companies to academics. There have been very few field trials for the use of genetically engineered microbes for bioremediation; therefore there is a need for this type of research to be moved from the lab to the field. The United states EPA (Environmental Protection Agency) started regulating genetically engineered microbes 30 years ago and have not been commercialized, this is due to possible reasons such as cost or complexity.

There was high resistance from the public and scientists to unleashing modified microbes for bioremediation due to a variety of risks. One major concern is containment, specifically ‘how can microorganisms be contained following completion of the bioremediation process?’ these concerns come from the potential of genetically engineered microorganisms to disrupt the environment they persist in after the desired pollutant has been depleted. Containment of the microbes, involves programming the death of genetically engineered microorganisms after the depletion of the concerning pollutant. This removes the risk and associated concerns of use, generally associated with introducing genetically engineered microorganisms into the environment.

These proposals were made by the authors to resolve the failure of the research to commercialization cycle in the use of genetically engineered microorganisms for bioremediation:

• The EPA should emphasize risk stratification in the use and application of genetically engineered microorganisms for bioremediation in its TSCA regulations.
• Scientists working on bioremediation research should take note of existing regulatory frameworks at the onset of their research in order to facilitate their application and commercialization of their research products.  The EPA should not presume that all genetic manipulation is high risk and focus their regulatory effects on high risk organisms and their uses.
• Regulations should continually be updated by to the EPA to take into account the rapidly evolving containment technologies and new information in genetic engineering development.
• Scientists should focus efforts on the application of technical safeguards in the design of GM microbes for bioremediation. 
• The EPA should initiate programs that support start-up bioremediation companies to try their product in field trials.

This paper is analyses why there is a gap present between the research and commercialization of microorganisms in bioremediation. The application of genetically engineered microbes in bioremediation requires safeguards (containment) and assessment of risks (risk-based regulation) of each genetically modified microorganism and the surrounding environment. A noticeable issue in commercializing genetically engineered microbes was the inadequacy of biotechnology to contain the genetically modified bacteria once released; this may however improve with time and funding. 

I found this paper to be very thorough in research; it provided a background and was sectioned well. For further research into this area of study it would be interesting to include limitations different countries have on the use of bioremediation microbes and possibly any case studies showing the harm a genetically engineered bacterium has been known to cause in the past and whether incidences like ‘The Deep-water Horizon oil spill’ has increased the research and funding since 2010.

Ezeizika, O.C., Singer, P.A. (2010), Genetically engineered oil-eating microbes for bioremediation: prospects and regulatory challenges, Technology in society, Volume 32, Pages 332-335

Oil degradation by Ebro Delta microbial mats

The application of natural microbial communities for the cleanup of contaminated ecosystems after oil spills is of particular contemporary interest. Cyanobacterial mats contain phototrophic and heterotrophic bacteria and are thought to be promising bioremediation agents due to their capability to colonize contaminated coastal zones and their high metabolic diversity within a few millimeters depth. Llirós et al. (2008) aimed to determine the effect of two types of crude oil, Casablanca and Maya, on Ebro Delta microbial mat communities (a pristine marine benthic ecosystem, found at the sediment-water interface) in experimental microcosms. Casablanca has a low sulphur content, is similar to Arabian light and spreads easily and swiftly while Maya has a high sulphur content and viscosity and is similar to Aegyptian oil. Five microcosms were used, each with mechanical movement simulating a tidal cycle and a diel light cycle. Microcosms consisted of a control containing just the microbial mat (unpolluted); microbial mat and Casablanca oil (Casablanca); microbial mat and Maya oil (Maya); just Casablanca and just Maya (mechanical controls). They determined chlorophyll a (chl a) concentrations as a measure of oxygenic phototrophic biomass and protein content as an indicator of total biomass and cyanobacterial identification and total cultivable heterotrophic bacteria counts were carried out under aerobic and anaerobic conditions.

Llirós et al. (2008) found that the unpolluted microcosm showed no qualitative significant changes in diversity, chl a or protein content over time and at least seven cyanobacterial morphotypes were found. These were filamentous (Microcoleus chthonoplastes-like, Phormidium-like and two types of Oscillatoria-like morphotypes), ovoid (Synechococcus-like morphotypes) or spherical (Synechocystis-like or Gloeocapsa-like morphotypes). After 28 days the Casablanca microcosm showed diminished abundance of the filamentous forms and the disappearance of Gloeocapsa-like morphotypes, however there were no marked changes in Synechococcus- or Synechocystis-like morphotypes. Only the Gloeocapsa-like morphotypes were not present after 28 days exposure to Maya, but by the end of the experiment (50 days) the only filamentous cyanobacteria present was the M. chthonoplastes-like morphotypes and there was a noticeable decrease in the thickness of the photosynthetic layer of the mat. There were differences in counts of anaerobic heterotrophic bacteria between the polluted and unpolluted microcosms; significantly higher counts were found in Casablanca and significantly lower in Maya. While there were fluctuations, there were no significant differences in aerobic heterotrophic bacteria, oxygenic phototrophic biomass or total biomass. Lower molecular weight constituents of the oils were degraded to a higher extent by the microbial mats than higher weight.

These results show that microbial mat communities have a significant potential to degrade the components of oils, however different types of crude oils have distinct effects on the community structure. Gloeocapsa-like morphotypes were found to be the most sensitive to both types of oil, and M. chthonoplastes-like morphotypes show the highest tolerance for both, with a previous study showing that they are capable of surviving anaerobically on Maya as a sole carbon source. It would be interesting for further study to look at the effects of the individual components of crude oil on the microbial community however this shows great potential for microbial bioremediation of oil spills.

Llirós, M., Gaju, N., García de Oteyza, C., Grimalt, J., Esteve, I. & Martínez-Alonso, M. (2008) Microcosm experiments of oil degradation by microbial mats. II. The changes in microbial species. The Science of the Total Environment. 393, 39-49

The effects of diet on microbiota in cod larvae

Live feed is not a major determinant of the microbiota associated with cod larvae

J. Hinchcliffe

As described in the hologenome theory, all animals live in close symbioses with bacteria. The gastrointestinal (GI) microbiota in vertebrates is diverse and contributes to the host’s health and development in a number of various processes. For example, the colonization of the GI system of animals has been shown to be important for the development and function of the gut system and as defence mechanism against pathogenic bacteria.

For aquatic animals, there is usually no contact between parents and offspring, but eggs and newly hatched fish larvae are exposed to bacteria in the water. The shaping of the animal GI microbiota is a complex process, and various factors influence the composition of the GI microbiota. For fish, the water is a likely source of bacteria for the initial GI microbiota. In newly hatched larvae, before feeding, there is an active uptake of bacteria and microalgae, at a rate that exceeds the drinking rate by two orders of magnitude. A number of factors could potentially influence the establishment of the GI microbiota in fish larvae; these include, but are not limited to, water quality, temperature, salinity, diet, larval developmental stage and gut structure. It is generally accepted that diet has one of the largest impacts on the GI microbiota of individual fish larvae.

The objective of this study was to investigate the impact of diet on the microbiota associated with cod larvae. Larvae were fed three different live feed diets (Copepods and two differently cultivated Rotifer cultures), and reared in three replicate tanks for each feeding regime. So except for diet, the rearing conditions were identical among tanks. The microbial communities were assessed for individual larvae, live feed and water using a PCR / DGGE analysis. Analysis of the DGGE profiles showed that different diets did not cause major changes to the composition of the microbiota associated with cod larvae. However, rearing of larvae with identical diet, but in separate tanks, often resulted in significant differences in larval microbiota. This led Bakke et al (2013) to speculate that diet does not entail major changes to the composition of cod larval microbiota, but the meduim does.

Bakke et al acknowledge that developmental changes in the digestive system could influence the GI microbiota, for example a more developed gut could provide more niches for the gut bacteria. Therefore changes observed in the microbiota of developing fish larvae may be explained by factors other than diet.

There is a possibility that diets more distinct in nutritional value could cause larger effects on the larval microbiota. Bakke et al state that cod larva require live zooplankton as feed, and the potential to manipulate the nutrient composition of live feed is limited. The natural feed for cod larvae is copepods, which are considered to be superior in nutritional value to the Rotifer used in this experiment.  But in cultivation of marine fish larvae rotifers are often used because they are simpler to cultivate. Bakke state that Rotifers and Copepods represent the extremes in feed that results in normal development of cod larvae, so this gives a good comparison.  Bakke et al also tested the microbiota associated with the different live feed cultures used in the present study and found that they were very different from each other. This is striking because the live feeds used in this study were put on different diets also (Rotifers cultivated in a standard yeast/lipid diet and the algae (Rhodomonas baltica) which also were used for cultivation of copepods). So the results presented in this study do not support the hypothesis of live feed culture as a major cause of change in microbiota of cos larvae. But the microbiota in the live feed cultures were different, to me there could be so much more done with this. Was this difference in the micro biomes of the live feed affecting the cod larvae in any way? This could serve as an opportunity for opportunistic detrimental bacteria, but this is only speculation.