Partial description of bacteria in the gastrointestinal track of Haliotis midae, with the focus on potential probiotics

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The ocean’s fish resources are declining mainly because of irresponsible exploitation. Fish is a vital source of protein for humans, and growing world populations are threatening the sustainability of commercial fisheries. This has led to the rapid growth of aquaculture worldwide. In South Africa, aquaculture of both fresh and marine species is expanding and is now practised in all nine provinces of the country.

One of the major problems in aquaculture is financial loss as a result of disease. Viruses, bacteria, fungi and parasites are known to infect fish, with bacteria causing the majority of diseases. One of the largest groups of disease-causing bacteria in aquaculture comes from the family Vibrionaceae. The genus Vibrio includes more than 30 species and many are pathogenic. Major abalone pathogens from this genus include V. splendidus and V. harveyi. Disease outbreaks usually occur during the summer abalone spawning period, when there is a sharp increase in water temperature. During this period the energy reserves of the abalone are limited and their immune systems are relatively suppressed. Other stress-related factors, such as handling or poor water quality, may also contribute to disease outbreaks.

Antibiotics have commonly been used to control diseases; however, due to their negative impact on the environment the use of these agents is being questioned. One of the major concerns regarding dispersing antibiotics in aquatic environments is the development of antibiotic resistance in bacteria. Studies found that more than 70% of bacteria found in aquatic environments are resistant to at least one antibiotic. Furthermore, many bacteria have been shown to have resistance to multiple antibiotics. A major concern of antibiotic-resistant bacteria in aquatic environments is the transfer of resistance between aquatic and terrestrial environments. The spread of antibiotic resistance through aquacultural activities, therefore, holds a significant risk to human health.

This has led to the search for probiotics as an alternative way to control bacterial diseases in aquaculture. Probiotics used in aquatic environments can be defined as live microbial supplements which have beneficial effects on the host by altering the microbial communities associated with the host and the immediate environment. The use of probiotics in terrestrial vertebrates such as pigs, chickens and humans have been studied extensively. Probiotics for terrestrial animals have been applied in aquaculture with varied success. Studies found that probiotics isolated from the environments in which they will be applied, such as aquatic environments, yield better results. In order to obtain potential probiotics for aquaculture, candidate bacteria should ideally be isolated from the GIT of aquatic species. Research on the use of probiotics in aquaculture is growing rapidly and is showing promising possibilities.

Probiotics have a variety of mechanisms of action. These bacteria can eliminate or reduce colonisation of the host by pathogenic organisms through the secretion of antagonistic compounds. These compounds can be defined as chemical substances produced by microorganisms that inhibit or are toxic towards other microorganisms in the surrounding environment. Antagonistic compounds include the production of secondary metabolites such as antibiotics and bacteriocins, which are produced by some bacteria to kill or inhibit the growth of surrounding bacteria, fungi and viruses. Probiotic bacteria can produce a variety of compounds that can be beneficial to the host, such as enzymes, vitamins, proteins, short-chain fatty acids and carotenoids. Probiotic bacteria can also enhance the immune response of the host. Certain bacterial compounds are known to act as immunostimulants. These compounds elicit different host responses, namely phagocytic activity and lysozyme production. Phagocytic activity plays an important role in antibacterial defences. Before antibodies are produced, phagocytic activity serves as an early activator of the inflammatory response.

The aim of this study was to isolate and identify bacteria in the gastrointestinal track of the South African abalone Haliotis midae and to evaluate the isolates' potential as a probiotic based on existing literature. Abalone were collected from Abagold farm (Hermanus, South Africa). The gut of each abalone was removed under aseptic conditions and transferred into sterile saline solution (0,9% NaCl) containing acid-washed glass beads (Sigma, South Africa). Each gut sample was homogenised and plated out on to three different media, Tryptone Soya agar (Casein enzymic hydrolysate 17g/l, papaic digest of soya bean meal 3g/l, D-Glucose 2,5g/l, bile salts mixture 1,5g/l, dipotassium hydrogen phosphate 4g/l, sodium chloride 5g/l and agar 12g/l), De Man, Rogosa and Sharpe agar (Universal peptone 10g/l, meat extract 5g/l, yeast extract 5g/l, D(+)-Glucose 20g/l, dipotassium hydrogen phosphate 2g/l, diammonium hydrogen citrate 2g/l, sodium acetate 5g/l, magnesium sulfate 0,1g/l, manganous sulfate 0,05g/l and agar 12g/l] and Zobell agar (Polypeptone 5g/l, yeast extract 1g/l and agar 12g/l). Random colonies were picked from the growth on different plates and streaked out in order to obtain pure cultures. Based on Gram stains, catalase and oxidase tests that were performed, representative strains were selected for DNA extractions. After successful DNA extractions the 16S ribosomal RNA region was amplified by PCR and these products were digested with RsaI (Fermentas Life Science, division of Thermo Fisher Scientific, Massachusetts, USA). Based on the patterns obtained from restriction enzyme profiles, representative isolates from each pattern were selected for DNA sequencing for identification.

Nine different bacterial species were isolated and identified as Corynebacterium variabilei,Staphylococcus carnosus, Staphylococcus equorum, Staphylococcus cohniii, Vibrioaestuarianus, Vibrio nigripulchritudo, Vibrio cyclitrophicus, Photobacterium leiognathi andParacoccus marcusii. One of these isolates, P. marcusii, is able to produce the carotenoid astaxanthin, an orange-red pigment with a very high antioxidant activity. As a result of the production of this pigment, P. marcusii shows promising probiotic properties due to the high antioxidant activity that enhances essential biological functions of fish such as increasing the defence potential against oxidative stress and enhancing sexual maturity. This bacterial isolate also shows potential to be used as a pigmentation source due to the production of astaxanthin. Certain fish species, such as salmonids, red sea bream, trout, lobster and shrimp are unable to synthesise carotenoids and have to obtain the pigments from their natural diets. However, under aquacultural conditions these fish species do not come into contact with natural pigments and this results in dark grey meat, which is unappealing to consumers. It is, therefore, important to include these carotenoids in the diet of certain aquaculture fish species such as salmonids, red sea bream, trout, lobster and shrimp. To conclude, Paracoccus marcusii shows possible probiotic properties and industrial applications based on the production of the astaxanthin pigment. Further in vivo studies need to be conducted in order to determine if P. marcusii can be used as an effective aquacultural feed additive.

Keywords: aquaculture, antibiotic, probiotic, bacterium, isolate

Read the article in Afrikaans: Gedeeltelike beskrywing van bakterieë in die SVK van Haliotis midae, met die fokus op potensiële probiotika

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