Rhamnolipid, another application

I have spoken about the uses of rhamnolipids, a biosurfactant, before and this paper goes on to talk about their potential to disrupt biofilms from surfaces exposed to the marine environment, a simple but generally unexplored area. Specifically this paper focuses on the biofilm crated by Bacillus pumilus and the effect of rhamnolipid on its ability to adhere to steel. Many Bacillus species are resistant to harsh environmental conditions such as low nutrient availability and UV radiation and this hardiness also means they are able to resist anti- fouling biocides. Adhesion to steel and the production of sticky exopolysaccharides and organic acids by Bacillus biofilm leads to increased corrosion and thousands of dollars’ worth of damage. Primary colonizing bacteria are the first to adhere to such surfaces and so the killing and ideally the removal of such is an important problem in need of a solution, biosurfactants could act as that solution.

B. pumilus were allowed to adhere for 4 hours at 30°C. Different concentrations (0.05 – 100mM) of rhamnolipid were then added to the polystyrene microtitre plates and left for a further one hour. After staining the results were taken as a percentage of cell adhesion compared to control plates which were not treated with rhamnolipid.

Exopolymeric substances (EPS), which have been investigated in other blogs, are the basis for adhesion of cellular substances to surfaces underwater. They allow for the attachment of other species leading to a community of microbes ultimately creating a damaging biofilm. Rhamnolipids have been shown to have antimicrobial as well as surfactant properties against other bacteria such as B. subtilis and Staphylococcus epidermis at low minimum inhibitory concentrations (<1.6 mM MIC) however the growth of B. pumilis in this case was not inhibited until >1.6mM and therefore higher concentrations of rhamnolipid are needed compared to other Gram negative bacteria. In contrast to this, at low concentrations, rhamnolipid significantly inhibited the adhesion of 46 – 99% of B. pumilus, after one hour there was at least 80% inhibition of adhesion to the polystyrene surfaces which may be enough to prevent the formation of biofilm. Other tests with different bacteria also show the effectiveness of rhamnolipid as an anti – adhesive when it comes to biofilm.

EPS is thought to neutralize antimicrobial agents and therefore assist the microbial community form dangerous biofilm. Therefore the disruption of such EPS could stop such formation. At higher concentrations than the MIC value there was a significant dose – dependent increase in biofilm disruption after treatment with rhamnolipid. After one hour there was significant EPS disruption and 24hours treatment led to destruction of microcolonies. One problem with this report is the suggestion that rhamnolipid usage would be more successful with the help of biocides. In the marine environment the use of biocides in unattractive due to other environmental hazards this brings about. However, the treatment of rhamnolipids does have potential and should be investigated further.

Review of Dusane et al (2010) Rhamnolipid mediated disruption of marine Bacillus pumilus biofilms

Fugu no longer toxic!

A review of: Noguchi, T., Arakawa, O. and Takatani, T. 2006. Toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 1(1), 153-157.

Marine pufferfish of the family Tetraodontidae can be known to possess a neurotoxin (TTX/Tetrodotoxin). This toxin is poisonous to humans, causing paralysis and even death. In Japan ‘Fugu’ is a popular dish which is made from these pufferfish. Many believe the liver to be the tastiest part, however this is also the most toxic. Safe preparation of this dish requires a great amount of training and the serving within restaurants is strictly controlled. The serving of the liver has even been banned due to a frequent occurrence of food poisonings. No detailed studies have been made on the mechanisms of uptake, accumulation, metabolism and excretion of TTX in pufferfish. While they do have a TTX-secreting gland or cells within their skin which is thought to be a defence mechanism, only a small amount if any is ever produced and this does not intoxicate the liver. Previous studies have suggested that the pufferfish do not synthesise the TTX by themselves. Instead they are thought to accumulate it through the food chain. So this study hypothesised that non-toxic pufferfish should be able to be produced if cultured with a TTX-free diet.

Over 5000 species of the pufferfish, Takifugu rubripes were cultured by netcages at sea, and aquaria on land for up to three years. As they are known to accumulate the toxin mainly in the liver and ovary following ingestion, the liver was focussed on for testing, but other parts were also used. Toxicity was assessed using the Japanese official mouse assay method for TTX and also LC/MS analysis. Each tissue was extracted with 0.1% acetic acid and then examined for toxicity through intraperitoneal injection into male mice.

The results indicated that all parts of all pufferfish tested were considered to be ‘non-toxic’ in both the mouse assay where levels were less than 2MU/g and the LC/MS analysis where levels were less than 0.1MU/g. The expression MU is used where 1 MU is defined as the amount of toxin required to kill a mouse in 30 minutes after injection. Anything less than 10MU/g is considered to be non-toxic in food hygiene. This provides solid evidence that the pufferfish become intoxicated through the food chain and non-toxic fish can be successfully produced by netcage or land culture irrespective of culture area and season. If cultured in this way, the fish and in particular the liver can be considered safe to consume.

Lava Eaters

The aim of this study was to assess the abundance, species richness and phylogenetic diversity of endolithic and epilithic microbial communities inhabiting young, unsedimented ocean crust at the sea floor. Hence, basaltic lavas of various ages and alteration states were sampled by the authors from the East Pacific Rise (EPR) and around Hawaii and then analyzed using quantitative PCR, FISH and microscopy. The PCR measurements of the glassy rinds of lava flows showed that the total bacterial and archaeal cell densities were ranging from 3x10⁶ to 1x10⁹ cells/g and that bacteria were dominating (88–96%) all the rock samples examined. This results were then confirmed by the FISH analysis, which revealed dense populations of Bacteria (significantly more abundant than Archaea) exhibiting cell abundances of 3-4 orders of magnitude greater than in the overlying deep sea waters (8x10³ - 9x10⁴ cells/ml) where half of the cells were instead, Archaea.

Subsequently, in order to evaluate in more detail the community composition, the authors used fulllength 16S ribosomal RNA gene clone libraries constructed from basaltic lavas and surrounding sea water samples. These phylogenetic analyses revealed that both the basalt-hosted biospheres (EPR and Hawaii), were harbouring high-richness bacterial communities and that community membership was shared between these sites. A statistical approach was then used to evaluate the species richness (number of operational taxonomic units) as compared to other oceanic environments analyzed in other studies (e.g. an hydrothermal white smoker, the upper water column of the Sargasso Sea, hydrothermal fluids from the Mid-Atlantic Ridge and deep-subsurface sediments from the Nankai Trough). These comparative analysis, revealed that abundance, phylogenetic diversity and richness of Bacteria in these other deep-sea environments were clearly lower and much different than EPR and Hawaii deep-sea basalts. The 21 taxonomic groups recovered from basalt were dominated by Proteobacteria (68% and 66% of all sequences in EPR and Hawaii respectively), while non-Proteobacteria groups included Plantomycetes (8%/5%), Actinobacteria (7%/8%), Bacteroidetes (4%/1%), Acidobacteria (3%/4%) and Verrucomicrobia (2%/2%). Interestingly, the OTU richness for the two geographically separated basalt communities (EPR and Hawaii) showed considerable overlap in community membership, suggesting that oceanic basalt microbes are widely distributed among this biotope.

These differences in phylogenetic diversity, species richness, and total biomass between the basaltic lavas and overlying sea water raised questions about what energy source fuel this biosphere. Potential energy sources capable of sustaining microbial life in ocean crust include hydrothermal input of manganese and iron (chemolithoautotrophic growth) and dissolved organic carbon in sea water or hydrothermal fluids (heterotrophic growth). However, according to the authors, the most plausible explanation is that oceanic lithosphere exposed at the sea floor undergoes seawater-rock alteration reactions and these reactions are capable of supplying sufficient energy for chemolithoautotrophic microbial growth. Lava surfaces in fact, are composed predominantly of volcanic glass, a highly reactive rock component that contains reduced elemental species such as iron, sulphur and manganese. Oxygen and nitrate in deep sea water oxidize these  constituents and chemolithoautotrophic microorganisms can potentially exploit the free energy changes associated with these redox reactions for their metabolic requirements. Laboratory studies have already demonstrated that iron-oxidizing bacteria isolated from the sea floor are able to use rock and minerals, including glassy basalt, for metabolism and growth. The authors estimated that about 6x10⁷-6x10⁹ cells per g basalt may be supported through these reactions and actually, cell densities in EPR basalts were falling exactly within this range. So in conclusion, alteration reactions in the upper ocean crust may fuel microbial ecosystems at the sea floor, which constitute a trophic base of the basalt biotope, with important implications for deep-sea carbon cycling and chemical exchange between basalt and sea water. This hypothesis supports the understanding of the phylogenetically rich and distinct nature of the basalt biotope. The enrichment of taxa from diverse metabolic groups may result from the establishment of chemical microenvironments within or on rock cavities and surfaces during alteration, mineral precipitation and biofilm formation. This niche creation would allow for a greater variety of redox reactions and metabolic pathways (e.g. heterotrophic, anaerobic, or reductive) including those supporting complex organotrophic and mixotrophic communities. 

Reference:

Santelli, C.M., B.N. Orcutt, E. Banning, W. Bach, C.L. Moyer, M.L. Sogin, H. Staudigel, and K.J. Edwards. (2008). Abundance and diversity of microbial life in ocean crust. Nature 453:653-656.  

New Species of Bacteria Isolated from the RMS Titanic

A review of: Sánchez-Porro, C., Kaur, B., Mann, H., and Ventosa, A. (2010) Halomonas titanicae sp. nov., a halophilic bacterium isolated from the RMS Titanic. International Journal of Systematic and Evolutionary Microbiology 60:2768-2774

A new species of bacterium isolated from the RMS titanic is described in this paper. The bacterium was isolated from the rusticles on the ship, bioconcretious structures that look like icicles but are formed from oxidised iron. Various microorganisms live within these structures, which are the by-product of the microorganisms effectively feeding off the metal of the ship.

In this study, the authors used phylogenetic analysis alongside other methods including obtaining 16s rRNA though PCR to perform a BLAST search in order to try and identify the strain. The results of these methods showed that the strain isolated was most closely related to the genus Halomonas. Its most closely related species included Halomonas neptunia (98.6% sequence similarity), Halomonas variabilis (98.4%), Halomonas boliviensis (98.3%) and Halomonas sulfidaeris (97.5%), amongst others confirming it belonged in the Halomonas genus. However, several important differences, including phenotypic and chemotaxonomic differences features that confirmed the strain was a separate and distinct species not previously identified. The paper goes on to describe the new bacterium in detail.

The genus halomonas is heterogeneous, containing more than 60 species to date and are a member of the salt-loving Halomonadaceae family which are able to grow at salinities of 5 - 10% and who are considered generally non-pathogenic aerobes. The new bacterium is also gram negative, flagellated and motile.The discovery of this new bacterium is of particular interest as it may contribute more to the understanding of the mechanisms of rusticles, how they form etc. It also can have wider implications in understanding how to protect other submerged metal structures such as oil pipeline, oil rigs and the disposal of ships at sea for example.

Additional reference: BBC. (06/12/2010). New species of bacteria found in Titanic 'rusticles'.Available: http://www.bbc.co.uk/news/science-environment-11926932.

Prebiotics help for soybean meal bases, do they work?

Prebiotics like probiotics are used in aquaculture to maintain the health of a species, however prebiotics unlike probiotics are a non-digestible food ingredient which can benefit the host by stimulating the growth and activity of bacteria residing in the colon, promoting a healthier host organism. This study investigates the effects of prebiotics on nutrient digestibility of a soybean-meal-based diet by the Red Drum Sciaenops ocellatus (Linnaeus).

The authors used a recirculation system for the 35 sub adult Red Drum which were fed a control diet containing 40% crude protein, exclusively from menhaden fish meal, 0% lipid, and an estimated available energyof14.6 kJ g-1. Five experimental diets were to be similar to the control diet, but with approximately 50% of the protein supplied by menhaden fish meal and 50% provided by soybean meal. To four of the experimental diets, prebiotics were singularly added at 1% of dry weight in place of cellulose while the basal diet had no prebiotic supplementation. The prebiotics in use for the trial (a mix or individually) were mannanoligosaccharide (MOS), galactooligosaccharide (GOS) and inulin. The faecal matter was used for nutrient analysis.

The results showed that the basal diet mixed with MOS and GOS significantly increased protein (82% for the three tanks) and organic matter (69, 64 & 66% for the three tanks) in comparison to the control diet which showed 69% for protein and 49% for organic matter. However the lipid values were significantly decreased for fish fed with MOS, GOS and inulin (63, 61 & 61%) compared to the control of 77%. Energy values were the same for the fish fed with inulin as the control diet of 54%.

This is the first study to demonstrate that nutrient and energy digestibility of soybean-meal-based diets can be enhanced by prebiotics. The wider significance of this investigation is paramount to replacing a higher percentage of fishmeal in fish diets with soybean and or other protein replacements. The side effects such as gastroenteritis can be treated with probiotics and the protein enhancement can be treated with prebiotics, thus a mixture of both in diets would be the next step in further research.

A review of: Burr, G., Hume, M., Neill, W. H., & Gatlin III, D. M. (2008). Effects of prebiotics on nutrient digestibility of a soybean-meal-based diet by red drum Sciaenops ocellatus (Linnaeus). Aquaculture Research, 39, 1680-1686.

The occurrence of enteric viruses in shellfish

A review of: Suffredini, E., Corrain, C., Arcangeli, G., Fasolato, L., Manfrin, A., Rossetti, E., Biazzi, E., (2008), Occurrence of enteric viruses in shellfish and relation to climatic-environmental factors, Applied Microbiology, 47(5):467-474

Shellfish are considered to be potential vectors of foodborne diseases, due to their accumulation of pathogenic microorganisms through filter-feeding. Norovirus (NoV) is an enteric virus that has been linked to shellfish-associated disease outbreaks and is responsible for 60-80% of human gastroenteritis outbreaks. Similarly, shellfish are linked to the transmission of hepatitis A virus (HAV), and its consumption has been reported in 69% of HAV infected patients. In Italy, there have been numerous cases of these diseases related to seafood consumption registered in several cities.

It has been established that climate has direct and indirect effects on the occurrence of enteric viruses. For example, high precipitation can cause flooding and sewage runoff which are key factors in contamination of coastal water and shellfish harvesting areas.

The aim of this study was to assess the prevalence of HAV and NoV in shellfish harvested in a specific national production area, the deltic area of the river Po (North Italy). They also looked at the effect that environmental factors have on viral contamination in the production area.

A survey was carried out for 1 year on samples of shellfish (mussels and clams), taken every 15days from two areas, A (sea area) and B (lagoon area). Environmental parameters such as temperature, pH and salinity were noted as well as bacteriological analysis (for E.coli and Salmonella) and virological analysis (for NoV and HAV), using various PCR methods.

The results showed no significant differences in environmental paramaters in the two areas, with some predictable fluctuations due to seasonal variations. No Salmonella was detected and E.coli numbers were always below legislation limits. There was also no HAV found in both areas but NoV was detected in 10 of the 120 samples, all from area B. NoV was also present in samples ranging throughout the sampling period, with the majority of positive samples in spring and summer. This led the researchers to conclude that a definite association between NoV clinical cases arising during the winter period and shellfish cannot be established.

They also noted that the increase in viral contamination could be in relation to the flow of the tributary river, which could transport further viruses into the harvesting areas and moreover mixes and lifts the sediment at the bottom of the lagoon where viruses can deposit and survive for long periods.

The study highlights the importance of accurate classification of harvesting areas in assuring the safety of shellfish for direct consumption and is useful in helping to establish suitable prevention techniques, especially after meteorological events.

Novel adenovirus isolated from sea lions

Viruses of the family Adenoviridae have genomes consisting of double stranded DNA. They infect various species of vertebrates, including humans. Adenoviruses were first isolated in 1953 from human adenoids. Two types of canine adenoviruses are well known, type 1 and 2. Type 1 causes infectious canine hepatitis, a potentially serious disease involving vasculitis and hepatitis. Type 1 infection can also cause respiratory and eye infections. Canine adenovirus 2 (CAdV-2) is one of the potential causes of kennel cough. Viral hepatitis associated with adenoviral infection has been previously seen in free-ranging California sea lions Zalophus californianus. However previous isolation of viruses were unsuccessful and identification of this virus was ceased and no specific virus was documented. However because the morphological features seen under the microscope were quite similar to that of canine infectious hepatitis and since the virus has a wide host range, it was thought that perhaps the virus responsible for disease was CAdV-1.

The presence of adenoviral DNA was examined in tissue samples from 2 live stranded California sea lions that were admitted to the rehabilitation facility at Californian Marine Mammal Centre. The two rescued animals died with serious symptoms , first was diagnosed with arteritis , pneumonia and pulmonary haemorrhage. Also eosinophilic intranuclear inclusion bodies (sign of adenoviral infection) were discovered in few organs including lymph nodule and lungs. The other case died from severe viral hepatitis with intranuclear eosinophilic inclusions found within hepatocytes.

Tissues from both animals were first examined under electron microscope and then analysed using PCR with the use of specific primers (sense: 5’-GCG CAC TTA CTC ATC CAT TTC C-3’, antisense: 5’-GCT ATT TCT CCA CGC AGC GG-3’). The virus was isololated and compared with known adenoviruses.

The examination of lymph node from first animal and liver of the second one revealed the presence of adenoviral-like particles (70 to90 nm icosahedral) , within the nucleus of affected endothelial cells .The sera from both animals were negative for antibodies against both CAdV-1 and 2. The PCR to detect CAdV-1 and 2 was also negative. Sequencing confirmed the presence of a fragment of the DNA polymerase gene of a novel adenovirus and comparison of the sequence to known adenoviruses in GenBank showed that this was a novel virus from the Mastadenovirus genus. This virus was similar in 77% to tree shrew adenovirus 1 (TSAdV-1) CAdV-1 (72%) and 2 (74%) however in overall is was treated as independent lineage and species.

This was very interesting study and quite exciting that they managed to find new virus , although the more analysis is needed in order to establish if this virus is a primary pathogen causing death. It is quite surprising that although the symptoms of this disease were quite similar to those reported in many other cases , nobody actually managed to identify this virus before.

A review of Goldstein et al.2011 . Isolation of a novel adenovirus from California sea lions Zalophus californianus. Dis Aquat Org. 94: 243–248.