Blog entries closed for coursework

Many thanks for all your submissions. Students are welcome to continue adding posts and comments  but anything entered after 11 April  will not count towards the coursework mark (unless you are given referred coursework at the Assessment Panel).

Between October 2012 and April 2013 the class of 25 students wrote

231 posts
430 comments
177,108 words - that's about the same as JJR Tolkein's "Fellowship of the Ring"!

There were 13993 page views of the blog
59% of views were from the UK (mostly us?)
20% were from Europe, 16% from USA, and 6% from RoW

Here's a link to a Wordle, showing the most commonly used words in the blog.(apart from common words like and, the etc.)  Not surprisingly, "bacteria" and "bacterial" scored top. Not such a strong showing for "viruses" this year!

http://www.wordle.net/show/wrdl/6584057/MicrobesRuletheWaves2012

Are Gorgonian Corals a Source of Novel Antibiotics?

Quorum sensing (QS) is a method of cell-to-cell communication employed by a variety of bacteria and such signals are often used to facilitate colonisation of host organisms. Gorgonian corals are already known to possess compounds that mediate cellular interactions and may be used to regulate microbial colonisation. The researchers in this study used extracts of twelve species of gorgonian corals sampled from the Caribbean to search for compounds that exhibited antibacterial activity that could potentially be developed for bacterial control purposes.

The original coral samples were collected at depths of 5-10m from La Parguera, Puerto Rico and Looe Key Reef, Florida Keys, USA. The following species were sampled: Briareum sp., Eunicea laciniata, Eunicea tourneforti, Plexaura flexuosa, Plexaura homomalla, Pseudoplexaura porosa, Pseudopterogorgia americana, and Pseudopterogorgia acerosa, for Puerto Rico, and Gorgonia ventalina, Plexaurella sp., Muriceopsis flavida, and Eunicea mammosa for Florida Keys. In the Texas-based lab, ethanol extracts were prepared and antibacterial assays performed.

A variety of gram-negative and gram-positive bacteria of both marine and non marine origin were used for the assays, including known coral pathogens such as Serratia marcescens and Vibrio alginolyticus. Human pathogens were also used in the antibacterial assays. The assay itself was a bacteria turbidity assay which was conducted in a 96-well flat bottom plastic microplate, an assay chosen as it requires less of the coral sample than others, thus less damage to the reefs. The assays were simultaneously replicated three times with the controls of bacteria only, bacteria and antibiotic and bacteria and ethanol. Growth inhibition of bacteria was measured by drawing comparisons between the growth rates of the bacteria with the coral antibiotic extracts and the bacterial with ethanol as a control.

Bacteria chosen for the assays had to be isolated and identified, and shown to be culturable. Firstly, mucus samples were collected and cultured on glycerol agar. Bacteria that had grown was then subcultured in a marine broth an identified by species. PCR was used to amplify DNA and amplified sequences were compared with data from the GenBank database. Vibrio parahaemolyticus was the only bacteria identified and it was isolated from Pseudoplexaura porosa.

Bioassays to detect long chain N-acylhomoserine lactones (AHLs) in the ethanol extracts were carried out using Pseudomonas aeruginosa as a QS reporter strain. Inhibition or stimulation of quorum sensing was tested for, and P. aeruginosa is known to be sensitive to the long chain AHLs that result in these responses, making it an ideal reporter strain to reveal the compounds. Fluorescent signals that indicate transient gene expression were measured using spectrophotometry, this allowed for real-time QS detection.

Another biosensor strain, Chromobacterium violaceum, was used as an indicator organism in experiments to measure AHL presence by quantifying violacein synthesis. The synthesis of this pigment indicates a bacterium under QS control. The bioassay in this experiment was based again on spectrophotometry to compare absorbances at A₅₉₀ of the bacteria exposed to antibacterial compounds and the bacteria with ethanol as a control.

Their main findings were that the gorgonian corals possessed compounds that could inhibit and stimulate quorum sensing, suggesting selection on behalf of the corals of the bacteria that could colonize. P. americana, P. acerosa, and P. flexuosa displayed the highest QS inhibition and P. porosa displayed the highest QS stimulation. These differences in effects on QS hint at corals actively selecting the bacteria that can colonize the coral holobiont. More antimicrobial activity was noted against non-marine strains than marine strains of bacteria, suggesting that corals employ several mechanisms of bacterial control. Bacillus subtilis, vancomycin resistanct Enterococcus and methicillin-sensitive Staphylococcus aureus were most sensitive to compounds from the gorgonian extracts and can all be pathogenic to humans. Gram-negative strains of bacteria were less susceptible to gorgonian compounds than gram-positive strains from both marine and non-marine origins, suggesting that gorgonian antimicrobial activity is not broad-spectrum, this may be expected as they would have evolved mechanisms to deal with the bacteria present in the coral holobiont.

The researchers closed by calling for further work on the Caribbean gorgonians which exhibited strong antibacterial action against human pathogens, as these represent a potential for much needed novel antibiotics.

Reference

Hunt., L.R., Smith, S.M., Downum., K.R. and Mydlarz, L.D. (2012) Microbial regulation in gorgonian corals. Mar. Drugs 10: 1225-1243.

Degradation of dispersed crude oil

As we have found out in lectures dispersants can play an important role in aiding biodegradation of large oil spills. Many studies have demonstrated how effective the use of such dispersants but few studies have actually assessed the biodegradation of oil once it has been dispersed in environmentally relevant conditions. Once dispersants have been added oil droplets are very small and become diluted within the water column. This study looked at the degradation of oil at these low concentrations, close to those observed following a real spill when dispersants have been used in order to discover degradation is affected by the presence of a dispersant. 

This study is carried out in laboratory with sea water collected in April and January 2010 from the shore of New Jersey and stored in Carboys. No bacteria or nutrients were added so only indigenous bacteria and nutrients were present throughout the study in an attempt to mimic natural conditions for biodegradation. Oil was added to the samples, one lot of oil samples was lightly weathered in the laboratory by evaporation at room temperature until 20% of its weigh was lost (to mimic 24 hours exposure to sea water) and the other was fresh oil. To both types of the dispersant Corexit 9500 was added. A second set of experiments were carried out where no dispersant was added the sea water samples. The experiment was carried out in a cold room at 8 °C.

At designated times water was extracted from each Carboy 3 times and analysed using gas chromatography and mass spectrometry. Oil biodegradation was monitored with respect to 17α(H),21β(H)-hopane as a conserved internal marker within the oil. Oil was found to be degraded quickly and extensively (more than 80% after 60 days). The half times of the biodegradation were found to be similar with and without dispersant at 13.8 and 11 days respectively. The level of oil present was 2.5 ppm by volume which is clearly low and represents levels expected when dispersants have successfully been added. The authors note that this study cannot be used to assess the effectiveness of dispersants but the study shows that under natural conditions biodegradation is not affected by the presence of dispersants but is rapid and extensive.

I found this study interesting as it used natural nutrient levels and only indigenous bacteria. I think for future work it would be good to look at which bacteria are actually present in the sample and in what numbers to gain a more clear understanding of the whole process. It would also be good to compare the results from this study to with water taken from other areas as there would presumably be different naturally occurring bacteria and nutrient levels which would be likely to change the degradation rates.

Prince. R, McFarlin. K, Butler. J,  Febboe. E, Wang. F, Nedwed, T. 2013 The primary biodegradation of dispersed crude oil in the sea. Chemosphere. 90; 521-526. 

Inactivation rates in pathogens: lab vs in situ

Managed aquifer recharge (MAR) schemes involve water being recycled and stored underground in an aquifer for reuse and relieve water shortages. Microbial pathogens can be present and survive in the recharged water, which subsequently cause health hazards. The potential survival times of enteric pathogens can be assessed to alter the MAR scheme or determine the need for additional treatment. In this study the authors have evaluated the effectiveness of using in situ diffusion chambers for assessment of health risks associated with MAR, this involved comparing inactivation rates in both laboratory microcosms and in situ diffusion chambers.

Groundwater samples seeded with pathogens and indicators were tested in laboratory microcosms (50ml polypropylene centrifuge tubes) and in situ Teflon diffusion chambers. The chambers contained membranes with pore sizes 0.010µm and 0.025 µm and were suspended in a well for 50 days with the upper chambers 1 m below the water table to intercept flow of groundwater. The pH, temperature, redox potential, dissolved oxygen (DO) and EC were measured in situ while dissolved organic carbon (DOC) was analysed by a commercial laboratory. These parameters remained almost constant during the in situ study.

To tackle the issue of potential clogging in groundwater mentioned in previous studies, the chambers were recovered and Rhodamine WT (RWT) a stable fluorescent dye was used to measure diffusion of water across the constructed diffusion chambers and to determine whether clogging of membranes has reduced the flow of water across the chambers. No biological growth or biological clogging was observed on the membranes, so investigating the potential effect of clogging on water flow requires further studies that involve greater turbidity and nutrient concentrations.

All microorganisms tested in this study were observed to decay both in laboratory microcosms and in diffusion chambers. The bacteriophage MS2 was found to decay at much faster rate than adenovirus, which suggests that MS2 is not a suitable indicator for enteric virus inactivation in groundwater. The results are simplified in figure 3, showing that the inactivation times of seeded bacteria were lower when 0.010µm membranes were used compared to 0.025 µm membranes. The higher inactivation time found in the smaller pore size (0.010µm) suggests that reduced rate of water flow across the membranes does influence the rate of bacterial inactivation. The laboratory microcosm inactivation rates were similar to the 0.025 µm membranes, yet significantly different compared to the 0.010µm membranes.

Previous studies have used laboratory microcosms and in situ diffusion chambers however in situ studies are preferred for accurate assessment of pathogen inactivation. Reasons for this preference seem to be laboratory microcosms can cause an underestimation of inactivation rates. I agree with this preference because an in situ model is likely to simulate the amount of time the recharged water is contained  within the aquifer, as well as the conditions it is subjected to. The authors provided several possible factors (for example, low water flow or water temperature) that could have influenced the inactivation rates of the pathogens, but they were unable to explain several  mechanisms behind some of the variations in inactivation rates which suggest that further research into the factors associated with diffusion chambers is needed. 

Sidhu, J.P.S. and Toze, S. (2012), Assessment of pathogen survival potential during managed aquifer recharge with diffusion chambers. Journal of Applied Microbiology, Volume 113, pages 693–700. 

Potential Interactions of Particle-Associated Anammox Bacteria with Bacterial and Archaeal Partners in the Namibian Upwelling System

Nitrogen is often considered a factor that limits the growth rate of phytoplankton. A high proportion of nitrogen loss (30-50%) occurs in oxygen minimum zones (OMZ) such as the Nambian Shelf, partially due to micro-organisms that carry out anaerobic oxidation of ammonium (anamox species). Previous research has been carried out into anamox species that reside in the Nambian Shelf under conditions that are similar to that near sewage systems, which contain more ammonium than naturally occurs in these waters. Woebken et al (2007) investigated the distribution and particle association of anamoxx species to improve understanding of their occurrence in their natural habitat. This topic is worthy of investigation because of the importance of the phytoplankton species influenced by the activity of anamox species.

Water samples containing bacterioplankton were collected from 4 stations around the Nambian Shelf, and water at 6 stations along a transect (23.0°S from 14.36°E to 12.0°E) were investigated. Profiles were created in terms of turbidity, oxygen levels and ammonium levels. Correlation was seen between anamox numbers and particulate organic carbon, and particulate organic nitrogen. Anamox species were present in depths of 30 m down to the sediment, across suboxic to anoxic conditions but not oxic, with the highest concentrations occurring near the coast.
            DNA from collected water samples was isolated, then replicated using PCR with universal bacterial and archaeal primers for the 16S rRNA sequences. The results were used to choose specific primers for a second PCR and specific probes from FISH and CARD-FISH (Catalyzed reporter deposition Fluorescence In Situ Hybridization).
            From the second PCR, they created several bacterial clonal libraries from the water samples at station 182 (119 m and 130 m deep) and one archaeal library based on samples from station 182 (130 m deep). The bacterial diversity and species numbers found were greater than that of Archaeal species: 235 bacterial species versus 23 archaeal species, belonging to 5 major groups as opposed to only 2 major groups for archaeal species. Several sequences were found to belong to uncultured bacteria. The species identified occur worldwide.
            FISH and CARD-FISH performed using group- and species-specific probes found a majority of bacteria (56.3%) occurred in clusters; 24.5% attached to particles; and 19.3% were individual cells.
            In situ observations were also carried out in the water column through a remotely operated camera, showing high densities of macroparticles.

This research shows the ability for anamox species to inhabit a wide range of habitats, limited by high oxygen levels. Their reliance on nitrogen suggests that natural cycles in terms of population numbers occur due to their removal of nitrogen as their numbers increase. It would be interesting to investigate the interactions between the cells considering they seem to flourish in group situations, such as quorum sensing, co-operative metabolism and gene transfer. Perhaps it is inter-bacterial interactions that allow them to inhabit regions with varying environmental conditions? If these species prove problematic for valuable species of phytoplankton it may be beneficial to consider research into breaking down these aggregates. Future methods of collection that did not disturb the aggregates may provide a more accurate representation of the frequency of clusters.

Woebken, D; Fuchs,B. M; Kuypers,M. M.M. and Amann, R. (2007) Potential Interactions of Particle-Associated Anammox Bacteria with Bacterial and Archaeal Partners in the Namibian Upwelling System Appl Environ Microbiol. 73(14): 4648–4657.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932835/

Megan

Gradients in Microbial Methanol Uptake in Atlantic Waters

Methanol biogeochemistry is an emerging area of research with importance to understanding marine microbial carbon cycling. Methylotrophic bacteria have been known to exist in oceans for a number of years, but it is only recently that extensive research on them has been carried out. Researchers have begun to establish where methylotrophs are located, often in coastal waters, and understand the significant roles they, and bacteria of similar metabolism, play in carbon cycling, such as the heterotroph SAR11 Alphaproteobacteria’s oxidation of one-carbon compounds representing a source of CO₂ in the upper ocean. This and similar research shows how methanol turnover may be common and be integral in the marine environment.

The researchers (Dixon et al, 2013) in this study investigated the microbial demand for methanol along a gradient of metabolic productivity from highly productive coastal waters to comparatively arid oligotrophic gyres. They collected seawater samples along a transect in the Atlantic ocean during research cruises and started experiments within an hour of sampling.

Microbial methanol uptake was investigated by incubating the seawater samples with ¹⁴C labelled methanol for 5-10 hours at the temperature of their origin. Uptake of this labelled methanol into particulate cell biomass was assessed. Bacterial production rates were determined by measuring the incorporation of ³H-leucine into protein synthesis. ¹⁴C-bicarbonate incorporation was used as a measure of primary production and fluorometric analysis of acetone-extracted pigments was used to determine chlorophyll a concentrations. Flow cytometry on SYBR Green I DNA-stained bacterioplankton cells and unstained Prochlorococcus sp. and Synechococcus sp. cells was used to established community composition and plankton community composition was determined by inverted settlement microscopy. Inorganic phosphate and nitrite concentrations of the samples were also determined. Finally, DNA was extracted from the samples, amplified and analysed using PCR and sequences compared to databases to identify bacteria.

The researchers found that microbial methanol uptake varies between 0.1–24.8 nmol l^-1 d^-1 in coastal upwelling waters and decreased at depths below 20m, though overall methanol uptake was up to 10 times lower in samples from the gyres. Most of the assimilation of methanol in both upwelling waters and gyres was attributed to microbes of 0.2-2.0 mm in size. Leucine uptake rates varied between 190-2279 pmol Leu l^-1 d^-1 in the upwellings, and uptake was 3-7 lower in the gyres. Primary production rates of the upwelling waters decreased at depths below 20m and 99% of primary production in the top 20m of the waters was associated with larger cells such as flagellates and diatoms, the primary production rates of the gyre samples were again lower but were also associated with larger cells. Chlorophyll a concentrations ranged from 6.5 mg m^-3 in recently upwelled waters to 0.5mg m^-3 10 days later and seemed to correlate with methanol uptake. Prochlorococcus sp. were absent from the upwelling waters but Synechococcus was present as were pico-plankton and nano-phytoplankton. All microbes with the exception of pico-plankton (numbers of which were higher in the gyres than the upwellings) were less abundant in the gyres. Inorganic nitrate and phosphate concentrations in the upwelling samples initially ranged between 6.1–7.7 mM and 0.43–0.58 mM but lowered slightly after and were low in the gyre samples.

The results of this study were therefore mostly consistent with expectations, with higher nutrient uptake, primary production and assimilation rates noted in more biochemically active waters than in waters that were less so. Coastal waters showed the highest methanol carbon assimilation rates when compared to northern temperate and equatorial upwelling waters and and in contrast with oligotrophic gyres. Their results also suggested that all surface waters of upwellings may contain a population of methylotrophic microbes, or microbes that use methanol-derived carbon for growth. Preliminary characterization of bacteria in the equatorial waters revealed a variety of methylotrophs supporting this finding. Around 50-60% of the total methanol was assimilated into carbon biomass in upwelling waters, which was in contrast to 97% of methanol being used oligotrophic microbes in gyres as an energy source.

Lastly, the researchers suggested that correlations between methanol uptake and chlorophyll a concentrations in upwelling and coastal waters could be used by climate scientists to infer methanol biological loss rates by imaging chlorophyll a remotely. Leucine uptake was not as stongly correlate and therefore couldn’t offer a similar use. The researchers recognized that further work would be needed to refine a technique that could use chlorophyll a concentrations to effectively estimate methanol loss rates and that further research was needed to narrow down the microbial species which were utilizing methanol.

Reference

Dixon, J.L., Sargeant, S., Nightingale, P.D. and Murrell, J.C. (2013) Gradients in microbial methanol uptake: productive coastal upwelling waters to oligotrophic gyres in the Atlantic Ocean. J ISME 7: 568-580.

Furunculosis, Aeromonas salmonicida & Fish Farming

The disease furunculosis caused by the bacteria Aeromonas salmonicida, continues to be a major health problem for the growing salmonid aquaculture. Continuing to raise high concerns in European salmonid fishfarms due to high mortality rates leading to detrimental economic losses.

In spite of effective vaccination programs frequent outbreaks occur at the fish farms calling for repeated antibiotic treatment. However, side effects following oil-adjuvanted vaccination have raised a series of ethical and welfare questions connected to the use of vaccines. Additionally antimicrobial residues left from treating out breaks can persist in the environment and lead to resistance spreading to other microbes.

Lars Holten-Andersen et al (2012) hypothesized that a difference in natural susceptibility to this disease might exist between Baltic salmon and the widely used rainbow trout. The researchers tested the hypothesis using a cohabitation challenge model; this was used to investigate the relative susceptibility to infection with A. salmonicida in rainbow trout and Baltic salmon. They monitored the course of the infection on a daily basis over the course 30-day period post challenge summerising the results as mortality curves.

The cohabitation infection model proved to be effective in terms of disease

transmission. Bacteriological examinations provide confirmation that the mortalities occurred as a result of the infection. This verifies the transmission of the disease from East-Atlantic salmon to Baltic salmon and rainbow trout.  Lars Holten-Andersen and co found that survival at day 30 was 6.2% and 34.0% for rainbow trout and Baltic salmon, respectively.  The differences in susceptibility to A. salmonicida were significant between the two salmonids. The risk of dying from the infection was found to be 3.36 higher Baltic salmon compared to rainbow trout.

FINAL NOTES:

Studies such as this one provide valuable data and evidence that we can use to make better-informed decisions in the future. For example, which particular species of fish would one select for cultivation? If a fish already possessing naturally high resistance was selected this might enable us to reduce the amount of antibiotic residues left in the environment do to lower frequencies of outbreaks. 

Full article here :) 

Holten-Andersen L, Dalsgaard I, Buchmann K (2012) Baltic Salmon, Salmo salar, from Swedish River Lule A ̈ lv Is More Resistant to Furunculosis Compared to Rainbow Trout. PLoS ONE 7(1):