Wednesday, 2 April 2014

Bivalve haemolymphatic bacteria: a potential source of probiotics

Recently, with an increase in antibiotic resistance genes, there is an-going search for alternatives to antibiotic use against pathogens in aquaculture. Many organisms play host to a variety of microorganisms many of which have antibacterial properties involved in maintaining the health of their host. With such properties, they are a potential source of probiotics for use in aquaculture.

As filter feeders bivalves are exposed to and vulnerable to a large number of pathogenic bacteria. Bivalves in a healthy state are known to harbour a diverse community of microorganisms within their haemolymph thought to confer a health benefit to the host. This study addressed the anti-microbial activity of cultivable haemolymph-associated bacteria within four species of bivalves: oyster Crassostrea gigas; pink clam Tapes rhomboids; blue mussel Mytilus edulis and the scallop Pecten maximus and their potential role as probiotics.
Haemolymph was extracted from individuals and distinguishable bacterial colonies were isolated, sub-cultured and prepared for long-term storage. These isolates were assayed against 12 common aquaculture pathogens for antibacterial activity.

Overall 843 strains of haemolymph-associated bacteria were isolated though bacteria were species and individual dependant. Fixed bivalves, oyster and mussels, had higher haemolymph bacterial concentrations than mobile bivalves with many individual clams and scallops possessing bacterial concentrations below detectable levels. 26 of the 843 strains showed antibacterial activity against at least 1 of the tested pathogens, though antibacterial activity was limited Gram-negative bacteria. None of the isolated strains were sourced from the clam.
Due to a loss in cultivability and bioactivity only 12 strains of remaining bioactive strains could be quantified for antibacterial potency. Anitbacterial activity to an additional 5 vibrio strains was tested and minimal inhibitory concentrations for all tested pathogens was assessed. All isolates from Crassostrea gigas, and 2 strains from Mytilus edulis were able to inhibit 100% of the growth of at least 1 tested pathogen after a 64-fold dilution, and 8 isolates inhibited at least 5 out of 7 tested Vibrio species.
16S rRNA genes of antimicrobially active isolated were compared with the GenBank database and were found to belong to the following: Alteromonadales, Vibrionales, Pseudoalteromonas and Thalassomonas.  Both Vibrio spp. and Pseudoalteromonas spp. antimicrobial and probiotic ability is concurrent with previous literature; both previously have been demonstrated to protect or enhance the survival of several bivalve species.  A new cluster of Pseudoalteromonas spp. was found, phylogenetically distinct from known probiotic strains and thus may have unique probiotic properties. Similarly Thalassomonas is a genus with no previously described antibacterial activity and thus both of these are potentially novel sources of probiotics.
Three Pseudoalteromonas strains of oyster haemolymph bacteria found in the current study, and 2 previously found antimicrobial producing strains were investigated for cytotoxicity. After exposure of haemocytes to several concentrations of the 5 bacterial strains, analyses by flow cytometry found that 2 strains of didn’t differ significantly from control values and 3 strains significantly reduced haemocyte mortality.
In order to limit the development of bacterial resistance probiotics, as a substitute of antibiotics, shouldn’t harbour antibiotic resistant genes. By disk diffusion method the antibiotic sensitivity of haemolymph-associated bacteria was assessed in response to amoxiciin, colistin, enrofaxin, florfenicol, fumequin, tetracycline and tripmethoprim/sulphamethoxazole. No resistance was found in the 5 tested oyster strains.
From the study is can be said that bivalve haemolymph-associated microbiota is evidenced to infer a health benefit to the host and represents a potential source of future probiotics. In my opinion of all the strains found and tested, from this study, those Pseudoalteromonas found in Crassostra gigas haemolymph, are the only ones that hold potential.

As a non-mobile species it has a high bacterial concentration and all strains from this species showed antibacterial potency against at least 5Vibrio spp. which holds huge importance as Vibro pss. are pathogenic to many economically important species including fish, molluscs and crustaceans.
Furthermore the 5 Pseudoalteromonas oyster strains were the only ones tested for cytotoxicity and antibiotic resistance. All these factors together suggest that Pseudoalteromonas isolated from Crassostrea gigas haemolymph is potentially an effective probiotic for aquaculture.
With that said, the fact that their antibacterial activity is solely directed toward Gram-negative bacteria, it would likely have to be used in conjunction with another probiotic, with antibacterial properties to Gram-positive bacteria. This would incur a lot of other factors including the potential interactions between the two, and whether this would reduce the effectiveness of either.  A lot more work needs to be conducted within this area.
Desriac F., Chevalier P., Brillet B., Leguerinel I., Thuillier B., Paillard C. and Fleury Y. (2013) Exploring the hologenome concept in marine bivalvia: haemolymph microbiota as a pertinent source of probiotics for aquaculture. FEMS Microbiology Letters, 350, 107-116

1 comment:

  1. From 843 to just 12 strains which could assessed for antimicrobial activity is an enormous loss. To me this suggests either very low abundances or that many were unculturable because they were obligate symbionts. Perhaps we are missing out on the really beneficial probiotic species because they have intricate life cycles, possibility with culturable free living stages we have not yet identified.
    Do you think that antimicrobial activity is good indicator of probiotic potential? I am unsure whether it is more important than competitive exclusion of pathogens or immune stimulation, but my cynical guess is that antimicrobial activity is simply the easiest to measure.