Why don’t you attach to my mucus?

The use of antibiotics in aquaculture has tight regulations as their discharge causes resistant bacterial strains, increasing the potential use of probiotics. Probiotic bacteria contribute to the health and well-being of their host by attaching themselves to the intestinal mucosa of the gut, thereby influencing the gastrointestinal (GI) microflora (Vine et al, 2004).

Although bacteria produce metabolites, the stage at which they do and their efficiency in competing for attachment isn’t clear (Vine et al, 2004). Vine et al (2006) suggests that metabolite producing bacteria (at stationary growth phase) are flushed out constantly in the gut if they do not attach. Adhesion to the gut may allow the probiotic to implement beneficial effects or may enhance their antagonistic activity against pathogens, inhibiting them.

To understand the interaction between the probiotic and pathogens, and the factors mediating competition, five unstudied probionts (AP1–AP5) isolated from clownfish, Amphiprion percula and two fish pathogens Vibrio alginolyticus (a good colonizer of the GI tract) and Aeromonas hydrophila (contains adhesin, mediating attachment to cells) were cultured by Vine et al (2004). The cultures were radioactively labelled using two radioactive isotopes to quantify competition and attachment, which was measured by liquid scintillation (counting ³H and ¹⁴C).

Probiotic bacteria were added to the wells containing pathogens and vice versa for 4h to allow competition for attachment. The number of adhering probionts and pathogens were assessed by observing the radioactivity recovered from the wells and comparing it with the radioactivity of the original bacterial suspension.

After statitstical analysis, all probionts (except A1) in their results showed greater attachment to intestinal mucus than to the surface of the uncoated well. For the controls, the average attachment was significantly different for the probiotics, but not the pathogens. The effects of sequence of addition, probiotic and pathogen attachment were all significant as were the interactions between them. In all combinations, attachment was improved in those where bacteria were added second, except AP5 and AP4 with V. alginolyticus showing <100% attachment.

Factors such as growth curves, attachment ability, and the effect of the sequence of addition on their ability to adhere are important when selecting probionts, suggesting that AP5 is the most favorable to the intestinal microflora. Probiotic attachment was greater when they were added after the pathogens and pathogen attachment was enhanced when they were added after the probiotics. Based on this observation, the paper suggests that the attached bacteria may have modified the mucus structure, creating more or better attachment sites. However, the addition of probiotics after the pathogens results in reduced pathogen attachment relative to the controls due to production of antibiotics, siderophores or antagonistic antimicrobial metabolites which may restrict the access of pathogens to tissue receptors. Alternatively, the bacteria added second may have competed with the attached bacteria for sites – measured using different radiolabels. It was hypothesized that the existence of AP5 in the digestive tract may reduce the attachment ability of the pathogens. However, if the host undergoes stress, pathogens may have the chance to compete. Some pathogens may also adapt other invasive strategies – for eg, Vibrio anguillarum O2 which has poor attachment but is highly virulent (Vine et al, 2006).

Clownfish ensures selective colonization of the intestinal tract by exogenous feeding and may have applications in therapy and preventing diseases (Vine et al, 2004). Advantageous probionts that aid in enhancing attachment should be determined that can be used for larviculture.

This paper has taken a novel approach as they used unstudied probiont candidates and although some assays are based on previously used protocols, the assay used to assess competition of attachment and radioactivity were termed ‘different’ by the authors.

The paper also highlights the importance of gastrointestinal fauna and how crucial some bacteria are to avoid the attachment of other, potentially harmful bacteria. A very interesting point mentioned in this paper is that post hatching, larvae tend to drink seawater to osmoregulate, therefore ingesting suspended bacteria. Thus, delivery of probiotics at early stages of larval development (even prior to exogenous feeding) may be very important. Aquatic animals mainly home Gram-negative aerobic, obligate anaerobic and facultative anaerobic bacteria and since factors such as stress, diet or age can cause a shift in microflora (Vine et al, 2006), it is important to understand and determine the most advantageous time for probiotic addition to maximize attachment. Probiotics also need to thrive in different parts of the digestive system in order to be efficient when it arrives to the gut (Vine et al, 2006), thus the nature of the probiotic within the host needs to be understood. Also, in my opinion, the combination of prebiotics with probiotics would be an interesting implementation to further allow the growth of the probiotic and as prebiotics have also been shown to prevent pathogen adhesion (Vine et al, 2006).

Paper reviewed:

Vine et al (2004, May 28). Competition for attachment of aquaculture candidate probiotic and pathogenic bacteria on fish intestinal mucus. Journal of fish diseases , 27(6), 319–326. http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2761.2004.00542.x/full

Additional references:

Vine et al (2006, May 1). Probiotics in marine larviculture. FEMS Microbiology Reviews, 30(3), 404-427. https://doi.org/10.1111/j.1574-6976.2006.00017.x