Bacteria not the primary cause of coral bleaching

Stress factors that cause the breakdown of symbiosis between corals and their endosymbionts is what causes coral bleaching to occur. Rises in sea temperature have been correlated to mass coral bleaching events, however it has been suggested that bacterial infection of corals may also cause bleaching. Ainsworth et al. (2008) investigated the microbial ecology of the coral Oculina patagonica during an annual bleaching event to map bacterial populations within the tissue levels and determine whether they were related to the bleaching process.

The study was conducted at Sdot Yam on the Mediterranean coast of Israel between June and August 2005 during the annual bleaching event. Samples from 140 Oculina patagonica corals were taken every two weeks, with three replicate core samples from each tissue region (bleached and unbleached) from each coral colony. Control samples were collected from unbleached colonies in the surrounding area. Fluorescence in situ hybridization was conducted to analyse the identity and structure of the microbial communities in association with the coral tissue. Transmission electron microscopy was also used to assess the structure of the coral tissue and the location of the endosymbionts within the tissue.

The main finding was that Vibrio shiloi was not present when coral bleaching occurred or during coral death as a result of bleaching and therefore does not support the Bacterial Bleaching Hypothesis. They did find endolithic bacterial communities to associate with coral tissue, with a shift occurring from cyanobacterial dominated communities to green algal dominated communities during the process of bleaching. This demonstrates the complex interactions of microbial communities of the Oculina patagonica, and although are thought to be of importance, the specific nature of these bacterial colonies still remains unknown. Nonetheless, it is thought that environmental stressors are the main trigger of coral bleaching and the subsequent colonisation of opportunistic bacteria may also play an important role. Although this study showed Vibrio shiloi to not be involved in the primary cause of coral bleaching, further studies are required to determine if bacteria perhaps have a secondary role in the bleaching process and whether microbial treatments would be effective to decrease the severity of coral bleaching.

Ainsworth, T.D., Fine, M., Roff, G. and Hoegeh-Guldberg, O. (2008). Bacteria are not the primary cause of bleaching in the Mediterranean coral Oculina patogonica. The ISME Journal. 2, 67-73.

http://www.nature.com/ismej/journal/v2/n1/full/ismej200788a.html

Hydrothermal vent mercury detoxification

Hydrothermal vent fluids of mid-oceanic ridge systems are highly enriched with both nutrients and heavy metals, and the concentrations of these metals may reach concentrations that are toxic to most living organisms. A gradient in metal toxicity surrounding the hydrothermal vents often exists where the vent fluids are diluted with the surrounding cold, oxygenated seawater. As the fluid mixes with oxygen the metals may become oxidized, more soluble and therefore more bioavailable and toxic. Microorganisms transform mercury between its three oxidation states (0, +1 and +2) and it is therefore expected that microbes found in the areas around hydrothermal vents will have high resistance to mercury and other metals. Mercuric reductase (MR) reduces ionic mercury [Hg(II)] to elemental mercury [Hg(0)], a less toxic form, and is encoded by the mer (bacterial mercury resistance) operon. Vetriani et al. (2005) investigated the mercury resistance, mer genes, MR activities and phylogenetic relatedness of one mesophile (EPR3; T_opt 28^oC), three moderate thermophiles (EPR6, EPR7 and EPR8; T_opt 45^oC) and psychrophilic strains found nearby as controls (T_opt 4^oC), that were all isolated at various distances from diffuse flow vents on the East Pacific Rise.

Vetriani et al. (2005) found that most moderately thermophilic strains were related to the genus Alcanivorax, and were highly resistance to Hg(II) and reduced it to Hg(0). They found that their mer genes formed a unique, previously unknown cluster that was most closely related to the clade that includes the best characterized MR and they differed from each other by one to three amino acid residues. The mesophilic strains were most closely related to Pseudoalteromonas, Halomonas, Pseudomonas, Marinobacter and unclassified Rhizobiales and removed Hg(II) before any growth occurred, showing moderate resistance to Hg(II). The psychrophilic strains were related to the genus’ Moritella, Psychrobacter and Photobacterium and were very sensitive to Hg(II). The activities of the MR tested corresponded to the ambient environmental temperature in all except Escherichia coli, which was used as a control. The E. coli MR exhibited maximal activity between 55 and 65^oC when extracted from the cell, but maximum rates were at 37^oC when inside the cell. This is characteristic of a thermophilic enzyme and may be a relic of evolution in a higher temperature, supporting the hypothesis of a hyperthermophilic origination of all life. A clear spatial relationship between distance from the vent and resistance to Hg(II) was shown, as would be expected, and it is likely that the bacteria here allow other vent organisms to exist by detoxifying the environment.

It would be interesting for further study to look at whether other vent organisms are capable of surviving the toxic mercury in their environment without the presence of these microbes as to whether the microbes really are allowing the existence of the whole ecosystem. Also, if anyone else is taking the Ecotoxicology module then this paper also links well with some of the concepts there, in particular there was a comment at the end of one of the lectures about how organisms cope with such high pressure and contamination…

Vetriani, C., Chew, Y., Miller, S., Yagi, J., Coombs, J., Lutz, R. & Barkay, T. (2005) Mercury Adaptation among Bacteria from a Deep-Sea Hydrothermal Vent. Applied and Environmental Microbiology. 71, 220-226

Pigs, Fish & Antimicrobial Resistance

Pigs, Fish & Antimicrobial Resistance

Integrated livestock-fish aquaculture commonly uses livestock manure (animal excreta, urine, and feed leftovers) as pond fertilizers in Southeast Asia. This process aids planktonic and microorganism growth that the fish are able to feed on.

The animal waste often contains antimicrobial residues and resistant bacteria, which are subsequently released into fishponds; residues often originate from antibiotics incorporated within the livestock feed. This may favor selection of antimicrobial-resistant bacteria, with a potential risk of resistance genes being transmitted to a range of aquatic bacteria.

Son Thi Thanh Dang and friends (2011) aimed to determine the association between the provision of pig feed, containing antimicrobials, and the development of antimicrobial resistance. Researchers measured resistance in E. coli and Enterococcus strains, isolated from pig manure and water-sediment pond samples, from an experimental pig-fish farm located in periurban Hanoi, Vietnam. Strains of Enterococcus spp. and Escherichia coli were chosen because they are often used as indicators of fecal contamination and to monitor antimicrobial resistance, since they are common in environments contaminated by human and animal feces.

The antibiotics used were tetracycline (TET) and enrofloxacin (ENR), these were incorporated as part of the feed provided to nine piglets over a course of four months (see below for feeding regime).

Month 1, feed without antimicrobials

Month 2, feed with TET and ENR

Month 3, feed without antimicrobials

Month 4, feed with TET and ENR

As well as testing resistance to ENR and TET, resistance to nalidixic acid (NAL) was examined in E. coli, since resistance to fluoroquinolones is believed to be a two-step process, in which the first step is NAL resistance caused be a single mutation.

MIC values, (minimum inhibitory concentration) for NAL and ENR showed that E. coli and Enterococcus spp. displayed significantly higher frequencies of resistance toward NAL and ENR during the months when pigs were provided feed containing antimicrobials, in both manure and water-sediment samples.

However, the study found TET resistance for both strains was high throughout the study period, indicating that TET-resistant E. coli and Enterococcus spp. were present in the piglets prior to the start of the experiment. PCR-identification revealed related occurrences of several Enterococcus spp. in the water-sediment and manure samples - suggesting that these species may have originated from the pig manure. 

In summary, this paper shows that E. coli and Enterococcus spp. isolated from manure and water-sediment samples developed resistance to NAL and ENR, but not to TET, and this was connected with the provisioning of feed containing the two antibiotics. However, the spread of antimicrobial resistance to other aquatic bacteria in such integrated farms needs further assessment regarding the possible human health and food safety. 

Click here to view full article  

Son Thi Thanh Dang, Andreas Petersen, Dung Van Truong, Huong Thi Thanh Chu and Anders Dalsgaard4. (2011). Impact of Medicated Feed on the Development of Antimicrobial Resistance in Bacteria at Integrated Pig-Fish Farms in Vietnam. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 77 (11), 4494–4498.

Water Quality Indicators and the Risk of Illness at Beaches With Nonpoint Sources of Faecal Contamination

In the past many studies have found a relationship between the presence of faecal indicator bacteria and swimming-related illness in people exposed to water.  Although these studies are useful they were mostly conducted in areas where human sewage was the main source of pollution.  Currently used indicator bacteria may not be useful for non-point source contamination of water for example from animals because they may excrete bacterial indicators without human pathogens. There is a further problem relating to viruses as we have discussed in lectures. Even though there are some promising viral indicators they haven’t really been studies as predictors of health effects in bathers. The aim of this study was to look at the health effects experienced by bathers in relation to the water quality where nonpoint source contamination dominates.

The study was designed to look at whether the risk of illness increases with increased levels of traditionally used indicators and if contact with water increased the risk of illness for 2 weeks after contact. Participants were recruited on the same day as sampling, and their current health and degree of exposure to the water were recorded.  Three traditional indicators were used (enterococcus, total coliforms, fecal coliforms) which were measured by membrane filtration along with chromogenic substrates. Enterococcus was also measured using qPCR methods. Other indicators were measured  including Bacteroides, somatic coliphage, male-specific coliphage, adenovirus, and norovirus.

1892 samples were analysed, approximately 16% of these samples failed to meet water quality standards with 96% of these coming from enterococcus. Swimmers experienced more symptoms such as diarrhoea and a skin rash than non-swimmers and incidence of these symptoms increased with  different exposure categories i.e. those swallowing water showed highest percentage of symptoms. This increased risk was not seen for more severe symptoms such as vomiting or fever. The latter symptoms have been considered more relevant as it is difficult to tell if exposure to sea water and subsequent irritation could be responsible for symptoms such as a skin rash.

No correlation was observed between indicator levels and the risk of illness. In previous studies a link has been show but this is usually when human faecal contamination has been shown to be present. As the area is not known to have high level of human faecal contamination  the authors conclude that this lack of correlation may be attributed to this lack of human faecal indicators leading them to suggest that alternative bacterial indicators should be used where non-point source contamination is present.

I chose to review this paper as I thought it was interesting that the authors focused on non-point source contamination and compared it to the risk of illness. As with any study involving reports of illness from people it is dependent on how severe the people thought their symptoms were. Although the authors did a lot in an attempt to avoid problems with results for the human health outcomes but can never be totally be avoided.. I think that is interesting that the authors found no correlation between illness and levels of indicator bacteria. It is difficult to conclude that the symptoms seen were definitely due to exposure to sea water but  it highlights another problem with the use of certain indicators when non-point source contamination is a problem. This could be antoerh thing which should be taken into account with the EU bathing regulations.

Colford, J. Wade, T. Schiff, K. Wright, C. Griffith, J. Sandhu, S. Burns, S. Sobsey, M. Lovelace, G. Weisberg, S. 2007.  Water quality indicators and the risk of illness at beaches with nonpoint sources of fecal contamination.  Epidimiology 18; 27-35

Multi-level symbiotic system: anenomes, fish and cleaner shrimps

Cleaner shrimps form mutualisms with fish for the removal of ectoparasites, however have been far less studied than cleaner fish. Unlike cleaner fish, cleaner shrimps range in colour and behavioural signals among species. They have also been observed to form associations with anemones and corals, with such a multi-level symbiotic system further complicating attempts to understand their relationships with fish. Huebner and Chadwick (2012) aimed to investigate the visual role of the sea anemone Bartholomea annulata and the shrimp Ancylomenes pedersoni on the cleaning interactions formed with fish and how the fish responded to manipulation of these factors.

The study was conducted at two coral reef sites located at Brewers Bay and Flat Cay, located next to the Virgin Islands in America. Divers collected visual observations across 77 fish cleaning stations from 6:30 – 8:30 over a week during March, July and November 2009 and February 2010. They recorded the number of fish visits in addition to the visual features of the anemones including body size, height of tentacles, distance tentacles extended beyond the surrounding substratum, the number of shrimps and the total number of symbiotic crustaceans. Some cleaning stations were manipulated with a brown coloured mesh covering anemones and shrimps to analyse their visual effects on cleaning interactions, including both covered, only one covered and a control of none covered.

They found increased fish visits to anemones with a larger body size and total symbiotic crustaceans, although these two variables were not related, however when the covers were added there was an inconsistent response. Fish only posed for cleaning when the anemones were visible, regardless of shrimp visibility, meaning a decreased number of cleaning interactions were recorded when the anemones were covered. This suggests the anemones act as beneficial symbionts to the shrimps as they are a visual cue for the cleaning of fish. Fish were also observed repeatedly searching around the anemones that had been covered, suggesting they may act as a landmark that represent cleaning opportunities for fish. This was further indicated as fish posed at visible anemones even when the shrimps were covered, despite being close enough to be able to visually detect no shrimps were available. This may be related to the fact shrimps are sometimes hidden in crevices around the anemones, therefore the act of posing by the fish may cause the shrimps to emerge, further suggesting the anemones play an important role in the symbiosis of cleaning interactions. Nonetheless further research is required to determine the specific visual cues the anemones provide to these symbionts and the benefits obtained by each member in this complex system.

Huebner, L.K. and Chadwick, N.E. (2012). Reef fishes use sea anemones as visual cues for cleaning interactions with shrimp. Journal of Experimental Marine Biology and Ecology. 416, 237-242.

http://www.sciencedirect.com/science/article/pii/S0022098112000068

Macroalgal-associated bacterial communities: species vs genes

A previous study by Burke et al. (2011a) analysed the species composition of Ulva australis associated bacterial communities (covered in my earlier blog). Surprisingly, it was found that there is very little phylogenetic similarity between bacterial communities on different U. australis samples (15% similarity). To explain this variability in community structure, Burke et al. proposed a competitive lottery hypothesis which states that community composition is determined by the functional roles needed to colonise ecological niches. In terms of bacteria, this means there are ‘pools’ of species which share functional roles and any species from within these pools may colonise an ecological niche, depending on who gets there first. Importantly, members of each pool may be phylogenetically related or unrelated.

To test the competitive lottery model, Burke et al. (2011b) set out to show that algal-associated bacterial communities are functionally distinct from the surrounding seawater, but contain a core of functional genes which are present across all algal samples.

Bacterial DNA was extracted from six individual U. australis samples and eight seawater samples (corresponding to samples from Burke et al. (2011a)). DNA samples were then cut down to a small size (around 2 kb) and large scale shotgun sequencing was then performed (a method of sampling an entire genome by aligning the overlap among small samples of DNA).

Results showed that algal-associated communities were functionally distinct from those in the surrounding seawater. Furthermore, 70% similarity in functional composition was found between U. australis-associated bacterial communities, indicating that despite the large differences in species composition between hosts (found in the previous study) many functions are shared, supporting the competitive lottery model. As a result of these shared functions, authors were able to define a set of ‘core functions’ to U. australis-associated bacterial communities, these were; detection and movement toward host surfaces, attachment and biofilm formation, response to algal host environment, regulation in response to environmental stimuli, lateral gene transfer and defense. Finally, after phylogenetic and taxonomic analysis, it was found that core functions were not restricted to a particular taxonomic group, suggesting functional equivalence between taxa and further supporting the competitive lottery model.

This key study by Burke and colleagues highlights the underestimated importance of genes/gene clusters in understanding community assembly in bacterial systems. Currently, most theory regarding the understanding of microbial diversity comes from the study of eukaryote ecology, where genetic coherence within eukaryotic species can be assumed; therefore the unit of species is relevant. However, results from this study and an earlier publication Burke et al. (2011a) suggest the analysis of species composition may tell us little about bacterial community structure due to the massive amount of genetic exchange between taxonomically distinct bacteria. Finally, the model used in this study could also be applicable to other complex host-associated microbial communities, where species composition is highly variable between hosts, such as the human microbiome.

Burke, C., Thomas, T., Lewis, M., Steinberg, P. and Kjelleberg, S. (2011) Composition, uniqueness and variability of the epiphytic bacterial community of the green alga Ulva asutralis. The International Society for Mircobial Ecology journal  5. 590-600.

Burke, C., Steinberg, P., Rusch, D., Kjelleberg, S. and Thomans, T. (2011) Bacterial community assembly based on functional genes rather than species. Proceedings of the National Academy of Sciences 108. 14288-14293.

Surprising variability among macroalgal-associated bacteria

Macroalgae are crucial members of coastal ecosystems, contributing to primary production, offering protective environments for invertebrate species and providing living surfaces for the colonisation of microbial communities. Despite the importance of macroalgae and the importance of associated-bacterial communities in other sessile eukaryotes, the interactions between algal host-microbe and why these associations form still remain a mystery. Recently, studies have begun to ascertain the roles of individual microbial species (settlement of algal spores, antifouling and pathogenic defence), indicating the importance of these bacterial communities to the algal hosts normal function. 

This key paper by Burke et al. (2011) set out to perform an in-depth analysis of the bacterial communities of Ulva australis, then compare this with that of the surrounding seawater and finally, establish the extent of specificity in community composition between these habitats.

Bacterial DNA was extracted from the surface of U. australis fronds (sampled over space and time) and the surrounding seawater (200 litres per U. australis sample). Large 16S rRNA gene libraries of nearly full length sequences (≥1200 base pairs) were prepared from DNA extracted from all samples, sequences less than 1200 bp in length were removed. Sequences were then grouped in to operational taxonomic units (OTUs), for comparison.

An earlier publication by Tujula et al. (2010) reported a small sub-population of continually occurring species, termed ‘core microbiota’, associated with U. australis (based on DGGE (denaturing gradient gel electrophoresis) analysis). Results in this study did indicate a small number of OTUs (six) were present in all U. australis libraries, however, none of the consistently detected OTUs showed ≥97% similarity to core microbiota sequences found by Tujula et al. (2010). Furthermore, a large degree of variability was found among the U. australis libraries, the average level of similarity found to be less than 20% at the species level. Finally, as expected, U. australis-associated communities were shown to be distinct from communities present in the surrounding seawater (less than 2% of total OTUs occurring in both seawater and U. australis).

The authors conclude that the high degree of variability observed is most likely due to the deeper level of sequencing used, which identified many rare OTUs unlikely to be detected by DGGE. In opposition to the findings of Tujula et al., the results found by Burke and her colleagues suggest there does not appear to be a core microbiota present within U. australis communities. Furthermore, the authors propose that the observed variability between samples of U. australis could be explained by the lottery hypothesis, which states that species with similar functional roles, will occupy space within an ecosystem based on who gets there first. To test this theory, an analysis of the functional genes present in U. australis-associated bacterial communities is needed.

The methods used by burke and her colleagues makes this study the most comprehensive assessment of macroalgal-associated bacterial communities to date and hence, my reason for reviewing. The conclusions regarding the importance of species composition vs the function of genes present within a community interested me greatly, with potential applications in other host-microbe associations, such as corals, sponges and humans.

Tujula, N.A., Crocetti, G.R., Burke, C., Thomas, T., Holmstróm, C. and Kjelleberg, S. (2010) Variability and abundance of the epiphytic bacterial communitiy associated with a green marine Ulvacean alga. The International Society for Mircobial Ecology journal 4. 301-311. 

Burke, C., Thomas, T., Lewis, M., Steinberg, P. and Kjelleberg, S. (2011) Composition, uniqueness and variability of the epiphytic bacterial community of the green alga Ulva asutralis. The International Society for Mircobial Ecology journal  5. 590-600.