Human intestinal tract - the driving force of evolution of Vibrio parahaemolyticus?

Gastroenteritis obtained from undercooked or contaminated shellfish is a problem worldwide. One of the key pathogens that cause this infection is Vibrio parahaemolyticus. Theethakaew et al. (2013) studied the genetic and evolutionary relationships between isolates obtained in Thailand from clinical, human carriers and environmental sources. Since there is such a high consumption of seafood in Thailand, knowledge concerning this pathogen is of importance to allow the development of effective intervention strategies that prevent risk of infection of the public and economic costs to seafood producers.

Multilocus sequence typing (MLST) has been used in many other studies to analyse population genetics and molecular epidemiology of bacterial pathogens. This study used a modified version of MLST to identify potential sources of infection by analysing the population structure of V. parahaemolyticus. They were unable to find the most likely epidemiological sources of human carrier isolates, but determined that they were not always connected with seafood sources. There were five human carrier isolates that were identical to clinical isolates, indicating a genetic link between the two. Therefore human carriers may potentially transmit pathogens directly or indirectly to others or may contaminate seafood during production methods. However, 10 other human carrier isolates were also found, suggesting the human intestinal tract may act as a reservoir for novel V. parahaemolyticus strains.

A Bayesain analysis was also used. Although this was unable to differentiate between the isolates of the different epidemiological sources, they found the protein recA has an important role in the population structure of V. parahaemolyticus. Two divergent recA alleles were present. Nucelotide Blast analysis demonstrated these alleles to be closely linked to V. cincinnatiensis, a human bacterial pathogen and V. halioticoli, a bacterial pathogen found in the guts of the abalone mollusc, confirming these alleles had been obtained via horizontal gene transfer from other species. Other studies have also recorded a high diversity of recA in a range of Vibrio species, suggesting this is not a suitable marker for evolutionary analysis.

As recA has an intricate mosaic structure, intragenic recombination was also found to be an important aspect in the evolution of V. parahaemolyticus. Other species of Vibrio have likewise demonstrated intragenic recombination of recA, suggesting this may be a common source of recombination in this species. Since the study found human carrier and clinical isolates to contain mosaic recA proteins most frequently it could be suggested recombination occurs in the human intestinal tract more often than in the environment. Other studies investigating human carriers in seafood factories found a low number of carriers, however these individuals were found to harbour multiple strains of V. parahaemolyticus for a prolonged period of time. The existence of concurrent multiple strains supplies evidence for the exchange of DNA both within and between strains when in the intestinal tract, suggesting this may be an important driving force in the evolution of V. parahaemolyticus. Nonetheless, further studies are needed to determine the residence time of this pathogens in humans, the rate of horizontal gene transfer and ultimately the frequency at which new strains are evolving in order to develop successful intervention strategies of this pathogen.

Genetic Relationships of Vibrio parahaemolyticus Isolates from Clinical, Human Carrier, and Environmental Sources in Thailand, Determined by Multilocus Sequence Analysis

Chonchanok Theethakaew^a, Edward J. Feil^c,  Santiago Castillo-Ramírez^c, David M. Aanensen^d,  Orasa Suthienkul^e,  Douglas M. Neil^b and Robert L. Davies^a

http://aem.asm.org/content/79/7/2358.full?sid=46f4d79f-c54e-425e-b474-3221917da9b4

Arctic Wastewater Treatment (A review)

Wastewater typically goes through a three or four stage treatment process, however most Arctic regions, some of the most pristine environments, have either inadequate or completely lacking wastewater treatment. Wastewater can contain anthropogenic pollutants such as oil, grease, pharmaceuticals and personal care products as well as pathogenic microorganisms and parasites and antibiotic resistant bacteria, which may have even more severe consequences due to the low diversity and temperatures found in the Arctic. Gunnarsdóttir et al. (2013) reviewed the problems of the current lack of wastewater treatment processes as well as suggesting solutions to go forward with.

Gunnarsdóttir et al. (2013) state that bacterial pathogens, including enteric bacteria and indigenous aquatic bacteria, viral pathogens and protozoan parasites are commonly found in wastewater and sewage. Relationships between inadequate sanitation and higher rates of respiratory tract, skin and gastrointentestinal tract infections have previously been demonstrated in both the Arctic and other regions, as well as outbreaks of bronchitis, impetigo, ear infections, meningitis and hepatitis A and B. One of the primary concerns is that the combination of bacterial pathogens and pharmaceuticals may lead to the development of antibiotic resistant bacteria, as well as a higher incidence of antibiotic resistance genes found in hospital sewage anyway. Due to limited sunlight in the Arctic winter these genes are more likely to persist in the environment than in lower latitudes. There have also been incidences of zoonotic pathogens, such as Salmonella, which could have severe consequences if there were to pick up antibiotic resistance genes. They suggest a number of standard sewage treatment processes, some modified for the freezing temperatures of the Arctic regions, as well as suggesting that freezing could potentially be used in the treatment of wastewater.

This review is particularly useful to give an introduction to sewage treatment processes and a general overview of the issues of improper or lacking sewage treatment and highlights the importance of sewage treatment processes. A further study could look at areas before and after the implementation of adequate wastewater and sewage treatment as a comparison of the bacteria and viruses present.

Gunnarsdóttir, R., Jenssen, P., Jensen, P., Villumsen, A. & Kallenborn, R. (2013) A review of wastewater handling in the Arctic with special reference to pharmaceuticals and personal care products (PPCPs) and microbial pollution. Ecological Engineering. 50, 76-85

Will ocean acidification affect marine microbes?

Increasing quantities of carbon dioxide (CO2) in the atmosphere, combined with a decrease in dissolved oxygen (O) in the ocean as a result of temperature increases, is leading to theories of ocean acidification. If this is occurring, it is vital to understand how the functioning of microbes will be influenced due to their importance in marine productivity and planetary habitability. This review collected research surrounding this issue.

Ocean pH has never been constant as a result of natural fluctuations. These fluctuations depend on the location within the ocean in terms of depth and containment of water: deviations of 0.77 pH points have been found at 350m, and steep declines in pH occur within areas that are more contained, such as waters near estuaries. The pH levels in marine environments can fluctuate on a time-basis. Short-term fluctuations occur constantly as a result of gaseous exchanges of CO2 and O, however a majority is linked to microbial activity: high respiration to photosynthesis ratios cause accumulation of CO2, particularly in calm waters and during the night. Biological and physical processes (e.g. temperature), cause fluctuations over intermediate time periods, such as seasons. Although this suggests that natural changes still enable stability the terms of the microbial assemblage, the scale of this variation is almost 13 times lower than the permanent change predicted by 2300. There is evidence that once a pH change is initiated, it will increase expontentially as a result of the microbial communities: a phytoplankton bloom, initiated by an increase in CO2, will increase the concentration of CO2 further.

Of course these are only predictions, and considering numerous factors influencing pH, this may never become reality. One paper states that pH has not varied by more than 0.6 pH units for 350 million years, whilst another states a greater than expected change was observed in the central Pacific.

Research is conflicting in terms of how marine microbes will respond. Some clades are present across varying pH levels, however the species and function changes. For example, calcification rates of phytoplankton have been effected, however the direction of the effect is strain dependant.  Subtle variations in photosynthetic rates have been observed in species that do not have carbon concentrating mechanisms. The quantities of some bacteria have been shown to increase, such as phytoplankton. Steep ecological changes, including algal communities and invertebrates, have occurred alongside pH changes in estuaries.

It is possible for species, such as some representatives of the alphaproteobacteria and the gammaproteobacteria groups, to survive out of and within the core acidified zone: 200m – 4000m deep. I wonder what traits the species exhibit that enables them to survive? Considering organisms that live within the acidic zone live in close proximity to those that do not, and the ability of microbes to transfer genes, it would be interesting to determine at what point the genes for these traits were gained. Perhaps some microbes have a threshold point after reaching acidic conditions in which they must acquire the appropriate genes for survival from other microbes already present. Alternatively, these genes may be present yet not expressed until environmental stimulation is experienced.

In freshwater systems, some microbes persist through pH fluctuations of 2-3pH points daily. However, these microbes are adapted to these conditions, unlike those in marine environments, which are less adapted due to the higher buffering capacity, therefore lower natural variation levels of ~0.3 pH points.  This posed the question: Are the genomes of marine microbes flexible enough to allow them to acclimatize or can they accumulate new genes fast enough to enable them to survive ocean acidification? If so,  how will their function be influenced?

If the genomes of microbes in the water column are not adequate to allow said microbes to adapt, I wonder if this extends to symbiotic bacteria that are inherited directly from the parents, considering the hosts regulation of the internal environment inhabited by the microbes.

Answers to some of the questions posed could be gained through experiments using model marine microbes; comparing marine, coastal and freshwater systems that hold similar initial communities; and comparing stored isolates and fresh isolates.  The responses would be considering to pH changes. Due to problems revolving around culturability of marine microbes and controlling pH of media, a more valid approach is the comparison of the genes and gene expression. Another problematic factor is the number of confounding factors that occur naturally. Therefore laboratory experiments will need to be multi-factorial in order to represent a greater proportion of factors.

Although it is vital that the functional effects of altering the microbial communities as a result of pH changes are understood, considering the natural fluctuations over several time periods and locations it is debatable whether a permanent change will occur. Potential effects of this on microbial communities, and more importantly function, must be understood. Such understanding can be predicted more accurately through comparisons between organisms taken from ‘real’ environments as opposed to laboratory experiementation.

Joint I, Donay SC, Karl DM (2011). Will ocean acidification affect marine microbes? The ISME J.;5:1–7 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105673/

The use of Modelling to predict and assess concentrations of Enterococci sp. within the water column

For bacteria to survive within the marine water column, they must contend with several environmental factors: light, salinity, pH, Temperature and Hydrostatic pressure. In some cases bacteria enter into a ‘viable but not culturable’ (VBNC) state; the bacteria maintain a low metabolic rate and will not divide until induced by the presence of favorable conditions. This is a risky survival tactic. The dormant state makes bacteria are more vulnerable to threats; predation from surrounding phages. Some bacteria do not take the VBNC route and instead seek refuge and residence within the organic-rich sediments, settling and waiting for favorable conditions to then re-suspend into the water column.                                                                                    Gao et al. (2013) investigated the effect of the re-suspension of marine sediment bacteria: enterococci sp. on the bathing water quality in the Severn Estuary, UK. Currently the 2006 EU Bathing Directive regulates bathing water quality by using enterococci sp. as one of the  indicator organisms. Within this study the weather and tidal conditions were recorded. To test sediment-bacteria interaction a model was used, this was previously developed by the authors (Gao et al. 2011). The results indicated that concentrations of enterococci sp. were linked to sediment transport processes: deposition and re-suspension of particles (including that of marine sediment bacteria). The accuracy of this model was then tested. Using field measurements, the predictions of enterococci sp. concentrations in the Bristol Channel were subsequently supported.

This study was chosen as it demonstrates how accurate sophisticated modeling can be in quantifying the concentrations of potentially hazardous bacteria in coastal waters. The investigation also stressed the effect adverse weather conditions have on the dispersal of sediment residing bacteria. The conclusions from this study are particularly poignant as the new EU Bathing Directive plans to have all bathing waters reach a classification of ‘sufficient’ by 2015. These constructed models could play a significant role in rapid detection and monitoring of high bacterial concentrations of indicator bacteria. This would allow a swift implementation of public health warnings to bathers within affected coastal areas.                                                                                                                                                     Gao, G. Falconer, R. A. and Lin, B. (2013) Modelling importance of sediment effects on fate and transport of enterococci in the Severn Estuary, UK. Marine Pollution Bulletin. 67: 45-54.                                                                                    

Biofilm disruption by Laser Beams. Because we're all big kids at heart.

Biofilms cause many problems in many different aspects of life, both biotically and abiotically. They cause biodeterioration of useful structures leading to extensive maintenance costs, reduced efficacy of antibiotics in disease systems and dental plaques leading to tooth decay.  In disease systems, biofilm recalcitrance often means that antibiotic dosage needs to be 100-1000 fold higher in order to be effective, which is impractical as it would then be toxic to the host.

This article studies the effect of lasers on bacterial death and the disruption of biofilms. Using a Q-switched (pulsed output) ND:YAG (the crystal that emits the ionic activity of the beam) laser beam, the authors bombarded Staphylococcus epidermis biofilms grown on thin polycarbonate substrates, for a duration of <5ns with a peak stress of >50MPa at a wavelength of 1064nm (i.e. a very short but high powered beam). This beam specification was chosen due to earlier testing which showed that it is sufficient to kill bacterial lawns but not powerful enough to cause thermal damage to the surrounding (host) tissue.  

Using this methodology, the authors observed a 55% reduction in bacterial load in respect to control cultures, but state in the discussion that higher bacterial reduction can be achieved through stiffer backing plates, better positioning, and decrease in presence of air bubbles.

Personally I feel that this method is hardly going to replace toothbrushing, but shows promising application in clinical therapy and indeed, the authors conclude that the results of this study are the first steps towards a clinically viable therapeutic method in infected wound treatment, where antibiotics have proved ineffective. However it is important to note that, to my best knowledge, I cannot find many studies on the effects of this type of laser beam on human tissue, and due to the intense heat and ionising damage caused by the laser, it is difficult to refrain from immediately thinking of cancer. Nonetheless, the results of this study show the first steps towards the potential clinical application of lasers in biofilm disruption, which is heavily needed with the growing prevalence of antibiotic resistance. Moreover lasers could also have a potential usage in the disruption of biofilms outside disease systems.

REF: Taylor, Z.D., Navarro, A., Kealey, C.P., Beenhouwer, D., Haake, D.A., Grundfest, W.S., Gupta, V. (2010) Bacterial biofilm disruption using laser generated shockwaves. Conference Proceedings of the Engineering in the Medicine and Biology Society, 1028-1032.

ACCESSED FROM: http://www.ncbi.nlm.nih.gov/pubmed/21096997

Viruses in Sewage

One of the infectious causative agents of epidemic gastroenteritis are noroviruses, of which there are five genogroups, each including several genotypes; GGI (including Norwalk and Southampton genotypes), GGII (Hawaii and others), GGIII (Jena), GGIV (Alphatron) and GGV (found in mice). Inadequate or failing treatment of sewage leads to the insufficient removal of viruses and the discharge of this sewage may significantly enhance the concentration of viruses in the environment, potentially leading to virus-contaminated drinking or recreational water. Lodder & Husman (2005) investigated the contamination present in the Waal and Maas Rivers in the Netherlands and compared this to raw and treated sewage from the Apeldoorn pumping-engine station, Netherlands, and to norovirus stool sample specimens. They used molecular methods for enumeration and sequencing of the virus particles as well as cell culture for enumeration of plaque forming units. The River Waal is a tributary of the River Rhine and is composed of rain and melt water and approximately 30 million people depend upon it for drinking water, while the River Maas is made of just rainwater and approximately 5 million people depend upon it for drinking water.

Lodder & Husman (2005) found that each river was positive for the presence of F-specific and somatic phages, noroviruses, rotaviruses, reoviruses and enteroviruses and seven norovirus genotype strains were found out of a total of 38 clones. Four different norovirus genotypes were found in the River Maas; QueensArms, Mexico, Lordsdale and the Maas/Waal strain, while five norovirus genotypes were found in the River Waal; Southampton, Rotterdam, Lordsdale, Leeds and the Maas/Waal strain. The average virus concentrations were lower in treated sewage than raw sewage, except for rotaviruses, and virus removal at the treatment plant was 1.6, 1.1, 1.4, 1.3, 1.8 and 0.2 log₁₀ units for F-specific phages, somatic phages, enteroviruses, reoviruses, noroviruses and rotaviruses, respectively. Each sewage sample contained at least one strain of norovirus; the raw sewage samples contained six norovirus genotype strains while the treated sewage samples contained five norovirus genotype strains and Southampton, Mexico and Lordsdale genotypes were present in both.

This study highlights the need for more rigorous procedures aimed at removing viruses from sewage at treatment plants. Viruses can be effectively removed or inactivated by slow sand filtration and soil passage, however they are more resistant to UV and coagulation combined with sedimentation. This shows that consumption of drinking water or exposure to surface waters through recreation or shellfish could pose a risk if it coincides with failed treatment, however the screening period of the rivers was not extensive as it spanned only November through to April, when Noroviruses are known to be more prevalent so further investigation needs to determine whether the risk still occurs in the summer.

Lodder, W. & Husman, A. (2005) Presence of Noroviruses and other enteric viruses in sewage and surface waters in the Netherlands. Applied and Environmental Microbiology. 71, 1453-1461

An evaluation of bacterial source tracking of faecal bathing water pollution in the Kingsbridge estuary, UK.

In this study, Hussein et al. (2012) look at the microbial quality of water at South Sands, Salcombe. In the past, South Sands has experienced some faecal indicator bacteria (FIB) contamination; however it generally meets the requirements for European Union (EU) bathing waters. The beach is well known for human recreational activities.

Three samples of water and sediment from three locations were collected. One location was the beach; another was a pond just back from the beach and the last one a stream which runs into the sea. Human faeces were collected from healthy volunteers, whereas animal faeces were obtained from farms close to the study area. First of all membrane filtration was carried out, in order to detect and enumerate Enterococci, Escherichia coli and Bacteroides. DNA was extracted from the water and faeces samples for use in the Polymerase Chain Reaction (PCR), which was used to detect Bacteroides-Prevotella 16S rDNA gene in the water, sediment and faecal samples. Previously designed primer pairs were used.

The bacterial cultures showed that South Sands had ‘excellant’ values for FIB (Enterococci and E. coli) at all times of sampling. Water values of FIB and Bacteroides were higher in the stream than in the sea and higher still in the pond at all times. All sediment samples show a loading of both FIB and Bacteroides which also shows a significant increase out of the bathing season. The general Bacteroides primer set (Bac32F and Bac708R) confirmed that there were Bacteroides in all water, sediment and faecal samples. The human primer gave one positive result in the stream, while the cow primer gave a positive reaction with water and sediment from the stream and sediment from the beach. The horse and pig primers gave negative results.

This study revealed that the bathing water at South Sands was excellent based on the EU Bathing Water Directive 2006 for Enterococci and E. coli. The higher levels of bacteria in the stream and pond may be due to runoff from agricultural land, as it has previously been shown that this is a significant source of contamination. The increase in bacterial contamination in the sediment from the stream and pond, in comparison to the beach, is probably due to different sediment types, as the beach is composed of mobile sand. PCR was successful in showing although human faecal markers were not present on the beach; they were in present in the stream indicating a possible hazard for bathers, as the stream runs into the sea.

Overall, I think that this is a good study showing the successful use of microbial source tracking. More work needs to go into the specificity and persistence of Bacteroides markers in the environment, as this not known to any great degree. Once this has been confirmed, Bacteroides could be considered for a future EU Bathing Water Directive.

Hussein K.R, Bradley G, Glegg G. 2012. ‘An Evaluation of Bacterial Source Tracking of Faecal Bathing Water Pollution in the Kingsbridge Estuary, UK’. The Significance of Faecal Indicators in Water: A Global Perspective. Ed Kays and Fricker. Royal Society of Chemistry, UK.

http://books.google.co.uk/books?hl=en&lr=&id=5ZK9NHwmL7cC&oi=fnd&pg=PA1&dq=An+Evaluation+of+Bacterial+Source+Tracking+of+Faecal+Bathing+Water+Pollution+in+the+Kingsbridge+Estuary,+UK%E2%80%99.+&ots=pL2D_0zZ5k&sig=dDuRV0AxICspsF86n7jMIIdYWFc#v=onepage&q=An%20Evaluation%20of%20Bacterial%20Source%20Tracking%20of%20Faecal%20Bathing%20Water%20Pollution%20in%20the%20Kingsbridge%20Estuary%2C%20UK%E2%80%99.&f=false

Life’s a Beach: Testing the Waters Before you Dive In!

Bathing in the sea at beaches which are not impacted by known point source pollution is a growing concern worldwide. Globally, over 120 million cases of gastrointestinal disease and over 50 million cases of respiratory diseases per year are sourced back to bathing in wastewater-polluted coastal waters. The authors of this paper evaluated the presence of pollution indicator microbes at a bathing beach in South Florida known to be impacted by non-point sources of pollution by taking twelve water samples and eight sand samples and analysing them. EPA (the Environmental Protection Agency) recommend the microbial indicator species Enterococci, as it shows a correlation between its abundance and cases of illness at marine bathing beaches impacted by point source pollution. However, this correlation is not consistant with Enterococci abundance and marine bathing-related illnesses on beaches impacted by non-point source pollution, which lead the authors to question the true extent of how well Enterococci works as an indicator species for predictions of presence of pathogens.

The chosen experimental location was a beach in Miami-Dade County, Florida, where background descriptions state that dogs are allowed on the beach year-round, and that after an extensive search, there are no point sources of pollution. The beach is admission free, and busy in summer months. It is open for the vast majority of the year with the exception of around 4 days a year where it is recommended that bathers do not enter the water due to an increase in pathogens in the water, violating water quality legislations. Water samples were collected in 20 litre sterile containers, and sand samples were collected in the upper 2.5cm and placed into sterile Whirlpak bags; wet sand samples were collected from the intertidal zone, and dry sand samples from above the high tide line. The sand samples were placed in containers filled with 10 litres of phosphate-buffered saline, and this was mixed vigorously. The supernatant from the sand samples along with the seawater samples were then analysed, and the microorganisms were identified and enumerated. Environmental conditions such as weather, temperature, wind speed, water turbidity and the presence or absence of bathers and dogs were also taken into account.

The results of the study revealed that using Enterococci as an indicator showed a variety of results depending on which method was used to enumerate the bacteria. This is important when considering the uses of Enterococci as an indicator species at bathing beaches as differences in enumeration could lead to the unnecessary closure, or the unsafe opening of bathing beaches.Spatial and temporal variations in the area of study also show a difference in enumeration, which could also lead to different management decisions at bathing beaches. Presence of faecal indicator bacteria in the water and the sand samples were predominantly consistent with the Enterococcus results, showing the possibility that the environmental conditions which affect Enterococci also affect the faecal indicator organisms. The pathogens impacting the water is highly intermittent depending on illnesses carried by humans and animals such as dogs contributing to the pollution source entering the beach. Finally, the environmental conditions recorded during water and sand sampling periods show that on the sunnier days where sampling took place, there were less microbes as the UV is known to inactivate them. Sea temperatures and tidal height also played a part in the fluctuation of detection of microbes.

I enjoyed reading this paper as it was interesting to see how beaches which are not directly affected by point source pollution are affected by non-point source pollution from other areas.

Abdelzaher, A. M., Wright, M. E., Ortega,C., Solo-Gabriele, H. M., Miller, M., Elmir, S., Newman, X., Shih, P., Bonilla, J. A., Bonilla, T. D., Palmer, C. J., Scott, T., Lukasik, J., Harwood, V. J., McQuaig, S., Sinigalliano, C., Gidley, M., Plano, L. R. W., Zhu, X., Wang, J. D. & Fleming, L. E. (2010) Presence of Pathogens and Indicator Microbes at a Non-Point Source Subtropical Recreational Marine Beach. Applied and Environmental Microbiology. 76 (3): 724-732.

Vibrio cholerae exploits sub-lethal concentrations of a competitor-produced antibiotic to avoid toxic interactions

Vibrio cholerae exploits sub-lethal concentrations of a competitor-produced antibiotic to avoid toxic interactions

Vibrio cholerae a gram negative bacterium known to cause the disease cholera is transported in to the water column and human food mainly via detritus and planktonic organisms. These pathogens are shown to be inhibited by Vibrionales bacterium SWAT3, an exceptionally broad range inhibitor of other pelagic marine bacterial pathogens. It is able to achieve this pelagic inhibition via the secretion of the antibiotic andrimid.

This paper looks at the reaction pattern of V.cholerae to different concentrations of Andrimid, and to what levels pathogen can demonstrate avoidance behaviour before the antibiotic presence becomes lethal and the cells succumb to toxic concentrations.

The Vibrionales bacterium SWAT3 is also known to be a competitor for the same resources as the V.cholerae, this particle colonising competitor and antibiotic producer influence heavily the micro scale distribution of bacterial species. The study by Graff et al (2013) has shown V.cholerae colonising ability to be inhibited in the presence of SWAT3 to be statistically significant.  The antibiotic produced by the SWAT3 blocks the carboxyl-transfer reaction of acetyl-CoA carboxylase which the Andrimid producers show resistance.

In this study by Graff et al (2013) a growth inhibition and minimal inhibitory concentration of andrimid in agar was carried out, this showed that V.cholerae wasn’t able to colonise agar that contained SWAT3, but agar plates containing  SWAT3-III (a non-antibiotic producing mutant of SWAT3) the presence of V.cholerae was observed, demonstrating the effectiveness of SWAT3 Andrimid production on growth inhibition of these pathogenic marine bacterium.

In order to hypothesise whether andrimids function and interaction can work as chemical signal that deters V.cholerae from colonising particles; a chemotaxis assay assay was used to quantify swimming behaviour (speed and turning rates) by use of polydimethylsiloxane (PDMS) microchannels. In natural marine environments Andrimid produced by SWAT3 would act as “interspecific signalling molecules and deter particle colonisation by V.cholerae”.

Results demonstrated that swimming speeds, when exposed to sub-lethal levels of andrimid, were initially increased as a response to the inhibitor, but in lethal doses speed decreased suggesting the pathogens inability to cope, and succumbing to these toxic concentrations. A significant shift of trajectory was seen between control media and the andrimid treated media (P = 0.0006) but no significance was seen when subjected to SWAT3-III (P = 0.18).

This study was interesting in the fact that it looks at a bacteria V.cholerae, a pathogen effecting not only the marine environment but humans as well. The introduction of andrimid in to its life cycle inhibits its growth and demonstrates avoidance behavior. Such chemically mediated cell– cell interaction has direct implications for elemental cycling in the ocean as well as the spread or outbreak of diseases. This interaction has the ability to control harmful algal blooms and could aid useful future applications.

Graff, J. R., Forschner-Dancause, S. R., Menden-Deuer, S., Long, R. A., & Rowley, D. C.. 2013. Vibrio cholerae exploits sub-lethal concentrations of a competitor-produced antibiotic to avoid toxic interactions. Frontiers in microbiology, 4.

Microcosms to Study Climate Change

Microcosms to Study Climate Change

As discussed in previous blogs, climate change has the potential to change marine microbial communities. A change in microbial communities could in turn affect oceanic and truly global processes such as carbon and nitrogen cycling. A lot of previous work has focussed on long term microbial surveys and correlations with sea surface temperatures. So far this work had been somewhat convincing as strong correlations and patterns are clear to see, in particular with regard to increased temperature and vibrios. However, correlations are not causations, and as we’ve discussed climate change per se is multifaceted and complex. Krause et al. (2012) were particularly interested in ocean acidification, an element of climate change which is only just beginning to be researched. Ocean acidification describes the process of increased dissolution of CO₂ into the sea; CO₂ then disassociates into bicarbonate, carbonate, carbonic acid and hydrogen ions, overall reducing pH. Since the beginning of the industrial period, the oceans have taken up approximately one-third of anthropogenic CO₂ emissions which has already lead to a reduction in pH of 0.1 units. Given that oceanic pH has remained above 8.1 for the last 23 million years, recent and predicted ocean acidification is causing concern amongst many marine biologists.

Krause et al. (2012) specifically aimed to investigate the direct effects of reduced pH on microbial communities. The study consisted of highly-replicated (n=5 microcosms per treatment) microcosm laboratory acidification experiments with a natural bacterial community taken from the North Sea. Seasonal variability was accounted for by repeating the experiment in spring, summer, autumn and winter. An interesting dilution approach was used to select for different ecological strategies, i.e. oligotrophic vs high nutrients. Three pH levels were investigated:  “in situ” (pH 8.12-8.22) was the current day control, pH 7.82 and pH 7.67 represented two predicted scenarios for the North Sea by 2100 and were the “ocean acidification” treatments. After four weeks in the microcosms the community structure was analysed using automated ribosomal intergenic spacer analysis (ARISA) and 16S ribosomal amplicon pyrosequencing. Overall season and dilution treatment had the biggest effect on community structure, however pH was also responsible for some of the changes. The response to reduced pH was context-dependent, i.e it was different in different seasons and dilution treatments, and pH did not affect the abundance of microorganisms.

The reviewed paper represents the first highly-replicated, statistically convincing evidence that projected ocean acidification will change bacterial community structure. Unlike previous surveys, the high amount of control in this experiment allows true causation to be assigned to ocean acidification. Whilst the controlled microcosms allowed for a tentative causal relationship to be established, it should also be noted that they didn’t truly replicate what is likely to occur in the field. Firstly microcosms were kept in the dark, secondly the seawater was acidified using HCL rather than CO₂ (resulting in differences in alkalinity) and thirdly many microorganisms were filtered leaving only the bacteria. The issue of environmental relevance however does not detract from the main finding that reduced pH changes bacterial community structure. Furthermore the highly controlled experiment reviewed here can now be integrated into the current survey data to increase our overall understanding of the biological affects of ocean acidification.

Krause, E., Wichels, A., Giménez, L., Lunau, M., Schilhabel, M. B., & Gerdts, G. (2012). Small changes in pH have direct effects on marine bacterial community composition: a microcosm approach. PloS one, 7(10), e47035.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0047035

 

 

 

 

 

 

Could Phage Therapy be Effective Against Coral Disease?

The dramatic, global deterioration of tropical coral reefs has been connected to anthropogenic stressors such as poor water quality (including sedimentation, nutrification and pollution), direct physical damage (e.g. dynamite fishing), overexploitation of keystone species and elevated sea surface temperatures (SSTs).  Such factors are correlated to increasingly widespread and severe incidences of coral disease.  Vibrio spp. have been linked to temperature dependant bleaching and tissue necrosis in many species of tropical corals.  For example, the putative coral pathogen Vibrio coralliilyticus has been implicated in the coral disease ‘White Syndrome’ (WS), in which tissue necrosis is preceded by the loss of Symbiodinium.  Previously, it has been shown that V. coralliilyticus causes inactivation of Symbiodinium photosystem (PS) II via production of the proteolytic enzyme Zinc-metalloprotease, leading to inhibition of photosynthetic activity (and subsequent loss of Symbiodinium).  Such virulence is thought to be multifactorial, dependant on the general structure of the host microbiota (e.g. Symbiodinium susceptibility varies via clade) and temperature (which contributes to host density and Zn-metalloprotease production).

                Cohen et al. (2013) were interested in the potential for phage therapy treatment of V. coralliilyticus-based disease outbreaks on the Great Barrier Reef (GBR), and began by isolating a bacteriophage infectious to the V. coralliilyticus strain P1 (previously isolated from diseased corals).  The phage (named bacteriophage YC) was isolated from the waters around Magnetic Island on the GBR, where strain P1 was also originally isolated.  A series of enrichments and plaque assays was conducted, producing a high titre, pure phage stock.  The morphology of phage YC was observed as consistent with the Myoviridae family (indicated via Electron micrograph images), and the rate of phage absorption on to strain P1 was determined to be rapid (ascertained by adsorption kinetics experiments).  In order to test how phage YC might influence the affect of strain P1, the authors grew up various concentrations of strain P1, both without the addition of phage YC, and with phage added after 0, 2, 8 and 18 h (after the beginning of bacterial growth), and treated Symbiodinium cells and coral (Acropora millepora) juveniles with filtered and centrifuged ‘P1 supernatant’.

                The authors observed that Symbiodinium treated with P1 supernatant derived from bacterial cultures incubated with YC phage demonstrate significantly less photosynthetic inhibition (reduced quantum yield values), compared to Symbiodinium treated with P1 supernatant derived from bacterial cultures to which YC phage had not been added (>90% reduction quantum yield), suggesting higher PSII inactivation in this treatment.  Similar results were observed from the coral juveniles.  Expulsion of Symbiodinium, tissue lesions, and eventual total tissue loss coincided with quantum yield measurements of zero, 9 h after treatment with P1 supernatant produced without the addition of YC phage.  Conversely, in juveniles with P1 supernatant derived from bacterial cultures with YC phage administered after 2 h of growth, no change in appearance or signs of Symbiodinium expulsion were observed, and no decline in quantum yields was recorded.  However, juveniles treated with P1 supernatant derived from bacterial cultures incubated with YC phage added after 8, and 18 h of growth suffered a very similar fate to juvenilles treated with P1 supernatant produced without the addition of YC phage (i.e. total tissue loss etc).

This investigation is unusual because: a) the authors applied their isolated phage to the putative pathogen before applying it to the Symbiodinium/coral juveniles; b) they actually removed bacterial cells before applying the P1 supernatant to the Symbiodinium/juveniles.  Previous phage therapy investigations have generally added both the putative bacteria and the phage to the corals, either together (Efrony et al., 2006), or with an interval before adding the phage (Efrony et al., 2009).  Significantly, the high densities of V. coralliilyticus employed in this investigation have been recorded in the coral host, rather than in seawater, yet in this investigation the role of the host in bacteria-bacteriophage interaction has been excluded.  Additionally, it is hard to imagine a scenario where the coral host would experience only the secondary metabolites of a pathogen, rather than the bacterium itself. For these reasons, it appears that the relevance of these findings to what may occur in the field may be questionable.    

Cohen et al. (2013) conclude that as Zn-metalloprotease production is known to occur at high V. coralliilyticus densities, and because the addition of phage was observed to cause bacterial lysis, the results they observed were due to the presence/absence of Zn-metalloprotease expression in consequence of the presence/absence of phage activity.  Perhaps the removal of bacterial cells in this investigation was undertaken in order to provide evidence for the role of phage in reducing Zn-metalloprotease production.  This might be investigated further with the application of some sort of Zn-metalloprotease assay.  It appears that, although the authors have shown that the phage they isolated was effective against strain P1, more work needs to be undertaken before the relevance of this finding to the prevention of coral disease on the GBR can be ascertained.

Cohen, Y., Joseph Pollock, F., Rosenberg, E. & Bourne, D. G. (2013) Phage therapy treatment of the coral pathogen Vibrio coralliilyticus. MicrobiologyOpen 2: 64–74.