THE CHANGES IN COD INTESTINAL MICROBIOTA UPON CAPTURE

This study documents the intestinal changes in the gut of wild Atlantic Cod, Gaduas morhua, upon capture and artificial feeding. Over the years, this species has become increasingly important for mariculture in the northern hemisphere. Presently, wild Cod are caught and brought to sea-pens were they are fed an artificial diet to increase weight and size before slaughter. This practice also supplies a year-round Cod source. Past studies indicate that marine fish caught from the wild and fed artificial feed may change the intestinal microbiota in the caught stock. It is well-established that the microbiota of fish intestines plays an important role in the growth, survival, health and nutrition of a host, supplying nutrients, fatty acids, extracellular enzymes and vitamins. The intestinal communities of wild-caught and penned Scottish/Norweigian Salmon has been documented but here the changing intestinal communities of wild Atlantic Cod acclimatizing to artificial rearing will be assessed for the first time.

70 cod were caught off the coast of Norway and brought to a large indoor tank supplied with 250m seawater. Initial intestinal samples were collected from 8 fish before the remaining were distributed into 6 different tanks. They were left to acclimatize for 1 week before two different feeding trials began for 5 weeks. 3 tanks were fed with a formulated powder mixed with sausage and herring, these were fed to satiation, and 3 tanks were starved for 5 weeks. 8 fish were sampled from each feeding trail. After being euthanized, the intestinal tract was removed using aseptic procedure. The fore, mid and posterior sections were isolated. These were washed in a sterile o.o1 M phosphate-buffered saline solution and the contents was removed. These were kept on ice until analysis. The empty intestine was then flushed dissected into small pieces. This remained sterile for molecular analysis.

In order to collected quantitative data, the intestine contents were subject to a ten-fold dilution and plated onto Difco marine agar. The colonize that formed were observed for 3 weeks and counted. In addition a DAPI flurochrome stain was applied to fixed samples and photographed under a microscope.

Qualitative data was collected using DGGE (Denaturing gradient gel electronphoresis) and PCR-amplified genes coding for 16S rRNA. These would allow analysis of any intestinal community changes. DNA was isolated from the intestinal wall and content, and a primer amplified the 550-bp fragment in the rDNA. An annealing temperature gradient PCR was performed to find optimum temperature of amplification to be 55.8°C. The products of the PCR were used to produce a DGGE. There were four different analysis conducted; the posterior content, posterior wall, mid content and mid wall samples. The gels were stained with a diluted SYBR green stain. These were also photographed. Products were also purified using a PCR centrifuged filter and subjected to BLAST.

It was recorded that there was an increase both types of counts conducted when comparing the mid to the posterior intestine. This was recorded in all three feeding groups. Only the mid content compared to the mid wall showed a decrease in abundance and this only occurred in the starved trial. The band numbers in the DGGE were less in the fed group than the initial-capture group. The two groups showed similarity values of less than 50% however. The starved group however had almost the same number of bands as the initial group; this suggested that starvation had not effected the microbial structure.

From the quantitative studies, it was seen that there was no difference between the number of directly counted groups when comparing the initial-caught group with the fed and starved group. It was presumed that the resistance to change was caused by stability within the intestinal environment, as has been shown in previous studies. It was also discussed that the starved Cod were still found to contain hard digested matter, suggesting that a longer period of starvation than the proposed 42 day period should be implemented in later studies. This study reveals that the microbial community of Atlantic Cod remains at a relatively constant level despite dietary changes. The paper further suggests that the persistent microbiota might be ‘sentinal organisms’.

The qualitative data determined that there was a difference in the number of bands between the initial-capture group and the fed group. This was unexpected as the number of counts showed no significant difference in the quantitative data set. It was proposed that the abundance of microbiota  remained the same but the community shifted. This is consistent with previous studies conducted upon various freshwater and marine fish.

Finally the paper notes that the number of culturable microbes were very low when compared to the number of microbes identified by the DGGE. This was shown to be down to the number of anaerobic bacteria present in the intestine.

This paper is intriguing as it uses an array of techniques to consider the microbial composition of wild cod intestines in association with a change in diet.

Anushu et al. 2011. Changes in the Intestinal Microbiota of Wild Atlatnic cod Gadus morhua L. upon captive rearing, Microb. Ecol. 61: 20-30

Symbiosis Regulation in Microgravity

Whilst space flight’s microgravity is no good for animals, causing bone loss, muscle atrophy and immune dysfunction, microbes seem to flourish in it. At least, pathogens do, with increased growth rates, antibiotic resistance and gene transfer. How microgravity affects symbiotic microbes has not yet been considered.

Microgravity is demonstrated to alter the expression of the global regulator Hfq, an RNA-binding protein that regulates the translation of mRNA according to environmental cues.

Half of all bacteria have an Hfq homologue, including members of prominent symbiont groups. The hfq gene in pathogenic species is down-regulated by microgravity, by mechanisms unknown. To determine the role of hfq in symbioses and how microgravity influences this regulator, the symbiosis between Euprymna scolopes and Vibrio fischeri was used as a model.

V. fischeri colonization of the host topples a sequence of developmental dominoes, leading to the controlled apoptosis of the ciliated epithelial appendages (CEA), once cilial beating has gathered enough bacteria for bioluminescence. These apoptotic cells normally show condensed nuclei after bacterial colonization and CEA regression can be observed within 24 hours, making it an ideal study model.

Microgravity conditions were created using rotating wall vessel bioreactors and Hfq deficient mutants were compared to the wild-type to assess colonization and CEA apoptosis-inducing abilities. These phenotypic characters were also tested in the presence or absence of microgravity.

Expression of hfq in V. fischeri was confirmed to be down-regulated by microgravity, causing acceleration of CEA apoptosis. It was also found to serve no role in colonization of the host light organ. Mutants lacking Hfq showed that the complete absence of this regulator reduced the apoptosis-accelerating effect of microgravity, but yet sped up CEA regression.

Aspects of future research in this topic will need to investigate the changes in transcription between the wild-type and the Hfq-mutants, in order to explore the full role of Hfq. In addition, there is a need for identification of the stimulon (the collection of genes under regulation by the same stimulus) influenced by gravity in both pathogenesis and symbiosis. Hfq’s role in symbiosis development indicates a research gap in which regulators control the diel cycle of this squid-microbe symbiosis, as microgravity appeared to have no effect on the regrowth of V. fischeri populations following expulsion. Faster CEA regression highlights the need to investigate the role of Hfq in quorum sensing; there is also an undetermined possibility that microgravity can regulate genes for extracellular matrix molecules, which may compromise membrane integrity and hasten apoptosis.

This is also an excellent example of how understanding of a marine microbe and its symbiosis is an effective tool for addressing fundamental questions which may apply to many other taxa. The apoptosis shown here to be encouraged by Hfq down-regulation in V. fischeri is part of the bobtail squid’s normal life cycle, but it is not unreasonable to posit that regulation of apoptotic effects may also be something to consider for pathogenic microbes.

Grant, K. C., Khodadad, C. L., & Foster, J. S. (2014). Role of Hfq in an animal–microbe symbiosis under simulated microgravity conditions. International Journal of Astrobiology, 1-9.

CARNIVOROUS FISH CAN USE PLANT-BASED DIETS EFFICIENTLY IF SUPPLEMENTED WITH CERTAIN BACTERIA

Successful and profitable aquaculture of a carnivorous fish requires ecologically and economically feasible supply of feeds containing high quality proteins. This requirement has conventionally been met by fish-meals from wild-caught fisheries, containing such high quality proteins. However, considering the problem of overfishing, this is not a sustainable source of high-quality proteins. For many ecological and economic reasons, there is a trend of replacing fish-meals with plant-derived proteins. However, plant-based diets are problematic to carnivorous fish as they contain large amounts of cellulose and starch, which carnivorous fish finds difficult to digest and use efficiently.

Cellulolytic microbes have the ability to break complex lingo-cellolosic bond in plant-based diets. Similarly, amylolytic microbes can convert starch to simple sugar by secreting digestive enzymes in the gut. Such cellololytic and amylolytic activities of gut microbes have been shown from the gastrointestinal tract of freshwater and brackish water fish in many studies. In vitro investigation on bacterial strains secreting cellulase, amylase and protease have shown reduction in the levels of fibre and carbohydrate and, increase in the protein content of the diets. Hence the idea is to use such types of microbes as supplements in the plant-based diets of aquaculture fish to improve the nutrient utilization and growth of fish.

This study examined the effectiveness of the selected cellulolytic and amylolytic bacterial strains as feed supplements for juveniles of Asian seabass (Lates calcarifer). Both of the bacterial strains used, belonged to the genus Bacillus, which were isolated from the intestines of brackish water fish. The juveniles of Asian seabass were fed plant-based diet, supplemented with the bacteria of those selected strains of Bacillus and then compared with the fish at control, that were also fed the same diet, but without the supplementation of bacteria. Apart from control, there was one treatment group supplemented with only cellulolytic bacteria (Bacillus sp.), another group supplemented with only amylolytic bacteria (Bacillus subtilis) and the last group supplemented with a mixture of both strains. 

Comparison with the control revealed that all of the bacteria supplemented groups had higher weight gain, improved nutrient digestibility and survival. Similarly, they showed higher protein efficiency ratio, specific growth rate and lower feed conversion rate. Thus, as we have learned in Daniel’s lectures, the findings of this study clearly showed the importance of gut microbiota of fish as well as the benefits of using probiotic bacteria in fish farming, which has been discussed in details by the authors. Colonisation of such beneficial microbes in the gut of fish, causes increased activity of digestive enzymes, better nutrient digestibility and increased nutrient absorption. Gut microbes play key roles in the digestion process by inducing the endogenous enzyme secretion and also by increasing the total enzyme activity, as this study reported increased levels of amylase and cellulase activity in treatment groups supplemented with bacteria, compared to controls. Besides that, gut microbes also help their host by producing other nutrients such as vitamins, essential amino acids and fatty acids. Vibrio population was lower in the water of bacteria supplemented treatment groups compared to controls, suggesting that the beneficial gut microbes also help their host against pathogens like Vibrio spp.

The benefits of increased activity of digestive enzymes were more pronounced in the fish which were supplemented with the mixture of amylolytic and cellulolytic bacteria, rather than those which were supplemented with either type of bacteria. Compared to controls, protein content of the supplemented groups was also higher, which was again highest in the fish supplemented with the mixture of both strains of bacteria. This highlights the importance of supplementing a cocktail of bacterial strains, rather than providing single strain/type of bacteria to the fish.

De, D., Ghoshal, T. K., Ananda, Raja, R., & Kumar, S. (2013). Growth performance, nutrient digestibility and digestive enzyme activity in Asian seabass, Lates calcarifer juveniles fed diets supplemented with cellulolytic and amylolytic gut bacteria isolated from brackishwater fish. Aquaculture Research 1-11; doi: 10.1111/are.12325. 

Can intraguild predation explain how Vibrio cholerae rapidly reach such high numbers in human hosts during cholera infection?

As we’ve learnt, Vibrio cholerae is a marine bacterium common in estuarine environments and is the causative agent of cholera.  V. cholerae enters the human gastro intestinal (GI) tract through contaminated water, passing the gastric acid barrier and mucin layer of the small intestine and adheres to the epithelial lining.  Once here, the bacteria quickly reproduces and becomes pathogenic, secreting the cholera toxin and causing acute diarrhoea in the host.  Each diarrheal episode purges huge numbers of V. cholerae, reaching counts of up to 10⁹ CFUs ml^-1, leading to cholera epidemics and responsible for over 120,000 fatalities each year.   In addition to diarrhoea, cholera also frequently causes vomiting and it is reported that symptoms are more severe in people suffering from malnutrition.  It is therefore very unlikely that digested food is a key nutrient source for V. cholerae cells in the human host, begging the question; how does V. cholerae reach such high numbers so quickly?  

During the onset of colonisation, energy sources such as the mucus layer coating the GI tract and sialic acids of mucous membranes are likely, however it is not clear whether these would provide sufficient requirements for the rapid multiplication and growth of V. cholerae in between diarrheal purges.  Pukatzki & Provenzano (2013) hypothesise that V. cholerae employ a Type VI secretion system (T6SS) to engage in intraguild predation (e.g. predation of neighbouring species or strains competing for the same resources) to supplement their high energy requirements.  

Predatory bacteria have been well documented and are best characterised by Bdellovibrionaceae that infiltrate the periplasm of gram negative bacteria and consume the macromolecules as a nutrient source.   Myxobacteria have also been shown to illicit bacteriocidal tendencies, killing and converting prey cells into growth straits and T6SS genes have been identified in Myxococcus Xanthus.  All V. cholerae strains sequenced to date have genes for a highly conserved TS66 and they have also been found in around 25% of all sequenced Proteobacteria, indicating that the secretion system has ancestral origins that precede the evolutionary divergence of Vibrio lineages.  

T6SS was linked to pathogenesis when it was first discovered in V. cholerae in 2007, documented by an effector that crosslinks actin and causes toxicity in another bacterium, Dictyostelium discoideium.  Whilst only a small number of strains have expressed T6SS in the lab, T6SS genes have been transcribed in human volunteers (using non-toxigenic strains!) using in vivo expression technology (IVET).  During infection of V. cholerae, the host microbiome undergoes a radical transformation, with ‘good’ bacteria rapidly being replaced by ‘bad’ bacteria.  Intraguild predation would allow V. cholerae to benefit both directly, through consumption of the prey cells and indirectly, from effectively eliminating the competition occupying the same niche.

This hypothesis appears highly plausible and would explain how V. cholerae are able to grow and multiply so rapidly.  In contrast to Roberto’s post (4^th March 2014) where Vibrio species are being predated, here it is the Vibrio species predating.  Could this be an example of an evolutionary arms race? Interestingly, all gram positive bacteria investigated to date are resistant to the bactericidal attempts of V. cholerae T6SS and understanding why this should be may allow development of treatments for cholera.  Exploring the triggers and relationship between V. cholerae and T6SS further should prove to be a promising area for future studies.

Pukatzki, S., & Provenzano, D. (2013). Vibrio cholerae as a predator: lessons from evolutionary principles. Frontiers in microbiology, 4 (384) 1-5   doi: 10.3389/fmicb.2013.00384

Life in the „Plastisphere“: Microbial Communities of Plastic Marine Debris

We all know that plastic waste in the oceans is a major problem affecting marine life. It is estimated that around 35 kg per year per person of the world population are produced, and a part of it finds its way into the sea.  Most of the studies carried out focus on the threat to marine macrofauna such as turtles and birds (entanglement, ingestions), and this study tried to address the bacteria associated to plastic. Pieces of plastic last much longer in the environment than other types of substrates to which the microorganisms can attach themselves to and it is assumed that plastic represents a vector transporting the microbes over a long distance. 

In order to investigate the bacteria associated with the plastic debris, plastics as well as seawater samples were collected and total DNA extracted from the samples. Plastics were identified as polyethylene and polypropylene, and DNA analysis revealed that the microbial communities were consistently distinct from each other between the plastics and were also different from the seawater samples. Cyanobacteria Phormidium and Rivularia sp. occurred on the plastics but not in seawater samples and Pelagibacter sp. dominated the water samples as heterotrophic representatives, and showed varied abundances in the plastic samples. Curiously, the authors found radiolarian OTUs on both plastic types, which is an unusual since these protists are generally not assumed to be substrate associated. The authors furthermore analysed the hydrocarbon degrading bacteria associated with the plastics and discovered among the the taxa identified Oceaniserpentilla, which is one of the major taxa related to the OTUs found in the Deepwater Horizon oil spill. Although the presence of these bacteria does not imply that they are involved in plastic degradation, it offers an opportunity for testing this. 

According to the paper, the most striking finding was the abundance of Vibrio OTUs which was found to be as high as 24% of the communities associated with the plastic. Vibrio sp. (with a few exceptions such as V. harveyi) are usually not found in such high concentrations within a community (often not higher than 1%), and the fast growth rate of this genus could be a possible explanation for this. I think this is a very interesting finding; we have recently discussed the persistence of Vibrios in the oceans. The authors have found diatoms attached to some of the plastic samples, and with the background that Vibrio sp. have been found to attach themselves to diatom chitin, the presence of diatoms could be an explanation for the high concentrations of Vibrio OTUs. Plastics could also have an effect on the environmental persistence of other bacteria likewise, and therefore could also influence the spread of harmful pathogens. 

Zettler ER, Mincer TJ & Amaral-Zettler LA (2013). Life in the ”Plastisphere”: Microbial Communities on Plastic Marine Debris

Predatory bacteria as natural modulators of Vibrio parahaemolyticus and Vibrio vulnificus in seawater and oysters.

Start searching for selective advantage and becoming predation dynamics.

Vibrio parahaemoliticus is one of the principal sources of seafood-associated human illness worldwide. Eating contaminated raw seafood or shellfish (for example oysters) can lead a diarrheal disease. Different serotypes were identified during last decade pandemic episodes and V. parahaemoliticus serotype O3:K6 was one of the most diffused. Although high infecting dose of vibrios is required to lead outbreak, not only the number of ingested organisms are important but also the presence of virulence factors in them. Virulence factors are genes able to encode toxins, enzymes or regulatory proteins that can enhance the infectivity and the pathogenicity of the strain that show them.

Vibrio genus is widely spread as symbiont and pathogen but is also a prey of other organisms. It’s known that Vibrio can be predated by a class of gram- bacteria: Bdellovibrio and like organisms (BALOs). This group is phylogenetically diverse and has complex life cycles with both host-dependent and host-independent replication. BALOs exploit and kill hosts attacking on them and using host cell content as nutrient reservoir for replication.

Richards et al. in this article evaluate whether rpoS and toxRS genes confer advantages in uptake or colonization of vibrios in Eastern oysters Crassostrea virginica or survival of vibrios in seawater. The rpoS and toxRS genes are involved in alternative stress response and virulence regulation respectively so are considered integrally involved in Vibrio survival and virulence. Authors hypothesized that knockout mutant for these genes (ΔrpoS and ΔtoxRS) could have different colonization or survival success in respect to wildtype O3:K6. They also evaluate growth and persistence of V. parahaemolyticus O1:KUT (strand for K untypeable) and V. vulnificus both associated with outbreaks of seafood-associated illness or wound infections.

They set separate experiments to count number of each O3:K6, ΔrpoS and ΔtoxRS strain at 0, 24, 48, and 72h after same inoculation quantity in: (A) tanks of natural seawater (NSW) with oyster and live microalgae (to feed oysters); (B) in oyster directly (kept and fed in same conditions of A). The result was a decrease of more than 96% after 48h and values of less 1% after 72h in A; and after a peak of accumulation at 24h a following decrease of 91-97% at 72h in B for every vibrios strain inoculated. Since there were no significant differences between three strains, authors proposed that toxRS and rpoS genes not give any selective advantages in colonization and persistence in oysters.

To evaluate whether this failure to thrive was unique to this species and serotype (O3:K6), they test in the same way (A and B design) V. parahaemolyticus O1:KUT and V. vulnificus comparing uptake and persistence in seawater and oysters. Authors obtained same failure to survival: significant decrease number after 24h and negligible level after 72h in A; significant decrease after 72h in B. This result showed the inability of all these pathogens to persist in NSW or oysters.

At this point to determine if Vibrio level decrease in seawater was due to the filtering process of oysters, O3:K6 was inoculated in NSW (without oysters and added microalgae). The result was again a significant decrease to negligible level after 72h. Indeed some factor in the seawater was responsible for vibrios reduction.

A different interesting result was obtained testing the same inoculation of O3:K6 in autoclaved seawater (ASW) and NSW. There was an increase of 1000-fold for the former and a decrease of 47-fold for the second, this last result was coherent with all the previous results.

This step gives the idea of a heat-labile inhibitor. Authors devised a plaque assay in order to quantify VPB and to obtain scanning microscopy images of them. Furthermore was set the same counting experiment (0-24-48-72h for A and B) monitoring the number of O3:K6 and VPB. Here they found an interesting prey-predator relationship (FIG 1-A) in NSW but not in oysters because methods to detect and count VPB within oysters are not yet available. Microscopy scanning images were compared between O3:K6 plaque isolates (FIG 1-B) and Bdellovibrio bacterivorus and Bacterivorax stolpii cultured in E.coli host cell (both of them BALOs model species). This gave a good visual similarity allowing authors to identify BALOs in a variety of morphology near to Bd. bacterivorus-, Ba. stolpii- and M. aeruginosavorus-like cells.

 FIG 1 A) Comparison of mean counts of V. parahaemoliticus (Vp) O3:K6 and Vibrio predatory bacteria (VPB) over time in natural seawater. B) Image from scanning microscopy of VPB (BALO) entering a V. parahaemoliticus host cell.

At this point, authors assayed for VPB samples of NSW from 5 different sites on Atlantic and Pacific US coast and monthly in 4 sites in Delaware from October to March. They found the higher level of VPB in the site with higher salinity and average temperature, a marshland. Authors propose a possible association of VPB with high-productivity marshes and high salinity, although insufficient information were available to infer this correlation both with salinity and temperature. However they support this hypothesis citing other evidences of disappearance of V.vulnificus from costal NSW and oyster in North Carolina after 2 years of hard drought.

In conclusion in this article authors start wanting to determine if toxRS or rpoS gene were involved in colonization and persistence of V. parahaemoliticus O3:K6 in oysters giving a selective advantage to different strains. Step by step they excluded strain-specific and species-specific features arriving to the identification of a biotic factor as responsible for Vibrio decrease in seawater. Started as a lab molecular experiment, it become an inter-specific predation assay with also a screening of field samples. This work seems to me like some of the pioneering works done for discover etiologic causes of bacterial disease in past centuries. This article give useful details to better understand pandemic Vibrio pathogen outbreak, suggest that VPB should play an important role in Vibrio decline and maybe climatic conditions limit potential spread of this pathogen. In my opinion more investigation on the ecology of VPB should be done. Maybe revealing their presence in other pathogen bacteria it should discover more prey and predators. And VPB also being bacteria, what’s happens to them with antibiotics pollution?

Richards, G. P., Fay, J. P., Dickens, K. A., Parent, M. A., Soroka, D. S., & Boyd, E. F. (2012). Predatory bacteria as natural modulators of Vibrio parahaemolyticus and Vibrio vulnificus in seawater and oysters. Applied and environmental microbiology, 78(20), 7455-7466.

Assessing water quality: a modified technique for detecting viral pathogens

The microbial quality of water has often been used as a method to assess overall water quality, to ensure it is not harmful to human health. Esherichia coli whilst an efficient indicator of bacterial pathogens it is unable to be used as a direct indicator of viral pathogens. Due to this, and because viral contamination has been attributed to >50% of all waterborne illnesses since 1980’s, coliphages (viruses that infect coliform bacteria) are recommended to be used in conjunction with E. coli as bioindicators of faecal contamination and predictors of water quality. However, the additional testing for coliphages was not considered economically feasible by the 2006 Ground Water Rule (a prequalification of water quality for general human use).

Current methods for coliphage detection and enumeration are multi-step procedures which are costly and require 2-3 days for a result. Thus a quicker and more economically viable alternative viral indicator test is required.

This study attempted to simplify the current methodology by developing a same-day fluorescence assay to detect coliphages which meets the same standard of detection as the current methodology, Method 1601. This modified methodology was termed Fast Phage and used an isopropyl-β-D-1-thiogalactopyranoside (IPTG) enrichment medium to induce transcription of the lac operon in the host coliform bacteria E. coli. On this medium uninfected cells grew exponentially whereas the expression of the lac operon induced lysis of infected cells identified by a rapid release of beta-galactosidase enzyme. When this initial enrichment material was transferred to a secondary medium containing enzyme substrate 4-methylumbelliferyl-β-D-galactosidase (MUG-Gal), MUG-Gal was cleaved from the medium by infected cells; beta-galactosidase formed an enzyme-substrate complex with the MUG-Gal. Cleaved substrate liberated the MUG, a component which fluoresces under UV light. Positive fluorescence indicated coliphage presence, non-fluorescence indicated coliphage absence

20 replicate samples were analysed using Method 1601 and Fast Phage. Fast Phage met the performance criteria defined by Method 1601, and as fluorescence was shown occur within 1h it was able to reliably detect coliphage levels rapidly. This was demonstrated by all 5 laboratories that tested the modified methodology with no significant difference in results between Fast Phage, and the reference method, Method 1601.

Testing for coliphages as an indicator of viral pathogens alongside current bacterial indicators is important to more thoroughly assess water quality and further protect human health. This preliminary study does demonstrate that a quicker, cheaper yet still reliable method can potentially be used to detect coliphages in water. The modified method, Fast Phage, meets the established criteria for detecting coliphages. Using a fluorescence-based predictor is novel but can provide an early warning of viral pathogens, as it has demonstrated to yield results within a day. Though many more stringent studies will have to be conducted prior to this modified method being validated in the protocol for water quality testing, the data from this study can be used to support the concept of the Fast Phage method. With further tweaking of the current methodology, I feel there is definitely potential for fluorescence-based coliphage testing to be used in the future as a measure of viral pathogens.

*** I found this paper quite technical in terms of existing protocol and procedures for accepting a new methodology. For the ease of reading and understanding I have tried to draw the main conclusions from the paper, though the paper does contain a LOT more detailed methodologies, results and applications. ***

Salter R., Durbin G., Conklin E., Rosen J. and Clancy J. (2010) Proposed modification of environmental protection agency method 1601 for detection of coliphages in drinking water, with same-day fluorescence-based detection and evaluation by the performance-based measurement system and alternative test protocol validation approaches. Applied and Environmental Microbiology, 76(23), 7803-7810