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