Non-O1/O139, no problem? Fish as reservoirs and vectors of Vibrio cholerae

Vibrio cholerae exhibits over 200 serotypes, with two; O1 and O139 responsible for seven pandemics since 1817. Two critical virulence factors common to both ‘O1’ serotypes are genes coding for the production of the cholera cytotoxin (ctx) leading to secretory diarrhea and the toxin coregulated pilus (tcp) that facilitates colonisation of the small intestine. However, ‘non-O1/O139’ V. cholerae serotypes lacking tcp and ctx encoding genes can still cause gastrointestinal disease through possession of other virulence factors such as heat stable enterotoxin (stn), type III secretion systems (TTSS), and haemolysin toxin (hlyA). Identification of environmental V. cholerae populations is important to ascertain the risk posed to humans that may come into contact with the bacterium.

V. cholerae is a ubiquitous aquatic vibrio and has been found in association with copepods and chironomids acting as environmental reservoirs and vectors facilitating transmission to humans. Outbreaks of cholera have been associated with the consumption of raw, dried and salted fish (Forssman et al, 2007), however, precious little research has considered the importance of fish as an environmental reservoir and vector of V. cholerae. Senderovich et al (2010) surveyed and characterised V. cholerae found within the gastrointestinal tract of various freshwater and marine fish species in Israel.

Fish were obtained from fishermen selling ‘fresh fish’ for human consumption from lakes, fish ponds, rivers, and the Mediterranean sea within Israel (n=110). Middle or lower intestinal contents were commonly directly streaked out onto TCBS agar or rarely enriched in broth culture prior to streaking. V. cholerae suspect colonies were subcultured onto LB agar and subject to oxidase and string tests. The identity of suspect isolates was confirmed by multiplex PCR assay of a V. cholerae specific outer membrane protein (ompW) and the cholera cytotoxin (ctxA). Determination of O1/O139 serotype was achieved by slide agglutination with specific antisera. The presence of additional toxin genes was assayed for all strains. Chitinase activity was determined through a chitin degradation assay.

The majority of the fish species isolated from freshwater habitats (10 out of 14 species; 71 %, n=26) were positive for ‘non-O1/O139’ V. cholerae, compared to only 1 out of 44 species (2.3 %, n=1) of marine species sampled. 50 strains of V. cholerae were isolated from the gastrointestinal tract of the fish species in this study. None of the isolates possessed the genes to produce cholera cytotoxin (ctxA), toxin coregulated pilus (tcp), or non-O1 heat stable enterotoxin (stn/sto). However, all strains possessed the gene toxR, a regulon of the cholera cytotoxin and toxin coregulated pilus, and hapA, responsible for soluble haemagglutinin/protease production. 32 % of the strains were positive for the type III secretion system (TTSS) and almost all of these strains possessed the gene hylA encoding for haemolysin toxin. There was no correlation between fish species and the genotype of the isolated V. cholerae strain. Enumeration of V. cholerae was achieved for two freshwater fish species Sarotherodon galilaeus (St Peter’s fish), and Mugil cephalus (flathead grey mullet) by calculating the colony forming units per gram of intestinal content when cultured on TCBS agar overnight at 37 °C. St Peter’s fish intestine contained 4.8 x 10³ cfu V. cholerae per gram of intestinal contents, while the flathead grey mullet contained 1.4 x 10² cfu.

All V. cholerae isolates possessed the ability to degrade chitin, suggesting a possible benefit to the host through having a ‘commensal’ population of V. cholerae in its gastrointestinal tract, particularly for species consuming a diet high in chitin such as copepod/insect prey. It would be interesting to see if a high chitin diet correlated with an increase in V. cholerae in the G.I. tract.  

This study is the first of its kind to identify fish as reservoirs of V. cholerae. Indeed, the high levels –ca 5 x 10³ cfu- of V. cholerae isolated from St. Peter’s fish is indicative of its prevalence in the aquatic environment. Although all of the strains (n=50) isolated in this study were ‘non-O1/O139’, they may still cause gastrointestinal disease in humans through production of virulence factors. Furthermore, ‘non O1/O139’ serotypes inhabit the same environmental niche as their more pathogenic brothers, and may acquire ctx and tcp genes via horizontal gene transfer. There is therefore a need to be cautious when consuming fish from species known to harbour V. cholerae, to avoid eating raw, dried, or salted fish and ensure fish is thoroughly cooked before consumption.

The propensity for dissemination of V. cholerae by migratory fish species is a novel concept that may become increasingly topical as climate change leads to increased incidence of vibrio diseases such as cholera in subtropical and temperate regions e.g. increased incidence of cholera in the Baltic Sea. 

Main Reference:
Senderovich, Y., Izhaki, I., & Halpern, M. (2010). Fish as reservoirs and vectors of Vibrio cholerae. PLoS One, 5(1), e8607.

Additional Reference:
Forssman B, Mannes T, Musto J, Baumann B, Frei U, et al. (2007) Vibrio cholerae O1 El Tor cluster in Sydney linked to imported whitebait. MJA 187: 345–347.

Euprymna scolopes: an additional association

The Euprymna scolopes is known for its fascinating association with Vibrio fischerii, which provides the squid with counter illumination whilst hunting at night. What is less known is that another bacterial association is found in this squid. Bacteria in genus Roseobacter appear to be associated with the accessory nidamental glad (ANG) in female E. scolopes. The ANG is an integral part of the female reproductive system, and Rosebacter are thought to be transmitted vertically; integrated in the egg of the baby squid. But what it does there is largely unknown. An analysis of the genomes of Rosobacter sp. isolated from the ANG suggest that these bacteria may communicate with their host through quorum sensing, as well as provides evidence of a specialised type 6 secretory system and genes for siderophores synthesis.

The genome sequences of 59 Rosebacter isolated from five mature females of E. scolopes were large, reflecting the metabolic diversity of these bacteria. Little genome reduction has occurred in these bacteria, even though they are tightly associated with a host. Contrasting to previous results, all of the isolates had genes for nitrate reductase and denitrifying enzymes, showing they can survive anaerobically.

The Type 4 Secretion System often found in Roseobacter was not found in these bacteria, but instead all isolates contained genes for the Type 6 Secretion System (see my last blog). The author hypothesised that this may be used for protection of the egg masses, as the T6SS is a particularly nasty cellular weaponry. However, they also mention is could be used as a form of communication between the host and bacteria. As the bacteria are located in the ANG epithelium and hemocytes, the T6SS could be used in the transfer of effector molecules to the host or symbiont. Subsequently altering biological activity, such as gene expression, enzyme activity or cell signalling.

Two quorum sensing LuxI homologs, similar to the ssaIR and ssbIR in Ruegeria sp, were detected in the bacteria. Only two isolates had both pairs, the remaining contained only the ssbIR. In Ruegeria sp. the ssaIR and ssbIR are part of an interconnected quorum-sensing network, which regulates biofilm formation, whilst simultaneously controlling swimming motility. The function the ssbIR alone is therefore unknown, yet due to the frequency of its occurrence, the author hypothesises it must have an integral function. One hypothesis was based on the raiIR in Rhizobium etli, which controls growth and nitrogen fixation. The author suggests that the luxIR could be used to regulate the growth of other the bacteria on the ANG.

Siderophores genes were found in all the bacteria, which is not common for Roseobacter. When the bacteria were grown in iron-limited conditions, an increase in siderophore synthesis occurred. Iron is often limiting in the marine environment, therefore having these siderophore may function as a selective advantage for these bacteria in competition with other bacteria without siderophores.

This paper provides the first insight into the association between E. scolopes and Rosebacter, presenting some interesting specialisations in these Rosebacter. The function of these bacteria on the ANG are still unknown, but are thought to contribute to the protection and development of the young cephalopod.  In my opinion this paper could have had more detailed explanations of the quorum-sensing systems it was referring to as I found it a bit unclear, however the majority of the paper was very interesting.

Reference:

Collins, A. J., Fullmer, M. S., Gogarten, J. P., & Nyholm, S. V. (2015). Comparative genomics of Roseobacter clade bacteria isolated from the accessory nidamental gland of Euprymna scolopes. Frontiers in microbiology, 6.

BREAKING NEWS: Seagrasses a vector of coral disease under OA?

Apart from corals, seagrass meadows are a crucial component of reef ecosystems (fish nurseries, nutrient cyclers, organic carbon producers and sediment stabilisers). Similar to corals, seagrasses are colonised by microorganisms that form epiphytic biofilms on the leaves.  Therefore a seagrass plant and its epiphytic biofilm can be referred to as a seagrass holobiont. Ocean acidification (OA) on corals can create changes in the composition of the microbial biofilm associated with the coral reducing larval settlement and probably coral health. Seagrasses, on the other hand, are generally thought to benefit from OA due to the increased availability of CO2 and bicarbonate for photosynthesis. However, data on how the epiphytic biofilm on seagrass leaves might respond to OA and on the behaviour of the seagrass holobiont in future OA scenarios are still sparse.

Here Hassenrück et al, (2015) studied the epiphytic biofilm on the leaves of the seagrass Enhalus acroides at a natural CO₂ vent (pH values of 7.8) and a control site (pH values of 8.3) in Papua New Guinea. 18S ribosomal DNA sequences were used to make sure that all the seagrass shoots collected were of the same species. The leaf age was taken into consideration; carbon content of the seagrass leaves decreased with leaf age from approximately 33% to 26%. Epiphyte cover increased with leaf age. 

High taxonomic resolution provided by 16S and 18S amplicon sequencing showed that epiphyte communities seemed to be as diverse at the vent site than at the control site. Automated Ribosomal Intergenic Spacer Analysis (ARISA) identified bacterial and eukaryotic operational taxonomic units (OTU). Overall the results show that bacterial and eukaryotic epiphytes formed distinct communities at the CO₂-impacted site compared to the control site.This study went into a lot of depth on what type of bacteria was found and why, which was really interesting to read. For example OA seemed to decrease cyanobacterial abundance and diversity in microbial films. Also Reinekea, a genus of the Gammaproteobacteria, was mentioned to may play an important role in the degradation of organic matter after phytoplankton blooms. It had a reduced abundance at the vent site, which may have been caused by the decreased availability of degradable material presumably due to the lower percentage of epiphyte cover (which was also looked at). This study may of actually bought to light microbial organsisms, previously not considered in OA research.

Their biggest finding was that they detected an increased prevalence of microbial sequence types associated with coral diseases (Fusobacteria, Thalassomonas [white plague-like disease]) at the vent site under elevated pCO₂ conditions whereas eukaryotes such as certain coralline algae commonly related to healthy reefs were less diverse (which is no surprise!). This agrees with the hypothesis that coral reefs experiencing elevated pCO₂ levels will be more susceptible to diseases than reefs not yet exposed to OA. It further highlights the potential of seagrasses as vectors of coral pathogens and stresses the point that seagrasses should be viewed as a holobiont when making predictions about OA effects and ecological consequences in coral reefs.

 Reference: Hassenrück, C., Hofmann, L. C., Bischof, K., & Ramette, A. (2015). Seagrass biofilm communities at a naturally CO2‐rich vent. Environmental microbiology reports.

Can be found at: http://onlinelibrary.wiley.com/doi/10.1111/1758-2229.12282/abstract

Group Post; A plastic house for microbes?

It is well documented that plastic is the most common form of marine debris, and its presence in our waters continues to increase, thus the term ‘plastisphere’ has been introduced. The effects of plastics on animals are relatively well documented; the same however cannot be said for microbes. Plastics provide a substrate to which microbial communities can attach, that said plastics may elicit a negative impact on these communities, this has not been investigated. The aim of this study was to characterize the microbial communities (with particular focus on the bacteria) of two different plastics, and compare these to the open ocean.

Samples were collected from the surface waters of the open ocean. Plastic fragments were sorted in to two groups; those destined for SEM and those DNA analysis. In order to confirm the identity of the plastics, ramen spectroscopy was undertaken, these were identified as polypropylene (PP) and polyethylene (PE). Using amplicon pyrotag sequencing, the microbial communities were categorised through formation of operational taxonomic units (OTUs) and cluster analysis was employed.

Zettler et al. (2013), analysed 2 types of plastic; polypropylene and polyethylene. Both plastics showed signs of degradation in terms of cracks and pitting. These pits contained unidentified round cells of 2µm diameter with signs of division, suggesting active growth. They named the communities associated with the plastic the ‘plastisphere’. In this plastisphere they found rich eukaryotic and microbial community with evidence of phototrophy, symbiosis, heterotrophy and predation. Diatoms and filaments were the most common morphotype observed on the plastic. As expected there was a distinction between the communities associated with the plastic compared to that of the seawater. A high concentration of Vibrio species were found in the plastishpere communities, which whilst not identified to species level, could show possibility of animal or human pathogens. This could show potential for the transmission of disease via these plastishpere communities and the importance of finding out more about plastic associated communities.

From the analysis, the authors identified a huge range of diversity or OTUs from a single fragment of PE and PP. They observed two distinct differences between diversity patterns between the plastics fragments and surrounding seawater. Firstly, the average observed richness was much higher in surrounding water compared to plastics. Secondly, seawater have higher average richness and polyethylene with the lowest.  However, plastic substrates showed greater evenness than seawater.Nevertheless, there is much ambiguity in quantifying the relative richness between seawater and plastic substrates. First off, sample size difference might be one of the big considerations which would result in the skew of the data. Even with normalisation of the results with respect to sampling effort, it still raises the question if the way of quantification is appropriate. Richness correlates with substrate area. When comparing plastic substrate of different sizes, they observed bigger size plastic to have higher diversity. Evenness on the other hand, was consistently higher on plastics compared to seawater and the brown alga Sargassum.    

Some of the bacteria identified in the ‘plastosphere’ were thought to be capable of degrading hydrocarbons. The identified taxa were recognised as being associated with hydrocarbon degradation such in the environment, such as in the horizon oil spill. The author suggested that the bacteria had formed a network that together was degrading the plastic. They indicated that through either physical or metabolic processes, bacteria degradation could be a possible sink for plastic in the ocean.

Jack, Freya, Li and Kat

Zettler, E.R., Mincer, T.J. and Amaral-Zettler, L.A. (2013) Life in the ‘Plastisphere’: Microbial Communities on Plastic Marine Debris. Environmental Science and Technology. 47, pp7137-7146.

Shallow water vent bacteria cured my… herpes?

Introduction

Extreme environments can lead to some highly diverse marine microorganisms. With this diversity comes a whole range of biomolecules that may not be produced by their less extreme relatives, which can be of use, for example, in microbial biotechnology or pharmaceuticals. Gugliandolo, et al. (2013) used the example of the herpes simplex virus type 2 (HSV-2) which is attributed as one of the most common and continuously occurring viral infections in humans, particularly due to the persistent latency of the virus after the first infection. Unfortunately with recurrent use of drug treatments, viral resistance comes hand in hand. This requires new variations of HSV fighting treatments to battle the new resistant strains. Isolation of a novel strain of Bacillus licheniformis T14 can yield new exopolysaccharides (EPSs) with antiviral properties useful for the production of these alternate treatments. The benefits extend to the thermophilic nature of the vent-dwelling bacteria, where proliferation rates would be much greater than its mesophilic or psychrophilic counterparts.

Methods

Isolation of B. lichenformis strain T14 occurred from a thermal fluid sample obtained from a shallow submarine vent of Panarea Island in the Eolian Islands, Italy. EPS1 was obtained after 48h of incubation from culture medium and purified using gel chromatography before characterisation of physiochemical properties. Human peripheral blood mononuclear cells (PBMC) were collected from healthy blood donors, washed in RPMI 1640 medium and cultured at 37°C. The PBMC were treated with 200, 300 and 400 micrograms per millilitre (ug ml^-1) of EPS1 and incubated for 24h at 37°C. Cytoxicity tests were done on PBMC and WISH cells, and colorimetric assays were done to determine the effect of the concentrations on the viability of the cells. In another series of experiments, the PBMC and WISH cells were plated and treated with EPS1 at the concentrations of 200, 300 and 400 ug ml^-1 and incubated at 37°C for 24h. The plates were then frozen and thawed three times to cause the intracellular virus to release.

Results

PBMC show a dose-dependent cytoxicity relationship with EPS1. 48h post treatment indicates that cytoxicity was not found at concentrations of 400 ug ml^-1 or below, but above cytoxicity increased with higher concentrations of EPS1. EPS1 exhibited antiviral properties on HSV-2 (PMBC) at 300 and 400 ug ml^-1 but not with lower concentrations, whereas with the WISH cell line, no significant inhibition was found at any concentration. High levels of cytokines were detected in EPS1-treated PBMC supernatants but on samples then infected with HSV-2, there was a significant down regulation of cytokine production.

Discussion

The EPS that B. licheniformis T14 produces contains both fructose and fucose. Fucose containing polysaccharides are highly marketable due to their rarity and value in pharmaceuticals and cosmetics, and have attributes including anti-inflammatory, anticarcinogenic, and immunomodulatory properties. EPS1 provides a knowledge that damaging immunological disorders caused by HSV-2 could be reversed. The key of this biomolecule is the relative ease of cultivation, particularly with B. licheniformis T14’s affinity to thrive at variable temperatures, pH’s and salinities due to its high stress tolerance which is attributed to organisms that exist in such extreme environments. The potential of this research is huge, and this paper presents vital data for the deriving of novel biomolecules for use in medicine and biotechnology, especially with resistant pathogenic strains on the rise. The author’s conclusions send a positive message to those afflicted with reoccurring viral infections and also the potential of strains of microorganisms yet undiscovered in the oceans and the ability they may have to produce biomolecules that can be used to aid in medical and pharmaceutical applications.

Ref: Gugliandolo, C., Spanò, A., Lentini, V., Arena, A. & Maugeri, T. L. (2013) Antiviral and immunomodulatory effects of a novel bacterial exopolysaccharide of shallow marine vent origin. Journal of Applied Microbiology. 116(4), 1028-1034. doi:10.1111/jam.12422

Group post: Composition and function of bacterial communities

Heterotrophic microbial communities are responsible for remineralising and transforming a considerable fraction of organic matter in marine systems and help shape the nature and quantity of carbon and nutrients that pass from surface waters to the deep ocean. The first step in this transformation is the release of extracellular enzymes to hydrolyse high molecular weight (MW) compounds to smaller substrates. A focal point for investigation has been the differences in the nature and composition of aggregate-associated and free living microbial communities. Low MW substrates have previously been used in these investigations to act as proxies when assessing the capabilities of microbial communities. However, these proxies provide no information on many enzyme processes. A study by D’Ambrosio et al. (2014) instead used high MW substrates and, for the first time, measured potential enzyme activity directly from the same filters used for community analysis. The focal point of this study was the potential polysaccharide hydrolysing endo-acting enzymes as high MW carbohydrates constitute a large percentage of phytoplankton detritus as well as marine POM and DOM.

Carboys were used to hold water samples collected from sampling sites. Inshore samples were collected at 3-4m depth (surface) on the North shore of Bogue Sound in late October and offshore samples were collected in early December at depths of 2m (surface), 146m (midwater), and 505m (deep/bottom water). The differences in sampling times and depths may have caused discrepancies in the consistency of the microbial communities collected due to high turnover rates. Changes in surface depth samples may also alter community composition.

Samples collected both on and offshore were gravity filtered over 17 hours to separate free living and particle associated communities. Onshore samples were filtered immediately after collection whereas offshore samples were filtered after 10 days. This again may result in changes to composition and enzyme function due to a long storage time.

Hydrolysis measurements were used to assess enzyme activities and specific substrate hydrolysis. Six different fluorescently labelled polysaccharides were used, however, the paper does not describe where they were found or at what concentrations they are normally found in the environment. This would allow for more accurate comparisons for abundance or hydrolysis rates. Potential rates were compared at day 2 and day 7 for the offshore samples and day 8 for coastal.

The experimental design of this study was able to link the potential substrate hydrolysis rates of a community functions to community composition using clone libraries which provided a measure of the extent and active members of the community at initiation of the hydrolysis experiments.

After two days of incubation, summed hydrolysis rates in whole water were higher in coastal than in offshore waters. The same pattern of hydrolysis rates can also be seen after 7/8 days of incubation. At the coastal station all six substrates were hydrolysed in whole water after 2 days while at the offshore station only 3/4 were hydrolysed depending on depth. Even after extended incubation, the offshore communities hydrolysed a narrower spectrum of substrate. Similar to the patterns of enzyme activities, patterns of 16S rDNA and rRNA sequences showed spatial distinctions. rDNA and rRNA clone libraries from coastal surface waters were dominated by SAR11 and Roseobacter respectively but offshore surface  libraries were dominated by the groups Pseudoalteromonas (particle-associated DNA library) and Thalassospira (free-living RNA library). The mid-depth and bottom libraries from colder water offshore were dominated by Gammaproteobacteria.

Although the bacterial communities changed extensively with depth, the ability to hydrolyse a particular substrate changed more gradually. Bottom water communities only hydrolysed laminarin, xylan and chondroitin. Depth-related changes in microbial community composition were quite distinct with a strong partitioning between surface water and the deepwater column communities. The DNA and RNA libraries also indicate bacterial populations with attenuated activity and rRNA content or entirely inactive populations.

It was found that compositional differences between particle-associated and free-living communities did not change with depth. The two types exhibited similar summed hydrolysis rates but the time point for enzyme detection differed. Overall, the particle and free-living communities hydrolysed the same spectrum of substrates as the unfiltered fraction.

Functional redundancy occurs when groups of bacteria share the capacity to use a certain substrate, so not all of the bacteria have to produce the enzyme to hydrolyse it otherwise it could result in a waste of resources. Laminarin, for example, was hydrolysed rapidly by all the samples used in the study because it has a wide distribution so many microbial phyla have the ability to hydrolyse it. Also it was found that cultured marine bacteria in close relation to Gamma- and Alpha-proteobacteria in the clone libraries can grow on laminarin and are able to use it as a sole carbon source.

Although the authors of this paper assume that the long filtration time is unlikely to affect the results it is possible that it might cause changes to the community composition and enzyme activity. Different filtration methods should be compared to ensure that this does not affect community composition and function. The samples were also kept for 10 days after collection at 4^oC. This could also affect the community structure and composition as many papers have showed that within a few days of collection bacterial community structure is likely to change drastically. There is also very little information present as to why these six carbon sources were chosen and their relevance. While laminarin and fuciodan might be very wide spread compounds, the other compounds are likely to be very rare so few bacteria would be expected to hydrolyse them.

(Group: B. Oliver, B. Sockett, M. Nisulescu)

D'Ambrosio, L., Ziervogel, K., MacGregor, B., Teske, A., & Arnosti, C. (2014). Composition and enzymatic function of particle-associated and free-living bacteria: a coastal/offshore comparison. The ISME journal.

http://www.nature.com/ismej/journal/v8/n11/full/ismej201467a.html

Is Mucus Maximising Microlayer Microbes?

The Sea-surface Microlayer (SML) is a gelatinous biofilm-like habitat with a distinct microbial community teaming with bacteria, flagellates, algae and fungi. These communities are significantly 'enriched' compared to the water below due to rising TEPs (Transparent Exopolymer Particles) that supply nutrition and attached microbes to the microlayer habitat. Although TEP is largely formed from phytoplankton DOM, coral mucus may play a similar role. Corals produce vast amounts of mucus, some of which rises to the surface as transparent filaments and strings, due to trapped air bubbles. This could potentially supply both microorganisms and nutrients to the microlayer in waters above reefs. Therefore, it may be that the communities on the SML above reefs could be more enriched with bacteria than other ecosystems and enrichment may increase as coral coverage does.

To test this, Nakajima et al. sampled a fringing coral reef off Bidong Island, Malaysia for three consecutive days in June at three sites. Two shallow sites, one with high coral cover and the other with low coral cover (dominated by Acropora nobilis and A. formosa) and also an offshore site, where I assume coral was absent. The SML was sampled using a metal mesh sampler, which collected the upper 250µm of water and subsurface water samples were taken a depth of 10cm. Water samples were immediately brought to the lab on ice. DOC and Chlorophyll a concentrations were measured. Microbes were enumerated using epi-fluorescence after filtration onto membrane filter, 0.2µm for heterotrophic bacteria and 0.8µm for cyanobacteria, heterotrophic and autotrophic nanoflagellates. Bacterial (heterotrophic) production was the measurement of bromodeoxyuridine incorporation in the dark and bacterial growth rate was calculated. Enrichment factors were calculated for all parameters and multivariate statistics carried out. Unsurprisingly, the SML was significantly enriched over every parameter bar bacterial production and growth rate. More interestingly, enrichment factors for all the microbial groups maybe higher than in other marine habitats. These higher enrichment factors could have implications for the exchange of gases, if matched to an increased metabolism. However, it is difficult to confirm this due to varying methodologies used in different studies. Remarkably, the high coral cover site had the highest enrichment of heterotrophic bacteria. This may support the hypothesis that coral mucus transports trapped pelagic and coral-associated bacteria into the SML. Furthermore, transport into the SML may be more important than in-situ production given a low bacterial growth rate. Additionally, at the high coral sites there was a very low ratio of heterotrophic flagellates to bacteria/cyanobacteria possibly suggesting that there could be a higher level of grazing above the high coral site.

I found the study very interesting, but it can be summarised as being very 'old school', given that there was no focus on taxonomy. A full phylogenetic analysis using 16S/18S rRNA sequences would have allowed direct comparison between the SML communities above the coral with other marine systems and the coral microbes themselves. It would also have been much more informative to sample a number of sites with differing coral coverage to give a clearer picture of whether the enrichment really increased with increasing coverage. Although I am cautious about some of the conclusions made, I think that the study does raise interesting questions and provides a good starting point for future studies where more powerful techniques could be used.  

Ref: Nakajima, R., Tsuchiya, K., Nakatomi, N., Yoshida, T., Tada, Y. Konno, F., Toda, T., Kuwahara, V.S., Hamasaki, K., Othman, B.H.R., Segaran, T.C., Effendy, A.W.M. (2013). Enrichment of microbial abundance in the sea-surface microlayer over a coral reef: implications for biogeochemical cycles in reef ecosystems. Marine Ecology Progress Series, 490, 11-22.