FEATURE
The global surveillance of
ANTIMICROBIAL
RESISTANCE
WHOs (World Health Organization)
2014 report on global surveillance of
antimicrobial resistance reveals that anti-
biotic resistance is no longer a prediction
for the future; it is happening right now,
across the world, and is putting at risk the
ability to treat common infections in the
community and hospitals.
O
ne of the stress points being
placed in the sometimes
abusive use of antibiotics is
in animal production. The
common trend in all countries is to reduce or
even ban the use of these valuable molecules
to avoid an apocalyptic post-antibiotic era, in
which common infections and minor injuries,
which have been treatable for decades, can
once again kill.
As an estimation of the situation nowadays
we can state that in Europe 25,000 deaths
per year can be attributed to this antimicro-
bial resistant superbugs and 38,000 deaths
in Thailand and 23,000 deaths per year in
the EEUU. The overall society cost impact is
estimated to be US$ 35 billion in the EEUU
(sources: ECDC 2007, Pumart et al 2012, US
CDC 2013)
New strategies are needed therefore in the
fight to the common pathologies suffered by
production animals. And quorum quenching
A damaged
hepatopancreas
in a AHPNS
infected shrimp
(disruption of quorum sensing) is getting more
and more attention by scientists as many of
its characteristics makes possible to conjugate
pathogens control with a non resistances-
promoting therapy.
But what is quorum sensing?
The first indication of bacterial cell-cell
communication was introduced in 1965, when
Tomasz suggested that the regulation of com-
petence in Streptococcus pneumoniae was
aided by a hormone-like extracellular product
(Tomasz and Beiser, 1965).
However, cell-cell signaling and coordinated
16 | INTERNATIONAL AQUAFEED | September-October 2014
by Alvaro Ortiz,
Norel, S.A
microbial group behavior was officially
ascertained by Nealson and co-workers,
who reported that the bioluminescence
developed by the marine bacterium
Vibrio fischeri (formerly Photobacterium
fischeri) in its symbiotic relationship with
the Hawaiian squid Euprymna scolopes
(E. scolopes) was controlled by one or more
signaling molecules accumulating in the extra-
cellular milieu as a function of cell growth
(Nealson et al., 1970a). V. fischeri infects the
light organ of the squid, where the cell density
reaches 1010-1011 cells mL-1, then the signal
molecules can accumulate to an adequate con-
centration to trigger the transcription of genes
encoding luminescence enzymes.
Microbial cell-cell signaling has become
known as quorum sensing (QS), this system
allows microorganisms to sense its own popu-
lation density and when the external signal
(known as autoinducers) reaches a threshold
or quorum a number of target genes are
activated or repressed in order to synchronize
processes such as bioluminescence, antibiotic
production, conjugative DNA transfer, sporula-
tion, virulence, biofilm formation, etc.
The best-known autoinducer is N-acyl-
homoserine lactone (AHL)
It has been widely accepted that Gram-
negative bacteria utilise various AHLs to regu-
late the mechanisms that help them to adapt
to changes in the environment. AHL signals
appear to be dedicated molecules produced
Table 1: Inhibition halo expressed in mm
Control Test 1 Test 2
7 h (pH 6.4) 9 11
24 h (pH 8.6) 11 13
24 h (pH 6.2) 11 12
48 h (pH 8.6) 9 9
48 h (pH 6.2) 9 9
with the sole purpose of mediating specific
quorum sensing processes.
Different AHLs are usually characterized
by acyl chains with variable length, saturation
level and oxidation state. In AHL-dependent
quorum sensing systems the specificity of the
transcriptional activator protein for its cognate
AHL depends on both the length of the acyl
side chain and chemical modification at the
-position of the HSL ring.
Therefore, AHLs require an N-acyl side
chain of at least four carbons in length in
order to be functional under physiological
conditions in mammalian tissue fluids, and the
Barnase-Barstar
complex.
Source: Mazen
Ahmad,
Center of
Bioinformatics,
longer the acyl side chain Germany
the more stable the AHL
signal molecule.
It is also well known, that this autoinducers
regulates the expression of virulence genes
responsible for the production of extracellular
proteases, extracellular hemolysin and other
extracellular factors contributing to cytotoxic
activity.
Some enzymes are able to deactivate these
molecules and subsequently block the com-
munication, disrupting the quorum sensing, this
effect is known as quorum quenching (QQ).
Only two enzyme families in the micro-
organism have the capability of cutting AHL
structures; the AHL-lactonases and the AHL-
acylases have been demonstrated to be
involved in the real cleavage of the QS signal
molecules for quenching QS.
The interference with the quorum sensing
system by the quorum quenching enzyme is a
potential strategy for replacing traditional anti-
biotics because the quorum quenching strategy
does not aim to kill the pathogen or limit cell
growth but to shut down the expression of
the pathogenic gene. Therefore they do not
create a selection pressure on the organisms
and as result do not promote the appearance
of resistances.
QQ can be developed as a technique for
disrupting the ability of a pathogen to sense
its cell density and disable or diminish the
capability of triggering the virulent expression.
This capability ensures that the host has time
to eradicate the pathogens naturally through
normal immune system function. Additionally,
AHL-mediated signaling mechanisms are wide-
spread and highly conserved in many patho-
genic bacteria, being an attractive target for
novel anti-infective therapies.
Some microbes not only produce QQ
enzymes as a defence strategy against their
competitors but also utilize AHL and its enzy-
matic degradation products as the sole carbon
and nitrogen sources for cell growth.
As this QS mechanism was first discovered
in the marine bacterium Vibrio fischeri, the
acyl-homoserine lactone (AHL) system in this
microorganism is well characterized and is used
as a model system for many AHL-producing
Gram-negative bacteria.
 FEATURE

The genus Vibrio includes more than 30
species, many of which are associated with
human diseases, and have described QS sys-
tems for both inter-bacterial and intra-bacterial
communication. Among them AHL system is
present in many Vibrio species, including V.
harveyi (closely related species such as Vibrio
campbellii and Vibrio parahaemolyticus) and
V. anguillarum which are amongst the most
important bacterial pathogens in the intensive
rearing of mollusks, finfish and especially shrimp.
Therefore it seems to be a natural conclu-
sion that QQ could be a promising alternative
to antibiotics in aquaculture, especially against
Vibrios and particularly against V. parahaemo-
lyticus, known etiologic agent in EMS/AHPNS.
Probiotics here arise as an optimum solu-
tion; these products combine different modes
of action and some of them also show quorum
quenching activity.
The AHL-degrading enzyme was first identi-
fied in Bacillus and recently, B. amyloliquefaciens
was confirmed to have AHL-degrading activity
(Yin, X.T.; Xu, L.; Fan, S.S.; Xu, L.N.; Li, D.C.;
Liu, Z.Y. Isolation and characterization of an
AHL lactonase gene from Bacillus amylolique-
faciens. World J. Microbiol. Biotechnol. 2010,
26, 13611367.)
The probiotic ECOBIOL produced by
NOREL is based on this bacillus strain: B.
amyloliquefaciens CECT 5940. It is capable
to secrete 0.9 g of lactic acid per g of sugar
present in the environment; this lactic acid
will reduce pathogenic bacteria. In addition,
the vegetative form of B. amyloliquefaciens
produces barnase, a bacteriocine, which has an
antibiotic effect.
Another mode of action reported in
ECOBIOL is the ability to stimulate the immune
system (not based on antibodies) by increasing
the activity of phagocityc cells and lysozyme.
So, the combination of the quorum quench-
ing activity which switches off the virulent
expression together with all the above men-
tioned pathogens fighting tools, have been
demonstrated to be effective against many
harmful microorganisms.
In vitro study conducted at NOREL lab,
it could be observed
how the growth of
Vibrio parahaemo-
lyticus was inhibited
by the presence of
B. amyloliquefaciens
CECT 5940 culture
supernatant (Table 1)
In the study, after culture of the probiotic,
cells free extracts of B. amyloliquefaciens CECT
5940 were obtained by centrifugation and used
to test the bactericide effect using the inhibition
halo technique.
Bacillus amyloliquefaciens supernatant
showed inhibition halos around the mugs. It
is hence concluded that ECOBIOL is able to
control the population growth of this kind of
pathogenic bacteria.
In the FAO Fisheries and Aquaculture
Report No. 1053, probiotics were recom-
mended as a tool in the control of AHPNS.
Ecobiol based on B. amyloliquefaciens CECT
5940 not only features quorum quenching
activity, additionally it has, as a kind of Swiss
army knife, more mechanisms capable of mod-
ulate and control the growth and effects of
typical aquaculture pathogens.
Aquaculture producers must have probiot-
ics in mind when trying to cope with the more
and more difficult task of producing food for
feeding 7 billion people in a sustainable way.
www.norel.es

Ecobiol Aqua
A highly effective new generation probiotic

Die and roll re-working machines

Pathogenic bacteria antagonist

Proven activity against V. parahaemolitycus
Enhances friendly intestinal microflora
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Helps mantain optimal health status
Reduces mortality in the ponds

www.oj-hojtryk.dk
Phone: +45 75 14 22 55
Fax: +45 82 28 91 41
mail: info@oj-hojtryk.dk
O&J Hjtryk A/S
rnevej 1, DK-6705
Esbjerg Head Office and Orders Jess Aprendiz, 19 1 A-B 28007 Madrid SPAIN
T. +34 915 014 041 norel@norel.es www.norel.es
CVR.: 73 66 86 11

September-October 2014 | INTERNATIONAL AQUAFEED | 17