The little Bacillus that could: disruption of biofilms in aquaculture.

Introduction

Microbial films are capable of resisting antimicrobial agents which makes them an excellent study for aquaculture treatments.  This paper by Hamza, et al. (2015) examined a culture of Bacillus licheniformis D1 isolated from the surface of the green mussel, Perna viridis from Tamil Nadu, India. The resulting isolate contained BLDZ1 which is an antimicrobial protein. It was tested for its antibiofilm potential against two known pathogens, Vibrio harveyi and Pseudomonas aeruginosa. These two pathogens play an important role in diseases associated with marine life. Vibrios, for example, infect marine fish and crustaceans, whilst Pseudomonas spp. can cause diseases such as black spot necrosis in prawns. Despite the fact that antibacterials are used widely, microorganisms can evolve resistance over time and biofilms in particular provide substantial protection against them. This study’s aims were to: i) investigate V. harveyi and P. aeruginosa’s biofilm forming abilities on different surfaces of polystyrene and glass, ii) to study the use of cell free supernatants (CFS) to disrupt biofilm production and iii) to discover the biological activities associated with biofilm disruption.

Materials and methods

The CFS was prepared by using the isolate B. licheniformis D1, identified using 16S rDNA analysis and Genbank sequence database. 16S rDNA can be used for identification of microorganisms that can be classed as unculturable, rare, slow-growing or bacteria with an unusual phenotypic profile. The bacteria was grown in LB broth at 30 °C with shaking for 36 hours. It was then centrifuged and filtered. For this study, the CFS produced was then used without further purification. Scanning electron microscope (SEM) analysis was used to examine the effect of the CFS on incubation of the biofilms for both polystyrene and glass surfaces.

Polystyrene surface

Test cultures of V. harveyi and P. aeruginosa were co-cultured in polystyrene microtiter plates alongside 100 μl CFS to examine the effects of biofilm inhibition, with a control of both pathogens cultured without CFS, both incubated for 24 hours. Pre-formed biofilms were also used, and biofilms were grown for 24 hours on polystyrene surfaces before 100 μl of CFS was added and incubated for a further 24 hours.

Glass surface

V. harveyi and P. aeruginosa were incubated with either 100 μl or 200 μl of CFS and biofilm formation occurred on sterilised microscopic glass slides treated with LB medium for 24 hours. For pre-formed biofilms, both cultures were inoculated in LB medium and biofilm formation allowed to occur for 24 hours before the slides were put in CFS containing medium for a further 24 hours. Controls were done for both, following the same procedure but without introduction of CFS.

Results

Both of the test cultures formed biofilms on polystyrene surfaces and were inhibited by CFS substantially, V. harveyi by around 80.5% and P. aeruginosa by around 77.5% and preformed biofilms were disrupted by CFS by 73.08% and 73.76% respectively. Both showed statistical significance with a p-value of <0.001.

SEM analysis showed CFS caused significant inhibition and removal of mature biofilms on glass surfaces depending on the concentration (100 μl or 200 μl CFS) shown in the figures in the report. Exopolymeric substances (EPS) were found in the control biofilms but not in CFS treated samples. 

Biological activities

CFS displayed anti-adhesive abilities which prevented attachment of culture cells to polystyrene surfaces, and caused cytoplasmic membrane permeabilisation leading to leaking of cytoplasmic material and ultimately cell death.

Discussion

With the rise of resistance to antibiotics, new methods must be put in place to minimise the damage that pathogens can have on the aquaculture industry. Antibiotics use the same avenue when attacking pathogenic cells, but by deploying natural epibiotic bacteria, novel methods of inhibition by production of bioactive compounds can open up an entirely new capacity for antimicrobial methods. This experiment also presented the knowledge that inhibition can not only occur at the immature biofilm stage, and even upon extensive growth the pathogens can be hindered and potentially removed altogether. However, the experimental growth was only allowed for 24 hours which means that further growth may prove more difficult to overcome by antibiotics. On the other hand, regular checking and cleaning of aquaculture tanks may prevent biofilm growth from occurring for such lengths of time but this depends on the particular facility, resources and size. This is made more difficult by biofilms growing in difficult to see/reach places such as in pipework. By lacing the CFS along the pipework regularly, this would prevent adhesion by the biofilm from ever happening. This is indicated by the presence of EPS in the control, which acts as a core structural component in biofilms. 

Aquaculture allows a safer method of supplying fish that does not damage natural population densities by overfishing or the environment through invasive fishing practices. Inclusion of probiotics into the community also aids humans who consume them, especially when wild populations of marine organisms have been found with increasing levels of harmful elements. Poisons such as mercury or endocrine disrupters such as bisphenol-A are but a few that are capable of bio-accumulating and working their way up the food chain. In conclusion, this report presents a novel pathway in the elimination of certain aquaculture pathogens and therefore reduces the mortality rate of the organisms kept, leading to increased revenue for facilities.

Ref: Hamza, F., Kumar, A. R. & Zinjarde, S. (2015) Antibiofilm potential of a tropical marine Bacillus licheniformis isolate: role in disruption of aquaculture associated biofilms. Aquaculture Research. 1-9. doi: 10.1111/are.12716 

Karen Wynbergs Seminar: Coral reefs go viral

Karen Wynberg works at the Australian institute of Marine Science in Townsville Australia, studying the role of viruses on coral reefs. I had the pleasure of getting to know Karen and her co-worker Elisha Wood-Charlson at AIMS, and honestly these two worked regularly around the clock in a specialised lab with their own flow cytometer, to isolate, sequence and portray the virome of a coral reef.  Some of results of this work was published last year, see reference below.

Karen’s seminar was based upon work she conducted when she started at AIMS, regarding what part viruses played in coral bleaching (if any)? But first she presented some fun facts on the importance of viruses on the reef 1) they can play a role in preventing disease, for example bacteriophages that infect pathogenic bacteria. 2) They are crucial in nutrients cycling (the viral shunt) in oligotrophic water and 3) are a key part in horizontal gene transfer, one possible mechanisms which allowed corals to become resistant to V. shilloi in the Red Sea,  as we discussed in the lecture.

Why corals expel their zooxanthellae is still under debate (heated debate between coral biologist) if you then ask a virologist, they’ll put this spin on it:
It’s a virus infecting the zooxanthellae, causing the corals to bleach.

What Karen hypothesised was that the symbiodinium harboured a lysogenic virus that under stress, such as elevated temperature or UV, entered the lytic cycle. She tested her hypothesis using cultured symbiodinium (looking at them with transmission electron microscope) and found filamentous virus like (VLP) particles within the nucleus and cytoplasm. The size and morphology of the VLP were similar to plant viruses, maybe it was a viruses causing bleaching? Unfortunately, no nature paper here.  When she looked at fresh symbiodinium from thin sections of corals, the VLP were never present. The virus must only infect the symbiodinium when it is without the protection of the coral.

She also talked about the possibility of using viruses as biological control in cases of serious disease outbreak, which is something her PhD student is currently working on. They are currently looking into phages that infect cyanobacteria, to use a remedy in the initial stages of black band disease (which starts as a cyanobacteria lesion – see previous post on BBD).

Crown of thorns starfish (COTS) are listed, as one of the largest threats to the Great Barrier Reef, so another biological controls they are looking into is a virus that specifically infects the Ancanthaster planci. If they could isolate such a virus, it could be used instead of the current method, which is injecting a nasty cocktail of chemicals into the COTS and leaving it to die.

This was a great seminar from Karen, and an eye opener into the role of viruses on the reef, where my focus had previously only got as far as bacteria. Virology is a fascinating subject, with a lot to still learn, making it that bit more intriguing. I always admired the two virologists in their lab at the end of the corridor and it was great to be presented with some of Karen’s’ previous ideas, and those to come. I highly recommend keeping an eye on this author if you are interested in reef virology.

Karen's paper mentioned:

Weynberg, K. D., Wood-Charlson, E. M., Suttle, C. A., & van Oppen, M. J. (2014). Generating viral metagenomes from the coral holobiont. Frontiers in microbiology, 5.

Cons of Probiotics?

The use of probiotics as a therapeutic treatment for human disease and illness has long been established. Pioneered by Élie Metchnikoff in 1907, probiotics now form the basis of a multi-million, if not billion pound industry, which has now been applied in aquaculture. A vast array of literature has been published focussing on probiotics, with researchers claiming a diverse range of benefits; reduction of gastrointestinal pathology, protection of biomacromolecules from oxidative damage and improvement of the immune system, to name a few. Probiotics are can be defined as live micro-organisms which confer benefits to the host when consumed.The term, and relative definition of probiotic has been subject to much conjecture in the literature; the original definition fails to take into account the viability of the microbial consortia constituting the probiotic. It has been reported that even non-viable microbes can confer benefits to the host-organism; thus the term paraprobiotic was conceived –  non-viable microbial cells, which confer benefits to the host in sufficient amounts. Recently, Dash et al. (2015) investigated the effect of probiotics, specifically paraprobiotics on the immune response in the prawn, Macrobrachium rosenbergii.

The paraprobiotic strain, Lactobacillus plantarum was heat-killed and added to the diet of the shrimp, M. rosenbergii at three concentrations; 10⁷, 10⁸ and 10⁹ cfu g^-1 diet. These were compared to a control group of shrimp, which had no supplementation with paraprobiotics. Standard aquaculture feed trial measures, such as weight gain, growth rate, feed conversion ratio and food conversion ratios were taken at the end of the 90-day investigation period. Through extraction of haemolymph, immunological/biochemical assays were undertaken including; haemocyte counts, phenoloxidase activity, respiratory burst activity and bacterial clearance efficiency. M. rosenbergii were immune challenged with the pathogenic bacteria, Aeromonas hydrophilia.

A. hydrophilia is a problem for a diverse range of organisms (particularly crustaceans) because of their ability to produce cytoxic enterotoxins that can induce tissue damage. This bacterium is also considered to be resistant to a number of antibiotics, thus finding a way to manage infection of this bacterium, without causing deleterious changes in the host’s microflora is greatly beneficial.

Phenoloxidase activity (an antimicrobial phenol and component of the invertebrate immune system, hemocyte count, respiratory burst activity (rapid release of ROS; proxy of immune cell activity) and bacterial clearance efficiency of M. rosenbergii all increased when supplemented  with paraprobiotics. Also, reduction in mortality was observed in organisms supplemented with the paraprobiotic; those subject to higher concentrations of the paraprobiotic displayed less mortality, thus showing an increased disease resistance capacity. Little change in the growth parameters was observed throughout the investigation.

 A definitive mechanistic understanding as to how probiotics enhance immunity in invertebrates is non-existent. That said, it is evident that probiotics do provide benefits to the host particularly immunological function and disease resistance. Elucidating the host-microflora-probiotic cross-talk would be incredibly interesting and revealing, furthermore observing how this cross-talk changes in response to a number of factors, from pollutants to xenobiotics to other bacterial strains could introduce a novel use for probiotic supplementation. However, in a more ecological context, do ‘natural’ probiotics exist and have existing studies merely designated these as constituents of the microflora? 

Jack 

Dash, G., Raman, R.P., Prasad, K.P., Makesh, M., Pradeep, M.A. and Sen, S. (2015). Evaluation of paraprobiotic applicability of Lactobacillus plantarum in improving the immune response and disease protection in giant freshwater prawn, Macrobrachium rosenbergii (de Man, 1879), Fish & Shellfish Immunology, 43(1). p167-174.

Bacterial Septicemia in Cancer pagarus

Vibriosis is a serious condition that affects many species in the marine environment, it is though that susceptibility in marine species changes depending on the life stage of the organism. Crustaceans have seen to be susceptible to many vibrio-induced infections and are prone to attack from a wide range of species including V. alginolyticus, V. fluvialis, V. harveyi and V. vulnificus. However, our knowledge of the importance of vibrios in Crustacea is limited to few studies only using a small number of species, including shrimp and the blue crab. This study looks at the importance of vibrios in infection of juvenile stages of the edible crab, Cancer pagurus. It has been previously suggested that the juvenile life stage is a ‘bottleneck’ for survival to adulthood, meaning the importance of susceptibility to virbriosis is highest at this point, allowing for optimal infection and bacterial septicaemia to persist.

Juvenile C. pagarus were collected from Mumbles Head and Gower Penisula, Swansea, UK. Their haemolymph was extracted and aseptically plated onto saline tryptone soya agar (TSA) and thiosulfate citrate bile salts sucrose agar (TCBS). Colonies were left to form at 25ᵒC for 2-5 days and a baseline of 2000 colony forming units (CFU) mL^-1 was used as an indication of the extent of bacterial septicaemia, below this level it was assumed to be low severity infection.

After incubation, the morphological characteristics of the bacteria were recorded, this showed the bacterial colonies from the haemolymph to be highly variable. 33% of the samples had vibrio-like bacteria culturable on TSA, however only 1% were of high severity (>2000 CFU mL-1). This bacterial susceptibility was shown to have no influence by carapace damage or limb loss which contrasts to previous studies considering the blue crab, which was shown to be more prone to bacterial infection if damaged or missing limbs. The extent of low level infection was shown to be influenced by the stage of moult, post-moult crabs with softer, more vulnerable cuticles were shown to have significantly higher levels of bacterial haemolymph infection. Infection was also shown to be of higher persistence in the summer to autumn months when sea surface temperatures were highest. This has been shown to be typical of Virbio species as it has been shown that higher water temperatures often favour their replication.

Gill damage was measured using histological studies, it showed correlation between cases of high level of haemolymph infection, this is suggested to be either the route of entry of the infecting bacteria or a resulting defect of septicaemia. Previous studies have shown nodule formation in Carcinus maenus to be a defence mechanism for dealing with bacterial septicaemia.

This study also looked at the effect of the dinoflagellate, Hematodinium, on bacterial infection. The presence of Hematodinium in the crabs hepatopancreas correlated with reduced bacterial infection, it is therefore thought that a competitive interaction exists between this dinoflagellate and bacterial infection as a possible result of phagocytic action.

Molecular analysis of 16s rRNA was used to identify the infecting agents colonies with the most potential for vibrio species from 3 separate samples. The colonies were amplified with PCR, sequenced by MWG eurofins operon and searched using BLAST and the Ribosomal Database project II using an identity threshold of 99%. All three of the tested colonies showed highest similarity to Virbio spp, with two out of the three being most similar to Vibrio pectenicida. These V.pectenicida colonies were only cultureable on the TSA agar, suggesting that this study may have had limitations by using TCBS agar which may have led to an underestimation of vibrio species in the samples.

This study showed Vibrio species to be present in juvenile C. pagarus, however there was little identification of the species contributing to infection in the blood. To further analyse the crab septicaemia, identification of the more prevalent colonies and correlation between the damage to the crabs and the bacteria present would be needed. However, they did show that bacterial infection is significantly reduced with the presence of the dinoflagellate Hematodinium spp., showing there is a potential of competitive and perhaps phagocytic activity between these two communities, further study into the relationship between them would be recommended.

Smith, A., Whitten, M., Hirschle, L., Pope, E., Wootton, E., Vogan, C. and Rowley, A. (2013). Bacterial septicaemia in prerecruit edible crabs, Cancer pagurus L. Journal of Fish Disease, 37, 8, 729-737.

Are Salmon Lice like Marine Mosquitos?

Lice infestations are an inevitable consequence of rearing high densities of fish in aquaculture. As salmon lice, Lepeophtheirus salmonis, graze on the skin they create a route for opportunistic pathogens to enter such as the bacterium Pasteurella, which causes pasteurelliosis. In addition, the lice have been shown to harbour aquatic pathogens themselves, for example Aeromonas salmonicida. However, until recently their role as vectors had never been proven. Jakobs et al. (2011) provided the first evidence that lice act as vectors, in this case for Infectious Haematopoietic Necrosis Virus (IHNV). This virus has been previously blamed for several deadly epidemics in North American aquaculture of Atlantic salmon, Salmo salar.

IHNV naïve adult lice from farmed salmon and salmon parr (a juvenile stage) were collected for use in the experiment. The IHNV strain used was from an epidemic that occurred in the 90s. Infection was confirmed throughout the experiment via RT-PCR and tissue culture methods. Firstly, it was confirmed that lice were able to acquire IHNV from exposure to the virus present in a water bath. The virus remained associated with the lice for 24hrs after the exposure. Secondly, it was found that lice were able to obtain IHNV from exposure to fish injected with the virus. However, in this case the virus was only associated with lice for 12hrs after removal. Eventual loss of the virus from the lice indicates that the virus is unable to replicate within the crustacean. This is unlike other diseases such as malaria where the trypanosome replicates within the mosquito. It was then shown that exposure of lice to naive salmon led to high mortalities and infection with the virus. Exposure to lice, which had gained the virus through water-bath exposure, led to 70.6% mortality and 13 out of 17 fish testing positive for IHNV. Similarly, exposure to lice that had gained IHNV from infected salmon lead to comparable levels of infection and mortality in naïve salmon. An important question was also, are lice able to transmit the virus to fish via direct contact or through the water? In short, the ability of IHNV to infect salmon without attachment of the lice was zero. Interestingly, in this case exposure to IHNV infected lice led to lower mortalities than the first set of experiments. Which could be due to differences in host susceptibility or virus load.

Overall, this study provides some evidence that salmon lice are able to transmit the virus IHNV to naive salmon although this affect was highly variable in the study. Also, given the short time of persistence of the virus within the lice, it is unlikely that lice are important vectors of IHNV between farms and wild populations over large distances. Instead, lice may help to transport the virus between pens along with water bourn dispersal of virions during an outbreak. However, if other pathogens are able to persist within the lice they could act as vectors. Further research should focus on determining whether lice can act as a vector for other diseases such as Aeromonas salmonicida.

Ref: Jakob, E., Barker, D.E. and Garver, K.A (2011). Vector potential of the salmon louse Lepeophtheirus salmonis in the transmission of infectious haematopoietic necrosis virus (IHNV). Diseases of Aquatic Organisms, 97, 155-165.