Probiotics Enhancing Gut Performance
The fish microbiome
The microbiome is the totality of the microbiota and their genes in a given environment. The study of the composition and dynamics of the microbiome of humans and other animal hosts has been made possible by the development of high-throughput sequencing technologies and associated bioinformatics. These studies have revealed that an organism’s microbiome, through cross-talk and various interactions at the mucosal surfaces, plays key roles in regulating host development, digestive function, homeostasis, metabolism, welfare, behavior and health. Despite a plethora of studies available (for review see Llewellyn et al., 2014, Romero et al., 2014), our knowledge of the composition and relevance of the microbiomes of fish is primitive in comparison to that of humans and other mammals, where it is increasingly clear that the microbiome is intimately involved in multiple aspects of both nutrition and health (Thavagnanam et al., 2008, Cani and Delzenne, 2009). From the studies to date, we know that the microbiomes of fish are dominated by complex and diverse communities of bacteria, and to a lesser extent yeasts and Archaea. These communities inhabit the skin, gills and gastrointestinal tract and influence various host functions including digestion, development, nutrition, immunity and disease resistance. For example, zebrafish larvae reared in germ-free environments, with no microbial interactions, show a degenerative phenotype (Bates et al., 2006, Rawls et al., 2007). The digestive tract fails to differentiate properly and the intestine displays: reduced levels of enteroendocrine cells and goblet cells, a lack of brush border intestinal alkaline phosphatase activity, reduced epithelial cell turnover, immature patterns of glycan expression on enterocytes and a loss of epidermal integrity (Rawls et al., 2004, Bates et al., 2006). These characteristics ultimately led to a failure to uptake protein macromolecules. Further, 212 genes in the gastrointestinal tract were regulated by the microbial communities; these genes were involved in numerous processes including nutrition (e.g. genes involved in lipid metabolism, Cpt1a, Ctp1b and Fbp1), cell division and DNA replication (e.g. minichromosome maintenance genes and Pcna) and immunity (e.g. Saa1, Crp, C3 and Socs3) (Rawls et al., 2004). The commensal and beneficial microbes of the microbiome are also important in mediating barrier function (i.e. excluding foreign pathogens), through competition for adhesion sites and nutrients and via the production of various antimicrobial compounds.
As well as these important roles in promoting host development and supporting immunological functionality, the gut microbiota also has an important nutritional role through the production of vitamins, extracellular digestive enzymes and short chain fatty acids (SCFAs). For instance, SCFAs, produced as end products of fermentation of dietary fiber by the gut microbiota, can be utilized directly by the enterocytes as an energy source or transported across the intestine to the vascular system (Clements, 1991, Clements and Choat, 1995, Mountfort et al., 2002). SCFAs contribute considerably to the host energy requirements of mammals and it is not unreasonable to assume that this could also be the case for fish species’. Despite these benefits, the microbiome will also contain opportunistic pathogens which can infect immunocompromised hosts, and may also contain low levels of primary pathogens, which are suppressed under normal conditions, by the antagonistic actions of the commensal/symbiotic microbes and the host’s localized immunity. Much of the available data on the microbiota of fish is derived from culture-based methods. Though such studies provide useful information, in some fish species they capture <1% of the total microbial community (Navarrete et al., 2009) and therefore molecular ecology techniques such as fingerprinting methods (e.g. denaturing gradient gel electrophoresis (DGGE)) have become more commonly used in the last decade to investigate the gut microbiome of fish. Still, these approaches are not quantitative and do not detect minor members of the microbial community. In more recent studies, high-throughput sequencing approaches have been used to generate 16S rRNA libraries which extend our knowledge of the “rare biosphere” of the fish gut and provide a fascinating insight into the effects of diet on some aspects of the microbiome (Standen et al., 2015, Falcinelli et al., 2015, 2016, Apper et al., 2016).
As aquatic animals live in a microbial laden aqueous environment, the definition of the term probiotic in the aquaculture context has been a source of debate (Merrifield et al., 2010). The rearing water itself therefore is a target site that friendly bacteria could colonize resulting in a modulated environmental microbial community. Additionally, the rearing water could be used as a vector for providing friendly microbes to the target organism. As such, the evolution of the probiotic definition, away from that originally proposed for terrestrial animals, has occurred concomitantly with other terms such as biocontrol and bioremediation agents. Indeed, there is quite some overlap between these terms, and the term probiotic is often used for any live microbial intervention applied via the rearing water or feed with the aim of improving the microbial balance of the host organism or its immediate rearing environment. Research on the application of probiotic microorganisms in aquaculture began around three decades ago. The main reason for this was the need for alternative prophylactic methods to remedy the survival problems encountered at different stages of intensive fish rearing. Subsequently, it has become increasingly common to use probiotics, or other microbial modifiers, in aquaculture practices. The application of probiotics in aquaculture has provided several beneficial effects, namely modulation of the host immune system, as well as enhanced survival, feed utilization and disease resistance.
Probiotics for gut health
Attachment of probiotics, pathogens and commensal microbes to the mucosal surfaces is to a large extent influenced by the mucous and mucosal properties of the host. Host-microbe cross talk at the mucosal interface is a complex process which determines the nature of the subsequent host-microbe relationship. The mechanisms involved in mammals are well described, and a number of key molecules have been identified. Although poorly described in fish, with the exception of studies on specific probiotics and pathogens, some of these molecules and processes, or their analogous counterparts, have been identified in fish (for reviews see: Foey and Picchietti, 2014, Salinas and Parra, 2015). In the gut (and other mucosal surfaces), microbial recognition occurs via pattern recognition receptors (PRRs), of which the toll-like receptors (TLRs) have received most attention. Several studies in fish have demonstrated that bacterial probiotics may upregulate the gene expression of TLRs (e.g. TLR-2) in the gut. Upon TLR activation a complex cascade including adaptor molecules (e.g. Myd88) and the transcription factor NFĸB, results in the production of pro-inflammatory cytokines (e.g. IL-1β, IL-8 and TNFα). Several studies have demonstrated modulated expression of different components of this pathway in fish fed probiotics. This pro-inflammatory response is characterized by increased recruitment of leucocytes to the intestinal epithelium, increased goblet cell production and upregulated expression of immune and mucin genes (Carnevali et al., 2014, Lauzon et al., 2014). However, teleosts have evolved a suite of mechanisms to prevent excessive inflammatory responses towards commensal and symbiotic microbes. These processes are partly described, and include the production of IAP, anti-inflammatory cytokines (TGF-β and IL-10) and a number of regulatory molecules (e.g. DIGIRR and Tollip). Concomitant with the observed pro-inflammatory response in the intestine of probiotic fed fish, studies have revealed an increased expression of anti-inflammatory genes (e.g. IL-10), which may indicate an elevated immune-readiness of the intestine without inducing an excess inflammatory response. This has often been confirmed by histological examinations which verify that probiotic applications do not manifest intestinal pathologies or lesions, and in some cases may impart favorable morphological changes such as increased fold length and elevated microvilli length/density yielding improved absorptive surface area (Carnevali et al., 2014, Lauzon et al., 2014).
The current literature however is somewhat mixed – for every study that reveals probiotic benefits, either locally (at the gut level) or systemically, there are several other studies which do not report such benefits. In many cases this is due to the exploration of a novel (and unsuccessful) probiotic strain, inappropriate dosage or vector of provision. However, there remains contradictory data in different studies which have used the same probiotic species/strains with the same fish species. This highlights a unique challenge with the application of probiotics in aquaculture, not faced by applications in humans, other mammals or poultry. Fish are poikilothermic animals and thus their metabolism, and the metabolism of their microbiota (including the embedded probiotic), is heavily dependent on environmental conditions. Although there is sufficient evidence to conclude that most fish species will harbor a core microbiome in their gastrointestinal tracts (Roeselers et al., 2011, Romero et al., 2014, Falcinelli et al., 2015, 2016), evidence also suggests that individual fish of the same species may develop different microbiome phenotypes when reared under different conditions, at different seasons, at different life stages, and/or when fed different diets (Romero et al., 2014, Merrifield and Rodiles, 2015). The different environmental conditions, as well as the different microbiome phenotypes, are therefore likely to greatly influence the efficacy of probiotic applications in aquaculture operations. This presents quite a challenge when attempting to develop optimal probiotic application strategies.
Future research focus
Future research efforts must focus on three main themes. Firstly, gaining a better understanding of the normal microbiomes of fish; for a given fish species: to what extent does the microbiome composition, abundance and diversity vary across life history stages? Do different fish genotypes harbor different microbiomes? Do different fish phenotypes (i.e. dominant vs subordinate; fast growers vs slow growers; robust vs disease susceptible) harbor different microbiomes? Secondly, a better understanding of the functional attributes of the microbiome is required. Not just which microbes are present in the gut, but what are they doing? With the ever depreciating costs of sequencing, and with the anticipated improvement of databases to include more strains commonly found in fish, this can be achieved through metagenomics and metatranscriptomics in the coming decade. Lastly, a better understanding of the localized host responses to the microbiome, and how such responses may change when the microbiome is modulated or manipulated. This is beginning to be addressed through the use of transcriptomic appraisal of intestinal samples derived from probiotic fed fish. Further research in this vein, along with proteomic appraisal of intestinal mucus of probiotic fed fish, is warranted. With such information, we can then make informed decisions as to which probiotics are appropriate for which species, as well as when and how to use them.