FEATURE
The global surveillance of
ANTIMICROBIAL RESISTANCE
by Alvaro Ortiz, Norel, S.A
WHO’s (World Health Organization) 2014 report on global surveillance of antimicrobial resistance reveals that antibiotic 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 antimicrobial 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
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 extracellular 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 concentration 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 population 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, sporulation, virulence, biofilm formation, etc. The best-known autoinducer is N-acylhomoserine lactone (AHL) It has been widely accepted that Gramnegative bacteria utilise various AHLs to regulate 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
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 resistancespromoting 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 competence in Streptococcus pneumoniae was aided by a hormone-like extracellular product (Tomasz and Beiser, 1965). However, cell-cell signaling and coordinated
Test 1
Test 2
7 h (pH 6.4)
9
11
24 h (pH 8.6)
11
13
24 h (pH 6.2)
11
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48 h (pH 8.6)
9
9
48 h (pH 6.2)
9
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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 β-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, Germany
longer the acyl side chain 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 communication, disrupting the quorum sensing, this effect is known as quorum quenching (QQ). Only two enzyme families in the microorganism have the capability of cutting AHL structures; the AHL-lactonases and the AHLacylases 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 antibiotics 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 widespread and highly conserved in many pathogenic 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 enzymatic 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.
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FEATURE
The genus Vibrio includes more than 30 species, many of which are associated with human diseases, and have described QS systems 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 conclusion that QQ could be a promising alternative to antibiotics in aquaculture, especially against Vibrios and particularly against V. parahaemolyticus, known etiologic agent in EMS/AHPNS. Probiotics here arise as an optimum solution; these products combine different modes of action and some of them also show quorum quenching activity. The AHL-degrading enzyme was first identified 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 amyloliquefaciens. World J. Microbiol. Biotechnol. 2010, 26, 1361–1367.) 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 quenching activity which switches off the virulent expression together with all the above mentioned 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 parahaemolyticus 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 recommended 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 modulate and control the growth and effects of typical aquaculture pathogens. Aquaculture producers must have probiotics 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
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