Xenobiotic Metabolizing Bacteria of the Indian Ocean - Getting More Common?

A review of: Rodrigues, J., Madhukar, A., Kumar, N., Sangodkar, U.M.X., (2010), Isolation and characterization of a marine bacterium belonging to the genus Alkalignes capable of the complete mineralization of the dibenzothiopene, Indian Journal of Geo Marine Sciences, 40, 3, 391-397

With a rising demand for fossil fuels from the ever expanding human population, there is always a need for the transportation of products between countries, this is most commonly done via tanker on the worlds oceans. But in recent years this method of transport has resulted in the contamination and near destruction of many of the worlds marine ecosystems. In many places around the world this is a huge threat, but considering that there are many areas with a vast concentration of marine life bordered and regulated by developing countries, this threat can be further increased. 

This study directly looks at the western coast of India region of Goa, where, in 1995, the M.V. Sea transporter ran aground during a cyclone, resulting in the spillage of furnace oil. A marine microbe of genus Alkaligenes was found to be live here and was observed to completely mineralize the xenobiotic compound dibenzothiopene (DBT), an aromatic hydrocarbon found in the tar balls of the contaminated site. The purpose of this test is to look directly at the relationship between the Alkaligenes and the DBT.

During the study, sequential enrichment techniques were favoured, whereby cultures of the bacteria were grown in artificial sea water and to this, 1g of DBT was added and mixed thoroughly to simulate conditions found at the site of contamination. Cultures were then acidified at different times and extracted using an ethyl acetate extract and thoroughly dried. Preparative silica gel G was used to apply the extracts to TLC plates and then exposed to UV light. Residual chemicals were then compared using  industry standard chemicals. The different strains were then separated and tested further against the DBT.

Results show that there are 5 strains of the bacteria that utilizes the DBT, and out of these, the JR110 strain. This was then sent to the IMTECH facility in Chandigarh, India for detailed testing with results as follows, the JR110 strain was seen to degrade 8 different aromatic compounds also with degradation of DBT intermediates to form a red pigmented colony. Tests carried out exposing only the JR110 strain to the DBT were largely successful, showing a clean sigmoid curve of reduced contamination as the growth of the bacteria increases.

Conclusions are drawn that due to the highly contaminated nature of the west coast of India, there is likely to be continuous evolution of degradation processes of xenobiotic compounds, which is believed to be due to the intermediates of benzoate utilizing the pathways in marine bacteria, thus exerting a positive pressure on the mineralization of xenobiotic compounds such as DBT.

Monitoring sewage pollution with sea fans

A review of: Baker, D. M., Jorda’n-Dahlgren, E., Maldonado, M. A. and Harvell, C. D. 2010. Sea fan corals provide a stable isotope baseline for assessing sewage pollution in the Mexican Caribbean. Limnology and oceanography, 55(5), 2139-2149.

As populations continue to rise, especially on the coastlines, sewage pollution is becoming a greater threat to coastal marine systems. The input of contaminants, especially nutrients such as nitrogen and phosphate can greatly disturb the environment. Increases in primary productivity can smother critical species such as sea grasses and reef building corals, pathogens can cause disease especially in corals and there is a generally common result of lowered biodiversity. While monitoring of this kind of activity is important everywhere, it is especially important that it is studied and controlled in developing regions which are often dependent on their ecosystem’s health and wellbeing.

This study hypothesised that sewage-derived nitrogen inputs are detectable and more severe in developed areas along the Mesoamerican barrier reef of Mexico. To test their hypothesis in this area they compared the stable nitrogen isotope (δ15N) values from the common Caribbean sea fan, Gorgonia ventalina, collected from a developed and undeveloped are of the coastline. Akumal coast was selected as the developed site as there are a great number of residents and a huge influx of tourists. The shoreline of Mahahual was selected to be the undeveloped site since there were few residents and tourists and a sewage treatment infrastructure.

The isotopic ratio of 15N:14N is regarded as an effective and direct indicator of human nitrogen pollution. Enriched isotope values arise from the accumulation and degradation of human and animal wastes and are easily distinguishable from other sources. Perpendicular to the shore samples of sea fans were taken 1km from the shore. 2cm-squared fragments were cut that are
likely to represent the previous year of growth. Stable isotope analysis was performed on the samples. The prevalence of Enterococcus was sampled at sites adjacent to where the sea fans were sampled. Enterococcus assays were used to determine if the nitrogen isotope analysis values were correlated with sewage pollution. Positive results for this test would rule out the possibility of enrichment due to denitrification. Statistical analysis was then performed.

Results found that samples from the developed site sea fans were enriched in δ15N (as high as 7.7‰ near shore) and were ≈3.5‰ greater than sea fan samples from the undeveloped site. The δ15N values were also positively correlated with faecal Enterococcus counts from the seawater. This confirms that the enrichments are associated with sewage and not denitrification. This study suggests that data collected from the undeveloped site which is relatively pristine could now be used as an isotopic baseline for monitoring the Mesoamerican barrier reef at sites where increased development is planned or underway. Another interesting find of this study was that the highest Enterococci counts were found to be from a lagoon popular with bathing tourists. The counts were approximately 59CFU per 100mL. The presence of faecal Enterococci is well above US Environmental Protection Agency standards for recreational waters which is 35CPU per 100mL.

This method of stable isotope analysis in sea fans could be a promising tool for monitoring changes in the contribution of human nitrogen sources to nearby ecosystems, especially in developing regions where water quality monitoring programs are not established.

Not just for the Bathroom...

Marine sponges are well known for harbouring a wide variety of microbes which have been found to have a mixture of roles, including protection, pathogens and competitors.  Sponges are also the most prolific marine producers of novel compounds, many of which are of pharmaceutical and biotechnological importance. The structural similarity between the compounds found in sponges and compounds found in sponge microbiota suggest that they could in fact be of microbial origin. Sponge-associated microorganisms have recently received renewed attention, with much research focusing on the production of bioactive compounds. This research has found that bacteria belonging to the actinobacteria genera are the largest producers of these secondary metabolites.

The aim of this investigation is to analyse the compounds produced by bacteria from two species of sponge (Suberites carnosus – non-calcarious, and Leucosolenia sp. - calcarious) in an effort to produce novel antibacterials to combat drug resistant pathogens, which are becoming a big problem in the medical industry.

Samples of the sponge species were collected at a 15m depth from Lough Hayne, Co. Cork in November 2008. Bacteria from these samples were cultivated on agar over a period of two months, during which time they were incubated at 18^oC and checked regularly for distinguishable colonies.  After the incubation period, the cultivated bacteria underwent deferred antagonism and well diffusion assays, PCR and phylogenetic analysis of the 16S rRNA gene.

For both sponge species, analysis of 16S rRNA sequences revealed that the dominant phylum of bacteria found was Protebacteria, and the most abundant in both cases was γ – Proteobacteria. The genetic identity of over 98% of the isolates found in both sponge species are already known to science. However, some isolates, especially from Leucosolenia sp. are not. A result like this is not unexpected, as calcarious sponges have been subject to little research before now. A wide range of the bacteria found in both sponge species displayed antimicrobial activity during analysis, though more of the isolates from the sponge species S. carnosus showed this than those from Leucosolenia sp. This can be explained by different genera and species isolated in both, as the dominant genera of bacteria found was different in each species of sponge (Pseudoalteromonas and Vibrio in Leucosolenia sp., and Pseudovibrio and Spongiobacter in S. carnosus).

The authors summarised that in both sponge species, a high level of anti-microbial activity was found. Leucosolenia sp was found to produce more effective antifungal compounds, whereas S. carnosus appeared to contain more microbes with antibacterial properties. The researchers did struggle to isolate the compounds responsible for this activity in many cases however, meaning that the paper lends itself easily to further research. Overall, the findings from this paper may prove to be useful in future, particularly to the medical industry.

A review of Flemer B., Kenedy J., Margassery L.M., Morrissey J.P., O’Gara F. and Dobson A.D.W. (2011) Diversity and Antimicrobial Activities of Microbes from Two Irish Marine Sponges, Suberites carnosus and Leucosolenia sp., Journal of Applied Microbiology, 112, 289-301

Nitrification in the OMZ of the Arabian Sea (OMG!)

A review of: Newell SE, Babbin AR, Jayakumar A, Ward BB (2011) Ammonia oxidation rates and nitrification in the Arabian Sea. Global Biogeochemical Cycles, 25: 1-10.

A major rate limiting nutrient in the marine environment is nitrogen and oxidation of ammonia derived from organic matter is postulated to support 12-32% of primary production globally. The involvement of ammonia oxidising archaea (AOA) from the phylum Crenarcheota in nitrogen recyling was a relatively recent discovery. It was previously thought that ammonia oxidation was restricted to groups of β and γ proteobacteria (AOB), however more recent studies reveal AOA numbers that are considerably higher than AOB. The rates of archaea and bacteria ammonia oxidation in the oceans have not been quantified separately and archaea metabolism in the mesopelagic zone is yet to be fully elucidated. The Arabian Sea oxygen minimum zone (OMZ) is the largest in the world and the nitrogen cycle within this body of water has been the focus of many recent studies. The aim of the investigation by Newell et al (2011) was to explore the factors that influence the rates of ammonia oxidation and the abundance of AOA and AOB on the surface of the Arabian Sea, in low oxygen regions and in the mesopelagic zone below the OMZ.

Specific primers to identify the gene that encodes the ammonia monooxygenase enzyme (amoA) were used to estimate abundance of AOA and AOB using qPCR. Bacteria and archaea amoA genes were detected at every sampling depth in three locations, revealing AOA to be 35 to 216 times more abundant than AOB. Ammonia oxidation rates ranged from undetectable to 21.1 ± 0.1 nmol l^-1 d^-1; with the highest measurement near the surface layer (40 to 80 m in depth). The rates of ammonia oxidation within the oxycline (95-136 m in depth) above the OMZ were between 1.5 ± 0.2 and 4.6 ± 0.4 nmol l^-1 d^-1, while a low average of 0.07 ± 0.05 nmol l^-1 d^-1 was detected  in the mesopelagic zone (900 to 1000 m in depth)directly below the OMZ.

AOA appear to be the dominant ammonia oxidisers in the Arabian Sea, significantly contributing to nitrite production within the primary nitrite maximum (PNM) zone above 100 m and may be responsible for 10-100% of nitrite supply required to maintain the PNM. The role of ammonia oxidising Crenarchaeota in the deep ocean remains unclear however amoA gene abundance of 10⁵ copies ml^-1 at around 1000 m indicates a large population that substantially impacts nitrogen and carbon cycling. AOA nitrification in the photic zone provides recycled nitrate for primary production and may be a significant source of nitrous oxide (a greenhouse gas) which warrants further investigation. The role of AOA in OMZs and the metabolic pathways that sustain growth in hypoxic and anoxic conditions also requires elucidation, along with microbial diversity (via 16S gene analysis) and community dynamics. Furthermore, the abundance of amoA genes in the mesopelagic zone suggests approximately 50% of microbial biomass could be autotrophic and nitrification may be the main process that utilises most of the ammonium produced from organic carbon fluxes.         

Bioactive potential of Seagrass bacteria against human bacterial pathogens

The discovery of drugs from natural sources is an increasing trend. Researchers are searching for more novel chemical entities, in the hope they will provide new leads for disease treatment. The marine environment is particularly interesting for researchers because of the harsh physical and chemical conditions experienced by many organisms; yet despite this and the biodiversity of the oceans far outweighing terrestrial biodiversity research in this area remains in its infancy.
In particular, marine plants are known to produce a large number of structurally diverse secondary metabolites. Seagrasses are the only angiosperms to successfully grow in sub-tidal and tidal conditions. Several species of Seagrass have obligate microbial populations within their roots, leaves and rhizomes. Some medicines and chemicals are already prepared from Seagrasses and their associates.
An even greater concern than bacteria which are resistant to a single antibiotic is bacteria which are resistant to multiple antibiotics. As antibiotic resistance has developed, researchers have developed alternative antibiotics and combination therapies. However, the constant overuse of antibiotics in humans and their livestock has led to many bacteria being resistant to many antibiotics. The problem of resistance demands renewed efforts to seek antibacterial agents effective against pathogenic bacteria.
Two species of Seagrass from the SE coast of India were sampled and returned to the lab under sterile conditions, for the isolation of epiphytic and endophytic heterotrophic bacteria. 32 strains of endo/epiphytic bacteria were tested for their antagonistic activities against 5 antibiotic resistant human pathogens, of these, 10 were found to be antagonistic against one or more human pathogens. The authors go on to discuss the minimum inhibitory concentrations (MIC) as well as minimum bacterial concentrations (MBC) for the 32 tested strains.
The outcome of this research is that the endophytic bacteria isolated from the Seagrasses showed maximum sensitivity against several of the human pathogens compared with the epiphytic bacteria. And also that the bioactive compounds from the endophytic bacteria show maximum sensitivity with MIC than the bioactive compounds from the epiphytic bacteria.
From the outcomes of this research steps have been taken to find out the reason for the maxium activity of endophytic bacteria from Seagrasses.

There were two main reasons I choose to review this paper 1) The use of natural sources in the fight against humans pathogens is something that I find interesting and 2) In another module I have studies Seagrasses and like to see an over-lap/relation between modules. That being said, I found this paper very hard to read. It appears to me that the authors first language is defiantly not English, the paper did not read smoothly and the sentences seemed very disjointed, this I feel should have been addressed further. There were also seemingly simple mistakes made, for example species names were not italicised – I think these should have been picked up on. Simple changes would have made for a better read.
Despite this, the results of the paper are encouraging and the above mentioned points shouldn’t distract from the outcome. I hope that more antibiotics which can be used against both human and animal pathogens are found, as the problem of antibiotic resistance does appear to be rising.

A review of:
Ravikumar, S., Thajuddin, N., Suganthi, P., Inbaneson, S. J. and Vinodkumar, T. (2010) Bioactive potential of seagraa bacteria against human bacterial pathogens. Journal of Environmental Biology. 31 387-389.

Biofilms lead the way

Biofilms are thought to stimulate the attachment of invertebrates and algae to marine surfaces. This is known as biofouling, which refers to the accumulation of organisms and biogenic structures on marine surfaces. In particular sessile organisms compose the fouling assemblage. Many organisms such as barnacles and tubeworms produce shells and other firm structures during growth, which allow for the attachment of other organisms. This results in a multilayered fouling community. A central issue to the build up of biofouling on ships is an increases fractional drag, biofouling covers oceanographic equipment, coats floating structures and promotes structural deterioration. Biofouling costs industry billions of dollars per year due to prevention costs, maintenance and additional fuel consumption. Biofouling begins with the formation of biofilms followed by the aggregation of other diatoms and other micro-organisms bound together by extracellular polymeric substances. Biofilms can form within hours of immersion and rapidly increase in density and structural complexity. Furthering this, algae and invertebrates and aggregate. Biofouling is thought to be facilitated by the detection of appropriate sub strata and adhesion of larvae. It is thought that chemical cues play a pivotal role in invertebrate settlement. Microbial biofilms produce chemical signals that attract settlement. Antifouling research has provided information about the structure and function of biofilms, in particular with reference to understanding the adhesion and settling of invertebrate influence by microbial films. Zardus et al.'s research investigates the influence of microbial films on the adhesion of newly settled invertebrate larvae, they compared the removal rate of settlers from glass surfaces with and without natural biofilm coatings after exposure to controlled forces of shear. Their research was carried out on four marine fouling organisms: polychaete worm Hydroides elegans, barnacle Balanus Amphitrite, bryozoan Bugula neritina and a tunicate Phallusia nigra.

Larvae of the four invertebrate species were obtained from field collected adults and cultures were maintained in the laboratory following standard protocols. Trials were carried out in a turbulent channel flow apparatus and replicates of treatments were tested on the different invertebrates.

Larvae settlement was much greater on biofilm glass than on clear glass surfaces for Hydroides elegans, same was the case for Balanus amphitrite. Biofilms also had a positive effect on the adhesion strength for some of the settlement stages. Therefore bioflims increase invertebrates shear strength.

Adhesion of larvae is facilitated by microbial biofilms providing a connection between biofouling and biofilms. The mechanisms that cause this relationship require further research. Invertebrates use viscoelastic gels that have an adhesive nature in the presence of biofilms and form complex interactions, the adhesive strength is dependent on the taxa involved. It is suggested that biofilms may also stimulate increases and decreases in the adhesive produced by invertebrates, which has consequences for its structural strength. The trend observed was that with age invertebrates showed stronger adhesive forces. This discovery that biofilms facilitate invertebrate adhesion requires further research. Investigations are called for into the physical mechanisms of the biofilm invertebrate connection, paying particular attention on how these adhesives are modulated.

It is understandable that biofouling is undesirable for ships, however if structures such as marine renewable energy devices attract biofouling, this may have trophic food chain benefits supporting local biodiversity. This may be a desirable outcome provided biofilms don't cause any structural corrosion.

References:

Zardus, J. D., Nedved, B. T., Huang, Y., Tran, C. & Hadfield, M. G. (2008). "Microbial biofilms facilitate adhesion in biofouling invertebrates." The Biological bulletin 214: 91-98

Sorry Gastroenteritis. Theres just no relationship between us. From Entrococci

The purpose of this study was to evaluate the risk of exposing bathers to human pathogens in sub tropical recreational marine waters with non-point source of sewage and other pollutants. The authors also examined possible relationships between microbial densities and random symptoms in human subjects by questioning bathers at random and following up with microbial monitoring.

A group of regular adult bathers were recruited and divided into two separate groups : bathers and non bathers. Bathers were required to spend 15 minutes in knee deep water across the beach and submerge their heads every 5 minutes. Water samples were also collected by the bathers which were assayed for enterococci by membrane filtration. The non bathers group were not allowed to enter the water and were restricted to sitting on a plastic seat in a covered roped off area distant from water and sand exposure for 15 minutes at a time. This was followed up 7 days later with a phone questionnaire to determine if any of the bathers and non bathers had any illnesses.

From the water sampling it was determined that all bathers were exposed to an average of 71 enterococci/100ml of water however there was a very large range between samples. Symptoms reported after a week included gastrointestinal, skin, eye and ear infections which would normally be associated with exposure to contaminated bathing waters. Bathers reported more gastrointestinal, respiratory and skin infections compared to the non-bathers group and also reported a significantly shorter onset time of illness.

This paper confirms that people bathing are more likely to acquire infections from subtropical recreational marine waters compared to those that do not bathe even when there is no source point.

It was also found that the skin illnesses reported were dose dependant, as expected, however there was no relationship between the degree of exposure to enterococci and severity of the respiratory and gastrointestinal infections with the causative agents unknown. This could have further implications as, currently, many U.S. authorities monitor water systems using gastrointestinal illnesses as an indicator to water quality. Therefore, if there is no direct correlation between dose and outcome of gastrointestinal illness, the advice given by these authorities may not be completely accurate.

Although this was the first paper of its kind, all the participants were previously healthy adults and does not represent children or anyone with a compromised immune system.

A review of: Fleisher, J.M., et.al.; 2010; The BEACHES Study: health effects and exposures from non-point source microbial contaminants in subtropical recreastional marine waters; Int. J. Epidemiol.; 39(5): pp1291-98.