The use of antibiotics is being discouraged in many countries of the world
due to their residues in milk or meat and possible resistance in bacteria of
human and animal origin (Butaye et al., 2003).
Consequently, there is growing interest in finding viable alternatives for disease
prevention and enhancement of production in farm animals. The preponderance
of research data in this field suggests the likelihood beneficial changes due
to exposure to probiotics. The most important advantage of probiotics to antibiotics
is that the former is free of any residues in meat or milk of farm animals which
might have serious health implications for consumers. Probiotics has been defined
in several ways. In simple term, probiotic means for life. Lilly
and Stillwell (1965) were the first to define this term as those substances
which are produced by one microorganisim and stimulate the growth of another.
Later, Parker (1974) described the term as organisms
and substances which contribute to intestinal microflora balance.
Fuller (1989) modified the definition as a live microbial feed supplement
which beneficially affects the host animal by improving its microbial balance.
This definition avoids the too broad term substance which could even include
antibiotics. In more modern definition, Salminen et al.
(1998) defined probiotics as food containing live bacteria which are beneficial
for health whereas, Marteu et al. (2004) define
it as microbial cell preparations or components of microbial cells that have
a beneficial effect on the health and well being. Despite these numerous theoretical
definitions, a practical question arises what is the criteria of selection of
an organism to be considered as probiotics. Several characteristics of probiotics
have been described which shown as follow:
Characteristics of a good probiotic:
||Should be acid and bile resistant
||Be strain specific and possess high ability to multiply in the gut
||Should not have any side effect; should neither be pathogenic nor toxic
to the host
||Culture should have a strong adhesive capability with the digestive tract
of the host
||Be durable enough to withstand the duress of commercial manufacturing,
processing and distribution
||Should have the ability to reduce the pathogenic microorganisms
The most commonly used probiotics are strains of lactic acid bacteria such
as Lactobacillus, Bifadobacterium and Streptococcus. These bacteria are known
to resist gastric acid secretion and bile salts enzymes effects, adhere well
to the colonic mucosa and readily colonize intestinal tract (Jin
et al., 1998). Moreover, these bacteria have shown strong in vitro
inhibition against Salmonella typhimurium, Staphylococcus aureus,
Escherichia coli and Clostridium perfringens (Fioramonti
et al., 2003).
Some yeast preparation such as Aspergillus oryzae, Saccharomyces
boulardii and S. cerevisiae have also been used as probiotic agent
due to its wide range characteristics like inhibit the growth of several microbial
pathogens, survive throughout the intestinal tract and it is unaffected by antibiotic
therapy (Fuller, 1999; Saegusa
et al., 2004; Zhu et al., 2009; Vila
et al., 2010). Microorgnisms commonly used as probiotics for livestock
animals (Ohashi and Ushida, 2009):
POSTULATED MECHANISMS OF PROBIOTICS ACTION
Enhancement of epithelial barrier integrity: Probiotic bacteria can
thwart the potential pathogen bacteria by enhancement of intestinal barrier
function through modulation of cytoskeletal and epithelial tight junction in
the intestinal mucosa (Chichlowski et al., 2007;
Ng et al., 2009). Under normal physiological
conditions, intestinal barrier is maintained by several factors like mucus production,
water and chloride secretion and epithelial cells that form tight junction (Ng
et al., 2009).
Disruption of epithelial barrier has been reported in several clinical conditions
such as enteric infections, celiac diseases and infection bowl disease (Ng
et al., 2009). Enhancement of epithelial barrier integrity may be
an important mechanism through which the probiotic bacteria benefit the host
in these disease conditions. According to Chichlowski et
al. (2007), the process of enhancement of epithelial integrity is accomplished
by two mechanisms. First, the enterocytes produce a thick blanket of mucus,
secreted by goblet cells which are dispersed throughout the luminal epithelium
of the intestines.
The probiotic bacteria have been reported to increase the secretion of mucus
by triggering inflammation in enterocytes of the intestines (Mack
et al., 1999; Chichlowski et al., 2007).
Caballero-Franco et al. (2007) reported that
treatment of probiotic bacteria triggered an increase of 60% of basal luminal
mucin contents by up-regulation of MUC2 gene expression. In the same
experiment, an increase in number of goblet cells were detected as an effect
of probiotic treatment. Similar results were recorded by Chichlowski
et al. (2007) who found greater number of goblet cells on chicken
intestinal villi in response to probiotics treatment and suggested that metabolites
produced during bacterial fermentation may play a role in the growth and maturation
of goblet cells. These observations were strengthened by the study of Montalto
et al. (2004) who reported increase production of mucin (MUC3) after
treatment with several strains of Lactobacillus.
The 2nd mechanism that ensures epithelial barrier integrity is associated with a unique structure called tight junction. It is unbroken, contiguous biological barrier which prevents the entrance of macromolecules and pathogenic bacteria. The tight junction proteins are dynamic structure subject to changes that dictates their function.
The action of probiotics on cytoskeleton of tight junction came to lime light
when Shen et al. (2006) demonstrated intact epithelial
cell junction by using electron microscopy. Tight junction permeability is influenced
by zonulin which is involved in the movement of molecules from intestine into
the blood stream and vice versa (Shen et al., 2006).
The protective action of zonulin was reported by Buts
et al. (2002) in response to administration of non-steroidal anti-inflammatory
drugs when the animals were dosed with Lactobacillus. However, this mechanism
is till vague and not fully understood. Some bacteria have been found to limit
water and chloride secretion such as S. thermophilus and L. acidophilus
reverse the E. coli induced chloride secretion by epithelial cells (Resta-Lenert
and Barrrett, 2003). Tight junction protein called zonula occludens-1 is
disturbed when exposed to pathogenic bacteria such as S. dublin.
The cytoskeleton arrangement was restored under the treatment of probiotic
(VSL#3), suggesting the role of probiotics in preservation of barrier function
and cytoskeleton architecture (Ng et al., 2009).
Similarly, other probiotic bacteria such as L. acidophilus, S. thermophilus,
individually or collectively, maintain or stabilize other cytoskeleton structures
like actin, ZO-1 and occludin when disrupted by pathogenic bacteria (Resta-Lenert
and Barrrett, 2003; Ng et al., 2009). For
some bacteria, antioxidative properties have also been shown. For example, Bifadobacterium
longum and L. acidophilus have been demonstrated to scavenge α,
α-diphenyl-β-picrylhydrazyl (DPPH) radical which lead to the inhibition
of lipid peroxidation and reduction of DNA oxidative damage in intestinal epithelial
cells (Lin and Chang, 2000).
The competition for space to adhere between indigenous bacteria and exogenous
pathogens result in the competitive exclusion of pathogenic bacteria (Ohashi
and Ushida, 2009). Competitive exclusion refers to physical blocking of
pathogenic bacteria colonization by probiotic bacteria from their favourite
site such as intestinal villus, goblet cells and colonic crypts (Chichlowski
et al., 2007).
The probiotic bacteria alter the physical environment of the intestines in
such a way that pathogenic bacteria cannot survive. Probiotic bacteria exclude
the opportunistic bacteria in two ways. First, the probiotic bacteria compete
with pathogenic bacteria for nutrients and energy source thus, preventing them
from acquiring energy r equired for growth and proliferation of pathogenic bacteria
in the gut environment (Cummings and Macfarlane, 1997).
Second, probiotics produce several organic acid and Volatile Fatty Acids (VFA)
as a result of their metabolism and fermentation. Consequently, the pH of the
gut is lowered below that essential for survival of pathogenic bacteria such
as E. coli and Salmonella (Marteu et al.,
2004; Chichlowski et al., 2007). Probiotic
bacteria also eject the colonization of pathogenic bacteria by attaching themselves
to the surface of the gut thus preventing the adhesion of the pathogenic bacteria
to gastrointestinal epithelium.
Probiotic bacteria such as Lactobacillus plantarum induces the transcription
and excretion of the mucins, MUC2 and MUC3 from goblet cells, thereby inhibits
the adherence of enteropathogenic such as E. coli to the intestinal
wall (Fooks and Gibson, 2002). Other such examples include
the detachment of Salmonella typhimurium, Shigella flexneri,
Clostridium difficile and other pathogens (Mead, 1989;
Isolauri et al., 2004).
SECRETION OF BACTERIOCINS
Lactobacilli and B. cereus have been reported to produce various metabolites
which have inhibitory effect on pathogenic bacteria (Oscariz
et al., 1999; Vila et al., 2010).
Lactobacillus acidophilus has been reported to produce acidophilin, lactocidin
and acidolin and L. plantarum produces lactolin (Vila
et al., 2010). Nicin and diplococcin are among the anti-metabolites
produce by Streptococci.
Bacillus cereus produces bacteriocin like substances which presents
high activity in the pH range 2.0-9.0. (Risoen et al.,
2004). These bacteriocins have been demonstrated in vitro experiments
for their inhibitory action against range of bacteria like Bacillus,
Klebecella, Pseudomonas, Proteus, Salmonella, Shigella,
Staphylococcus, Vibrio species and E. coli (Vila
et al., 2010).
INTERFERENCE WITH QUORUM SENSING SIGNALLING AGENTS
From the recent research, it has been concluded that quorum sensing regulates
the virulence expression in probiotics which may interfere with the signalling
system avoiding the onset of virulence in pathogenic bacteria (Vila
et al., 2010). Bacteria communicate with each other as well as with
their surrounding environment through chemical signalling molecules called auto-inducers
(Schauder and Bassler, 2001; Vila
et al., 2010).
This phenomenon is called quorum sensing. The probiotic bacteria such as Lactobacillus, Bifadobacterium and B. cereus strains degrade the auto-inducers of pathogenic bacteria by enzymatic secretion or production of auto-inducer antagonists which render the quorum sensing bacteria mute and deaf.
Medellin-Pena et al. (2007) demonstrated that
Lactobacillus acidophilus secretes a molecule that inhibits the quorum
sensing signalling or directly interact with bacterial transcription of E.
coli O157 gene, involved in colonization and thus, bacterial toxicity is
thwarted. Medina-Martinez et al. (2007) and
Cerda-Cuellar et al. (2009) recorded similar
conclusions using B. cereus and B. toyoi probiotic bacteria.
EFFECTS OF PROBIOTICS ON EPITHELIAL CELLS
One of the major question before the bacteriologist was how the intestinal
epithelial cells distinguish probiotic and pathogenic bacteria. This concept
was cleared by Lammers et al. (2002) and Otte
and Podolsky (2004) who concluded that distinction is based upon the production
These researchers found that probiotic bacteria did not induce IL-8 secretion by epithelial cells compared with intestinal pathogens such as E. coli, Salmonella dublin, Shigella dysenteriae and Listeria monocytogenes. In addition, combined culture of pathogenic bacteria, S. dublin and probiotic, VSL≠3, decreased the production of IL-8 indicating that probiotic bacteria can override the effect of pathogenic bacteria.
Another effect of probiotic bacteria on epithelial cells is recognition of
these bacteria through production of Toll-like receptors such as TLR-2 and TLR-4.
Such interaction result in production of protective cytokines that enhances
epithelial cell regeneration and inhibition of epithelial apoptosis (Rakoff-Nahoum
et al., 2004; Ng et al., 2009).
ANTI-INFLAMMATORY EFFECT OF PROBIOTIC BACTERIA
Ng et al. (2009) described that pathogenic bacteria
induce proinflammatory response in intestinal cells by activating the transcription
factor NF-kB. In contrast, non- pathogenic bacteria can attenuate the proinflammatory
response by secreting the counter-regulatory factor IkB. This phenomenon was
demonstrated in non pathogenic bacteria which attenuated IL-8 secretion elicited
by pathogenic S. typhimurium (Neish et al.,
2000). Kelly et al. (2004) demonstrated
in a case of Bacteroides thetaiotaomicron that the anti-inflammatory
response of the bacteria is achieved by blocking the transcription factor NF-kB
in the nucleus through nuclear hormone receptor, resulting in attenuation of
NF-kB-mediated inflammatory gene expression in pathogenic bacteria.
Other studies show that the ani-inflammatory response of the probiotic bacteria
has been achieved through variety of mechanisms like inhibitition of soluble
chymotrypsin-like activity of proteosome of the intestinal epithelial cells,
production of cytoprotective heat shock protein, delayed activation of NF-kB
and stabilizing level of IkB which result in attenuation of proinflammatory
affect of the pathogenic bacteria (Petrof et al.,
2004; Ng et al., 2009).
The gut is often referred as the largest immune organ of the body as more lymphocytes
reside in the gut than any other organ of the body (Chichlowski
et al., 2007). The enterocytes of intestines provide such a barrier
which prevents the passive loss of nutrients on one hand and on the other prevent
the access of pathogens into the body. The lamina properia of the intestines
is enriched with lymphocytes, macrophages, heterophils and dendritic cells all
of them fighting against the pathogens. Probiotics have the ability to enhance
the capacity of the host immune system against the pathogens and ultimately
improve their health.
Probiotic bacteria are able to influence the inflammatory response elicited
by pathogens through specific signalling pathway (Yurong
et al., 2005; Chichlowski et al., 2007).
Probiotic bacteria may exert its beneficial effects and modulate the immune
system of the host against potentially harmful antigens via activation of lymphocytes
and antibody production (Ng et al., 2009). For
example, L. rhamnosus admistration resulted in enhanced non-specific
humoral response reflected by an increase production of IgG, IgA and IgM from
Similar results were recorded by feeding yogurts containing L. acidophilus,
L. bulgaricus, S. thermophilus, B. bifidum and B. infantis
probiotic bacteria (Tejada-Simon et al., 1999).
Accumulated body of evidence have shown that the protective effect of probiotics
is associated with elevated humoral and cellular immune response which is achieved
through increased production of T lymphocytes, CD+cells and antibody
secreting cells, expression of pro and anti-inflammatory cytokines, interleukins,
IFN-γ, natural killer cells, antibody production, respiratory burst of
macrophages and delayed type hypersensitivity reaction (Panda
et al., 2003; Oyetayo and Oyetayo, 2005;
Chichlowski et al., 2007; Zhu
et al., 2009; Ng et al., 2009; Ohashi
and Ushida, 2009).
Probiotics are microbial cell culture that produces beneficial effect on the health and well-being of the host. The beneficial effects of probiotics are the consequence of their proposed mechanisms of action. Understanding the modes of probiotics may permit to improve the production of livestock and minimize the side-effects associated with the use of antibiotics.