Probiotic Applications in Crustaceans - Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics - page 303

Bacillus spp. have been linked to polymyxin, bacitracin and gramicidin antibiotic production
(Gullian et al. 2004). More specifically, the mechanisms by which Bacillus spp. control Vibrio
spp. and other pathogens in crustacean intestines are associated with their capacity to produce
antibiotic agents and to compete for nutrients and habitat with other bacteria (Moriarty 1998).
In the case of LAB, it is commonly accepted that the primary causative effect is the reduction
of pH via organic acid production and the removal of carbohydrates (Vázquez et al. 2005).
Nevertheless, it is also accepted that the probiotic effects of many LAB are also based on the
production of diverse antibacterial metabolites (bacteriocins in particular).
However, as emphasized by Kesarcodi-Watson et al. (2008), the in vitro screening for pro-
duction of inhibitory substances presents two major limitations: (1) it excludes other potential
probiotics which posses different modes of action that are undetected by agar plate meth-
ods; and (2) there is potentially no direct link between in vitro and in vivo assays as already
reported (Gram et al. 2001). Moreover, bacteria, such as Vibrio spp., might develop resistance
if the production of growth inhibitory compounds is the only mode of action. Based on this
statement, the European Union and the European Food Safety Association (EFSA) stated that
microorganisms intended for use as probiotics should not be able to produce any antimicrobial
substances used as antibiotics in humans or animals (Anadón 2006). Therefore, in vivo vali-
dations are absolutely necessary. A plethora of in vivo trials has been carried out with shrimp
species in order to (1) validate that in vitro properties of probiotics can be achieved in vivo
(Vaseeharan and Ramasamy 2003; Castex et al. 2008; Boonthai et al. 2011); (2) assess the
possible pathogenicity of a probiotic to the host (Chythanya et al. 2002); and (3) evaluate the
degree of protection a probiotic may confer during a challenge with a pathogen (Rengpipat
et al. 1998; Ajitha et al. 2004; Gullian et al. 2004; Balcázar et al. 2007; Castex et al. 2010).
For instance, Vaseeharan and Ramasamy (2003) demonstrated inhibitory effects of cell-free
extracts of B. subtilis BT23 against V. harveyi and then confirmed in vivo that this strain,
when administered at 10 6 -10 8 CFU ml -1 for 6 days, confers a 90% reduction in accumulated
mortality of P. monodon challenged with V. harveyi . In a recent study, Boonthai et al. (2011)
observed, over a 120 day trial in tanks, that a dietary combination of several Bacillus species
led to a log 3 increase in the total Bacillus counts in the intestine and digestive gland of P.

monodon within 30 days, subsequently associated with a significant control of the cultivable
Vibrio levels after the 30 days. Similar results were also reported in L. stylirostris with an LAB
probiotic over a 10 week experiment in commercial ponds (Castex et al. 2008). Other studies
have tried to elucidate the molecules involved in the antagonistic activity. Chythanya et al.
(2002) reported the inhibitory activity of cell-free extract of Ps. aeruginosa I-2 and showed
that the extract, when applied at

≥

20 mg ml -1 , was able to reduce V. harveyi concentrations
in rearing water by over a log unit. This effect was associated with the inhibitory effect of
the chloroform extract of Pseudomonas I-2 cell-free supernatant and the author suggested that
pyocyanine could be the antibacterial agent responsible for this effect.

≥

11.3.3 Interference with quorum sensing
Another putative mode of action recently studied concerns the inhibition of virulence of
pathogenic bacteria via interference of quorum sensing. 'Quorum sensing' is the process by
which bacteria communicate and coordinate the expression of certain genes in response to
signal molecules. These quorum sensing signal molecules were found to be involved in the
regulation of virulence factors in many pathogenic bacteria, including the fish and crustacean
pathogen V. harveyi . Recently, Defoirdt et al. (2005) and Tinh et al. (2007) demonstrated