Tuesday, 8 April 2014

Vibrio cholerae also afraid of something and he run away.

- Maybe the deadline is over but I was working on this post so I posted it anyway, peace and love-

The ocean water is an heterogeneous environment where microorganisms must navigate to locate resource and conditions suitable for their growth. Chemotaxis is a fundamental mechanism that allow planktonic microorganism to aggregate around phytoplankton cell, colloidal particle, plume of organic substrates and resources. In such environment specific secondary metabolite can influence the behaviour of motile marine bacteria and a good example can be the quorum sensing for intraspecific signalling or antibiotic compounds for interspecific competition.

It’s knew that Vibrio SWAT3-wild-type (SWAT3-wt) and the human phatogen Vibrio cholerae are both particle-attached bacteria and the first one express antibiosis. SWAT3-wt can produce andrimid (inhibitor of acetyl-CoA carboxylase, an essential enzyme) that instead SWAT3-111 (a mutant) cannot produce. The andrimid potently inhibits the growth of V. cholerae but there are few informations on the effect of such molecule in much real natural condition as sub-lethal concentration.

V. cholerae is a particle attached bacterium and the authors in this work try to understand if andrimid can have a role in antagonistic interations with the other Vibrio species. They develop a chemotaxis assay combining a microchannel and a diffusion disk (on an agar surface Fig.1) to quantify the swimming behaviour, speed and turning rate. They used microvideography and cell tracking methods to monitor the swimming pattern of V. cholerae when exposed to lethal concentration of andrimid (i.e. cell stop swimming) and when exposed to sub-lethal concentration, either of the pure compound or when produced by a colony of SWAT3-wt.

To have a control reference, the authors performed two assays, one (1) with no chemical stimuli (to test the effect of the microchannel) and another (2) with a growing colony of SWAT3-111 (to be sure that other secondary metabolites don’t give effects). For both controls the swimming behaviour of V. cholerae were not related to the position in the microchannel and distance from the growing colony. They measured swimming speeds of 52.6 and 53.2 μm/s for (1) and (2) respectively. Then exposing V. cholerea in two andrimid gradients (one from pure antibiotics and one from a growing colony of SWAT3-wt) they found similar values of motility between controls and in a zone called non-lethal zone (1-1.2 cm distant from the source). In contrast, in a nearer zone to the sourse (called sub-lethal) they found significantly altered swimming behavior. V. cholerae was faster of about 25% (67.4-65.6 μm/s) in comparison to the controls. Also was defined a lethal zone where the bacteria showed a slower swimming speed (35% less than controls). Another interesting results were the turning rate and the run length (30% more) both increased in the sub-lethal zone meaning that V. cholerae run away from the danger. Were also measured swimming trajectories and the result was significantly shifted way 180° direction, so again away from the source of andrimid.

This results gave more information on species-specific competition for resources since the two Vibrio species tested here are both reported to colonize particulate organic matter in planktonic environments. The authors indeed suggest a mechanism to explain the observed decrease attachement by V. cholerae to particle previously colonized by SWAT3. So this rise some questions in my mind: how abbundant is SWAT3 in nature? May it explain also the fluctuation of V. cholerae outbreak in particular location? Such interaction can take place near nutrient rich particles likely determine the winner in the competition for space and resources and ultimately the abundance of V. cholerae in environment. Obviously more molecular study on this field could give additional data on this chemotaxis related competition. Maybe monitoring the abundance of SWAT3 in natural samples can also give some more light on the difficult manage of harmful outbreak of disease related to V. cholerae.

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.

Monday, 7 April 2014

The risks of Domoic acid poisoning in humans.

Domoic acid (DA) is a biotoxin produced by the marine diatom from the genera Pseudo-nitzschia.  It is known to cause amnesic shellfish poisoning in humans, it has neurological and gastrointestinal effects but may also result in seizure or coma.  It is ingested through consumption of shellfish, which are known to accumulate the toxin for longer periods of time than other fish or shellfish.  Previous studies on scallops have found the toxin to be present in levels exceeding the regulatory limit of 20 μg/kg in up to 17% of tested samples within the EU. 

Although there are measures in place to limit consumption by humans it is unknown what effect low levels of this toxin may have, if it will build up over time or even if it could be passed from mothers to their unborn children as has been seen in rats.  Although the tolerable daily intake (TDI) for humans is set at 0.075 ppm there have been no apparent clinical effects found for levels of less than 1.0 ppm.  That being said, other edible marine species such as razor clams and crabs have TDI’s of 19.4 and 31.5 ppm respectively.  Should these be accidentally consumed they could have dire consequences for humans.

A team in Belgium have analysed shellfish samples and data obtained from national food agencies to try to determine the actual risks to humans from eating scallops and oysters in particular. From their sample of shellfish (Oysters, Mussels and scallops), they discovered 22% of their scallops had DA levels higher than the regulatory limits, this was the highest percentage of all shellfish.  By extrapolating this data they calculated that actually less than 1% of the Belgian population would be at risk from amnesic shellfish poisoning due to eating shellfish with levels of DA that exceeded the acute dose. 

There are fairly stringent procedures in place to ensure acute exposure is limited however the effects of long term exposure are currently unknown.  It is also not known if cooking definitely reduces concentrations of DA in all species and this is certainly an area that could be fairly easily evaluated.  The study also highlighted the fact that long term exposure could actually affect as many as 5% of the population and confirmed that certain groups of the population such as pregnant women, young children or those with any form of reduced liver function could be at greater risk of poisoning. 

Given that DA poisoning can not only effect humans but also some marine mammals and birds it is certain that research into accumulation of DA and the way it may move through the food chain is essential.  There have been fairly recent advances in ways to detect the poison using enzyme-linked immunosorbent assay (ELISA), which now means that testing is much cheaper than it has been previously.  Currently there seem to be only isolated outbreaks amnesic shellfish poisoning but going forward it will be important to discover what effects environmental changes such as global warming may have on both the diatoms themselves and also on the species that accumulate the poison.  We must also consider the fact that as availability of fish populations changes shellfish may grow in popularity which would again increase the risk of outbreaks and isolated cases.

Andjelkovic, M., Vandevijvere, S., Van Klaveren, J., Van Oyen, H., & Van Loco, J. (2012). Exposure to domoic acid through shellfish consumption in Belgium. Environment international, 49, 115-119.

New digestive symbiosis in the hydrothermal vent amphipoda Ventiella sulfuris

New digestive symbiosis in the hydrothermal vent amphipoda Ventiella sulfuris

Ventiella sulfuris is the most abundant amphipod species inhabiting the Eastern Pacific Rise vent field, and is commonly found near population of the Pompeii worm (Alvinella pompejana)

Usually, species living on/near hydrothermal vents survive due to their symbiotic relationships, commonly with chemoautotrophic bacteria. These symbionts often aid their hosts by supplying them with nutrition, which they themselves obtain through sulphur or methane energy sources. For this reason the authors collected specimens of V. sulfuris with the objective of understanding and documenting the bacterial symbiosis occurring in the species (if it did occur), and to hypothesise how this amphipod gains nutrition.

Gut contents of V. sulfuris contained bacterial traces (empty walls or sheaths) and non-degraded Alvinella pompejana cuticle, suggesting that these amphipods feed on A. pompejana and/or their respective symbionts. A previously hypothesis suggested that V. sulfuris grazed directly on the epibiotic bacteria, found on the external surface of the Pompeii worm. This theory is supported by gut condense as they discovered no inner tissue from the worm, only surface tissue; alongside an abundance bacterial traces.

Light (LM) and electron microscopy (SEM and TEM) revealed the outer body, surface, gills and appendages of the host are free from micro-organisms; however the digestive system (the midgut and the hind gut) contains two major microbial communities which were observed in all organisms examined.

In amphipods the midgut and digestive gland are the major organs involved in digestion and absorption of nutrients. In this species electron microscopy revealed the posterior section of the midgut harboured a community of Gram-negative, single-cell, long rod-shaped bacteria. Some of these bacteria were in contact with the endodermal cell membrane, and high magnification revealed numerous membrane vesicles and debris between the bacterium. This is an unusual location, as this is a highly absorptive part of the digestive tract. Additionally, there was an absence of bacteria in the digestive gland.

In the hindgut, the authors observed that some cells exhibited numerous mitochondria that seemed larger than those found in other regions of the digestive system. Electron microscopy revealed densely packed epimural rod-shaped bacteria that (at high magnification) exhibited two morphotypes (electron-lucent short rods, and electron-dense thin-long rods). Both forms were tightly attached to the cuticle or citicular spines, and formed thick mats embedded in a dense organic matter.

16S rRNA gene diversity of all bacterial communities found in host were molecularly analysed to revealed 12 phylogenetic groups; the two most common of which included the Epsilonproteobacteria and the Firmicutes. Interestingly, many of the ID’ed bacterium were closely related to mammalian gut microflora. However some phyla, such as Epsilonproteobacteria are commonly associated with deep sea and/or hydrothermal vent symbiosis.

Sequencing analyses of 16S rRNA genes revealed that the bacteria found in the digestive system of V. sulfuris belong to six phyla: Epsilonproteobacteria, Firmicutes and Cytophaga– Flavobacter–Bacteroides, Gammaproteobacteria, Betaproteobacteria and Alphaproteobacteria. Most of these sequences are close to symbiotic bacteria of hydrothermal vent organisms, and bacteria involved in digestive symbiosis.

Due to a low sample size and an unsuitable fixation of specimens, bacterial DNA was partly degraded and therefore FISH (a technique that could have successfully correlated bacteria phylogenetic affiliation to their location) the authors were unable to specifically ID the two bacterial groups that were discovered in the gut. They did speculate that the midgut community was probably Epsilonproteobacteria, and the foregut phyla were likely to be Alphaproteobacteria, Firmicutes and/or CFB.

Overall an interesting study, but a real shame they couldn't pinpoint the exact phyla/species of the symbionts. However, they are one of the first studies to investigate this symbiotic relationship and for that credit is due.

Corbari, L., Durand, L., Cambon-Bonavita, M. A., Gaill, F., & Compère, P. (2012). New digestive symbiosis in the hydrothermal vent amphipoda Ventiella sulfurisComptes rendus biologies, 335(2), 142-154.

Heat-shock response of cyanobacteria

Cyanobacteria originated as a group of photoautotrophs nearly 3.5 billion years ago and were partially responsible for the initial oxygenation of the earth’s atmosphere. Today they are ubiquitous across aquatic ecosystems, even in extreme environments such as, hot springs, frozen lakes, salt ponds and deserts. They have the ability to survive in temperatures ranging from -60 to 74˚C. Studies on the stress responses of cyanobacteria are essential, as they play a major role in ecosystems as well as being used for human purposes in biotechnology and as biofertilizers.

Cyanobacteria are nitrogen fixers in which this process depends entirely on photosynthesis and both these processes are affected by heat and other stressors. Photosystems can become inactive as a result of a temperature shift, which has been shown in some unicellular cyanobacteria. Nitrogen fixation has also been found to be fragile at temperatures above 42˚C. These changes in metabolic capabilities in the presence of environmental stressors, emphasizes the importance of studying the heat shock response in cyanobacteria.

Cyanobacteria induce a group of proteins called heat-shock proteins (Hsps) by transcriptional activation and the degree of temperature increase determines the magnitude in which genes are upregulated. A typical heat-shock response in Synechocystis PCC6803 includes the upregulation of 90 genes after one hour of heat stress. The protein family Hsp100 are enhanced mainly by heat stress, whereas the Hsp90 family is associated with several abiotic stressors, a more general stress protein.

Molecular chaperones are essential components of many cellular functions: 1) folding of proteins after translation 2) transport of unfolded proteins 3) protein conformational homeostasis and 4) protection of the photosynthetic apparatus from stress induced damage. Hsps are present in low abundances under normal conditions, but are amplified when needed. These genes are then repressed by cis-elements or by trans-acting proteins. Maintenance of chaperone proteins in appropriate levels ensures there availability on demand and reduces any complications that might arise.

In conclusion, the roles of Hsp in cyanobacteria may help to enhance their stress tolerance, especially when subjected to sub-lethal levels prior to exposure. This was a fairly complex paper and went into a lot of detail about complicated pathways involving genes. The language used was also difficult to understand and maybe aimed mainly for geneticists.

Rajaram, H., Chaurasia, A.K. and Apte, S.K. (2014) Cyanobacterial heat-shock response: role and regulation of molecular chaperones. Microbiology. 160: 647-658

Gaping Jaw Disease - Are microbes to blame?

The Atlantic Hallibut (Hippoglossus hippoglossus) is an important commercial species, and is farmed in globally.  Although it has been commercially farmed for almost 40 years there are still issues arising in early developmet caused by opportunistic bacteria including Flexibacter ovolyticus, Vibrio salmonicida and Vibrio anguilarum.  One particular disease, known as “gaping jaw” is thought to be caused by bacteria infecting lesions caused by the rearing container in larvae.  s the larvae have very little immune system they are unable to fight it off and the disease itself eventually prevents effective feeding causing the animal to die from starvation. 

This paper looks at the analysis of the culturable bacteria found in larvae yolk sacs of those individuals exhibiting symptoms of gaping jaw.  Samples were taken from 40 larvae, grown on agar plates and amplified using PCR.  Although the results showed a heterogeneity for many bacteria, (32 out of 44 found), there were a number that were only found in larvae with gaping jaw.  These included species from Vibrio, Photobacterium, Flavobacterium and Bacillus genera suggesting that they may be linked to the jaw deformity.

The team also assessed the level of expression of three immune genes and found that expression was significantly elevated for two out of the three genes in larvae with gaping jaw.  One in particular, Hepcidin, was elevated 1196 times when compared to levels from healthy larvae.

One of the Vibrio species isolated had a high similarity to Vibrio alginolyticus which is a known pathogen.  In addition there are also previously described pathogens from the Photobacterium genera.  Even though Flavobacterium are generally harmless there are again a number that are associated with fish disease.  The presence of Bacillus however could be linked to the disease but there is also a possibility that they are there as an antimicrobial agent as they are able to produce bacteriocins.
The increase in levels of Hepcidin in the presence of the bacteria found on diseased larvae is interesting as it is know to up-regulate in other fish such as the Atlantic cod in response to bacterial infections.  Similar increases in levels of Hepcidin have also been seen in sea bass in response to infection from both Photobacterium and Vibrio species.

Given that previous studies into this disease have found little correlation between other environmental factors such as temperature, salinity and larval density it is possible that it is indeed caused by a bacterial infection.  Further study will now be required to determine which if any of these culturable bacteria may be responsible for the disease.  It would also be of interest to conduct metagenomic studies to determine which if any VBNC bacteria may be present and also complete individual tests to see what effect Hepcidin might have on the bacteria specific to the diseased larvae.

Urtubia, R., Gallardo, P., Lavin, P., Brown, N., & González, M. (2014). Characterization of culturable bacterial flora in yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.) with" gaping jaws" syndrome. Latin American Journal of Aquatic Research, 42(1).

Copepods act as vectors for the for the accumulation of domoic acid in the Arctic food web

Out of the 37 described species of the diatom genus Pseudo-nitzschia, 12 are known to produce the neurotoxin domoic acid (DA), which accumulates in organisms which graze on the toxic Pseudo-nitzschia, such as copepods, krill, blue mussels and scallops, and are then passed through food webs when animals at higher tropic levels consume these grazers, and these even include whales and seabirds. Symptoms have even been found in humans that include several types of neurological disorders, and may even lead to mortality. One of these domoic-acid producing species, Pseudo-nitzschia seriata, was first discovered in Arctic waters in western Greenland. However, some of the earlier records of this species could have also included the morphologically-similar Pseudo-nitzschia delicatissima, which does not produce the toxin. P. seriata is a component of the Arctic phytoplankton community, but there is a lack of information about the frequency and intensity of blooms that form within Arctic waters.

Three species of copepod, Calanus finmarchicus, Calanus glacialis and Calanus hyperboreus are known to be the most important metazoan grazers in Arctic Ocean and are key sources of prey to shrimps, including the commercially significant northern shrimp (Pandalus borealis) which is consumed by a large proportion of the local human population of Greenland and other countries which import them. It also has recently been shown that there is a risk of domoic acid accumulation in higher trophic levels via Calanus spp., as the North Atlantic right whale (Eubalaena glacialis) was found to be regularly exposed to domoic acid from its critical prey source, C. finmarchicus, and this has been suggested as one of the possible reasons for the reduction in reproduction success of this critically endangered whale population.

The authors of this current study looked at the risk of transferring DA through the Arctic food web via the three different species of zooplankton to determine whether they accumulate the toxin when fed with unialgal toxic cultures. They also examined whether the copepods are affected by DA and if this can be measured by looking for changes in grazing rates and/ or mortality, whether the three copepods vary in grazing rate and toxin accumulation, and finally if the copepods ingest toxic and non-toxic Pseudo-nitzschia at the same rate.

They found that the three species of Calanus graze on and ingest toxin-producing Pseudo-nitzschia seriata and that they can accumulate domoic acid after feeding, so they are indeed possible vectors. This paper is also the first to study the consequences of grazing on Pseudo-nitzschia spp. in Arctic waters, as previous studies have not attempted to find differences in clearance or ingestion rates between the toxic and non-toxic species. 

The weight-specific carbon ingestion rates of all three Calanus species were not significantly different when they fed on either toxic or non-toxic species, but the toxic P. seriata was egested/ cleared out of the gut and also ingested significantly less at 6-12 h than at 0-6 h by C. finmarchicus and C. hyperboreus. The amount of specific fecal pellet production (SPP) were very similar following each type of diet, which confirmed that both the toxic and non-toxic species were grazed by the copepods in roughly the same quantities. Although the copepods in this study were fed with unialgal cultures, the authors say one of the most critical ideas to investigate in future research is determining whether copepods graze on toxic Pseudo-nitzschia and accumulate DA when given a choice of food in the natural phytoplankton community. In addition, C. finmarchicus and C. hyperboreus seemed to stop grazing during the last 6 h of the experiment, which could be due to physiological incapacitation caused by toxin ingestion.

There may be an effect of size of the host on grazing rate, as C. finmarchicus was the smallest of the three species, which may be why it had the highest amount of DA per gram per copepod, but it also had the greatest weight-specific carbon ingestion of the toxic species. However, the different size ranges of the diatom species were not predicted to influence the grazing rate of any of the copepod species. Not all DA is egested by gut clearance but part of it is actually assimilated and accumulated into the vector’s tissues, so even if the guts of the copepods are clear of all toxic Pseudo-nitzschia species, it may still get transferred up into the higher trophic levels when these zooplankton are consumed by predators.

This study demonstrated a new and interesting insight into the effect of toxic microorganisms on the lower levels of the food web in Arctic waters. They mention that there is a threshold level where the Calanus species start to be affected by the toxin themselves, which seems to depend on a number of factors, including the size of copepod and concentration of domoic acid. In addition, the frequency and intensity of Pseudo-nitzschia species within Arctic waters is unknown. So I wonder whether certain external/ environmental conditions can enhance this impact, for instance in the face of global warming. An increase in sea temperature could cause a possible rise in phytoplankton blooms, and this could either cause a higher amount of DA to accumulate in copepods, or could lead to greater incidences of mortality. Either of these options could potentially impact the food web in some way, so the effect of temperature on the growth of blooms, and the subsequent influence of domoic acid on Calanus species and higher trophic levels, should be investigated.

Tammilehto, A., Nielson, T.G., Krock, B., Møller, E.F., and Lundholm, N. (2012) Calanus spp. – Vectors for the biotoxin, domoic acid, in the Arctic marine ecosystem? Harmful Algae, 20: 165-174

Bacterial Vesicles in Marine Ecosystems

Vesicles are known to be released from many heterotrophic organisms, gram negative bacteria release vesicles containing lipids, DNA, RNA and proteins during growth. Though the mechanism for this are not yet understood, though it can be influenced by many external factors. Vesicles have been shown to influence model organisms in many ways, but research up to this point has not uncovered their functions and abundance in marine ecosystems.
Biller et al. (2014) discovered what they deem to be a vesicle produced from the photoautotroph Prochlorochoccus  (and a strain of Synechoccus), and confirmed this finding using Negative stain and thin section micrographs and independently with nanoparticle tracking analysis of unperturbed cultures.

Prochlorochoccus are dominant photoautotrophs world wide with a global population of ~1027 cells and are the most abundant photoautotroph in ogliotrophic oceans (Synechoccus is the second).

Biller et al.’s experiments with the vesicles found that vesicles were produced during light and dark periods, with production consistent with other vesicle producing microbes. And the initial data predicts Prochlorochoccus produces 10^27 – 10^28 vesicles per day, though this is likely an overestimation.

Similarly to other vesicles Prochlorochoccuss’ contain lipopolysaccharides as well as normal cyanonbacterial lipids. They have rigid membranes, many proteins (though not known if all are functional), and importantly DNA.

Bacterial membrane vesicle release may account in part, for the relative abundance of membrane proteins among all dissolved proteins in seawater.

Supporting their findings with former field and lab data, observations of vesicle structures from coastal and ogliotrophic (Sagrasso sea) sites were classified as similar vesicles, though the origin was not confirmed through further analysis.

In the Sargasso Sea such vesicles were also found in and below the euphotic zone. Mechanisms dictating their distribution are unknown, but as vesicles and bacterial abundance correlate, they lean to the likelihood of microbial origin.

Prochlorochoccus are thought to produce large fractions of dissolved organic carbon (DOC) for oligotrophic regions, and these vesicles may be a contributing mechanism. Lower estimations of the initial data from this study predict Prochlorochoccus  supplies 10^4 – 10^5 tonnes of fixed C exported into the oceans per day. Also vesicle lipids have structural similarities to, and may account for a proportion of lipid DOC.

Biller et al. (2014) examined whether the Prochlorochoccus vesicles could support heterotrophic bacteria in culture and found that Alteromonas and Halomonas could both grow in sea water supplemented by Prochlorochoccus vesicles as the only carbon source. They contain about 1/100th of a cells carbon and likely influence carbon movements in the microbial food web. The presence of proteins and nucleic acids infer that they influence N & P cycles as well.

Prochlorochoccus has adapted to have very low phosphorus and nitrogen demands, though why an organism would supply vital materials in harsh oligotrophic environments is puzzling. Vesicle functions could be that the growth of Prochlorochoccus is positively influenced by the presence of heterotrophs,, so supporting them increases Prochlorochoccus fitness. Vesicles could be diversions for cyanophages – their use as such confirmed by Biller et al.s’ experiments.

As a purpose or side affect, they will also influence microbes through horizontal gene transfer. Vesicles have been shown to facilitate horizontal gene transfer before in Escherichia coli and Acinetobacter. Metagenomics of “wild vesicles found DNA pool of significant homology to 33 species phyla from the 3 domains. These sequences could reflect the export of prophage sequences within vesicles or DNA arising from phage infection of vesicles in the field. These vesicles could even assist microbes by providing a reactive surface!

It seems most likely that photoautotroph vesicles are important in many biotic and abiotic microbial processes in the sparse environment of ogliotrophic seas. I look forward to further research on this subject, and how this will fit in and help shape our knowledge of ocean processes.

Biller, S. J., Schubotz, F., Roggensack, S. E., Thompson, A. W., Summons, R. E., & Chisholm, S. W. (2014). Bacterial Vesicles in Marine Ecosystems. science, 343(6167), 183-186.

The EU Bathing Directive: More information required!

Although the current EU bathing directive has certainly had a positive effect on the condition of our beaches and waters there are still concerns, particularly when considering urban beaches, which are subjected to higher environmental pressure due to their location, but also experience seasonal increases in visitors increasing the risk of human exposure to possible infection.  One of the main concerns of the EU bathing directive has been to monitor and control pollution relating to sewage but when sampling methods have been designed, they have not taken into consideration possible small scale temporal variations.
Recent research has focused on this aspect in a study conducted in North West Portugal.  Four beaches were chosen, to be sampled hourly for a period of 11 hours, on several different occasions.  All samples were analysed to determine numbers of microbial indicators, Escherichia coli and intestinal enterococci. 

It was found that waters with lower salinity due to the beaches being closer to river run off had significantly higher numbers of both indicators.  In addition during the summer months, reduced rainfall lead to a higher concentration of microbes in the run off.  Wind direction was also found to have a significant influence on readings, driving contaminated surface waters from river mouths towards the monitored beaches at times.  As wind direction may change regularly, it is important that this be taken into consideration when samples are taken as this could have significant effects on the readings acquired.

Although water quality varied at all sample sites throughout the day, the highest readings for indicator species were generally found in the morning.  Readings could be as high as 5100 cfu 100 ml-1, and as low as 21 cfu 100 ml-1 with in one 11-hour period.  These readings could mean the difference between the beach being closed or being given a rating of excellent. 

The current directive requires a minimum of four samples to be taken from each bathing area during any season however there appears to be no specification regarding times that samples should be taken.  Previous studies have shown that if samples are taken five times per week they highlight 80% of non-conformity events however only 5% of these events were detected when sampling was monthly.

I believe that this study along with previous evidence highlights the need for not only an increase in sampling efforts to ensure that those beaches considered more at risk from pollution are maintained at a safe level but also a requirement to have a greater understanding of the local area.  If these studies were carried out on a more local basis rather than by a national organizations it would allow for a greater understanding of how daily variations and seasonal patterns may affect the cleanliness of bathing waters.  This could allow local authorities to better inform visitors highlighting the safest times for swimming or watersports.

Amorim, E., Ramos, S., & Bordalo, A. A. (2014). Relevance of temporal and spatial variability for monitoring the microbiological water quality in an urban bathing area. Ocean & Coastal Management, 91, 41-49.