Probiotics for sea cucumbers? More detail on sea cucumber gut bacteria reveals probiotic potential.

Holothurians or sea cucumbers are epi-benthic deposit feeding animals that use their tentacles to pass surface sediments containing organic matter into their mouths. Much of the organic component within sediments, however, is diluted by indigestible matter. The bacteria found within the sediments are an important constituent of the diet of sea cucumbers and are thought to provide a food source as well as a nutritional benefit to the host via provision of nutrients. The bacteria may also provide an additional benefit by degrading and breaking down indigestible components with extracellular enzymes. One particular holothurian species, Apostichopus japonicus inhabits the north-western Pacific Ocean and is an economically important species for aquaculture in China.

Previously, other techniques such as culture-dependent methods and Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) have been used to survey the composition of microorganisms within the digestive tracts of holothurians. This study by Gao et al., (2014) compared the bacterial community composition in the gut contents of sea cucumbers Apostichopus japonicus with that of the surrounding habitat sediment using 16S rRNA gene 454-pyrosequencing. The advantages of using pyrosequencing over more traditional methods is that is reveals a far greater proportion of the total community present. DGGE, for example may typically reveal less than 1% of the total taxa present and culturing methods even less. Pyrosequencing can produce many short sequences from very variable regions in rRNA genes, hence this revolutionary technique can vastly improve sampling effort. This is crucial for revealing more scarce taxa as well as breaking down communities to the lower taxonomic levels, such as genus level.

Sea cucumbers of similar weight were sampled from an area near Qingdao, Eastern China and sediment samples were taken from the same location as the sea-cucumbers. The sea cucumbers were stored on ice within an hour of being collected before being sterilized with 70% ethanol. The hindgut and foregut contents were sampled separately to avoid cross-contamination. DNA was extracted and amplified by PCR, from which amplicon libraries were generated and pyrosequencing conducted.

The results showed that the bacterial diversity of the gut contents of the sea cucumbers was lower that of the habitat sediments. Despite this, the foregut and hindgut contents harboured characteristic bacterial communities that differed from communities in the habitat sediments. Within these communities, the abundances of different groups differed in both the foregut and hindgut contents compared to the sediment. The authors suggest that selective feeding by A. japonicus could be responsible for this, although disappointingly, don’t go into further detail. The abundance of Proteobacteria was lower in the gut contents but Acidobacteria, Actinobacteria, Planctomycetes and Chloroflexi were significantly higher in the foregut contents than the habitat sediments. Furthermore, there were differences between the fore- and hindgut, with Acidobacteria, Actinobacteria and Chloroflexi being much less abundant in the hindgut than the foregut. Interestingly, Sulfate Reducing Bacteria (SRB) in the hindgut and Bacillus sp. were present in all samples. SRB are believed to provide nutrition for the host by nitrogen fixation and acetate production. SRB are also found in marine sediments, particularly anoxic ones. In addition, many of the genera analysed, including Bacillus, Lactobacillus, Lactococcus, Streptococcus, Micrococcus, Alteromonas, Pseudomonas and Vibrio are used as probiotics in aquaculture. These species offer potential as possible biological control agents within the aquaculture industry for an economically important species.

Holothurians are important deposit feeders and their feeding activity likely influences the microbial communities on and within marine sediments. This study was able to identify a greater diversity of different bacteria within the communities associated with the sea cucumber A. japonicus through pyrosequencing. These techniques have revolutionized microbial ecology in terms of exploring bacterial diversity and help enrich the results found by more traditional methods used in previous studies.

Reference: Gao, F., Li, H., Tan, J., Yan, J. and Sun, H. (2014) Bacterial community composition in the gut content and ambient sediment of sea cucumber Apostichopus japonicus revealed by 16S rRNA gene pyrosequencing, PLOS One, 9, (6), 1-10.

http://www.ncbi.nlm.nih.gov/pubmed/24967593

ARBS - A Polymicrobial Puzzle?

Both corals and Sponges possess vibrant microbial communities and so are both of interest to microbiologists. Unfortunately sponges also share another similarity with corals, reports of diseases in sponges are increasing. One particular disease is Aplysina red band syndrome or ARBS, which affects Aplysina cauliformis in the Caribbean. The disease is not lethal but does cause necrosis, lesions teaming with cyanobacteria, reduced growth and physiological and biochemical changes to the sponge.  But very little is known about what causes this affliction. or how the sponge’s microbiota responds. Olson et al. (2014) collected branches of visibly healthy and ARBS affected sponges from two sites using SCUBA, in Belize and the Bahamas. Samples were frozen, DNA extracted and clone libraries created by amplification of bacterial 16S rRNA. They also carried out a technique called terminal restriction fragment length polymorphism (TFLP), which allowed them to roughly enumerate the abundance of certain sequences and so bacteria (or groups of). They also cultured the cyanobacterium Leptolyngbya, which they thought may be a cause of the disease due to its presence in lesions and its bright red colour, which they used to infect healthy sponges in the field.

Generally they discovered that although there was a fairly large dissimilarity between the healthy and ARBS-affected sponge microbiota, a core community is maintained across both health states dominated by Chloroflexi and Proteobacteri, with high variation between location and collection year in community. Overall eight bacterial phyla were found, with their relative proportion varying on disease status. In terms of bacteria that were found to increase a sequence, Leptolyngbya spp. (and Phormidium) was found to increase along with two sequences of Gammaproteobacteria. Overall the photosynthetic symbiont Symbiodinium spongiarum declined, along with four (possibly) sponge related Gammaproteobacteria and a decrease in the group Chloroflexi. However in results of the transmission experiment showed that Leptolyngbya cannot cause the disease in healthy coral on its own. So what does? Unfortunately the study could shed little light on this, but it does suggest that the pathogen could be opportunistic, viral or eukaryote (not detected by 16S rRNA), or the disease may have an archaeal component which may not have been assayed effectively if the primers used did not target this group. Another possibility is that the infection has a polymicrobial basis and given the similarities to coral black-band disease (another polymicrobial infection), this strikes me as plausible. For example, cyanobacteria of the Leptolyngbya genus are present in black band that is also characterised by large communities of cyanobacteria at infection sites. In fact a red band disease does occur in corals in a similar way to black band (Munn 2011). So it might be fruitful to take some of the lessons learned from black band disease research and apply them to this problem. Either way, sponge diseases need to be studied to a greater degree than they currently are. We require much more intensive study on the ailments of this phyla especially in light of the decline of corals and the increasing role which sponges are likely to play due to this in future tropical ecosystems.

Munn, C.B. (2011). Marine Microbiology: Ecology and Applications, 2^nd ed. New York: Garland Science.

Olson, J.B., Thacker, R.W. and Gochfeld D.J. (2014). Molecular community profiling reveals impacts of time, space, and disease status on the bacterial community associated with the Caribbean sponge Aplysina cauliformis. FEMS Microbial Ecology, 87, 268-279.

Probiotics for larvae; Bacillus as a beneficial gut-bug

Rearing organisms in the laboratory can be a delicate process. Some are hardier than others but there are ways of boosting survivability rates. The larval period is often the most vulnerable of the life cycle and poor environmental conditions or food can negatively affect body size and development. Marine organisms are especially at risk due to their osmoregulatory function. The constant ingestion of the water around them means they don’t have much choice in what microflora they’re taking in. In fact, the bacteria they first ingest is what primarily colonises their once-sterile digestive tract. And they better be happy about what they’ve got because, even after metamorphosis occurs, those are the ones that stick around. Therefore, having a healthy microflora gives the developing larvae a higher chance of survival compared with those that don’t.

This is where probiotics come into play, Bacillus sp. being of particular interest to Hauville et al. (2015) due to its spore-forming ability. Spores are sensitive to environment changes despite their inactive metabolism and are resistant to physical/chemical stress, are heat stable and can survive in low pH. This coupled with their low productive cost means that it’s very cheap and easy to produce lots in labs with minimal effort. Previous studies have found that introduction of Bacillus can cause an increase in gene expression associated with growth metabolism and animal welfare, whilst others show the potential for increased resistance to vibrios. In this one, Hauville et al. (2015) test the effects of a commercial mix of Bacillus on growth and digestive enzyme in Florida pompano, common snook and red drum larvae.

In the control, all three organisms were fed rotifers enriched with Algamac 3050. In treatment 1 (PB) the rotifers were enriched with Algamac 3050 and also a commercial mix of 0.5g of Bacillus spp. per litre. Treatment 2 (PB+) consisted of the same as treatment 1 but with an additional 5g m^-3 of probiotics.

The results showed no significant difference in survival between treatments and species, but survival was significantly higher in pompano and red drum compared with snook larvae. PB and PB+ pompano larvae had significantly greater standard length and body depth than the pompano control and the same occurred for snook larvae although there was no significant difference in body length. Red drum showed no differenced observed between the treatments. Although for some reason, the authors don’t go on to describe what difference occurs in common snook which is odd. For pompano, it was found that the counts of colony forming units (CFU) per larvae were higher on marine agar media for PB and PB+ when compared with the control. The lack of description occurs again for snook and red drum where no comparisons are made and in table 2 it shows only pompano being tested for CFU. It seems incomplete to only include one species. The number of vibrios on the TCBS media were low and there was no significant difference between treatments. In terms of enzyme activity, pompano and snook showed trypsin specific activities which were significantly higher in PB and PB+ in comparison to the control. Amylase activity was different only in snook, with PB+ higher than in the control. Alkaline phosphatase (AP) of pompano (PB and PB+) and snook (PB+) was higher than their controls.

The results show that Bacillus as a probiotic has beneficial effects on growth and digestive enzyme activity. The Bacillus mix given for this study involved three different strains; B. pumilus, B. lichenformis and B. amyloliquefaciens and are all known to be closely related to B. subtilis. These strains have different metabolic abilities. B. pumilus produces amylase and cellulose whilst also having strong inhibition factors against Vibrio sp. and B. lichenformis has antiviral properties and B. amyloliquefaciens is closely related to B. subtilis. Hauville et al. (2015) discuss previous studies where B. subtilis, B. lichenformis or B. pumilus were added to the diet of olive flounder. B. subtilis enhanced growth, B. subtilis and B. pumilus increased survival rate and B. pumilus and B. lichenformis increase superoxide dismutase activity and disease resistance. This showed how beneficial it was to supplement the diets with multiple strains of the bacteria simultaneously.

This paper is looking at the benefit of using Bacillus sp. as a probiotic in three different organisms, but in many of their tables and figures they only include comparisons with either one, or two and use a different table or figure for another. This seems a very convoluted way of presenting the information rather than just using separate comparison tables for e.g. standard body length and specific activities separately. However, it could be due to the fact that there were no statistical differences observed in red drum larvae as opposed to snook and pompano. 

Ref; Hauville, M. R., Zambonino-Infante, J. L., Gordon Bell, J., Migaud, H. & Main, K. L. (2015) Effects of a mix of Bacillus sp. as a potential probiotic for Florida pompano, common snook and red drum larvae performances and digestive enzyme activities. Aquaculture nutrition. 

doi: 10.1111/anu.12226

A Bacterium for all Seasons

Bacterial presence within living organisms has been investigated on a microbiological level and has been deemed as a crucial component of a host’s health and success. The presence of varying bacterial populations within coral is required to fulfil different symbiotic roles. The bacterial community can be found within the tissue, mucus and skeleton of coral, pointing towards the need for bacterial functions in different formats within the organism’s structure. The fluctuation of bacterial populations, occurring in conjunction with seasonal environmental changes, have previously been investigated, however, no significant patterns have been identified.   

Within the study by Li et al, 2014 the aspect of bacterial community temporal fluctuations in the coral Porites lutea are addressed. Through sample gathering of P.lutea over a period of one year two questions were addressed; whether bacterial communities changed in composition in P.lutea with changes in season and whether seasonal change in bacteria composition occurred within the three compartments: tissue, skeleton and mucus. The samples of P.lutea were gathered at a depth of 3-5 metres, during February, May, August and November of 2012, offering a balanced span of the seasons. At the same time of coral sample collection samples of surrounding seawater were also made for analysis of bacterial assemblage.

Identification of bacterial assemblage was conducted through sequencing of the V1 and V2 regions of the 16s rRNA gene. The highest count of bacterial species identified through sequencing was found in the coral skeleton during February, with the lowest count being found within the coral mucus during May. Through investigation of the coral structure dominant species were found within certain temporal samples and diversity varied throughout the seasons. The different niches provided by P.lutea also demonstrated variations, with the bacterial assemblage of the mucus, skeleton and tissue showing greater species diversity within the winter months compared to summer months.  The dominant bacterial groups also varied when examined at a lower taxonomic level, with similarities between each compartment being calculated at just 24%-46%. In correlation with the coral 16s rRNA sequencing results and using Shannon Weiner Index, the seawater diversity of bacterial communities was highest during the month of February. However, in contrast to the coral, surrounding seawater bacterial communities were considerably stable throughout the different seasons, with Flavobacteria, Alphaproteobacteria and Cyanobacteria being the dominant classes.

These findings support the theory of P.lutea experiencing bacterial community fluctuations due to seasonal environmental factors requiring different symbiotic relationships. Further investigation of environmental parameters and the corresponding response of bacterial community change were also investigated. Two main factors which displayed a direct effect on bacterial community assemblage were dissolved oxygen (DO) and rain fall. The change to an increase of Bacilli during August corresponds with the increase of rainfall within the summer months, and leads to the conclusion that the Bacilli could be introduced to the coral through terrestrial fresh water runoff.

There were some seasonally and compartmentally unrelated changes in bacterial community assemblage which were observed during the study, these may point to numerous other influencing factors upon the coral holobiont and its bacterial assemblage. The impact of microbial communities from other surrounding organisms and seawater may additionally affect the bacterial assemblage within the coral, along with spatial differences on a longer term basis. Further study could be conducted within these areas and possibly lead to a simplistic multidimensional model being created, allowing an insight into the bacterial community assemblage of varying coral holobiont communities which are yet to be investigated.  

Li, J., Chen, Q., Long, L., Dong, J., Yang, J. and Zhang, S. (2014) Bacterial dynamics within the mucus, tissue and skeleton of the coral Porites lutea during different seasons. Scientific Reports. 4. 7320.

http://www.nature.com/srep/2014/141205/srep07320/full/srep07320.html

So whats still bleaching Oculina patagonica if Vibrio shiloi's not?

Note: this blog post is a tad longer than normal, but all information I felt was important to mention.

Even though the coral Oculina patagonica has developed resistance to infection by the coral pathogen Vibrio shiloi, it continues to demonstrate seasonal bleaching. These findings raise 2 important questions: (1) What is the current mechanism of bleaching of O. patagonica every summer in the eastern Mediterranean Sea? (2) How did the coral become resistant to V. shiloi infection and bleaching? Mills et al., 2013 provide evidence to explain the role of bacteria in the bleaching process of O. patagonica. It must be remembered that V. shiloi still adhere to the coral and penetrate the tissue, but it is then rapidly killed showing the corals defence mechanisms; which is surprising, because corals do not produce antibodies and are considered to lack an adaptive immune system.

A bleaching survey was first carried out in at Sdot Yam, Mediterranean off the coast of Israel which showed a clear correlation with bleaching profiles previously documented when V. shiloi was the infectant. Bleached colonies were often found next to or surrounded by apparently healthy colonies and bleached corals were on average larger than unbleached corals. This could lead to assumptions and further research that infections could be linked to coral age and it may not be passed on very easily.

Healthy fragments of O. patagonica in 11 aquaria were exposed to Nalidixic acid (Appendix I) for 24 hours, which was then removed and then the temperature was increased gradually (1°C) until it reached 31°C.  Infections with V. shiloi and strain EM1 were performed after the antibiotic had been removed (these strains were isolated from crushed fragments of O. patagonica) and temperatures had reached 29°C.  Also 8 aquaria were just exposed to the gradual increase of temperature. Both types of aquaria were left for 2-3 weeks. Zooxanthellae were also counted from tissue samples.

To test the hypotheses that corals now contained a bleaching bacterium other than V. shiloi and acquired one or more strains of bacteria that inhibit the growth of V. shiloi, several bacterial strains were isolated from crushed O. patagonica and checked their ability to bleach and/or their anti-V. shiloi activity. Out of 5 isolates tested, only 1 strain, EM1, bleached antibiotic-treated corals and reduced the zooxanthellae count by 74% (Table 1).  Strain EM1 were plated on TCBS Agar and based on its 16S rRNA gene sequence EM1 is a strain of Vibrio coralliilyticus (showing 99.8% identity to V. coralliilyticus). These data are suggestive but insufficient to demonstrate that V. coralliilyticus is the causative agent of the seasonal bleaching disease of O. patagonica. It was also clearly shown in (Table 1) that the beneficial bacteria that had caused the corals to become resistant to V. shiloi infection and bleaching were killed by nalidixic acid. Zooxanthellae counts were much lower in the EM1 strain treatment (Table 1) possibly indicating that the it is a more damaging bacteria than V. shiloi.

Table 1 presents the results. As you can see there is something else bleaching Oculina patagonica other than V. shiloi which could be the EM1 strain. 

One other strain, referred to as EM3, consistently inhibited the growth of V. shiloi after inhibition was observed on streak-plates of DNA this indicates the diffusion of an antibacterial compound. The inhibition by EM3 appeared to be specific to V. shiloi, but the chemical structure and mode of action of this material is not yet known. Further investigation into this strain would determine whether this was the antibiotic that saved the corals from V. shiloi bleaching.

This study perfectly links to the previously disputed coral probiotic hypothesis that stems from the concept of the coral holobiont. This is when the coral functions as the sum of the coral host and all of its symbiotic microorganisms, which means here that the coral acquired beneficial bacteria that inhibit infection and prevent bleaching.

It must be kept in mind that other mechanisms could be involved, such as temperature-induced virulence of certain viruses. Clearly, further multidisciplinary research, including a combination of coral microbiology together with coral host physiology is required to clarify the coral bleaching disease process. It will be interesting to determine whether antibiotics inhibit the bleaching of other corals too. But overall, the possibility that V. coralliilyticus could be involved in the bleaching is interesting as it is previously been know to be involved in necrosis, which shows the adaptive abilities of bacteria.

Reference: Mills, E., Shechtman, K., Loya, Y., & Rosenberg, E. (2013). Bacteria appear to play important roles both causing and preventing the bleaching of the coral Oculina patagonica. MEPS, 489, 155-162.

Can be found at: http://www.int-res.com/abstracts/meps/v489/p155-162/

Appendix I: Nalidixic acid was chosen as the antibiotic of choice after preliminary experiments indicated that it was efficient at killing coral-associated bacteria and had no apparent deleterious effect on the corals.

Hey Colin! Here's some more Vibrio for you. The use of the Type Six Secretion System to stab neighbouring bacteria and take their DNA

Horizontal gene transfer (HGT) occurs in bacteria and archaea through many different processes. Natural competence for transformation is a common mode of HGT and allows bacteria to take up free DNA from the environment. Vibrio cholerae is a mainly aquatic organism mostly found in marine habitats associating with plankton blooms as well as shellfish and is considered by the WHO to be the most important Vibrio sp. to humans. When growing on chitinous surfaces, V. cholerae can initiate natural competence through the production a regulatory protein, TfoX, due to the presence of chitin and its degradation products. The type VI secretion system (T6SS) is the most recently discovered mechanism for effector secretion that is now understood in gram-negative bacteria. Its structure and function resembles intracellular and membrane-attached phage tails and can pierce neighbouring bacterial and eukaryotic cells. A study by Borgeaud et. al. (2015) demonstrated the use of the T6SS by V. cholerae as part of the TfoX competence regulon and its expression when on chitinous surfaces.

V. cholerae was grown in the absence and presence of chitin-induced expression of TfoX. RNA-sequencing was then used to understand the extent of the competence regulon and enable an accurate assessment of the bacterial transcriptome. Two strains of V. cholerae were also used to simulate a predator-prey relationship to investigate whether competence induced T6SS-mediated killing affects transformation. Live-cell fluorescence microscopy imaging was used to visualise prey lysis and the transfer of genetic material.

Three T6SS encoding gene clusters were observed to be up-regulated upon the induction of TfoX, the major gene cluster and two auxiliary clusters. They concluded that the RNA-seq data suggests TfoX initiates the transcription of these clusters in the presence of a chitinous substrate and the T6SS genes were elevated after growth of V. cholerae on chitin flakes. The functionality of the T6SS was then assessed in an interspecies killing assay. V. cholerae strains with an inducible copy of TfoX were shown to have a significant killing behaviour toward Escherichia coli due to the activation of T6SS. They also found upon TfoX induction, in predator-prey relationship tests, natural transformants were readily obtained in predator cells and never or rarely found in T6SS-defective strains. In conclusion, upon competence induction V. cholerae induced the T6SS, this lead to the killing and lysis of non-immune neighbouring bacteria. This caused the release of DNA which transforms competent predatory cells. This technique resembles bacterial fractricide as described in Streptococcus pneumonia but, in contrast, V. cholerae seems to target strains with no compatible effector-immunity and is dependent on contact. Live-cell fluorescence microscopy showed high T6SS activity in predator cells followed by cell rounding and lysis of the prey. The close proximity observed of competent bacteria to lysed cells with the distinctive formation of certain proteins is indicative of DNA translocation into the predator cell. These gene transfer events were only observed in T6SS-positive strains.

These findings show the importance of the T6SS as a method of HGT in V. cholerae. Similar to S. pneumoniae and other bacteria, this uptake of DNA from the environment could lead to the emergence of multidrug resistant strains. This could have particular significance with antibiotics entering the water column, environmental V. cholerae could become resistant to these leading to cholera outbreaks which could be harder to fight and control. However, this papers findings could be used to develop new ways to fight V. cholerae; if a method could be found to interfere with the HGT pathways, similar to the effects of CRISPR on Staphylococcus epidermidis, then this may significantly reduce its evolution and thus the spread of antibiotic resistance. Borgeaud et. al also suggests that chitin-induced expression may also enhance the virulence potential of this pathogen in the human gut. Therefore, any method found to reduce the HGT of V. cholerae may have an added bonus of also reducing the severity or its ability to cause disease.

Borgeaud, S., Metzger, L. C., Scrignari, T., & Blokesch, M. (2015). The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer. Science, 347(6217), 63-67.

http://www.sciencemag.org/content/347/6217/63.short

Special thanks to Prof. Blokesch for access to this report