Alternatives to antibiotics to control bacterial diseases in aquaculture

Aquaculture is the fastest growing food producing sector worldwide, outbreaks of disease can cause losses of several billion us$ per year. The aquatic environment is more supportive of pathogenic bacteria than the terrestrial one, due to the ease of the bacteria being able to live outside the host organism.

The routine use of antibiotics in aquaculture in an attempt to control bacterial disease has led to widespread resistance of pathogenic bacteria to antibiotics. An example of widespread antibiotic use is of 76 shrimp farmers interviewed in Thailand, 56 used antibiotics, 10 different antibiotics and most used these used antibiotics prophylactically (as a prevention method not a treatment). Furthermore few countered monitor the quality of antibiotics used, and only a select few countries have regulations on the use of antibiotics in aquaculture. However many governments have enforced a Maximum Residue Levels for aquaculture products, meaning that countries which do not have adequate regulations on antibiotic use need to comply to the stricter regulations of the countries that the products are being exported to.

The widespread use of antibiotics in aquaculture has led to resistance development in some pathogenic aquaculture bacteria. For example the pathogen Vibrio harveyi which causes mass mortality in Penaeus monodon (giant tiger prawn) has strains which were resistant to chloramphenicol, erythromycin, cotrimoxazole and streptomycin. Of these antibiotics both cortimoxazole and chloramphenicol have been used a prophylactics.

An explanation for widespread antibiotic resistance, is that the resistance genes are often found on transferable plasmids and integrons of pathogenic bacteria. These resistance determining genes can then be passed to other bacteria via horizontal gene transfer. As antibiotics have a wide range of target organisms, including detrimental and beneficial bacteria the pool of bacteria which can develop resistance is greater and therefore widespread resistance is more common.

As antibiotics are rapidly becoming ineffective to aquaculture diseases, alternative methods are being explored into preventing diseases, but also as prevention is not always achievable controlling the pathogenic bacteria in order to make aquaculture more sustainable. This is not only important in order to save money but also as many of the bacteria are resistant to antimicrobials which are critically important in human medicine. It is likely that horizontal gene transfer may occur from aquatic bacteria into terrestrial human and animal pathogens.

One alternative method in controlling pathogenic bacteria is the use of bacteriophages; these are specific antimicrobial agents unlike the broad spectrum antibiotics. Bacteriophages are viruses which can infect and kill bacteria, bacteriophages are not only just species specific but also many are strain specific. This would be an ideal strategy as not only would the beneficial bacteria not be affected but the pool of bacteria in which resistance can develop is smaller and therefore the risk of resistance developing lower.

A phage from shrimp farm water which was found to have lytic effects against 50 V.harvyi strains, when tested in a commercial tiger prawn fishery the survival of the shrimp increased from 17 to 86% after 17 days. This rate is much higher than that of the addition of 5mg/L of oxytetracyclin and 10mg/l kanamycin daily, in which the shrimp survival was only 40%. The success of phage therapy would depend on the amount of strains of the pathogen species the phage can infect. A further problem with phage therapy would be the potential for phages to carry virulence genes and also the resistance of bacteria to phage attachment, the author suggests a variety of ways that the can be overcome including using phage components not intact phages.

A second technique into pathogen control is specific for Vibrio species, this is because the Vibrio genome is compartmentalised into two chromosomes. RctB is what causes the initiation of the replication of the smaller chromosome; this initiator is specific to the Vibrio family and would therefore be a good specific mode of bio control. A small molecule vibrepin was identified in a study to inhibit the growth of all vibrio species tested including aquaculture pathogens Vibrio parahaemolyticus and Vibrio vulnificus.

An alternative strategy to control pathogenic bacteria could be to inhibit their growth rather than killing them. Short chain fatty acids are currently used in animal feeds to control pathogens such as salmonella. Recent research has shown that shrimp suffering from vibriosis could have a higher survival as the short chain fatty acids reduce the survival rate of the pathogenic vibrios.

Another method which can be used as an alternative to killing cells is disrupting the functions needed for the cells to infect the host organism, also known as virulence factors. Bacteria can coordinate the expression of certain genes via small signal molecules; this is known as quorum sensing. There are two types of quorum sensing in bacteria, those that use N-acylhomoserine lactones (AHL’s) and signals are detected in the cytoplasm. Then there are the bacteria which are detected at the cell surface and use multi-channel systems, for example many vibrio species. Quorum sensing has been shown to regulate virulence factor gene expression; therefore disrupting quorum sensing can reduce the amount of bacterial cells which can cause infection.

One quorum sensing disrupting compound, halogenated furanones were discovered in a red marine alga. They disrupted quorum sensing gene expression in both types, however they also proved to be toxic to the test organisms at doses similar to those needed to disrupt quorum sensing. There are several molecules which have been found to interfere with quorum sensing, however more research is needed to see if these would be effective in practice.

Another method of disrupting quorum sensing is to inactivate the signal molecules, many bacteria have the ability to interfere with AHL quorum sensing through degrading signal molecules by degrading the lactone ring. However again several bacteria have been found to produce AHL degrading enzymes but more research is still needed into testing them in a host-pathogen setting.

Finally the ToxR gene in vibrios is found to control the expression of toxins, for example the cholera toxin in V.cholerae. a small molecule called virstatin has been reported to inhibit the transcriptional regulator of ToxT, this controls the ToxR regulon in vibrios. This method has also not been tested, along with suggestions that inhibiting specific virulence factors, eg: secretion systems and adhesion factors.

The author concludes by saying that the bio control methods outlines could be an important step forward in combating resistance to control in pathogenic aquaculture bacteria. This is because the methods outlines are more specific than antibiotics reducing the pool of bacteria which may develop resistance and therefore there are less bacteria to transfer the resistance genes. All of the methods outlined in the paper are still in the research phase, non-having been tested in a real life scenario. The author concludes by saying that the methods outlines are unlikely to be successful in all disease cases, therefore research needs to continue to develop ideas into overcoming disease in aquaculture.

A review of: Defoirdt, T., Sorgeloo, P., Bossier, P. 2011. Alternatives to antibiotics for the control of bacterial diseases in aquaculture. Current Opinion in Microbiology. 14, pp., 251-258.