Probiotic Modulation of the Gut Microbiota of Fish - Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics - page 186

Despite this, however, historically much of the research concerning probiotic applications in
aquatic animals has focused on the effects on the host at the system/whole-body level (e.g.
growth performance, immune status and disease resistance) and unfortunately the effect on
the gut microbiota has seldom been assessed. For example, of the 79 probiotic investigations
discussed in the recent reviews on probiotic applications in salmonids (Merrifield et al .
2010a) and Mediterranean fish species (Dimitroglou et al . 2011), only 35 investigations (or
44%) assessed the impact on the gut microbiota. Since the publication of these reviews, with
a greater appreciation and understanding of the importance of the gut microbes to the host,
more emphasis has been placed on understanding the effects of the probiotics on the gut
microbiota of aquatic animals.
In general studies which have incorporated microbiological analyses have typically adopted
one of several strategies (as summarized in Table 5.2):
(1) Most commonly, culture-dependent enumeration of the probiont levels within the diges-
tive tract (typically digesta based) has been undertaken, with or without quantifying total
viable populations. Typically, de Man, Ragosa and Sharpe (MRS) agar is used for the enu-
meration of LAB (e.g. Carnevali etal . 2006; Lv etal . 2007; Merrifield etal . 2010b; 2011),
Slanetz and Bartley medium is used for the enumeration of enterococci (Merrifield et al .
2010c; 2010d), and Bacillus spp. have been assessed after heat processing on tryptone soy
agar (TSA) (Ghosh et al . 2008) or a Bacillus selective medium containing polymixin B
(Daniels et al. 2010). Methods for determining probiont identity have included biochem-
ical and phenotypic analysis, 16S rRNA sequencing or RAPD analysis. Some studies of
this nature also enumerate the indigenous microbiota typically using a general purpose
medium such as TSA or nutrient agar.
(2) Less frequently, some studies have used culture-dependent approaches to assess probiont
levels as well as enumerating and identifying the indigenous microbiota (e.g. Chang and
Liu 2002; Aubin et al . 2005; Bagheri et al . 2008; Ghosh et al . 2008). In order to assess the
indigenous microbiota after probiotic supplementation, selective media have been utilized
for the enumeration of indigenous bacterial groups of particular interest (e.g. Carnevali
et al . 2004; 2006; Rollo et al . 2006; Ghosh et al . 2008; Silvi et al . 2008) or selected
isolates from general purpose media have been identified by traditional biochemical assays
and phenotypic observations (e.g. Chang and Liu 2002; Bagheri et al . 2008) or 16S rRNA
sequence analysis (e.g. Aubin et al . 2005).
(3) More recently, culture-independent assessment of the gut microbiota after probiotic
feeding using techniques such as denaturing gradient gel electrophoresis (DGGE)
has become more common (Kim and Austin 2006; Sáenz de Rodrigáñez et al . 2009;
Ferguson et al . 2010; Sun et al . 2011a) and to a lesser extent clone libraries (Zhou et al .
2012; Abid, Zhou and Merrifield unpublished), fluorescent in situ hybridization (FISH)
(Salinas et al . 2008; Del'Duca et al . 2013) and qPCR (Avella et al . 2010a; 2012) have
also been used. Due to the low cultivability of the gut microbiota of fish (Table 5.1),
culture-independent methodologies are useful tools in furthering our understanding of
fish intestinal microbiota. Therefore, methods for assessing the gut microbiota of fish,
including probiotic studies, should move away from exclusive use of culture-dependent
methods (i.e. approaches 1 and 2) and, in accordance with other more established areas
of environmental microbiology, derive a more complete picture of the extant microbial
diversity present within fish intestines by incorporating more sensitive molecular based
methods, including next-generation sequencing, into probiotic studies.