Aquaculture is one of the fastest growing sources of food production
and is not without its problems. Having organisms in such high densities
increases the risk of disease, to prevent this antibiotics are often used,
however this overuse of antibiotics can lead to bacterial resistance. A paper
by Santhakumari et al (2015), is the first to look at the anti-quorum sensing
potential of the marine cyanobacteria Synechoccus sp. on pathogenic marine
vibrio spp using the Q-25 extract. The aim of this study was to try suggest an
alternative to antibiotics used in aquaculture.
Bacterial pathogens can
develop resistance by the formation of biofilms and production of virulence
factors both of which are controlled by quorum sensing (QS) in the bacteria.
Swimming and swarming mobility is also regulated by QS. Quorum sensing allows coordination
of gene expression which uses signal molecule known as autoinducers. Biofilms
are the preferred lifestyle for bacteria as they enable enhanced growth and
allow the free entry of nutrients and prevent the entry of antibiotic. Extracts from marine cyanobacteria are
examples of anti-quorum sensing (anti-QS) compounds there have been previous
reports showing that cyanobacteria can interfere with QS system in pathogenic
bacteria. It is therefore suggested that by using cyanobacteria extract to
target these autoinducers it will inhibit the production of virulence enzymes
and also prevent biofilm formation to reduce bacterial infection.
Summary
of Method
- The Q-25 extract was obtained from strains of the marine cyanobacterium Synechococcus sp. 6 different bacterial species were used as test pathogens (Chromobacterium violaceum, Vibrio harveyi, Vibrio parahaemolyticus ,Vibrio alginolyticus ,Vibrio vulnificus and Aeromonas hydrophila).
- 110 marine cyanobacteria strains underwent qualitative analysis in order to find their anti-QS potential against the pathogen C. violaceum. C. violaceum synthesizes a violet-coloured pigment which is regulated by its QS mediated signal molecule. Treatments were observed for the reduction of this molecule with the addition of marine cyanobacteria containing Q-25. Strains which showed positive results were then subjected to quantitative analysis.
- Bioluminescence inhibition was studied using different concentration of Q-25 from 0, 50, 100, 150 and 200 μg mL−1 after 12 hours the intensity of bioluminescence was measured using a relative light unit using a luminometer.
- Gelatinase activity was observed using untreated and treaded cultures (50–200 μg mL−1) after 2-4 days the medium was flooded with HCL and HgCl2 solution, observation of a clear zone around the bacteria indicated gelatinase was inhibited
- Biofilm biomass was estimated using crystal violet staining assay both V. harveyi and V. vulnificus were investigated 200 μg mL−1 of Q-25 extract was added to broth containing each species after a 16 hour incubation period in order to remove any loosely attached cells not part of the biofilm the plates were washed with sterile-deionized water The absorbance was then measured using UV-visable spectrophometer, to allow Percentage inhibition of biofilm biomass to be calculated. After Staining both the control and non-controls were observed using light microscopy.
- · Swimming and swarming assay was also studied by using both a control and non-control containing Q-25 the bacteria which were observed over a 16 hour period.
Findings
Qualitative and quantitative assay showed that Q-25 had anti-QS potential and so the extracts form marine cyanobacteria which has shown positive quantitative analysis were used.the production of bioluminescence in the pathogen V.harvevi is positively regulated by QS systems the results found that Q-25 extract inhibited bioluminescence by up to 91%.
Gelatinase which is an example of a virulence factor was also
shown to be inhibited when Q-25 was at a concertation of 100 μg mL−1 furthermore comparisons between the treated and
untreated sample shows a significant reduction in both V. harveyi and V. vulnificus treated with Q-25.
Q-25 was found to be a concentration- dependent inhibitor,
for V. harveyi it was inhibited, up to 71% and for V. vulnificus
this was as high as 84%.
further observation by
light microscopic showed a visually difference between the control samples and those
in contact with Q-25 which showed both reduced efficiency to cover the glass
and reduced abundance in already mature biofilms.
Conclusion
The finding show that Q-25
extract was able to inhibit biofilm formation and significantly reduced the number
of micro-colonies which occurred in the biofilm. Showing that Q-25 is able to
both inhibit the development of biofilms most likely due to its ability to
reduce the swimming and swarming ability of bacteria, and also disrupt marine
biofilms. This is a significant finding because bacterial in mature biofilms
pose a greater threat being potentially 1000-fold more resistant to antibiotic treatment
compared to bacteria growing planktonically.
I like how this paper didn’t just focus on how one vibrio species
may be effected by Q-25 but included different species that have been shown to
be causes of disease in aquaculture. I think a strength of the paper is how it
looks in detail at 3 areas regulated by QS (swimming mobility, biofilms and virulence
factors production) as opposed to just focusing on one which gives a more in
depth picture. Although it’s the first to look at Q-25 extract it refers to
many examples of work on other sources of QSI further strengthening these
findings and potential applications for use in aquaculture.
Reference
Santhakumari, S., Kannappan, A., Pandian, S.,
Thajuddin, N., Rajendran, R. and Ravi, A. (2015). Inhibitory effect of marine
cyanobacterial extract on biofilm formation and virulence factor production of
bacterial pathogens causing vibriosis in aquaculture. J Appl Phycol, 28(1),
pp.313-324.
Hi Alisha,
ReplyDeleteThanks for the post! I agree that this study shows very interesting possibilities to maybe avoid antibiotics in aquaculture. If I got it right they tested the influence directly on the pathogenic bacteria. I think it would be interesting to see if the results change when the Q25 extract was added to infected fish or any other organisms grown in aquaculture. Would you expect any differences?
Thanks,
Eleni
Hi Eleni, yes they did test the influence directly on the pathogenic bacteria, they wanted to test the effect of the Q25 extract on 3 factors which were how Q25 might disrupt biofilm formation, prevent the production of virulence factors and see how the extract would influence swimming mobility in the bacteria due to the Q25 extract ability to disrupt quorum sensing in the bacteria.
DeleteTo my knowledge as of yet there has been no further investigation into how the Q25 extract may impact already infected fish and if there would be a difference. From my understanding I think the main aim of Q25 would be to prevent initial infection before fish became infected however there is no reason to say that further studies shouldn’t look at adding the extract to already infected fish I would assume that even within the fish the extract would still be able to disrupt the biofilms, and prevent pathogens from producing further virulence factors, a benefit to directly adding the extract to fish is that it would reduce the amount released directly into the water column i’m currently not sure if adding a high concentration of Q25 would have any negative effect to surrounding areas and is probably something that should be studied further before an field trail occur. It was mentioned that mature biofilms in some cases allowed pathogens to become up to 1000 fold more resistant to antibiotics maybe the combination of using the Q25 extract to disrupt the mature biofilms and then using antibiotics in a more controlled way at lower concentrations could be beneficial in curing infections this of course would have to be tested. I think one of the main reasons why the extract was added directly to the pathogen as opposed to infected fish was that it is much easier and quicker to see the impact of disease directly on the pathogens, however it would have taken more time and resources to see the effect of the pathogenic bacteria in infected fish, maybe now that Q25 has shown to disrupt quorum sensing in bacteria further research may investigate the effects in infected fish.
This comment has been removed by the author.
ReplyDeleteHi Alisha,
ReplyDeleteGreat post, have two small questions for you.
Quorum Quenching isn't really a wide spread technique in aquaculture, what do you think are the biggest hurdles it will have to get over?
Did the authors mention the use of any other alternative such as Bacteriocins, antimicrobial peptides, and bacteriophages? Most of these techniques are still in their early stages and have focuses mostly on poultry and cattle. Do you think quorum quenching is the correct route to take over these other methods?
Many thank,
Stefan
Hi Stephen,
DeleteIn my option I think it’s quite surprising that so far there hasn’t been more interest for quorum quenching as a wide spread technique in aquaculture I think this is most likely due to the overuse and reliance of antibiotics in aquaculture, this in itself I think is a big hurdle I think people may be reluctant to switch methods even if antibiotics lead to resistance. I think another hurdle is that even though the findings show that the Q25 extract is able to disrupt quorum sensing and reduce infection, the studies so far have all been lab based and so before the extract can be used in aquaculture the potential effects it has on the surrounding area would need to be investigated, what makes this an even bigger hurdle is how funding to investigate this will be obtained.
The authors did not mention or compare the method to any of the alternatives, From the findings I would say that quorum sensing is a good route to take a particular reason why I would argue that quorum quenching is a good route is that the extract was shown to be effective for a number of vibrio pathogens and therefore it could potentially be applied to prevent a range of disease simultaneously. On the other hand methods such antimicrobial peptides, bacteriophages and bacteriocins, are limited as they can only target specific strains for example bacteriophages can only inhibit the growth of similar or closely related bacterial strain it is true that phage cocktails can be used to target a number of stains simultaneously but again it would require the right ‘cocktail’ to be create I think a disadvantage of this specificity would be that areas that are not able to fund this research and find phages or bacteriophages that are specific to the disease that they face my continue to rely on antibiotics, however it appears that the Q25 extract would be able to be used much more generally due to its ability to dispute quorum sensing in a number of pathogens.
Hey Alisha,
ReplyDeleteA really insightful post - it's nice to read something that's not doom and gloom!I'm curious as to whether the authors mentioned anything about the effect of Q25 on bacteria which are associated with healthy individuals in aquaculture? Does this extract manage to pinpoint harmful bacteria, or is there a possibility that the healthy and necessary microbiota residing within and on the exterior of individuals will be negatively impacted? Also, there is no mention to the species of organism being 'cultured', or has the idea of Q-25 not progressed outside of the laboratory?
Thank you!
Harriet
Hi Harriet interesting question unfortunately the authors did not mention the potential impact it could have on bacteria that promote healthy aquaculture stock However by considering how the Q-25 extract works, it works by interrupting AHL mediated gene expression in quorum sensing this mediation by AHL to my knowledge appears to occur only in Gram-negative bacteria therefore I would expect that beneficial bacteria (excluding gram negative), cyanobacteria and fungi would not directly be effected by the Q-25 extract, however this still poses the question as to whether beneficial non-pathogenic gram negative bacteria would also be negatively affected by the Q-25 extract although unlike antibiotics the extract would not directly kill the bacteria it prevents factors controlled by quorum sensing including formation of biofilms, swimming mobility and productions of virulence factors the former two being important in non-pathogens, I think further study would be needed to assess the impact in a natural environment if the extract was found to effect beneficial gram negative bacteria adversely I think the extract would still be able to be used in aquaculture but the microbial community would have to first be considered to see if the microbiota associated with healthy individuals are gram-negative bacteria interactions or from other bacteria, cyanobacteria or fungi and if the gram-negative bacteria provide an interaction which is unable to be substituted for assuming it effects them negatively.
DeleteTo answer your second question currently the Q-25 extract has not been investigated out of the lab however the authors did investigate the extract on bacteria that have been known to cause disease in aquaculture for example they look at how the extract would affect V. harveyi this marine pathogen has been shown to cause mass mortalities in black tiger shrimp, so although currently the investigation has not progressed out of the lab I think it’s set the foundations for future field based studies
Hi Alisha,
ReplyDeleteIn your post you mention the virulence factors produced by the pathogenic vibrio spp. However, it's still not really clear to me what these exactly are. Gelatines are mentioned as an example, but do the virulence factors just include some kind of enzymes or is it broader than that? And how do these virulence factors support bacterial resistence?
I liked reading your post! Structure wise, it was easy to follow. And also, the promising results of these study are an interesting, positive development in the aquaculture industry. What did the authors say about further knowledge needed before considering practical application of these findings?
Thanks!
Thyrza
Hi Thyrza
DeleteAs you mentioned the enzyme gelatinase is an example of a virulence factor other examples found in Vibrios include caseinase and protease one benefits of these enzymes is they help the bacteria to resist the host immune response. A virulence factor can be describes simply as molecules that are produced by pathogens and contribute to the pathogenicity of the organism increasing their ability to colonize a host including by attachment to the cells. I did a bit of research into virulence factors and found they help the bacteria to invade the host, cause disease and evade the host defences this ability to evade the host defences therefore support bacteria resistance. As well as enzymes there are other types of virulence factors including: Adherence factors, which allow pathogens to adhere to cells by using cell surface proteins, pathogens may also release endotoxins all of these factors are important in increasing the pathogens potential to cause disease
The author mentions that further study should focus on applying the finding of this study including the anti-QS potential of the marine cyanobacterium Synechococcus sp. against bacterial pathogens to prevent bacteria diseases in aquaculture stock suggesting the next step should consider looking at if disease can be prevented in cultured aquaculture species and if it would be effective in the field