Lecture 7: Probiotic Bacteria

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Lecture 7 Probiotic Bacteria
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Lecture Outline

• Introduction/Definition of Probiotics
• Fundamental Questions
• Recent Findings
• Possible Modes of Action
• Rationale for Selecting Probiotics
• Conclusions and Further Directions

Source 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.
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Introduction

• As we know, aquaculture is growing faster than
  beef cattle production (10 vs. 3)
• Epizootics are a significant constraint to
  development
• Disease is major limiting factor in shrimp
  subsector
• 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 resistance
• Resistant 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

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Introduction

• Emphasis should be place on prevention
• More cost-effective than cure
• Antimicrobials, disinfectants and pesticides
  largely treat symptoms of the problem and not the
  cause
• Alternative strategies are just catching on
• Example 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

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Introduction

• Use 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 1992
• Reduced 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 quality
• Ultimate goal make aquaculture products more
  acceptable to consumers

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Definition of Probiotics

• Many proposed, however a live microbial feed
  supplement which beneficially affects the host
  animal by improving its intestinal balance
• Historically terrestrial animals, genus
  Lactobacillus
• Definition (above) requires some additional
  considerations
• 1) bacteria in aquatic medium influence
  composition of gut microbiota and vice versa
• 2) immediate ambient environment has much greater
  influence on microbiota than with terrestrials
• In aquatic environments, hosts and microorganisms
  share the ecosystem
• Terrestrials the gut represents a moist habitat
  in a water-limited world

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Definition of Probiotics

• Big Issue 1 aquatics are surrounded by an
  environment supporting their pathogens
  independently of the host animal
• Result opportunistic pathogens can reach high
  densities around the fish/shrimp
• Surrounding 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 larvae
• Big 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
  skin
• Point properties of bacteria in ambient water
  are very important

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Definition of Probiotics

• Better 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 environment
• Our focus response towards disease and
  improvement of the ambient environment
• Jobs 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.

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Can you Manipulate Microbial Communities?

• Difficult 1) discontinuous culture cycles 2)
  disinfection during pond prep 3) sudden
  increases in nutrients due to feeding
• Unlikely under intensive rearing
• Must consider deterministic factors (known
  response) salinity, temp, quality/quantity of
  feed
• Point the environment selects the range of
  microbes (axiom of environmental selection)
• Stochastic factors chance, right place/right
  time
• Evidence identical cultures started
  simultaneously yield different assemblages

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Can you Manipulate Microbial Communities?

• Concept instead of accidental colonization,
  start with a probiotic that is well adapted to
  prevailing environmental conditions
• This is probably better than competing with a
  dominant, well-established, non-probiotic
• Long-term exposure is often required to achieve a
  probiotic effect
• Does 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 stage
• Unless the host has been exposed to a limited
  range of microorganisms in its development, a
  single addition wont result in long-term
  colonization

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Recent Findings

• As mentioned, it all started with Yasuda and Taga
  (1980)
• Usually added in feed or to culture water as
  preventative agents against infection by
  pathogenic bacteria
• Nutritional benefits are usually secondary
• Typical genera Lactobacillus, Vibrio, Bacillus,
  Pseudomonas
• The following is a summary of findings based on
  various aquatic species

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Recent Findings fish eggs/larvae

• For incubators, use of antibiotics must be
  minimal
• Antibiotics dont represent control instead,
  unfavorable alteration of microbiota
• Goal establish colonization on the egg prior to
  pathogen colonization (i.e., no substrate)
• This, in turn, affects subsequent gut
  colonization
• Once 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 growth
• Mechanism? Production of antibiotics or
  siderophores (metal sequesterers)

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Recent Findings finfish

• Digestive 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)

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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.
  anguillarum
• Garcia-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
  controls
• Nikoskelainen et al. (2003) showed immune
  enhancement in rainbow trout via Lactobacillus
  rhamnosus supplemented in feeds

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Recent Findings shrimp

• Broad application in hatcheries, but few in-depth
  studies published
• often used as food source (e.g., soil bacteria
  for P. monodon nauplii)
• improved survival (57 vs. 0) after 13 days
  against V. anguillarum
• improved 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)

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Recent Findings bivalave molluscs

• Most research has focused on nutritional
  contributions to mollusc larvae
• most work in vitro wherein autochthonous strains
  have been isolated from scallops and have shown
  some inhibition to Vibrio sp. and Aeromonas
  hydrophila
• Bacillus sp. and Lactobacillus sp. shown to
  depurate oysters (Crassostrea virginica) against
  V. vulnificus (Williams et al., 2001)

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Part II. Modes of Action of Probiotic Bacteria
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Modes of Action

• Many publications about probiotics have emerged
  in aquaculture in the last decade
• most based on empirical arguments (simple)
• modes of action were largely circumstantial
• several modes of action have been proposed as a
  result of human and agricultural applications

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Modes of Action

• Info 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

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Modes of Action

• Also, if the candidate probiont cannot
  proliferate in the gut it probably wont exert a
  strong effect
• will need continuous application through the diet
  or via the water ()
• summary they must reach the location where
  effect is desired

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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

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(1) production of inhibitory compounds

• Release of chemicals having a bactericidal or
  bacteriostatic effect
• ultimate result competitive edge for
  nutrients/energy
• production sites in host intestine, on its
  surface, or in culture medium
• products antibiotics, bacteriocins,
  siderophores, lysozymes, proteases, hydrogen
  peroxide, organic acids (pH change)
• exact compound is seldom identified hence, the
  term inhibitory

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(1) production of inhibitory compounds

• Lactobacillus sp. produces bacteriocins (toxins)
• marine bacteria produce bacteriolytic enzymes
  against V. parahaemolyticus
• Alteromonas sp. produces monastatin, shown to be
  inhibitory against Aeromonas hydrophila
• inhibitory effects have been shown by probiotics
  against aquaculture pathogens
• no demonstration under in vivo conditions (oops!)
• more research required!!! (Didnt you mention
  this last time??)

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(2) Competition for Chemicals or Available Energy

• Explains how different microbial populations
  exist in same ecosystem
• it is likely that it occurs in the mammalian gut,
  but proof is lacking
• application of the principles of competition to
  natural situations is not easy
• microbial situation in ecosystems is usually
  controlled by heterotrophs competing for organic
  substrates as both carbon and energy sources
• if you know the factors affecting microbial
  composition of the microbiota, you can manipulate
  it

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(2) Competition for Chemicals or Available Energy

• All microorganisms require iron for growth
• siderophore low mw ferric ion-specific
  chelating agents
• dissolve precipitated Fe and make it available
  for microbial growth
• siderophores scavenge Fe and make it unavailable
  to other species
• this occurs at tissue level
• probiotics producing siderophores can outcompete
  pathogens for Fe, thus limiting pathogen growth
• works best with pathogens that also produce
  siderophores (e.g., V. anguillarum)

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(3) Competition for Adhesion Sites

• Competition for gut adhesion sites would limit
  colonization
• adhesion to enteric mucus is necessary for
  bacteria to become established in fish intestines
• this is probably the first probiotic effect
• adhesion 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

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(4) Enhancement of Immune Response

• Rem 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 animals
• lactic acid bacteria administered orally may
  induce increased resistance to enteric infections
  
• problem only shown with specific cell compounds
  or dead cells
• good indications, but no proof

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(5) Improvement of Water Quality

• Proposed as a mode of action as a result of
  monitoring water quality after addition of
  probiotics
• usually 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, POC
• so far, hasnt been demonstrated

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Rationale for Selecting Probiotics
Verschuere et al. (2000)
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Background Info

• Profound knowledge of culture
• Critical review of available literature
• Characterization of abiotic and biotic
  environment impacting culture
• Must know relationship between microbiota and
  host well-understood
• Relationship between microbiota and carrying
  capacity of environment

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Acquisition of Putative Probiotics

• Are 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

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Screening of Putatives In-vitro antagonism
testing

• Candidates are exposed to pathogen in liquid or
  solid medium
• screened for production of inhibitory compounds
  (bacteriocins), siderophores, or on competition
  for nutrients
• must be undertaken with extreme caution not all
  media are suitable
• probionts are finicky as to on which medium they
  produce inhibitory compounds (e.g., marine agar
  vs. TSB)

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Evaluation of Pathogenicity

• Probiotics must not be pathogenic to the host
  organism -- this must be confirmed prior to
  acceptance
• host must be challenged under stressed and
  non-stressed conditions
• usually accomplished by adding probiotic to the
  culture water
• proper 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)

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In-vivo Evaluations

• Involves introducing candidate species to host
  cultures and monitoring growth, survival,
  physiochemical parameters
• means of addition addition to artificial diet,
  addition to culture water, bathing, addition via
  live food
• next step experimental (allochthonous)
  infection of host via immersion
• needs long-term evaluation (is the pathogenic
  effect one of suppression or delay?)

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Conclusions/Future

• Current status of probiotics in aquaculture is
  really hazy
• Lack of knowledge on modes of actions is very
  evident
• more info on competitive processes between
  bacteria is required
• more info on relationship between bacteria and
  other microbiota required
• economic value/efficiency, anyone???