Monday, 31 March 2014

Antibiotic resistance as a result of salmon aquaculture in Chile

Antibiotic use in salmon aquaculture produces resistant bacteria with potential to transfer resistance to human pathogens

Chile is the world’s second largest producer of farmed salmon, after Norway. Pathogenic infections are a frequent source of mortality in intensive salmon aquaculture. As stocking densities increase in an effort to increase yield, increases in epizootic diseases are often the limiting factor of productivity. Unlike it Europe where the use of antibiotics in aquaculture is banned, where vaccination and hygienic animal husbandry are the most used methods of disease control, Chile’s primary method of disease control is the excessive use of antimicrobials. The emergence of antimicrobial-resistant pathogens and new pathogens has stimulated even greater use of antimicrobials.

Antimicrobials are placed within medicated feed. Antimicrobials in feed that is not consumed, along with unmetabolised antimicrobials in fish urine and faeces, end up in the surrounding environment, where they select for resistant bacteria and may also encourage horizontal gene transfer of antimicrobial-resistance genes (ARG). There is a potential flow of ARG between environmental bacteria and the pathogens of fish and humans.  

To find evidence of these potential effects of excessive antimicrobial use in aquaculture, this study investigated the antimicrobial resistance of 200 bacterial species sampled from aquaculture areas and non-aquaculture areas, searched within them for ARGs, and tested their ability to horizontally transfer ARGs to the human pathogen E.coli via conjugation.

They found that of the 200 bacterial isolates, 81% displayed antimicrobial resistance to at least one of the eight antimicrobials used. Resistance to the three most commonly used antimicrobials in Chile was 32%, 26% and 53% at the aquaculture site and 22%, 25% and 45% at the non-aquaculture site. There was no significant difference in antimicrobial resistance between the sites. They concluded from this that the effects of aquaculture antimicrobial use are far reaching (the ‘non-aquaculture site’ was located 8km from the aquaculture site).  I think that without additional samples from a much further distance from the aquaculture site, it is hard to say whether there is not just a background level of antimicrobial resistance in the environment. Specific resistance determinant DNA probes were used to identify ARGs within the bacterial isolates, 42 ARGs were confirmed in bacteria from the aquaculture site, whereas only 14 were confirmed in the non-aquaculture site bacteria. 9 multi-resistant bacteria, 6 from the aquaculture site and 3 from the non-aquaculture site were tested for the ability to horizontally transfer ARGs to E.coli, 2 bacteria from the aquaculture site successfully transferred the genes via conjugation.

The implications of this study are profound. They have found that levels of resistant bacteria are much higher in isolates from Chilean marine sediments than in other aquaculture-active countries around the world. They have found that resistance to specific antimicrobials can be as high as 58%, both in aquaculture and non-aquaculture sites. Comparison to other studies shows the severity of Chile’s situation, Chilean isolates showed 32% and 45% resistance to tetracycline and amoxicillin, whereas Washington isolates show 3-9% and 6-14% resistance respectively, and a Danish study showed 4.8% and 14.5% resistance respectively. Horizontal gene transfer of ARGs to E.coli is particularly hard hitting, as it highlights how the excessive use of antimicrobials in aquaculture can have a direct impact on human health. Hopefully the results of this study, which is yet to be published, will have an impact on the use of antimicrobials in Chilean aquaculture. It certainly backs up the EU’s restriction on their use.

Shah, S. Q. A., Cabello, F. C., L'Abée‐Lund, T. M., Tomova, A., Godfrey, H. P., Buschmann, A. H., & Sørum, H. (2014). Antimicrobial resistance and antimicrobial resistance genes in marine bacteria from salmon aquaculture and non‐aquaculture sites. Environmental microbiology.

Characterising the gut microbiota of economically important fish

Disease control is a major drag on aquaculture and one which antibiotics can only relieve in the short term, due to the rise of resistant strains. Probiotics are a promising alternative. They alter the host’s gut microbiota, improving health and fitness. Probiotic-enhanced gut communities can improve pathogen exclusion, normal gut development, nutrition and immune function.

At present, the favoured strategy is to use probiotics containing lactic acid and Bacillus bacteria, because we assume they are the best choice because they dominate terrestrial mammal guts. The extremely obvious problem with supplementing fish with these bacteria is reflected in the literature; the majority of studies have failed to demonstrate any fish health benefits from terrestrial lactic acid bacteria probiotics.

More modern research indicates that host species is a stronger determinant of gut community composition than the outside environment is. However, this body of information is very limited as nobody has bothered characterising the typical gut microbe communities of common aquaculture fish species.

Ictalurus punctatus (channel catfish), Micropterus salmoides (largemouth bass) and Leponis macrochirus (bluegill panfish) are the top aquaculture fish species in the USA, none of which have had their gut microbiota characterised. This study aimed to fill this information void, to provide a starting point for future probiotic design for these species.

After euthanization, all fish had the lower third of their intestines aseptically removed and the contents squeezed into a tube. These samples were then ran through pyrosequencing and 454 sequencing to determine community composition.

Most studies like this have used only 1 fish species. Multiple species comparison revealed a high specificity of association between host and microbial community, since all fish were from the same environment. M. salmoides is carnivorous, I. punctatus is carnivorous above a certain size and L. macrochirus is omnivorous. Though stomach contents were not analysed, gut community differences are likely due to diet.

Gut microbe diversity typically increases from carnivore to omnivore to herbivore, but this trend was not shown in these species. This may be because all species could have had similar diets, but without stomach content data this cannot be confirmed. The similarity between gut communities seems to suggest that diet should have been recorded.

The phylum Fusobacterium dominated the gut communities of all 3 species, with Proteobacteria in second place. Fusobacteria are anaerobic, gram-negative bacilli who produce butyrate by fermenting carbohydrates found in epithelial tissues. This short chain fatty acid can benefit the host by acting as the main energy source for gastrointestinal cells, aiding mucus production and immune functions. Butyrate is not expected to be prevalent in carnivore guts, given the low carbohydrate content of their diets. Butyrate has been shown to inhibit some freshwater pathogens and is used as a fish feed additive. However, as a supplement it has not been proven as beneficial to fish health. Perhaps Fusobacterium supplementation would be better, possibly by providing more appropriate doses or molecular forms of butyrate.

Over 70% of sequences were related to Cetobacterium somerae in all three species. C. somerae is an obscure, microaerotolerant  fermentation  bacteria found in many herbivorous fish. Though it can produce vitamin B12 and inhibit the growth of other strains, it has not been investigated as a potential probiotic microbe. The most abundant gut microbes found by this study seem like good candidates for improving fish health, providing a strong case for further characterisation of other commercially important species’ gut microbiotas.

Larsen, A. M., Mohammed, H. H., & Arias, C. R. (2014). Characterization of the gut microbiota of three commercially valuable warmwater fish species. Journal of applied microbiology.

The potential of Lactic Acid Bacteria for use as probiotics in aquaculture

Aquaculture is important, obviously. It has the potential to make a significant contribution to the ever increasing demand for seafood worldwide. However it faces many problems, one of which is disease control. The widespread use of antibiotics as a disease control measure has lead to the emergence of antibiotic-resistant pathogens, and has thus lead to a ban in Europe and more stringent regulations worldwide. Alternative methods of disease control have included better husbandry practices, vaccinations, immunostimulants, use of bacteriophages to target pathogens, quorum sensing disruption, prebiotics and probiotics.

Recently, a probiotic culture has been authorised for the first time for use in aquaculture by the EU, using Pediococcus acidilactici, a LAB. For a bacterium to be deemed safe for use as a feed additive in the EU, it must receive Qualified Presumption of Safety (QPS) status, show an absence of resistance to human and veterinary antibiotics, and also prove its effectiveness in its intended use in the food chain. Most LAB have already received QPS status, demonstration of their suitability as aquaculture probiotics therefore only requires proof of their antimicrobial activity against fish pathogens and a lack of resistance to antibiotics, this was the aim of this study.

Lactic Acid Bacteria (LAB) are a clade of bacteria grouped together for their common metabolic characteristics. Currently, strains of LAB are the most commonly used bacteria in human probiotics, specifically members of the genera Lactobacillus and Bifidobacterium. In this study, 99 LAB strains were isolated from fish, seafood and fish products, 59 enterococci and 40 non-enterococci. Their antimicrobial activity was tested against 8 indicator fish pathogens. They were also tested for antibiotic resistance against 8 antibiotics, and those that do not already possess QPS status were tested for virulence factors and detrimental enzymatic activity.

They found that every species tested showed antimicrobial activity against at least 4 of the 8 fish pathogens, which is a very successful result, the majority showed resistance against 5-7 pathogens. However, genus-specific safety concerns were highlighted by antibiotic resistance tests and virulence factor tests. Antibiotic resistance was found in 60% of Weisella, 44% of Pediococcus and 33% of Lactobacillus. 86% of Enterococcus were found to be unsafe due to resistance or virulence factors. Of the non-enterococci LAB, only 7.5% showed antibiotic resistance. There were several examples of resistances being described for the first time in certain genera. Detrimental enzymatic activity was described in some two Enterococcus species, such as gelatinase activity and haemolytic activity. No genera showed bile deconjugation, mucin degradation or other detrimental enzymatic activity.

The main use of this study is as large-scale preliminary selection of safe LAB species, to identify and select the most suitable candidates to be further evaluated as probiotics for aquaculture. They also described novel antibiotic resistances and virulence factors in a range of LAB species. Ultimately they have shown that antimicrobial activity against fish pathogens is a widespread property of Lactic Acid Bacteria, that many species show resistance to antibiotics, but there are many other species that are perfectly safe and would be potential candidates for use as probiotics in aquaculture.

Muñoz-Atienza, E., Gómez-Sala, B., Araújo, C., Campanero, C., Del Campo, R., Hernández, P. E., ... & Cintas, L. M. (2013). Antimicrobial activity, antibiotic susceptibility and virulence factors of Lactic Acid Bacteria of aquatic origin intended for use as probiotics in aquaculture. BMC microbiology13(1), 15.

Sunday, 30 March 2014

The Symbiotic Cyanobacteria in Mediterranean Sponges

Globally, sponges are known to harbour various symbiotic cyanobacteria or algae; although cyanobactiera are more prevalent. These organisms have a mutually beneficial relationship that can boost the metabolism and increase the supply of nutrients to a host, whilst the symbiont receives protection from grazers and reduced UV exposure, and also receives a source of nitrogen from the host. Some work suggests that cyanobacteria in particular are able to provide defensive secondary metabolites and can increase the adaptability and ecological function of sponges. The genetic diversity of these sponge-associated cyanobacteria consists of multiple linneages such as Synechoccus, Synechocystis, Oscillataria, Lyngbya and Cyanobacterium. The most common and widespread symbiont is Candidatus Synechococcus spongiarum, a single-celled cyanobacertium that exsists in the peripheral region of the sponge. This particular symbiont can be found globally from tropical to temperate regions and makes up 85% of sponge-photosymbiont associations. Recent molecular evidence has found hidden diversity within the populations of S. sporangium. The cyanobacterial symbionts Synechocystis and Prochloron have also been described from sponges however, genetic characterization of these photosymbionts has only been reported from 5 locations and the ecological importance of these species is unknown. This study examins the diversity and activity of cyanobacterial symbionts in the Mediterranean sponge species Ircinia fasciculate and Ircinia variabilis. I. fasciculate inhabits high-irradiance zones whilst I. variabilis inhabits low irradiance, shaded zones. This study compares the genetic diversity, morphology and chlorophyll a (chl a) content of the cyanobacterial symbionts in these two temperate sponges.

Six individuals of each sponge species were collected from two neighbouring sites. Tissues samples were preserved in enthanol and stored in -20°C for genetic analysis. Three individuals of each species were assessed for chl a by determining the various absorbance of supernatant aliquots and using equations from Parsons et al (1984). The ultrastructure of the cyanobacteria species was determined by using transmission electron microscopy (TEM). Tissue from the different sponger species was fixed and incubated overnight before being embedded in a Spurr resin. This was then sliced into ultra-thin sections. Only cells that exhibited a clear center and thylakoids were digitally analysed and measured. Three individuals of each sponge species were prepared for metagenomic analysis following Qiagen® protocol. DNA extracts were used in PCR amplification using the 3’ end of the 16s region, the entire 16s-23s ITS region and the 5’ end of the 23S region. A low annealing temperature was used to reduce PCR bias. The PCR products were purified and individual clones were screened. These were subject to BLAST searches. Phylogentic reconstructions were then built using the different recovered rRNA gene fragments. S. spongiarum was reconstructed using 16S-23S rRNA ITS sequence whilst Synechocystis was constructed using only 16S rRNA sequences.

Chl a in I. fasciculate averaged around 248.1±27.8 μg/g whilst I. variabilis averaged 131.0±15.1 μg/g. These differences were shown to be significant with I. fasciculate being nearly twice the level of I. variabilis. The dominant symbiont cell seen under TEM represented the cell Candidatus S. spongiarum. These cells were seen to be actively reproducing in the mesohyl layer of the sponges. They also appeared to be interacting with the hosts’ archeocyte cells. The S. spongiarum was occasionally seen to be engulfed by the host cell although there was evidence of consumption. These symbionts were seen to be significantly larger in I. variabilis than in I. fasciculate. I. fasciculate also exhibited a significantly higher abundance of glycogen granules and a second morphotype of S. spongiarum that was three times larger than the average S. spongiarum cell. Synechocystis was not seen to be reproducing and there was no obvious interaction between host and symbiont cells. The clone libraries revealed two different cyanobacterial symbionts in I. fasciculata and I. variabilis. 85% of genetic sequencing in I. fasciculate and 100% in I. variabilis was found to correspond to Candidatus S. spongiarum. The remaining percentage was found to be Synechocystis. The ITS markers from Synechocystis were found to be shorter than S. spongiarum. Additionally a novel clade, named clade ‘M’, of S. spongiarum was identified in both sponges whilst a novel clade was also discovered for Synechocystis in I. fasciculata.

Although the sponge species exhibited the similar species of cyanobacteria, the symbionts were composed of different chl a pigments and storage products that correlated to the irradiance of the environment from which the host was recovered. Additionally the symbionts in I. fasciculata were engrained with glycogen granules that indicated the transfer of surplus carbon stores to the host. The irradiance conditions were thought to play a role in dictating the activity of sponge-associated cyanobacteria. Both hosts were dominated by the novel ‘M’ S. spongiarum clade but Synechocystis was only observed in I. fasciculata, this is different to the Caribbean Ircinia spp hosts that do not contain Synechocystis and hold a different clade of S. spongiarum however, the different ITS markers (3’ 16S, 16S-23S and 5’ 23S) used produced different results on this matter. Yet past work on this topic has also eluded to distinct clades per region and the use of different ITS markers may further shed light on the hidden cryptic diversity of these cyanobacterial symbionts. The different cell sizes suggest morphological plasticity in response to ambient irradiance levels and the various irradiance gradients in these flexible organisms suggests an environmental gradient rather than an expulsion/compositional shift as in coral/zooxanthellae symbiosis. Furthermore, the dependence of the host upon the symbiont was not significant, despite high abundance of S. spongiarum. This is in contrast to most other sponge-cyanobacteria symbiosis. It was suggested that this may be due to the active regulation of cyanobacteria to avoid predation on cyanobacteria-rich sponges, and to reduce the oxidative stress produced by the symbiont.

Erwin P.M & López-Legentil S. 2012. Ultrastructure, Molecular Phylogenetics and Chlorophyll a Content of Novel Cyanobacterial Symbionts in Temperate Sponges, Microb. Ecol. 64: 771-783.

Gut microbiota manipulates fatty acid uptake in fish

It is clear that the regulation of intestinal fat absorption is critical to the energy balance of any animal. It is also clear from previous research that the intestinal microbiota of an animal impacts its energy balance. However, what is less clear is the role of the gut microbiota in metabolism of dietary fat. Understanding this link may have advantageous implications for fish health and nutrition and for aquaculture. Understanding the link between microbiota and fat absorption may also have implications for human health, in particular obesity and malnutrition.
In humans, dietary lipids supply 40-55% of the energy requirements in the typical western diet. For vertebrates in general, dietary fats come in the form of triglycerides. These are broken down into monoglycerides by lipases in the GI tract and absorbed by enterocytes in the intestinal epithelium. They are temporarily stored in the epithelium as Lipid Droplets before progressing to other tissues such as the liver.
The majority of previous work linking gut microbiota and metabolism focuses on microbial fermentation of otherwise indigestible carbohydrates and lipids that allows the (usually mammalian) host to make use of alternative energy sources, such as my post last term on microbe-assisted seaweed digestion. However, this study uses zebrafish to investigate how microbiota regulates lipid absorption. Specifically, they used fluorescently labelled fatty acids to view how epithelial Lipid Droplet abundance differed between zebrafish with different known microbiota, known as ‘gnotobiotic’ zebrafish.
Two fluorescent fatty acids were used, quantification of epithelial fluorescence showed that both fatty acids were absorbed more successfully in zebrafish containing a normal zebrafish microbiota compared to zebrafish that were raised ‘germ-free’.  Higher lumenal fluorescence in germ-free fish suggests that the microbiota plays a crucial role in absorption. Lipid Droplet number and size was also shown to increase in zebrafish with a conventional microbiota. However, these increases may not be due to increase absorption, but decreased ability to subsequently shift fatty acids from the epithelium to other tissues, such as the liver. They tested this and showed that the increase in epithelial Lipid Droplet size and number was not due to impaired intestinal lipid export, nor was it associated with ingestion rates, digestive organ size or differences in digestive enzyme activity. They isolated microbiota as the one variable responsible for increased fatty acid absorption and also increased export to other tissues.

This study differs from previous studies in that it uses a method that distinguishes between dietary and microbial produced fatty acids. They can therefore say with certainty that microbiota composition affects the absorption of dietary lipids, regardless of the fatty acids produced by the microbiota. The mechanistic understanding of this interaction remains incomplete, this study has only shown that it does happen, not how it happens. The implications of this study could potentially be applied to aquaculture, manipulation of fish gut microbiota via probiotics may allow increased efficiency of fatty acid uptake, producing fish that are more nutritionally valuable for human consumption. If applied to humans, greater understanding of our own microbiota could lead to probiotics that manipulate fatty acid uptake for treatment of disorders such as obesity and malnutrition.

Semova, I., Carten, J. D., Stombaugh, J., Mackey, L. C., Knight, R., Farber, S. A., & Rawls, J. F. (2012). Microbiota regulate intestinal absorption and metabolism of fatty acids in the zebrafish. Cell host & microbe12(3), 277-288.

Saturday, 29 March 2014

Invasive lionfish pose ciguatera health risk

Lionfish are native to the Indian Ocean, South Pacific and Red Sea, however, following their introduction into Florida waters in the 1990s they have rapidly spread across the northwestern Atlantic and Caribbean. They have spread so successfully due to a lack of natural predators, high reproductive rates and high growth rates.
This study focuses on the presence of lionfish in the U.S. Virgin Islands in the Caribbean, where lionfish were first reported in 2008. Their sudden abundance has caused dramatic ecological shifts as they outcompete native species of reef fish for resources. In an effort to reduce their numbers, they have become targeted by fisherman, and whilst this provides an economic opportunity for local communities, it may also pose a risk to human health if lion fish act as a vector for Ciguatera Fish Poisoning  (CFP).
A brief description of ciguatera: there are 500,000 new cases of CFP annually, it is caused by consumption of reef fish that have accumulated the precursors to ciguatoxins (CTX) produced by dinoflagellates. Symptoms can be gastrointestinal, neurological or cardiovascular. Chronic symptoms that last for years are reported in 20% of cases, acute symptoms are the result of CTX binding to cell membrane sodium channel and keeping them open, depolarising the cell, which can lead to cell death.
The risk of contracting CFP is minimised by local knowledge of which fish are hazardous and should be avoided in certain harvest areas. However, because lionfish are an invasive species and a recent addition to Caribbean fisheries, the level of risk they pose has not yet been established. Potential cases of CFP linked to consumption of lionfish instigated this study.
Lionfish were collected from waters around the U.S. Virgin Islands, and area described as ‘hyperendemic’ for CFP. Samples were tested used in a Neuroblastoma Cytotoxicity Assay, which tests for sodium channel toxins. All positive samples were concentrated and analysed by liquid chromatography and mass spectrometry to positively identify CTX.
Of the 153 lionfish tested, CTX presence tested positive in 40% of fish. 12% contained a level of CTX higher than the FDA ‘guidance level ‘of 0.1 μg/kg. The average level of CTX in fish above this level was 0.2 μg/kg. These rates are similar to those seen in other predatory reef fish that are currently avoided by local fisheries. Individual lionfish apparently consume an estimated 50,000 reef fish per year, fish from lower trophic levels that are an important step in the conversion of dinoflagellate- produced precursors to CTX, so bioaccumulation is to be expected.

This study it the first to document that lionfish are a vector of CTX in endemic regions. They have highlighted a potentially hazardous fish species that is currently fished and consumed in regions that have learnt to avoid other CFP-associated species. The novelty of the fish due to its invasive nature has led to local knowledge playing catch-up. However, with this sound evidence of the CFP risk, hopefully now communities will learn to avoid consumption of lionfish in CFP endemic regions.

Robertson, A., Garcia, A. C., Quintana, H. A. F., Smith, T. B., II, B. F. C., Gulli, J. A., ... & Plakas, S. M. (2013). Invasive Lionfish (Pterois volitans): A Potential Human Health Threat for Ciguatera Fish Poisoning in Tropical Waters.Marine drugs12(1), 88-97.

Friday, 28 March 2014

A review: The ability of symbiotic Vibrio fischeri to navigate through the multiple environments of Euprymna scolopes

Bacteria have the ability to effectively sense and adapt to a variety of different environments by using molecular signalling cascades to detect extracellular signals and activate intracellular molecular pathways, which is one of the reasons why they are one of the most successful domains of life. These signalling pathways allow bacteria to recognise environmental changes such as osmolarity, oxygen levels, antimicrobials and nutrient levels so they can establish a suitable niche within host organisms. A common and “simplified” model system that has been used in several studies to understand more about how these signalling pathways work in colonisation and infection is the symbiotic interactions between the bioluminescent marine bacterium, Vibrio fischeri, and its nocturnal squid host, Euprymna scolopes, as V. fischeri is the sole bacterium that can withstand the environmental conditions within the squid to colonise the specialised light organ and produce bioluminescent light.

For successful colonisation to occur, a number of steps are required which are facilitated by signalling pathways, and these can be experimentally tracked within V. fischeri. These include being taken via ventilation from the surrounding seawater by young squid that are initially aposymbiotic, followed by making contact with cilia on epithelial fields on the surface of the light organ to form biofilm-like aggregates around them in response to bacterial peptidoglycan. In the later stages of colonisation, the bacterial cells leave the aggregate, enter the ducts of the light organ, move on through the antechambers (which they are unable to colonise), and finally arrive within the crypts in the light organ.

Some of the responses of the host tissue to the presence of these symbiotic bacteria include the production of ROS and reactive nitrogen species (RNS), as well as certain types of antimicrobials. In all of the stages of colonisation, V. fischeri cells continually interact with antimicrobials, such as during initial biofilm aggregation outside the light organ and persistent colonisation within, so they have evolved specific mechanisms to counteract these molecules to maintain the symbiotic relationship.

To manage and reduce the levels of halide peroxide (HOCl), a toxic ROS to many bacteria, V. fischeri cells may produce a catalase enzyme to convert H2O2 to water and oxygen. The increase in iron levels can also result in a higher production of ROS, so V, fischeri use particular mechanisms to reduce these. In addition, nitric oxide (NO) might be initially toxic for V. fischeri in certain environmental types, but the bacterium may have evolved to sense and resist free NO, as V. fischeri encodes H-NOX. This is a protein that binds to NO and results in the detoxification of the molecule during colonisation. E. scolopes expresses a range of enzymes that are potentially antimicrobial, such as five peptidoglycan-recognition proteins (PGPRs).

They also can actively sense their destination and migrate towards it because of their motility from flagella and chemotaxis. V. fischeri cells are dependent on their flagella to colonise their host in the early steps, but tend to lose these appendages within the light organ, so motility may not be an important factor during all stages of colonisation. The variation in use and presence of these appendages may be because of certain environmental cues. Chemotaxis involves a series of “runs” (smooth swimming) and “tumbles” (for re-orientation). These events depend on a complicated TCS pathway involving methyl-accepting chemotaxis proteins (MCPs), but by controlling the activity of these molecules, V. fischeri is able to continually respond to signals from a chemogradient and increase the amount of “smooth” runs towards the site of colonisation.

Once bacteria reach the crypt space and reach a certain high cell density, they are able to fully establish the symbiosis with the squid host and start to produce light and bioluminesce. This bioluminescence is a critical component of the symbiotic relationship, as it provides a nutrient-rich environment for V. fischeri, and in return they produce counterillumination to the squid so that they can remain hidden from predators in the moonlight while they are hunting for their own food.

The lux operon encodes for the required structural proteins to generating light, and one of the genes in the operon, LuxI makes a pheromone, 3-oxo-C6-HSL that promotes lux transcription by binding to and activating LuxR. These regulators are part of a positive feedback loop, so that the LuxR-3-oxo-C6-HSL encourages the synthesis of both Lux enzymes, and more of 3-oxo-C6-HSL, which ultimately results in higher light generation. Bioluminescence may be affected from detected signals caused by changes in oxygen levels, osmolarity, Mg2+ levels and iron levels.

The review seems to mention how similar processes and mechanisms occur in other bacteria, including the closely related Vibrio cholerae on a few occasions (example), so this review seems to indicate how patchy the current knowledge is on the mechanisms involved in the establishment of the symbiosis between V. fischeri and E. scolopes. It also seems as though there have been many isolated studies that specifically focus on V. fischeri, but the authors of this current review keep raising questions that indicate that many functions of the main signals and mechanisms are still not completely understood, such as the need to identify the functions of MCPS to determine how V. fischeri cells can alter their movement towards particular colonisation sites.  

Norsworthy, A.N., and Visick, K.L. (2013) Gimme shelter: how Vibrio fischeri successfully navigates an animal’s multiple environments. Frontiers in Microbiology, 4 (356): 1-14

Thursday, 27 March 2014

Soybean protein concentrate as feed for Salmo salar

Fish meal is an expensive and unsustainable source of protein for fin fish diets in aquaculture. Many manufacturers are incorporating plant materials as a substitute, and soybean is a popular choice due to its availability and cheap cost. However carnivorous fish haven’t evolved the mechanisms for effectively digesting carbohydrates and anti-nutritional factors found in plant meals. As a result previous studies have it to cause inflammation of the intestine, impeding digestion and growth. This inflammation is caused by alcohol-removable components of the soybean. However even with these components removed to give soybean protein concentrate (SPC), the intestinal microbiota community is still affected, which likely exacerbates any inflammation.

This study looks at four Salmo salar diets containing different levels of SPC, fish meal, and the prebiotic mannan-ogiosaccharide (MOS). It’s also based in open sea pens at a commercial sea farm in Tasmania, Australia. This is unlike previous studies conducted in closed and stable environments. These previous studies also found that in late summer SPC fed fish had higher mortality, lower growth and apatite, and vomiting as well as diarrhoeic effects, termed summer gut syndrome (SGS).

The results found that bacteria increased in the directional tract in the late summer. Feeding SPC to salmon increased diversity of bacteria in the intestinal tract and the presence of two that have not been associated with marine fish before – Escherichia sp and Propionibacterium acnes. These two have only previously observed in fresh water fish. These two were also associated with SGS in S. salar.

Temporal changes also occurred and dominance of micro-organisms switched over the experiment. In contrast to past studies individual bacterial communities were found to be different between fish, though this is likely as the experiment was not in a closed environment. After comparisons with similar studies the author also found that the bacteria that colonise S. salar intestines include Betaand Gamma-Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Verrucomicrobia. Bacterial profiles couldn’t be compared between fish due to the large individual variation mentioned earlier; however what was detected was consistent with findings in similar studies of fish and crustaceans.

The Escherichia sp identified was most similar to four undifferentiated Shigella sp, and symptoms from diets without MOS infer that they caused vomiting and diarrhoeic conditions found in the late summer. The experiment also showed that though the cause of the disorder wasn’t identified, MOS does prevent the adverse affects and recommends further studies for its inclusion on soybean diets.

The use of antibiotics as preventative measures has hard restrictions in the EU due to developed resistance in bacteria, and does not seem to be a good recommendation. As well as this as the full function of MOS on the S. salar intestinal community is not known, and could suppress beneficial bacteria, and affecting S. salar health. A similar experiment with SPC in more temperate waters may not initiate SGS, and could need no prebiotic measures. As well as this branches of further research should include other preventative means such as probiotics and a wider sample group for comparison of bacterial community profiles.

- Green, T. J., Smullen, R., & Barnes, A. C. (2013). Dietary soybean protein concentrate-induced intestinal disorder in marine farmed Atlantic salmon, Salmo salar is associated with alterations in gut microbiota. Veterinary microbiology, 166(1), 286-292.

Microbial Alkane Biodegradation Genes In Chronically Polluted Sub-Antarctic Coastal Sediments

Petroleum is a very complex chemical containging thousands of hydrocarbon molecules. In the ocean, sediments are the natural sinks of these chemicals. However, in cold environments, petrol can be very persistent causing both short and long-term ecological effects. As oil exploration is becoming more common at higher latitudes it is important to increase our knowledge of cold-environment bioremediation microbes. Bioredemdiation is complex process in itself, involving various microbial organisms. However, particularly in sediments, our understanding of this pathway is disadvantaged as the biodiversity and heterogeneity of the habitat increases its complexity. Most bioremediation work has made significant advances in the both the  tropical and temperate environments. Saturated hydrocarbons include alkanes, branched alkanes and cycloalkanes; and pose the highest proportion in crude oil.  Some previous work has suggested that adding alkanes to environments polluted with other, less degradable chemicals my increase the break-down of these persistent substances. There are a surprising number of microbes that use alkanes as a carbon-source including Alcanivorax, Thalassiolituus, Oleiphilus and Oleispira. These organisms contain various alkane-activating enzymes known as alkB. It has been suggeste that alkB be used as biomarkers for the characterization of aerobic alkane-degrading bacterial populations.

This work studies the alkane-degrading genes and bacterial communities of Ushuaia Bay, South America; described as a chronically polluted environment where off-loading of petroleum is a daily occurrence.
Sediment samples were collected via coring along the low-tide line and at a depth of 11m. These were then stored at -80°C. There were three different treatments of the original sample (OR08). These were the sediment with added 0.46ml of crude oil (expOR08-O), the sediment with added crude oil, ammonium chloride and sodium phosphate (expOR08-ON) and finally, the control sediment (expOR08-c). In order to fully analyse the hydrocarbons, samples were extracted and treated with activated copper and evaporated until 0.2ml was reached. This was loaded into a alumina column to recover the aliphatic and aromatic fractions. Individual alkane (from n-C10 to n-C35) concentrations, Pristine and Phytane isoprenoid levels, total resolved alkanes and total aliphatic hydrocarbons were calculated for each sample.
DNA was also extracted from each sample using a FastDNA SPIN kit. Two DNA extractions were conducted per sample. The alkB gene fragments were then amplified using specific primers, these were cleaned, cloned and sequenced. The V4 hypervaribale region of the 16S rRNA was amplified using pyrosequencing and used to construct alkB gene libraries. The alkB sequences were screened against the GenBank database using BLAST. Alpha-diversity was calculated using OTU (operational taxanomic unit) analysis.

All sample collected from the bay were seen to contain alkane homologous series, isoprenoids pristine, isoprenoid phytane and unresolved complex mixtures. Biodegradation indices showed that biodegradation was an on-going process. A total of 30 OTUs were found and were estimated to cover 95% of the overall alkB gene diversity. Richness was found to be 39-48 OTUs but this was described as a conserved estimate. Groups found relating to alkB were mainly Proteobacteria. The most closely related (and the most common OUT) to alkB were deemed as uncultured microorganisms from cold marine sediments.
After 20 days expOR08-O showed a decease in the concentration of alkanes lower than C20. However expOR08-ON showed further degradation of alkanes.
5 alkB gene OTUs were present in expOR08-O, 4 of which were also present in the environmental samples taken; the most abundant in this treatment was most closely related to Actinobacteria however there were also Gammaproteobacteria. expOR08-ON showed only two OTUs; Kordiimonas gwangyangensis and Rhodococcus spp.
A total of 44,380 reads were obtained from the V hypervariblae region that could be amplified from the three treatments expOR08-O, expOR08-ON, expOR08-c and the environmental samples. All OR slurries showed high richness and low dominance values. Although the expOR08-c was 38% lower than the environmental sediments (attributed to the laboratory conditions), expOR08-O had a lower richness value still and the expOR08-ON was the most simplified.

The authors goes on to explain that this study indicates a high diversity of alkB genes in highly polluted areas. They say that this was consistent over time and space suggesting an ecological relevance to the microbial communities of the sub-Antarctic sediments. The closest matches to the alkB gene sequences were from uncultured microbes suggesting further study in this area. It is explained that different components such as the type of pollution present factors into the communities present at a site. It is hinted that more studies need to be conducted into various environmental factors and as to how these may affect the population dynamics. As the addition of crude oil to the samples resulted in faster degradation of the alkanes. Although these results are still considered controversial. Previous studies have before suggested that the alkB-carrying microbes are positively correlated to alkane concentration however, other studies have debunked this. However this paper the presence of oil results in a strong amplification of alkB genes. This helps to take the first steps toward using alkB genes as biomarkers

Guibert L.M, Loviso C.L, Marcos M.S, Commendatore M.G, Dionisi H.M & Lozada M. 2012. Alkane Biodegradation Genes from Chronically Polluted Subantarctic Coastal Sediments and Their Shifts in Response to Oil Exposure, Microb. Ecol. 64: 605-616.

Tuesday, 25 March 2014

Besides chitin, mannitol helps Vibrio cholerae to survive in the marine environment by activating its biofilm formation!

V. cholerae occurs in freshwater and marine environments in two states, the planktonic state and/or attached to a surface in a biofilm. One type of multilayer biofilm of V. cholera, consisting of multiple cell layers, depends on synthesis of an extracellular matrix comprised of the VPS exopolysaccharide and numerous VPS-associated proteins, all of which are encoded within VPS island of the V. cholerae genome. Various environmental cues, including quorum sensing autoinducers regulate transcription of genes on VPS island. Recent studies have shown that certain sugars such as glucose and mannose play key role in biofilm formation of V. cholerae. Transport of these sugars, including mannitol (a sugar alcohol) is dependent on phosphoenolpyruvate (PEP) phosphotransferase system (PTS). PTS is a conserved bacterial signal transduction pathway with various cellular functions, including transport and phosphorylation of certain sugars and their derivatives, detection of quorum sensing molecules and biofilm formation. The pathways of PTS and enzymes included in it are described in the introduction of this paper. Phosphorylation states of PTS depend on intracellular availability of PEP and the environmental availability of PTS-specific sugars, and therefore, they can be called as sensors of the nutritional status of the cell. The authors of this paper have already shown that mutations in PTS components of V. cholerae causes increased tendency of forming biofilms. PTS mutants showed increased transcription of mannitol-specific PTS component, compared to wild types and this PTS component was also induced under biofim activating conditions. This study investigated role of maninitol and mannitol-specific component of PTS in biofilm formation by V. cholerae.
The selected bacterial strains were cultured in mannitol containing medium. Mutants were created by using molecular methods and, wild and mutant strains were cultured and RNA of cells was isolated. This RNA was converted into cDNA by using reverse transcriptase and then this cDNA was used in microarray. The ectopic protein expression, total growth, biofilm formation and vpsL transcription, were measured.
Transcriptomic analysis revealed that compared to wild strain biofilm, 563 genes were differentially regulated in the PTS mutant biofilm. Same analysis between both wild and mutant types of planktonic state revealed differential regulation of only 218 genes. The majority of these differentially regulated genes were associated with transport or metabolism of carbohydrates such as sugars. Major differential regulation was with genes of certain (EII) components of PTS. These PTS components which were highly induced in both planktonic and biofilm cells, were previously found to be transcriptionally activated by the addition of PTS sugars, which increases transcription of biofilm genes. The authors hypothesized that abundance of PTS-sugars and mutation in PTS, both of these scenarios result in similar physiological state of activating biofilm formation in V. cholera.
Mannitol increases VPS-dependant biofilm formation and, it is dependent on mannitol transport in V. cholera. It was found that concentrations of mannitol needed to activate biofilm formation were lesser than mannitol concentrations naturally found in marine environments. Certain mannitol specific PTS component (EIIBMtl) activates biofilm formation in V. cholera. But it is dependant on phosphorylation state and, only unphosphorylated EIIBMtl activates biofilm formation. Mannitol and unphosphorylated EIIBMtl activate biofilm formation at transcriptional level.
Apart from its association with chitinaceous surfaces, very little is known about the natural habitats of V. cholerae in the marine environments. Various marine organisms use mannitol as osmoprotectant and it is also released during algal photosynthesis (e.g. brown algae). This study, with its robust molecular experiments showed that mannitol can induce V. cholera to form biofilms and additionally V. cholerae may also use it as a source of carbon to survive in the marine environment. This study highlights how complex the life cycle and/or surviving capabilities of this clinically important pathogen can be in the marine environment and how much still we need to explore about this pathogen in order to develop strategies for its effective control.

Ymele-Leki, P., Houot, L., & Watnick, P. I. (2013). Mannitol and the mannitol-specific enzyme IIB subunit activate Vibrio cholerae biofilm formation. Applied and environmental microbiology, 79(15), 4675-4683.

Marine-derived fungus as biosynthesizer of silver nanoparticles

Nano materials are of interest in a broad range of applications in various fields such as mechanics and biomedical sciences. The synthesis of nanoparticles with more diverse properties is of increasing interest; however it is somewhat limited due to the costs and the hazard of toxic chemicals involved. Novel and safer methods are therefore required, and recently marine microbial cells have been identified as potential sources for the synthesis of nanoparticles. Among these, marine-derived fungi in the Gulf of Khambhat have been found as promising metal nanoparticle producers, since many fungal species from this area have been previously recognised as efficient metal removers. 

The potential as silver nanoparticle (AgNP) synthesiser of the marine fungus Aspergillus flavus was analysed in this study which was isolated from the West Coast of India. After culturing, the fungus was added to different concentrations of AgNO3. An effect was observed after 24 h of incubation; change in the colouration of the mixtures indicated intracellular synthesis of AgNPs from AgNO3, and colouration and thus biomass of AgNPs increased with time. However, from spectrophotometric measurements it was suggested that a certain concentration is needed to detect significant amounts of AgNPs. 

Additionally, the effects of different pH to the biosynthesis activity of A. flavus were examined, and results revealed that more alkaline pH had significant impacts on the synthesis of AgNPs. At pH ranges from 3-7, intracellular biosynthesis occurred, whereas from pH 8 to 10, extracellular synthesis was concluded (indicated by different colouration) and higher pH generally showed faster changes of the solutions than more acidic ones. 

To summarise, the results from this study showed that the marine fungus A. flavus has the ability to synthesise AgNPs intracellularly from AgNO3. Moreover, alkaline pH increased the extracellular biosynthesis of these nano particles. The authors pointed out that this species is a potential candidate for an environmentally friendly producer of the economically important metal nanoparticles. However, with the increasing ocean acidification I can see a limitation of such applications in the field (e.g. in the with metals heavily polluted Gulf of Khambhat from ship yeards) but rather under laboratory controlled conditions. Undoubtedly, the utilisation of A. flavus for the production of AgNPs would be very beneficial, especially when the use of harmful chemicals could be avoided. Ideally, the waters could be “cleaned” of metals with the introduction of similar species and NPs exploited at the same time. Yet is unclear how this would affect the ecosystem in general, so precaution would be required. An interesting study nonetheless with potential for future uses.

Vala et al. (2014). Biogenesis of Silvernanoparticles by Marine-Derived Fungus Aspergillus flavus from Bhavnagar Coast, Gulf of Khambhat, India. J Mar Biol Oceanogr 3:1.