Potentially Human Pathogenic Vibrios in Marine and Fresh Bathing Waters Related to Environmental Conditions and Disease Outcome

Vibrios are more common in water temperatures over 17-20°C. They can survive in a range of adverse environmental conditions, in order to do this they enter a viable but non-culturable stage in which metabolic activities are minimal but they still retain pathogenicity. Several vibrio species are human pathogens and have been associated with wound infections (V. vulnificus) and ear infections (V. alginolyticus) after exposure to contaminated water, and gastroenteritis (V. parahaemolyticus and V. cholera) after consumption of contaminated food.

During the summer of 2006, four people developed V. alginolyticus infections after swimming in a large inlet on the North Sea, at separate but nearby locations. The water was then tested and both V. alginolyticus and V. parahaemolytis were found in the water samples. This study is follow up to previous finding of vibrio species in North Sea inlets. The quantification and typing of potentially human pathogenic vibrio species were looked at, as these areas had not been studied before. Vibrio numbers in bathing water were related to environmental conditions, for example water and salinity, and to European legislative requirements for bathing water quality.

In this study, Schet et al monitored four bathing sites in the Netherlands; samples at the bathing sites were taken biweekly or four-weekly from April to October. It was found that vibrio species were detected at all sites. There were 447 isolated vibrios from water samples found. 50.6% of these were V. alginolyticus and 8.5% were V. parahaemolyticus. The water temperatures ranged from approximately 10 to 21°C, no vibrio were detected in samples when the water was below 11°c. In the majority of the samples, faecal indicator levels were below the mandatory values for good water quality according to the European Bathing Water Directive. There was a correlation found between vibrio and E. coli concentration, however it was not a strong correlation. An even weaker correlation was found between intestinal enterococci and vibrio. Throughout the period of the study, only one case of a bathing water related vibrio infection was reported, swabs were taken and 2 V. clorae isolates were cultured from the wound.

Overall, potentially pathogenic vibrio species were detected, however lower numbers of vibrio were found than expected. This is due to colder bathing water temperatures than normal compared to other data looked at. This may be something to look at in the future and do the same experiments during another summer. The results underlie the need for further molecular studies on a larger number of clinical isolates and isolates from water samples to understand clonal distribution with seawater V. chloerae isolates in relation to bacterial pathogenicity, water salinity or environmental parameters. This study highlighted the need for education of public health workers, bathing water managers and the general public in order to prevent vibrio infections from bathing water exposure.

I chose to review this paper as I am interested in what actually is in bathing water; I was surprised to find these results especially as I have been swimming where some of the samples were taken from! I think that more studies need to address water quality in terms of vibrio content, especially as water temperatures could raise due to global warming and therefore have more favourable growth conditions for vibrio species.

http://www.sciencedirect.com/science/article/pii/S1438463911000526

F.M. Schets, H.H.J.L. van den Berg, A. Marchese, S. Garbom, A.M. de Roda Husman (2011). Potentially Human Pathogenic Vibrios in Marine and Fresh Bathing Waters Related to Environmental Conditions and Disease Outcome. International Journal of Hygiene and Environmental Health. Volume 214, Issue 5, September 2011, Pages 399–406.

How prey bacteria shape the community structure of their predators:

Predator and prey interactions have been noticed and studied since as far back as anyone can remember, unfortunately for microbiologists there havent been many studies as far down as microbes. This is due to numerous reasons; the most obvious being that it just isnt as easy due to the sheer range in sizes of them and that most microbes are minute, so compared to studying fish the difficulties are easy to spot.

Thankfully Huan Chen et al, decided to look at Bacteriovorax and how its prey; Vibrio vulnificus & Vibrio parahaemolyticus affect its population structure and number. The method that was used was to have flasks full of seawater and a specific concentration of the Bacteriovorax and either the V.vulnificus or the V.parahaemolyticus within the same flask as the predator and to monitor the abundance of the prey at specific time periods after the two were added together.

The results shown by this study showed that in the presence of a ready supply of prey a significant multiplication of abundance was demonstrated and this increase was extremely quick, the exact speed was a thousand fold increase within 24hours, from the 24 hour time mark to the 48 hour mark.  This increase led to a 2-4 fold log of prey reduction. Along with this it was shown that the Bacteriovorax had a faster predation on the V.p compared to the V.v. There were two clusters studied to see if there was a preferential prey for the predator and what was found was that V.v was preferential in one and V.p in the other, so although the rate of predation may have been quicker, given a choice of one or the other there isnt a favourite. Aswell as this as expected the paper showed that with an increase in prey this led to the standard predator-prey fluctuation where then predator levels increased and prey abundance decreased.

The reason why this paper is important and interesting is due to the fact that it is the start of a hopefully increasing area of study and it provides a backbone for the future studies. So it's important to the ever growing field of marine microbiology.

I hope more studies are done like this as it's always interesting to study predator and prey interactions and as there hasnt been much in this field it will be interesting to see just how far down the trophic scale predation plays a major part.

Here is the webpage if you want to read this paper:

http://www.nature.com/ismej/journal/v5/n8/pdf/ismej20114a.pdf

Huan Chen, Rana Athar, Guili Zheng and Henry N Williams

ISME J 5: 1314-1322; advance online publication, February 17, 2011; doi:10.1038/ismej.2011.4

Marine microalgae attack and feed on metazoans

The marine food web has been shown to be severely affected by mixotrophic dinoflagellates, with microalgal blooms causing the death of fish, copepods and other metazoans. Karlodinium is an example of a genus of toxic microalgae that form such blooms in eutrophic coastal waters. They work by releasing neurotoxins that stun their prey before feeding via myzocytosis. This investigation focused on the species Karlodinium armiger and Karlodinium veneficum and their effects on the motility and mortality of the copepod Acartia tonsa and other metazoans. Karlodinium veneficum is a well known ichtyotoxic bloom, which produces Karlotoxins to stun prey before ingestion. Karlodinium armiger is also believed to work in a similar manner, however has not been studied as extensively, so the specific neurotoxin used is currently unknown.

The interactions between the microalgae and copepods were observed using an inverted microscope. Immobilisation was characterised by copepods lying on the bottom of the microwell displaying erratic movements but with gut movements still occurring, and death was identified when gut movements stopped. Shortly after adding the copepods to the culture, Karlodinium armiger was attracted to them and first began to attach to the antennae and telson. These are important in sensing hydrodynamical disturbances in the surrounding area, which copepods then use to determine between prey and predator by size. As Karlodinium armiger is of a much smaller size in comparison, this suggests it is disguised as prey, allowing the attack of the copepod to occur whilst remaining unnoticed.

After 135 minutes Karlodinium armiger was shown to immobilise nearly all the copepods. Within 24 hours all copepods were dead and surrounded by swarms of Karlodinium armiger with feeding tubes attached. After 24 hours the copepods with the Karlodinium veneficum strains were alive and healthy, therefore further experiments used only Karlodinium armiger. The experiment was repeated with adult nematode, trochophore and late stage polychaete larvae, all of which experienced immobilisation and mortality, suggesting Karlodinium armiger affects a wide range of metazoans. Further experiments concerning cell density demonstrated that when Karlodinium armiger was below 1100 cells ml^-1 copepods were unaffected, however at 3500 cells ml^-1 they were immobilised and killed. As field studies have reported densities of Karlodinium armiger and Karlodinium veneficum between 10,000 and 100,000 cells ml^-1 in coastal areas, this suggests microalgal feeding is likely to have detrimental effects on metazoans.

Although Karlodinium armiger contains chloroplasts, when relying on only phototrophic growth, growth rates are very slow. However, feeding on prey such as copepods provides important growth factors and also stimulates photosynthesis, meaning consuming even a small amount of prey can result in a significant increase in growth rate. This was demonstrated in the 3500 cell ml^-1 cultures of Karlodinium armiger that contained copepods by an increase in 85% of the population growth rate in comparison to those without copepods.

This investigation has provided valuable insight into the flexibility of feeding exhibited by Karlodinium armiger. The fact that this species can consume a wide range of metazoan suggests competition for a variety of food sources may occur across many trophic levels, which could result in the disruption of the structure and function of the marine food web.  However, further studies are needed to establish the specific toxin produced by Karlodinium armiger and also into the behaviour of other such harmful species in order to reveal which organisms are most vulnerable.

Terje Berge, Louise K Poulsen, Morten Moldrup, Niels Daugbjerg and Per Juel Hansen

The ISME Journal 6, 1926-1936 (October 2012) | doi:10.1038/ismej.2012.29

http://www.nature.com/ismej/journal/v6/n10/full/ismej201229a.html

The “Cheshire Cat” escape strategy of the coccolihophore E. Huxleyi in response to viral infection. Frada et al (2008)

The theory of an ongoing evolutionary arms race that is taking place in biological systems has long been established. This is the idea that organisms develop defence mechanisms to out run, outwit or out perform their predators, and subsequently predators evolving strategies to overcome this. A similar dynamic can be observed in the relationship between organism and pathogen in cases of infection. This association has been described as the Red Queen dynamic due to the similarity with the Alice in Wonderland novel in which Alice is constantly running, yet remaining in the same place. However in some species this trend is questionable, for example in Emiliania Huxleyi there are no geographical subpopulations of host or virus; if local viruses only infected local hosts, geographical speciation would soon occur. This trend is not seen in E. huxleyi and its relationship with a giant phycodnaviruses called Emiliania huxleyi Virus (EhVs), as a virus strain isolated from the North Atlantic has been shown to be capable of easily infecting host cells originating from the Mediterranean Sea.

The coccolithophore E. huxleyi is one of the most successful eukaryotes in modern oceans and its blooms can be seen from space. This success can be attributed to a stage in its life-cycle which has exceptionally high phosphate uptake and very low photoinhibition of photosynthesis. At the end of its life-cycle these blooms are eradicated by a virus specific to E. huxleyi. This begs the question of why has little to no ‘evolutionary investment’ gone into a defence mechanism against this virus?

This paper shows that E. huxleyi occurs as a haplodiploid organism in which its lifecycle alternates between the two ploidies. The massive blooms occur as a calcified, coccolith-bearing diploid phase which can profoundly impact global biochemical equilibria, and a non-calcified flagellated haploid phase. This sexual cycling is temporarily separated by an as of yet unknown cause, however this paper hypothesises that viral infection may trigger meiosis and induces a shift from diploid to haploid. This viral escape mechanism has been described as a “Cheshire Cat” method of survival, due to a continuation of the Alice in Wonderland theme in which the Cheshire cat makes its body invisible in order to escape beheading. The susceptibility of diploid-stage and haploid-stage cells to EhVs was tested over a 50 day period and it was found that none of the haploid strains were sensitive, compared to a 100% infection rate of the diploid cells. From this it has been hypothesised that even if a diploid stage bloom is virally eradicated, the motile haploid stage is invisible to the virus and can therefore enable the continuity of the species.

As eukaryotic marine protists are responsible for nearly half of global primary productivity and carbonate production, further research in this area is essential to understand the genomic control and method of action of this escape strategy as this significantly advance assessment of marine eukaryotes on biogeochemical cycles and further reveal details of the control of the flux of matter between the atmosphere and the lithosphere.

Studying marine virus communities and distribution in their natural communities.

Marine viruses influence host community composition, however, the effect of viruses on communities of phytoplankton is sparsely studied. Natural algal-virus communities which infect and lyse marine primary producers are an abundant and active part of the marine ecosystem.

 

 To understand the dynamics & effects of phytoplankton viruses, the genetic composition of virus communities needs to be understood. Specifically viruses infecting the marine phytoplankton Micromonas pusillla lead to the development of the degenerate algal-virus-specific (AVS) PCR primers AVS1 and AVS2, which amplify a 700 base pair fragment of algal-virus DNA polymerase genes.

 Phylogenetic analysis of amplified DNA polymerase fragments showed that cultured algal-viruses formed a monophyletic group, compared to double-stranded DNA viruses.

 Algal-virus specific primers were used to amplify unknown algal virus DNA polymerase fragments from natural virus communities, showing that algal-virus diversity can be studied using molecular techniques. 

 DGGE analysis of PCR products that have been amplified bu AVS PCR primers can be used to examine natural algal-virus community diversity, for example, the banding patterns (fingerprints) in this experiment of samples taken from one location (Salmon Inlet) were identical and easily distinguished from other fingerprints; these samples were collected at the same time and location, but at different depths. However, the temperature and salinity at this site did not vary much with depth, indicating that the water and viruses were mixed well.

 On the opposite end of the spectrum, samples taken from a different location (Pendrell Sound) have differing banding patterns at the different depths. This is due to the salinity at depth being double that of surface salinity, meaning that the water and viruses here were had stratification.

 The DNA polymerase sequences derived from the PCR and DGGE were closely related to known algal-viruses, and remarkably, some 98% of sequences from samples taken at the Southern Ocean were identical to those from coastal British Columbia, despite how different the environments are.

 This paper showed how PCR and DGGE can be used to recover and identify unknown algal-virus DNA polymerase sequences from the natural environment, and that similar sequences can be recovered from different areas worldwide. 

 I found this paper interesting as it shows how the curiosity sparked by viruses infecting a specific species of marine phytoplankton lead to the development of new primers, and then onto being able to study marine virus community diversity in natural communities.

Sequence analysis of marine virus communities reveals that groups of related algal viruses are widely distributed in nature by Steven M. Short and Curtis A.Suttle, 2002

...here's the link if you'd like to read it http://ukpmc.ac.uk/articles/PMC123764/pdf/1471.pdf

Factors influencing viral distribution and abundance along a latitudinal transect of the North Atlantic Ocean at different depths.

It is estimated that there are 10³⁰ viruses in the ocean (approximately 10 times as many as bacteria). Viruses are not just abundant in number they are also genetically diverse.  They influence the composition of marine communities and play an important role in biochemical cycles. Moreover viruses control microbial mortality and may help maintain diversity.

The study by De Corte et al. (2012) looked at the factors controlling viral distribution, abundance and production to assess the potential variations in the relationship between viruses and prokaryotes across a latitudinal transect in the North Atlantic Ocean.

This study is important because currently most published material on virus-prokaryote interactions in marine environments focus on near shore waters whereas in this case a wider area over 4 North Atlantic provinces in different pelagic zones are used.  

Many factors may influence distribution of viruses throughout the ocean but in the end viral distribution depends on the availability of suitable host therefore both prokaryote production and abundance were measured as well as viral production and abundance. The aforementioned factors were assessed throughout the water column and at different depth to determine the potential variations in interactions. Samples were obtained from 24 depths at 33 stations from 5 different pelagic zones. Prokaryote and viral abundance was measured using flow cytometry standard procedure. Samples were stained with SBYR Green I (a fluorescent dye) after being shock frozen with N₂. Viral production was measured using the dilution approach. Prokaryote production was measured using an assay for radioactively labelled leucine incorporation. Radio activity of the samples and a blank were measured and after basic calculations the results were converted into the incorporation rate.

Results showed a significant decrease in abundance of viruses and prokaryotes with depth over all stations (abundance was negatively related to depth) Prokaryote production also decreased with depth. Lytic viral production decreased and lysogenic production stayed the same. Multivariate regression analysis was used to predict the factors explaining the variability of viral abundance between the different depths. Taking the whole data set into consideration the variation was mainly explained by prokaryote abundance, temperature and latitude which together accounted for 73% of total variation. Prokatyote abundance alone accounted for 46% of the variation in abundance.  Temperature and prokaryotic abundance were seen to be the main factors influencing the differences in viral abundance between the 4 provinces, 2 mainly influenced by temperature and 2 by viral abundance. Based on the above results by De Corte et al. (2012) concluded that virus-host interactions significantly change in different oceanic provinces in response to biological factors i.e. host availability but also to chemical and physical factors in the environment.

De Corte, Daniele Sintes, Eva Yokokawa, Taichi Reinthaler, Thomas Herndl, Gerhard J (2012) Links between viruses and prokaryotes throughout the water column along a North Atlantic latitudinal transect. The ISME Journal (2012) 6, 1566–1577.

http://www.nature.com/ismej/journal/v6/n8/full/ismej2011214a.html

Species-Specific Bacterial Symbionts in Hydra: Are Particular Microbial Communities Selected for and Maintained by the Host?

Cnidarians have a simple tissue grade organisation with a limited number of cell- and tissue-types, and mucus as the sole physical barrier between epithelial tissue and a habitat now known to support large numbers of microbes, including potential pathogens.  Without the benefit of defensive phagoctytes, what evidence exists for the selection of particular symbiotic bacteria within the Cnidarian surface tissue epithelium?

Interested in the evolution of metazoan microbial communities, Fraune and Bosche (2007) compared the microbiota of two closely related basal metazoan species: Hydra oligactis and Hydra vulgaris.  In order to examine microbiota evolution over time, hydra polyps were obtained both direct from the wild, and from cultures maintained in laboratories for over 30 years.  Microbial communities were surveyed via various means, including molecular methods (Restriction Fragment Length Polymorphism (RELP) analysis of 16S rRNA), Transmission Election Microscopy (TEM), phase contrast microscopy, and Fluorescent In-Situ Hybridisation (FISH).

RELP patterns indicated that the microbial communities of the two Hydra species were markedly different:  almost all lab-cultured H. oligactus samples shared one RELP pattern, whereas 16 different patterns were observed in H. vulgaris samples; this is a surprising result when considering the uniformity of living conditions (e.g. food, temperature) which these individuals experienced over the proceeding decades, and the authors cite selective constraints as being the probable cause (although care should be taken when interpreting such results, see below).  This result was echoed when wild polyps of both species where analysed: one dominant RELP pattern observed in H. oligactus samples (though different to that of cultured samples), and more diversity observed in the patterns of H. vulgaris samples.  It appears interspecies differences between wild and cultured polyps are far less pronounced than that between the microbial communities of the different hydra species.

Furthermore, species-specific phylotypes were observed via phylogenetic analysis of RELP derived sequences (phylotypes being defined as sequences of ≥97% similarity).  A particularly interesting α-proteobacteria phylotype specific only to H. oligactus was confirmed as a bacterial endosymbiont (via TEM and FISH techniques), previously unreported as extant in Hydra. Again, species’ differences were found to be larger than interspecies:  although no H. vulgaris samples were found to contain any bacterial endosymbiont, all cultured H. oligactus polyp epithelial cells and c. 20% of wild H. oligactus polyp cells were observed as containing this type of α-proteobacteria phylotype.  The authors conclude that the stark differences between the microbial communities of the two species of Hydra, together with the maintenance of a specific type of microbiota over time, suggests that selective pressures applied by the Hydra epithelium help to structure these microbial communities.

This study provides compelling evidence that cnidarian hosts do indeed have a role in shaping their microbiotas; some other investigations have found similarly species-specific microbial communities in other Anthozoans, including some analyses of the threatened stony schleractinians that build coral reefs.  However, observations of such clear cut species-specific differences in anthozoan microbiotas are not always evident, with many studies providing conflicting results; different molecular methods have their own advantages and disadvantages in the assessment of microbial diversity (e.g. the variability inherent to the RELP technique), which must be kept in mind when such investigations are interpreted. 

In addition, various abiotic and biotic factors have been suggested as having an important role in the structuring of microbial communities, including location, temperature, disease, and the influence of bacteriophage; these factors, alongside punitive selective pressures from the host itself, should be considered when designing further investigations into interactions between microbial communities and the metazoans which they inhabit.

Fraune, S. and Bosch, T. C. G. (2007) Long-term maintenance of species-specific bacterial microbiota in the basal metazoan Hydra. PNAS 104, pp 13146-13151.

http://www.pnas.org/content/104/32/13146