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Lecture 7: Probiotic

Bacteria
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.
Introduction
As 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 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
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

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
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


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
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.
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
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
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
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)
Recent Findings: finfish
Digestive tract of finfish contains 10
8
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. 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
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)

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)
Part II. Modes of Action of
Probiotic Bacteria
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

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
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

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

(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
(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)
(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

(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
(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 CO
2
than gram -
thus: phytoplankton blooms are more easily
maintained (interesting research area!)
monitor: DOC, POC
so far, hasnt been demonstrated
Rationale for
Selecting
Probiotics
Verschuere et al. (2000)
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

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

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)
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)
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?)

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???