Lecture 11 Probiotic Bacteria

Lecture 7: Probiotic Bacteria

Lecture OutlineIntroduction/Definition of ProbioticsFundamental QuestionsRecent FindingsPossible Modes of ActionRationale for Selecting ProbioticsConclusions and Further DirectionsSource of notes: Verschuere, L., Rombaut, G., Sorgeloos, P., and Verstraete, W., 2000. Probiotic bacteria as biological control agents in aquaculture. Microb. Mol. Biol. Rev., 64(4):655-671.

IntroductionAs we know, aquaculture is growing faster than beef cattle production (10% vs. 3%)Epizootics (disease) = major limiting factor in fish/shrimp developments!Disinfectants, antimicrobial drugs have had little effect in controlling disease.Massive use of antimicrobials increases selective pressure on microbes and encourages natural emergence of bacterial resistanceResistant bacteria thrive after non-resistant strains have been killed and can even pass on resistance genes to other bacteria that have not been exposed to antibiotics

IntroductionEmphasis should be place on preventionMore cost-effective than cure!Antimicrobials, disinfectants and pesticides largely treat symptoms of the problem and not the causeAlternative strategies are just catching onExample: via vaccination, Norway has reduced chemical therapeutant use from 50 MT in 1987 to less than 747 kg in 1997 (with 7x concomitant production increase!)Other: use of immunostimulants w/or w/out vaccines

IntroductionUse of bacteria as a food source and as a biological control agent of fish disease was first proposed by Yasuda and Taga (1980)Vibrio alginolyticus has been used as a probiotic in shrimp hatcheries in Ecuador since 1992Reduced hatchery down time from 7 days per month to less than 21 days per year!FAO has now designated use of probiotics as a major means for improvement of aquatic environmental qualityUltimate goal: make aquaculture products more acceptable to consumers

Definition of ProbioticsMany proposed, however: a live microbial feed supplement which beneficially affects the host animal by improving its intestinal balanceHistorically: terrestrial animals, genus LactobacillusDefinition (above) requires some additional considerations:1) bacteria in aquatic medium influence composition of gut microbiota and vice versa2) immediate ambient environment has much greater influence on microbiota than with terrestrialsIn aquatic environments, hosts and microorganisms share the ecosystemTerrestrials: the gut represents a moist habitat in a water-limited world

Definition of ProbioticsBig Issue 1: aquatics are surrounded by an environment supporting their pathogens independently of the host animalResult: opportunistic pathogens can reach high densities around the fish/shrimpSurrounding bacteria are commonly ingested with the feed or via drinking (maximum case: filter feeders)Research in probiotics began with fish juveniles but more attention recently given to shrimp and finfish larvaeBig Issue 2: terrestrials have inherent colonizing bacteria from the mother, aquatics largely spawned as axenic eggs (no further contact with parent)Ambient bacteria colonize eggs surface, young larvae often have no developed gut (e.g., shrimp), no microbial community in gut, gills or skinPoint: properties of bacteria in ambient water are very important

Definition of ProbioticsBetter definition: a live microbial adjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial community, by insuring improved use of feed or by enhancing its nutrition, by enhancing the host response towards disease, or by improving quality of the ambient environmentOur focus: response towards disease and improvement of the ambient environmentJobs of Microbial Adjuncts:1) microbial adjuncts preventing proliferation of pathogens in gut or elsewhere;2) improved digestibility;3) deliver improved nutrition to aquatics;4) enhancing host response to disease (acquired);5) improved environmental quality.

Can you Manipulate Microbial Communities?Difficult: 1) discontinuous culture cycles; 2) disinfection during pond prep; 3) sudden increases in nutrients due to feedingUnlikely under intensive rearingMust consider deterministic factors (known response): salinity, temp, quality/quantity of feedPoint: the environment selects the range of microbes (axiom of environmental selection)Stochastic factors: chance, right place/right timeEvidence: identical cultures started simultaneously yield different assemblages

Can you Manipulate Microbial Communities?Concept: instead of accidental colonization, start with a probiotic that is well adapted to prevailing environmental conditionsThis is probably better than competing with a dominant, well-established, non-probioticLong-term exposure is often required to achieve a probiotic effectDoes the probiotic have to be continuously introduced to the culture?Evidence: in most cases, yes (at least with Lactobacillus sp.)Most fish contain a specific intestinal microbiota established at the juvenile stageUnless the host has been exposed to a limited range of microorganisms in its development, a single addition wont result in long-term colonization

Recent FindingsAs mentioned, it all started with Yasuda and Taga (1980);Usually added in feed or to culture water as preventative agents against infection by pathogenic bacteriaNutritional benefits are usually secondaryTypical genera: Lactobacillus, Vibrio, Bacillus, PseudomonasThe following is a summary of findings based on various aquatic species

Recent Findings: fish eggs/larvaeFor incubators, use of antibiotics must be minimalAntibiotics dont represent control; instead, unfavorable alteration of microbiotaGoal: establish colonization on the egg prior to pathogen colonization (i.e., no substrate)This, in turn, affects subsequent gut colonizationOnce initial feeding has started, probiotics typically added to culture water or culture medium of live feed items (e.g., algae, rotifers, etc.)Result: improved survival, faster growthMechanism? Production of antibiotics or siderophores (metal sequesterers)

Recent Findings: finfishDigestive tract of finfish contains 108 cells/g (Ringo et al., 1995)For cod, Gadus gadus, gut is colonized by similar bacteria as found in eggs (Hansen and Olafsen, 1999)Putative probiotics added as soon as possible after hatching in order to colonize gut prior to feeding (Ringo and Vadstein, 1998)Turbot and dab harbor bacteria capable of suppressing growth of V. anguillarum (Ollson et al., 1992)V. alginolyticus was effective in reducing disease caused by Aeromonas salmonicida in Atlantic salmon (Austin et al., 1995)

Recent Findings: finfish Kennedy et al. (1998) showed addition of a Gram-positive probiotic increased survival, size uniformity, and growth rate of snook, red drum, spotted sea trout and striped mullet.Gram et al. (1999) reported a strain of Pseudomonas fluorescens reduced mortality of 40 g rainbow trout infected with pathogenic V. anguillarumGarcia-de-la-Banda et al. (1992) added Streptococcus lactis and Lactobacillus bulgaricus to rotifers and Artemia sp. nauplii and recorded 6x higher survival at weaning than untreated controlsNikoskelainen et al. (2003) showed immune enhancement in rainbow trout via Lactobacillus rhamnosus supplemented in feeds

Recent Findings: shrimp Broad application in hatcheries, but few in-depth studies publishedoften used as food source (e.g., soil bacteria for P. monodon nauplii)improved survival (57% vs. 0%) after 13 days against V. anguillarumimproved survival of L. vannamei PLs inoculated with V. alginolyticus (non-pathogenic) vs. oxytet and control (Garriques and Arivalo, 1995)Griffith (1995) reported that following the introduction of probiotics in Ecuador in 1992, hatchery down-time between batches was reduced from 7 days per month to 21 days per year, production volumes increased by 35% and antimicrobial use decreased by 94%In shrimp hatcheries in New Caledonia, a strain of Pseudoalteromonas piscicida was found to inhibit the growth of Vibrio sp. (Saulnier et al., 2000)

Recent Findings: bivalave molluscsMost research has focused on nutritional contributions to mollusc larvaemost work in vitro wherein autochthonous strains have been isolated from scallops and have shown some inhibition to Vibrio sp. and Aeromonas hydrophilaBacillus sp. and Lactobacillus sp. shown to depurate oysters (Crassostrea virginica) against V. vulnificus (Williams et al., 2001)

Part II. Modes of Action of Probiotic Bacteria

Modes of ActionMany publications about probiotics have emerged in aquaculture in the last decademost based on empirical arguments (simple)modes of action were largely circumstantialseveral modes of action have been proposed as a result of human and agricultural applications

Modes of ActionInfo on terrestrial investigations has been used for aquatics (esp. Lactobacillus sp.)

one problem: distinction between 1) the intrinsic ability of the probing to positively influence the host and 2) its ability to reach and maintain itself in the location where the effect is to be exerted

Point: does not matter if it produces siderophores or inhibitory compounds in the gut if its never ingested

can be very important: Mytilus edulis (Blue Muscle) can selectively ingest/digest microbes

Modes of ActionAlso, if the candidate probiont cannot proliferate in the gut it probably wont exert a strong effectwill need continuous application through the diet or via the water ($$$)summary: they must reach the location where effect is desired

Possible Modes of Action production of inhibitory compounds competition for chemicals/available energy competition for adhesion sites (exclusion) enhancement of the immune response improvement of water quality interaction with phytoplankton a source of macro- and micro-nutrients enzymatic contribution to digestion

(1) production of inhibitory compoundsRelease of chemicals having a bactericidal or bacteriostatic effectultimate result: competitive edge for nutrients/energyproduction sites: in host intestine, on its surface, or in culture mediumproducts: antibiotics, bacteriocins, siderophores, lysozymes, proteases, hydrogen peroxide, organic acids (pH change)exact compound is seldom identified: hence, the term inhibitory

(1) production of inhibitory compoundsLactobacillus sp. produces bacteriocins (toxins)marine bacteria produce bacteriolytic enzymes against V. parahaemolyticusAlteromonas sp. produces monastatin, shown to be inhibitory against Aeromonas hydrophilainhibitory effects have been shown by probiotics against aquaculture pathogensno demonstration under in vivo conditions (oops!)more research required!!! (Didnt you mention this last time??)

(2) Competition for Chemicals or Available EnergyExplains how different microbial populations exist in same ecosystemit is likely that it occurs in the mammalian gut, but proof is lackingapplication of the principles of competition to natural situations is not easymicrobial situation in ecosystems is usually controlled by heterotrophs competing for organic substrates as both carbon and energy sourcesif you know the factors affecting microbial composition of the microbiota, you can manipulate it

(2) Competition for Chemicals or Available EnergyAll microorganisms require iron for growthsiderophore: low mw ferric ion-specific chelating agentsdissolve precipitated Fe and make it available for microbial growthsiderophores scavenge Fe and make it unavailable to other speciesthis occurs at tissue levelprobiotics producing siderophores can outcompete pathogens for Fe, thus limiting pathogen growthworks best with pathogens that also produce siderophores (e.g., V. anguillarum)

(3) Competition for Adhesion SitesCompetition for gut adhesion sites would limit colonizationadhesion to enteric mucus is necessary for bacteria to become established in fish intestinesthis is probably the first probiotic effectadhesion can be specific (based on adhesin and receptor molecules) or non-specific (based on physiochemical factors)total probiotic effect is probably a mixture of site competition, production of inhibitory compounds and nutrient/energy competition

(4) Enhancement of Immune ResponseRem definition of an immunostimulant? Chemical compounds that activate the immune systems of animals and render them more resistant to infections by viruses, bacteria, fungi and parasites.Immune response varies in animalslactic acid bacteria administered orally may induce increased resistance to enteric infections problem: only shown with specific cell compounds or dead cellsgood indications, but no proof

(5) Improvement of Water QualityProposed as a mode of action as a result of monitoring water quality after addition of probioticsusually associated with Bacillus sp.Hook: gram + bacteria are better converters of organic matter back to CO2 than gram -thus: phytoplankton blooms are more easily maintained (interesting research area!)monitor: DOC, POCso far, hasnt been demonstrated

Rationale for Selecting ProbioticsVerschuere et al. (2000)

Background InfoProfound knowledge of culture Critical review of available literatureCharacterization of abiotic and biotic environment impacting cultureMust know relationship between microbiota and host well-understoodRelationship between microbiota and carrying capacity of environment

Acquisition of Putative ProbioticsAre putative probiotics (isolated from host or host environment) better than those from other sources?Putatives might be better due to fact they are already adhered to gut wall (i.e., could block pathogen adhesion)If a non-pathogenic bacterium exists at high density in typical culture water, it will be well-adapted to prevailing conditions and can probably compete efficiently with pathogens for nutrients

Screening of Putatives: In-vitro antagonism testingCandidates are exposed to pathogen in liquid or solid mediumscreened for production of inhibitory compounds (bacteriocins), siderophores, or on competition for nutrientsmust be undertaken with extreme caution: not all media are suitableprobionts are finicky as to on which medium they produce inhibitory compounds (e.g., marine agar vs. TSB)

Evaluation of PathogenicityProbiotics must not be pathogenic to the host organism -- this must be confirmed prior to acceptancehost must be challenged under stressed and non-stressed conditionsusually accomplished by adding probiotic to the culture waterproper way to do this under monoxenic conditions (only the probiont present)also look at interaction with other food organisms found simultaneously in culture (e.g., algae)

In-vivo EvaluationsInvolves introducing candidate species to host cultures and monitoring growth, survival, physiochemical parametersmeans of addition: addition to artificial diet, addition to culture water, bathing, addition via live foodnext step: experimental (allochthonous) infection of host via immersionneeds long-term evaluation (is the pathogenic effect one of suppression or delay?)

Conclusions/FutureCurrent status of probiotics in aquaculture is really hazyLack of knowledge on modes of actions is very evidentmore info on competitive processes between bacteria is requiredmore info on relationship between bacteria and other microbiota requiredeconomic value/efficiency, anyone???