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VYTAUTAS MAGNUS UNIVERSITY
FACULTY OF SCIENCE
BIOLOGY DEPARTMENT
Greta Mikučionytė
(MGM09017)
ANTIMICROBIAL ACTIVITY OF PROBIOTICS AND EFFECT OF
PREBIOTICS AGAINST ENTERIC PATHOGENS
Master thesis
Biology program, state code 61201B103
Biology study area
Supervisors : Assoc. Prof. Åsa Ljungh, Lund University ______________
(signature)
Prof. Torkel Wadström, Lund University ______________
(signature)
_______
(date)
_______
(date)
Prof. Limas Kupčinskas, Kaunas Medical University ______________
_______
(signature)
Adviser: Dr. Kanthi Kiran Kondepudi., M.Sc., Ph.D, Lund University _______
(date)
_______
(signature)
(date)
Opponent: Prof. Algimantas Paulauskas, Vytautas Magnus University _________ _______
(signature)
KAUNAS, 2011
(date)
Work has been carried out at Laboratory Medicine, Division of Bacteriology, Lund University,
Sölvegatan, SE 22362, Lund, Sweden.
Reviewer: Dr. B. Tamutė
Work to be presented: Vytautas Magnus University, Department of Biology, for public critisim in
auditorium 801, Vileikos 8, LT-44404 Kaunas, Lietuva, on June 2 at 9 o‘clock.
Number of Protocol:
Author of term paper: Greta Mikučionytė
Supervisors: Assoc. Prof. Åsa Ljungh; Prof. Torkel Wadström; Prof. Limas Kupčinskas.
Vytautas Magnus University, Biology Department.
Head of the department A. Sruoga _________________
2
TABLE OF CONTENTS
ABBREVIATIONS………………………………………………………………………………….4
SUMMARY………………………………………………………………………………………….5
ABSTRACT…………………………………………………………………………………………6
INTRODUCTION……………………………………………………………………………………8
1. LITERATURE REVIEW………………………...……………………………………………...9
1.1 History and definition………………………………………………………………………….9
1.2 Microorganisms used as probiotics……………………………………………………………9
1.2.1. Genus Lactobacillus…………………………………………………………………….10
1.2.2. Genus Bifidobacterium………………………………………………………………….10
1.3. Bacteriocins and acid production by lactic acid and bifidobacteria…………………………11
1.4. Mechanism of action of Probiotics…………………………………………………………..14
1.4.1. Modification of the microbial flora……………………………………………………..15
1.4.2. Enhancement of intestinal barrier function……………………………………………..16
1.4.3. Immunomodulation……………………………………………………………………..17
1.5. Antimicrobial activity by Lactobacilli and bifidobacteria against enteric pathogens……….18
1.6. Prebiotics…………………………………………………………………………………….22
2. MATERIALS AND METHODS……………………………………………………………….22
2.1. Growth Substrates…………………………………………………………………………...22
2.2. Bacterial strains……………………………………………………………………………...22
2.3. Bacterial growth conditions…………………………………………………………………23
2.4. Antibacterial activity of selected bacteria…………………………………………………...23
2.4.1. Effect of cell free filtrates of probiotic strains against E.coli, S. typhimurium, S.aureus
and B.cereus time course experiment…………………………………………………...23
2.4.2. Effect of prebiotics on the AMA of probiotic strains against E.coli, S. typhimurium,
S.aureus and B.cereus…………………………………………………………………..24
2.4.3. Effect of bile stress on AMA of probiotics strains against E.coli, S. typhimurium,
S.aureus and B.cereus…………………………………………………………………..24
2.4.4. Effect of probiotic strain combinations on AMA against E.coli, S. typhimurium,
S.aureus and B.cereus…………………………………………………………………..25
2.4.5. Partial purification of proteinecious antimicrobial substance…………………………..26
3. RESULTS………………………………………………………………………………………..26
3.1. Antimicrobial activity of LAB and bifidobacteria of growth of enteropathogens
(screening)…………………………………………………………………………………..26
3.2. A time course of antimicrobial activity of cell- free supernatant of probiotic bacteria against
enteric pathogens……………………………………………………………………………27
3.3. AMA studies of LAB and Bifidobacteria in presence of prebiotics………………………..30
3.4. Effect of Bile on AMA of probiotic strains…………………………………………………34
3.5. Effect of probiotic strain combinations on AMA against E.coli, S. typhimurium,
S.aureus and B.cereus……………………………………………………………………….36
3.6. AMA of partially purified bacteriocins from LAB and Bifidobacteria strains against enteric
pathogens……………………………………………………………………………………44
CONCLUSIONS…………………………………………………………………………………...45
ACKNOWLEDGEMENT…………………….…………………………………………………..46
REFERENCES…..………………………………………………………………………………...47
3
ABREVIATIONS
AMA – antimicrobial activity
CFS – cell free supernatant
EMP - Embden–Meyerhof–Parnas pathway
ESBL – beta spectrum lactamase– producing Escherichia coli
FOS – fructooligosaccharides
GI /GIT– gastrointestinal tract
GOS – galactooligosaccharides
IBD – bowel disease
IL – interleukin
IMOS – Isomaltooligosaccharides
LDH – lactate dehydrogenase
MHB – Muller-Hinton broth
MRSA - methicilin-resist Staphyloccocus aureus
MRS - De Man, Rogosa, Sharpe broth
MUC – mucin
NEC – necrotizing enterocolits
NF- κB - nuclear transcription factor
SOS - soybean-oligosaccharides
TLR – Toll – like receptors
TSS - toxic shock syndrome
XOS – xylooligosaccharides
4
SUMMARY
Author of term paper: Greta Mikučionytė
Full title of term paper: Antimicrobial activity of probiotics and effect of prebiotics against enteric
pathogens.
Supervisors: Assoc.Prof. Åsa Ljungh, Prof.Torkel Wadström, Prof. Limas Kupcinskas.
Adviser: Dr. Kanthi Kiran. Kondepudi. M.Sc., Ph.D
Work has been carried out at Laboratory Medicine, Department of Medical Microbiology, Division
of Bacteriology, Lund University, Sölvegatan, SE 22362, Lund, Sweden.
Number of pages: 53
Number of tables: 8
Number of figures: 32
Number of references: 76
5
ABSTRACT
In this study we have focused on 9 human probiotic strains, which were tested using
microtiter plate assays for their ability to inhibit the growth of some common enteropathogens:
Salmonella typhimurium, ESBL (see list of abbreviations), Staphylococcus aureus and Bacillus
cereus. Survivals at low pH, growth in the presence of bile and levels of inhibition of growth of
enteropathogens at specific time intervals have been also studied. Several tested probiotic strains
were combined with a range of prebiotic food supplements in an attempt to identify synbiotic
combinations. Subsequently all tested Lactobacillus and Bifidobacterium strains were examined in
co-culture experiment for their AMA to inhibit the growth of harmful bacteria. AMA of crude
proteins precipitated by ammonium sulphate from the cell free supernatants of 4 candidate probiotic
strains was also determined to define the possible role of proteinecious antimicrobial substances
(bacteriocins).
The results showed that four (2 Lactobacilli: L. paracasei F8, L. plantarum F44 and 2
Bifidobacteria: B. breve 46, B. lactis 88 of a total 9 Lactic acid and Bifidobacteria showed strong
antimicrobial activity when tested against some Gram-negative and Gram-positive pathogens.
In this study it was observed that AMA of collective candidate probiotic strains in different
time course against 4 enteric pathogens inhibited was highest with acid supernatants. When the
supernatants were neutralized to pH 6.0 there was a decrease of inhibition suggesting the possible
synergistic action of secreted acids and other proteinecious or non-proteinacious metabolites by the
probiotic strains (that inhibit the growth of pathogens).
Probiotics grown with different carbohydrate sources were tested in a microtiter plate assay
offered some inhibition of each of S. typhimurium, ESBL, S. aureus and B. cereus strains. The
extent of inhibition was dependent on the probiotic strain and on the carbohydrate source used.
AMA of top probiotics strains cell-free supernatants grown in presence of 5% porcine bile
was also determined since it has been shown that bile stress often enhance AMA of probiotic
strains. It was observed that bile stress did not abolish the AMA of LAB supernatants meanwhile
AMA against Gram-negative pathogens (ESBL and S .typhimurium) by Bifidobacteria cell free
supernatants was abolished when grown in presence of 5% porcine bile.
Laboratory studies have shown that at acid cell free supernatants of probiotic strain
combinations at 1:1 dilution inhibited the growth of S. typhimurium ESBL, S. aureus and B. cereus
more than 80 % meanwhile neutralized cell free supernatant did not show antimicrobial activity or
much less.
6
Crude proteins from two out of four candidate strains namely L. plantarum F44 and B.
lactis 8:8 showed maximum activity against enteric pathogens at lower dilutions indicating the role
of bacteriocins in AMA against 4 target enteric pathogens used in the present investigation.
In conclusion, this study identified 4 potential probiotic active strains (2 lactobacilli and 2
bifidobacteria) that can inhibit some important pathogenic gram-positive or gram-negative
enteropathogenic bacteria and could be potential candidate strains for in vivo studies in animal
models.
AIM OF THE PRESENT STUDY
1. To screen for AMA against human enteric pathogens by LAB and Bif strains and to
select the top strains for further in vitro studies.
2. To establish the time course of AMA against the target pathogens.
3. To investigate the effect of selected probiotic strains in combination with prebiotics on
human enteric pathogens.
4. To screen four top bile salt-resistatnt lactic acid bacteria for inhibitory activity against
enteropathogenic bacteria.
5. To study the effect of a co-culture of LAB and Bif strains on AMA to suppress the
growth of pathogens.
6. To fractionate proteinecious antimicrobial substances from probiotic strains using
ammonium
sulphate
precipitation
(salting
out)
and
test
these
against
the
enteropathogens.
7
INTRODUCTION
Gastrointestinal microflora consists of hundreds of different types of microorganisms and
is biologically important component of the gut to inhibit gut colonization by incoming pathogens. In
the 19th century, microbiologists identified the gastrointestinal (GI) microflora in the GI tract of
healthy individuals. The beneficial microflora found in the GI was termed probiotic microbes. The
term probiotic was defined more than 20 years ago and is usually defined as live microorganisms or
microbial food supplements that confer health benefits for the host, when administered in adequate
amounts (Reid et al., 2003). Most probiotics fall into the group of organisms known as lactic acid
producing bacteria such as Lactobacillus and Bifidobacterium which have a long safe history in the
manufacture of dairy products normally consumed in the form of yogurt, fermented milks or other
fermented foods.
The efficiency of these probiotic products on animal performance has been discussed
extensively, but the mode of action of probiotics still remains unclear. Number of mechanisms have
been proposed to explain their mode of action viz., decreasing luminal pH via the production of
volatile short chain fatty acids (SCFAs), competing for specific nutrients with pathogens,
modulation of the host immune system and producing specific inhibitory compounds such as
organic acids (lactic acid and acetic acid etc), oxygen catabolites (e.g. hydrogen peroxide) and
antibacterial proteinaceous compounds (e.g. bacteriocins) (Sanders, 1993; Helender et al., 1997;
Magnusson et all., 2003; Servin. 2004; Valerio et al., 2004). In the normal intestinal flora these
mechanisms are essential components of bacterial populations, with many advantages over
competing bacterial pathogens. However factors such as unhealthy diet, stress, microbial infections
and other diseases disturb the balance, which often lead to intestinal dysfunction by decreasing
number of viable lactobacilli and bifidobacteria (Fuller and Gibson, 1997).
For the last three decades the most extensively studied probiotics are Lactobacillus and
Bifidobacterium strains of many species, which contribute to inhibit a wide range of infections, such
as antibiotic-associated diarrhea (AAD), Helicobacter pylori gastroenteritis and urovaginal
infections have been demonstrated in both in vitro and in vivo experimental studies as well as
clinical trials (Sgouras et al., 2004; Wult et al., 2003).
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1. LITERATURE REVIEW
1.1 History and definition
Although the term of probiotics was not established until 1965 the history of probiotics is
as old as the consumption of fermented milk which first documented in The Old Testament and
exists for over 2000 years (JoMay, 2002). Before the term probiotics was even coined the Russian
scientist Ellie Metchnikoff in 1908 at the Pasteur Institute in Paris was one of the first developed
definable notion that fermented milk product might have beneficial effect on the intestine. He knew
that Bulgarian peasants who lived certain part of Bulgaria and consumed a large amount fermented
milk products were known for their extraordinary longevity. Metchnikoff was using a pure Grampositive rod shaped bacterium in those days called Bulgarian bacillus and latter Bacillus bulgaricus
of what is now called Lactobacills delbrueckii subsp. Bulgaricus which together with Steptococcus
thermophilus produce fermented milk products such as yoghurt, to modify the colonic microflora
through ingestion of soured milk. He established that normal microflora of the lower gut of humans
was having an adverse effect on the host and that consumption of soured milks reserved this effect
(Stiles and Holzaphel, 1996).
Probiotics, derived from the Greek and meaning “for life” , are defined as cultures of
viable microorganisms or microbial food ingredient when ingested in adequate amounts embrace
the antagonistic activity against gastrointestinal pathogens, stimulate the immune system and
beneficially effect the host by improving its intestinal microbial balance (Quigley and Quera, 2006;
Walker and Duffy, 1998). It was first used by Lilly and Stillwell in 1965 to describe as “microbial
substances secreted by one microorganism which stimulates the growth of another" and thus was
conversed with the term antibiotic (Lilly, 1965). Meanwhile Sperti (1971) designated it as tissue
extract which can improve the colonic bacterial growth. These conceptions did not establish.
The classical definition which is used until now was R.B Parker (1974) who defined
probiotics as “organisms and substances which contribute to intestinal microbial balance” (Parker,
1974). Subsequently in 1989, Fuller redefined probiotics by removing the “substance” which could
include antibiotics and microbial stimulant and revised it as "A live microbial feed supplement
which beneficially affects the host by improving its intestinal microbial balance (Chen and Walkerl,
2005; Fuller, 1989).
1.2 Microorganisms used as Probiotics
The group of microorganism most frequently regarded as probiotics consist of either yeast,
especially Saccharomyces, or bacteria, especially lactic acid bacteria which is the most commonly
9
used as nonpathogenic “viable cells”. An overall, traditional dairy strain of probiotics bacteria
belongs to the Lactobacillus and Bifidobacterium genera (in complete), but the yeast such as
Saccharomyces cerevisiae and some E. coli and Bacillus species are also used as probiotics
(Francisco et al., 2008).
1.2.1 Genus Lactobacillus
According to second edition of Bergey’s Manual of Systematic Bacteriology (2004)
published in a third volume (2009), the genus Lactobacillus belongs to the phylum Firmicutes,
class Bacilli, order Lactobacillales, family Lactobacillaceae and its closest relatives, being grouped
within the same family, are the genera Paralactobacillus and Pediococcus (Hammes and Hertel,
2009).
Lactobacilli are almost ubiquitous: they grow and found in environments where
carbohydrates are available, such as food: beer, fruit, grain mashes, marinated fish, sugar
processing, sour dough’s, milk, meat and meat products, fermented beverage, plants and plant
materials: soil, water, sewage and manure and human or animal habits: respiratory, oral cavity, GI
and genital tracts.
Lactobacilli are Gram-positive, non-sporeforming, non-motile facultative anaerobic or
microaerophilic organisms (Orla-Jensen, 1919) and can vary from long and slender, sometimes bent
rods to short, often coryneform coccobacilli (Hammes and Hertel, 2009). With a DNA base
composition lower than 54 mol% G+C content and not more than 10% range in G+C content exist
in a well-defined genus (Stiles and Holzapfel, 1996).
1.2.2 Genus Bifidobacterium
Before the 1960s, Bifidobacteria strains were first discovered in infant faces by Tisser in
1900 and named as "Bacillus bifidus" which later in 1920 was referred as "Lactobacillus bifidus"
by Holland. In 1924 Orla-Jensen recognized the existence of genus Bifidobacterium a separate
taxon but given their similarities of bifidobacteria with the genus Lactobacillus and bifidobacteria
were included in this genus as listed in the 7th edition of Bergey Manual of Determinative
Bacteriology (Biavati et al., 2000). In the eight edition of this manual that the genus
Bifidobacterium was included in the phylum Actinobacteria, class Actinobacteria, order
Bifidobacteriales, family Bifidobacteriaceae (Biavati and Mattarelli, 2006)
Bifidobacterium are Gram positive polymorphic branched anaerobic (although some
species can tolerate oxygen) rods that occur singly, in chains or clumps (Felis and Delaglio, 2007).
They are, non-motile, non-sporulating, non-filamentous and non-gas producing (produce only acid
10
from a variety of carbohydrates), and chemoorganotrophic, having a fermentative type of
metabolism (Ventura et al., 2004). The G+C content is quite high compared to Lactobacillus with a
value ranging from 42 to 67 mol% (Lee and O’Sullivan, 2010).
In the studies on the ecology of bifidobacteria described so far are grouped in six different
niches: the human intestine and vagina, dental carries and oral cavity, food (fermented milk), the
animal gastrointestinal tract, the insect intestine and sewage (Ventura et al., 2004).
1.3 Bacteriocins and acid production by lactic acid bacteria and
bifidobacteria
The earliest classifications of lactic acid bacteria are based on the production of speciesspecific stereoisomers of lactate from glucose fermentation as a means of identifying and
categorizing the various species in this group. Swedish scientist Schele in 1780 studied sour milk
and found that lactic acid is one of the most important organic acids produced by lactic acid bacteria
(LAB). Lactic acid exists in two optically active-stereo isomers, the L (+) and the D (-). Since
explored of D (-) lactic acid is harmful to humans, L (+) lactic acid is preferred isomer in food and
pharmaceutical industries as human have only L – lactate dehydrogenase (LDHs) that metabolizes
L ( +) lactic acid isomer normally found in the blood and irune (Reddy et al., 2008).
Lactic acid bacteria can be divided into groups on their type of fermentation, that bacterial
species are able to produce lactic acid as main half end-product of the fermentation of carbohydrates
which has beneficial effects. Three groups of lactobacilli fermentation have been established:
1.
Obligately homofermantive lactobacilli are able to ferment hexoses
into 85 % lactic acid by the Embden–Meyerhof–Parnas (EMP) pathway while pentoses and
gluconate are unable to ferment;
2.
Facultatively heterofermentative lactobacilli degrade hexoses only to
50 % lactic acid by the EMP pathway and may produce gas from gluconate but not from
glucose. They are also able to degrade pentoses and often gluconate as they possess both
aldolase and phosphoketolase;
3.
Obligately heterofermentative lactobacilli degrade hexoses by the
phosphogluconate pathway producing lactic acid, ethanol or acetic acid and carbon dioxide
(Connoly and Lonnerdal., 2004; Mayo and Sinderen., 2010).
It is currently been discussed that Lactobacilli has been attributed to the production of
metabolites such as organic acid (lactic and acetic acid), hydrogen peroxide, ethanol, diacetyl,
acetaldehyde which makes their environments acidic, and it may help to inhibit the growth of some
11
harmful pathogens or stabilize of the intestinal microflora. These microbes generate small
molecular metabolic products that exert beneficial regulatory influence on host biological
functions.These metabolic products are sometimes referred to as “postbiotics” and may function
biologically as immune modulators (Thomas et al., 2010).
It has been also observed that some of LAB produce low molecular mass (< 1000 Da)
peptides – bacteriocins (antimicrobial substances) or ribosomally synthesized peptides/ proteins
with bactericidal activity against related species or across genera. Bacteriocins serve as an important
mechanism of pathogen exclusion in fermented foods as well as in the gastrointestinal environment.
They are sub-divided into 3 or 4 classes. The most famous bacteriocins produces by lactic acid
bacteria which has beneficial effect in food industry is I class type A lantibiotic – nisin produced by
Lactococcus lactis. It is used to extend the shelf life of food products by inhibition of Gram-positive
bacteria, such as Actinomycetes, Bacillus, Clostridium, Corynebacterium, Enterococcus,
Gardnerella,
Lactococcus,
Listeria,
Micrococcus,
Mycobacterium,
Proprionbacterium,
Streptococcus and Staphylococcus (Suskovic et al., 2010; Mayo and Sinderen., 2010).
Table 1 shows the different species of Lactobacillus from animals and humans which has
most beneficial properties and predominantly used in probiotics based upon these criteria:
fermentation group, genome GC content, lactic acid isomer and antimicroial metabolites. Many of
these isolates were found in healthy human and animal gut and licensed as safe considering that
species differ between human and various animals.
Table 1. Subdivision and characteristics of Lactobacillus genus
Obligative
homofermentative
Facultative
heterofermentative
L. acidophilus NCFM
34.7
DL
B, C, E, H
1,2,3
L. johnsonii NCC533
L .salivarius subs.
salivarius UCC118
L. delbrueckii spp.
bulgaricus ATTCC
11842
L. casei ATTCC334
L. curvatus ATCC
33820
L. lactis IL1403
L. plantarum WCFS1
34.9
33.04
DL
L
B, C, E,
B, C, E,
4,5, 3
6, 5,3
49.7
D
B, C, E, H
6, 5,3
46.6
36.5
L
DL
A,B, C, E, G
A,B, C, E,G
7, 3
8, 9,3
35.4
45,6
L
DL
A,B, C, E,G
A,B, C, E,G,H,K,L,M
10,5, 3
4,9, 5, 3
L. casei Shirota
L. rhamnosus ATCC
53103
46.3
45-47
L
L
A,B, C, E, G
A,B, C, E, G,H
7, 3,11
5, 3
12
Table 1. – continued
Obligately
heterofermentative
L. fermentum CECT
5716
L. reuteri JCM1112
51.9
38.9
DL
DL
A,B, C, E, G
A, B, C, E, G,N,P
12,9, 3
13, 3
46.2
DL
A, B, C, E, G ,H
5, 3
L. brevis ATCC367
*A – acetic acid; B – lactic acid; C – diacetyl acetaldehyde acetoin; E – hydrogen peroxide, G - carbon dioxide, H – 3phenyllactic acid, 4-hydroxyphenyllactic acid; K – 3-hydroxy fatty acids; L – benzoic acid methylhydantoin
mevalonolactone; M - cyclic dipeptides; N – reuteryciclin; P – reuterin.
**1- Altermann et al., 2005; 2- Connolly and Leonnerdal, 2004; 3- Suskovic et al., 2010; 4- Boekhorst et al., 2004; 5Ljungh and Wadström, 2009; 6- Claesson et al., 2004; 7- Cai et al., 2009; 8- Felis et al., 2009; 9 - Felis and Dellaglio,
2007; 10- Bolotin et al., 2001; 11- Watanabe et al., 2009; 12 - Jime´nez et al., 2010; 13- Morita et al., 2008;
Although genus Bifidobacterium phylogenetically not related to LAB it is often grouped
part of the LAB for its beneficial health effects, including the regulation of intestinal microbial
homeostasis, the inhibition of pathogens and harmful bacteria, the modulation of local and systemic
immune responses, the repression of procarcinogenic enzymatic activities within the microbiota, the
production of vitamins, and the bioconversion of a number of dietary compounds into bioactive
molecules (Mayo and Sinderen., 2010; Rastall et al., 2005).
However much metabolic research of bifidobacteria has focused on carbohydrate
fermentation and they play an important role in
been
observed
that
genus
Bifidobacterium
oligosaccharide metabolism in the colon. It has
possesses
a
unique
fructose-6-phosphate
phosphoketolase pathway employed to ferment carbohydrates which produce lactic acid and
additional amount acetic acid, larger than the amounts secreted by lactobacilli, as the main product
of carbohydrate metabolism that inhibit various pathogenic bacteria (Biavati et al., 2000). L-isomer
produced by bifidobacteria is more potent than the D-isomer (Mayo and Sinderen, 2010).
Unfortunately it is unclear which active stereo-isomers are produced by different Bifidobacterium
species. Unlike lactobacilli, very little is known about the production of bacteriocins by
bifidobacteria. They produce at least six categories of inhibitory substances other than small
terminal metabolites or other non – peptide compounds. It is only clear that some types of
bacteriocins produced by bifidobacteria have the physiochemical stability making them suitable as
potential antimicrobial agents in food for inhibiting some gastrointestinal pathogens and possibly
provide an adjunct or alternative to antibiotic therapy. The most promising bacteriocins produced by
Bifidobacterium bifidum NCDO 1452 is bifidin and the second one called as bifidocin B is
produced by B. bifidum NCFM 1454 (Mayo and Sinderen, 2010). Table 2 shows the different
species of genus Bifidobacterium for human consumption which has most beneficial properties and
extensively used in commercial probiotic preparations. Many of these isolates were found in healthy
13
human gut and licensed as a safe considering that different species effects differ between species in
human or animal studies.
Table 2. Subdivision and characteristics of Bifidobacterium genus
Species
B.adolescentis:
ATCC15703;
L2-32;
B. angulatum DSM 20098
B.animalis subsp. lactic:
AD011
B1-04
DSM 10140
HMN019
B.bifidum:
S17;
PRL 2010;
NCIMB 41171
B.breve DSM 20213
B. catenulatum DSM 16992
B.dentium:
Bd1;
ATCC 27678;
B.gallicum DSM 20093
B.longum subsp. infantis:
ATCC15697;
ATCC 55813;
CCUG 52486;
B.longum subsp. longum:
DJO10A;
NCC 2705;
G+C
(mol %)
*Refernces
59.18
59
59
Ventura e al., 2007
Turoni et al., 2011
60.49
60.49
60.48
60.48
60.48
Kim et al., 2009
Lee and O’Sullivan
2010
Mayo and Sinderen, 2010
62
63
59
56
Turnoei et al., 2011
58.94
59
58
Lee et al., 2010
59.86
60.15
60.13
Lee and O’Sullivan
2010
Lee et al., 2010
Mayo and Sinderen, 2010
1.4 Mechanism of action of probiotics
Critera for Lactobacilli and Bifidobacteria strains to be called as probiotic are:
bacterium must survive the acidic conditions of the upper GIT and colonize the intestine
microbiota. Fermentation products and cell components must not contain pathogenic toxins,
mutagens or carcinogens and must be genetically stable. Finally it must be easily reproducible and
remain viable during processing and storage (Ouigley, 2010).
One of the most exciting areas of research is the mechanism of action of probiotics.
Mechanism by which probiotics strains affect the micro ecology of the intestinal tract are not well
understood with at least three modes of action: modification of the microbial flora, enhancement of
intestinal barrier function and stimulation of immune response have been observed (Table 3).
14
Table 3. Probiotic effects on the development of host defense
Mechanism of action of probiotics
Modification of the
Enhancement of
microbial floraintestinal barrier
antimicrobial activity
function
 Decrease luminal pH
 Increase mucus
production (Trefoil
 Produce inhibitory
factors)
compounds
 Enhance barrier
 Inhibit bacterial invasion
integrity (tight
(gene expression)
junctions) (epithelial
 Block bacterial adhesion
barrier function)
to epithelial cells.
Immunomodulation



Effect on epithelial cells
Effects on dendritic cells
Stimulation and effect on
macrophages
 Stimulation and effects on
lymphocytes:
- B lymphocytes
- NK cells
- T cells
- T cells redistribution
- Interferon
 Secrete polymeric IgA
secretion
1.4.1 Modification of the microbial flora
Probiotics possess the ability to colonize the GI tract by increasing the numbers of
beneficial microbes and decreasing the population of potentially pathogenic microorganisms and
creates a balance in the gut microbiota of the host. A front line of defense against pathogens is
antimicrobial effect:

By reducing luminal pH. Lactic acid bacteria produce organic acid the
most lactate and acetate, which create acidic microenvironment in the gut lumen and that is
inhibitory to virulent organism growth (Sherman et al., 2009).

By producing inhibitory (antimicrobial) compounds (substances).
Probiotics secrete antimicrobial compounds – inhibitory peptides such as : lantibiotics (class
I), peptide bacteriocins (class II), and bacteriolysins (class III) and may become active
participants in the fight against certain infections while others are potent anti-inflammatory
agents and produce (L.reuteri) antimicrobial-multi compound such as reuterins, which also
has broad spectrum activity against of some harmful Gram-negative and Gram-positive
bacteria (Suskovic et al. 2010). The general functions of effect are depicted in Figure 1.

By inhibiting bacterial invasion. It has been observed that some strains
of probiotics may influence virulence gene expression of microbial pathogens ( MedellinPena et al., 2007).
15

Inhibiting bacterial adherence. Some probiotics strains such as L.
crispatus and L. helveticus has a surface- protein layer ( S-layer) and are able to bind to
host cell surfaces of epithelial cells thereby competing with pathogenic bacteria for the
same glycoconjugate epithelial surface receptor as those used by pathogens as receptors for
adherence (Saulnier et al., 2009). The general functions of effect are depicted in Figure 2.
Fig. 1: Antibacterial substances
(Chen. and Walker, 2005)
Produce and secrete antimicrobial substances.
Fig. 2: Inhibition of adherence
(Chen. and Walker, 2005)
Compete with pathogens for glycoconjugate
receptors on the mucosal surface thereby
limiting adherence and colonization.
1.4.2. Enhancement of intestinal barrier function
The mucosal epithelial cell barrier is the first line of defense against pathogen attack on
that ground probiotic can help:

By enhancing the production and secretion of mucin (mucin gene expression- MUC2 or
MUC3) or reduction of gut permeability. Promoting mucin production thereby enhanced
mucus layer overlying the epithelial and reducing intestinal permeability it may serve as
antimicrobial shield that prevents penetration of pathogenic organism and toxic substances (
(Lindenet al., 2008). Mucin producing cells can easily secrete antimicrobial peptides –
treifol factors (human proteins) in response to microbial pathogens binding to the epithelial
cell surface (Sherman et al., 2009).

Direct effect on enhancing epithelial barrier function to modify mucus or chloride secretion
or changes in tight junction protein expression by epithelial cells. The general functions of
effect are depicted in Fig. 3.
16
Fig. 3: Strengthen tight junction
Enhance tight junction proteins to strengthen
against the mucosal barrier.
Fig. 4: innate immunomodulation
Stimulate specific mucosal host defenses
pathogens.
1.4.3 Immunomodulation
Probiotics are able to affect or suppress aspects of the immune response.

Some of the strains affect the epithelial cells and may decrease chemokine interleukin (IL)-8
secretion by epithelial cells compared with some intestinal pathogens such as enteropathogenic
E. coli and Salmonella dublin, Shigella dysenteriae and Listeria monocytogens and indicate that
probiotic bacteria may override the effects of pathogenic bacteria. On the other effect of
probiotic bacteria on epithelial cells is the ability to produce the innate immune system receptors
– Toll-like (TLR) such as TLR-2 and TLR-4 on the surface of epithelial cells and can induce the
production of cytokines that enhance epithelial cell regeneration and inhibit epithelial cell
apoptosis. Probiotic bacteria can also reduce a proinflammatory response in intestinal epithelial
cells by blocking phosphorylation and degradation by ubiquitination of IκB, meanwhile some
pathogenic bacteria induce proinflammatory response by activating the nuclear transcription
factor , NF-κB.( NF-κB is a protein complex that controls the transcription of DNA and plays a
key role in regulating the immune response to infection (Ng et al., 2009).

It has been also observed that probiotic bacteria can have effect on dendritic cells. Dendritic
as antigen-presenting cell (APCs) are important in bacterial recognition and subsequent T –
cell responses, but the most important aspect are their ability to recognize and responds
bacterial components, to initiate primary immune response, and to direct developing T- and
B-cells response (Walker, 2008).

Some of probiotic strains can increase IL-10, IL-12, IL-18, IFN-γ, TNFα synthesis and
secretion in macrophages (Ng et al., 2009).
17

Meanwhile others can elevate the B lymphocytes, NK cells, T cells and interferon activities.

Probiotics may also promote the differentiation of B cells into plasma cells and thereby
increase the production of secretory immunoglobulin A that plays a critical role in mucosal
immunity. IgA, in turn, coats the muscusal surface to control microbial and antigen
penetration (Walker, 2008; Sherman et al., 2009). The general functional affects are
depicted in Fig. 4.
1.5 Antimicrobial activity by lactobacilli and bifidobacteria against
enteric pathogens
Lactic acid bacteria play a key role in maintaining the balance of the normal gastrointestinal microflora (Quigley, 2010). However factors such as diet, stress, an enfeebled immunity
system, microbial infections, other diseases and a prolonged antibiotic usage disturbs the balance,
which often leads to a decrease of viable lactobacilli and bifidobacteria. The subsequent
uncontrolled proliferation of pathogens bacteria may often lead to viral or bacterial diarrhea,
inflammatory bowel disease (IBD), necrotizing enterocolits (NEC), cancer and other clinical
disorders.
There are a number of studies that suggest that lactic acid bacteria can decrease the
incidence, duration and severity of some pathogens which can cause gastric and intestinal illness.
Protective effects of probiotics against gastric and intestinal infections have been demonstrated in
well-designed in vitro and in vivo experimental studies (Table 4). These mechanisms may include
lowering of the pH, production of lactic acid and antimicrobial compounds such as bacteriocins,
hydrogen peroxide and competition for nutrients or adhesion receptors. In addition it is important to
mention that the antimicrobial activity of lactobacilli and bifidobacteria is a strain specific property
and cannot be extrapolated to other lactic acid bacteria that is why it is necessary to screen other
members of Lactobacillus and Bifidobacterium and select the most effective strains to prevent and
treat infectious bacterial and viral diarrhea, gastroenteritis or urovaginal infections.
We focused on four enteric pathogens: (a) two Gram negative enteropathogens; Salmonella
typhimurium which causes gastroenteritis in humans and other mammals; and extended-spectrum
beta-lactamase ( ESBL) – producing Escherichia coli which can various forrms of diarrhea and (b)
two Gram positive pathogens
methicilin-resist Staphyloccocus aureus (MRSA) which is
responsible for various diseases including: mild skin infections (impetigo, folliculitis, etc.), invasive
diseases (wound infections, osteomyelitis, bacteremia with metastatic complications, etc.), toxin
mediated diseases (food poisoning, toxic shock syndrome or TSS, scaled skin syndrome, etc.) and
spore-forming Bacillus cereus which causes two types of food poisoning in humans including
18
diarrheal syndrome and emetic syndrome. A representative summary of recent scientist research of
probiotics antimicrobial effects against S. typhimurium, ESBL, S. aureus and Bacillus cerues
infection is listed in Table 4.
Table 4. Inhibition effects of probiotics on S. typhimurium, ESBL, S. aureus and B.cerues
Barrier function and
Probiotics Tested
L. acidophilus:
LB
Pathogens
Epithelial adherence
S.typhimurium:
(SL 1344)
E.coli:
JPN15[pMAR7] and
S.typhimurium :
(CECT 1456),
(SL 1344), (C5)
Model
References
In vitro (Caco2/TC-7)
Coconnrie et al.,
1999;2000
+ in vivo (gem-free
mouse)
Santos et al., 2003
Bernet-Camard et
al., 1997
+ HT-29, HT29
MTX
Gopal et al., 2001
In vitro (Caco-2)
Nesser et al., 2000
In vitro
(intestine mucus)
In vitro (Caco-2 or
intestine mucus)
In vitro
(intestine mucus
Tuomola et all.,
1999
Lee et al., 2003,
Lee and Puong.,
2002
Tuomola et all.,
1999
Lee et al., 2003,
Lee and Puong.,
2002
Tuomola et all.,
1999
L.gasseri :
(UO 002)
L. kefiranofaciens:
( CYC 10058)
L.delbruckii:
( CYC 10048)
L.helveticus:
( R0052)
Bifidobacteria strains :
(CA1) and (F9)
E.coli :
(0157:H7)
E.coli:
(ETEC)-enterotoxigenic; (EPEC) –
enteropathogenic;
S.typhimurium:
(ATCC 14028)
E.coli:
(ATCC 11775), (TG-1), (0157)
S.typhimurium :
(ATCC 14028), (E10), (E12) SfaIIfimbrated E.coli HB101 (paZZ50),
E.coli:
(ATCC 11775), (TG-1), (0157)
S.typhimurium:
( ATCC 14028), (E10), (E12)
SfaII- fimbrated E.coli HB101
(paZZ50),
E.coli :
( 0157:H7)
E.coli :
(ETEC) (ATCC 31705)enterotoxigenic, (EIEC) IID 995enteroinvasive, (EPEC) IID 956 enteropathogenic
E.coli :
(0111)
S.typhimurium :
(CECT 1456),
E.coli :
(CECT 4076)
E.coli:
(0157:H7)
S.typhimurium:
(SL 1344)
lactis (DR10)
E.coli :
( 0157:H7)
In vitro
(Caco-2, HT-29,
HT29 MTX
(CYC 10051)
(LA1-SCS )
(HN017)
L.johnsonii :
(La1)
(LJ1)
L.casei subsp:
Shirota (Yakult;Singapore)
rhamnosus :
GG (ATCC 53103)
GG (ATCC 53103) and
(LC-705)
(DR20)
L.crispatus:
(JCM 5810)
In vitro (Caco-2)
In vitro
(intestine mucus)
In vitro
(Caco-2, HT-29,
HT29 MTX)
Gopal et al., 2001
In vitro
(Matrigel)
Horie et al., 2002
In vitro
(Caco-2)
In vitro
(Caco-2)
Fernanadez et al.,
2003
Santos et al., 2003
In vitro (Hep-2,
T84)
In vitro
(Caco-2)
and in vivo
(axenic mouse)
Johnson-Henry et
al., 2007
Lievin et al., 2000
Gopal et al., 2001
19
Table 4 - continued
Probiotic antimicrobial factors
P.acdilactici :
(NCIM 2292)
P.pentosaceous
(NCIM 2296)
P. cervisiae
(NCIM 217)
L.plantarum:
(42)
L.curvaus:
( DF 38)
P.pentosaceus :
(34)
L.salivarius:
(241)
L.casei LHS
L.acidopillus :
(CRL 1259),
(I 26) ,(I 16)
( YIT0070)
L.crispatus:
(I 12)
L.brevis:
(I 23), (I 211,) (I 218),
(C10), (C1, C17)
L.fermentum:
(I 24),( I 25), (C 16)
L.casei subsp:
Shirota:
(YT 9029)
(DN -114001)
rhamnosus:
(GR1)
(ATCC 4356)
casei :
(ATCC 393)
E. faecium:
(ATCC 19434)
L.johnsonii:
(La1)
L.sakei:
(CWB 030202)
S.aureus :
(MTCC 737) and
B.cereus :
(MTCC 1272)
E.coli, S.typhimurium
including strains isolated
from patients diagnosed
with HIV/AIDS
E.coli.:
(O1:K1), (O2:K1),
(O78:K80),
S.typhimurium
(0157:H7)
E.coli:
O1:K1, O2:K1, O78:K80
and S.typhimurium
E.coli :
(0157:H7)
S.typhimurium:
(SL 1344)
In vitro
Jamuna and
Jeevarantnam, 2004
In vitro
Brink et al., 2006
In vitro
Juarez et al., 2003
Jin et al., 1996
Ogawa et al., 2001
In vitro
Jin et al., 1996
In vitro
Ogawa et al., 2001
Fayol-Messaoudi et al.,
2005
Enteroaggregative E.coli
(EaggEC TN-2)
In vitro
Miyazaki et al., 2010
S.typhimurium:
(SL 1344)
In vitro
Fayol-Messaoudi et al.,
2005
20
1.6 Prebiotics
The term prebiotic, first coined by Gibson and Reberfroid, refers to non-digestible but
fermentable food ingredients that beneficially affect the host by selectively stimulating the growth
of a limited number of health-promoting commensal flora. A number of food ingredients could act
as prebiotics but the most commonly evaluated prebiotics are those stimulate probiotic
microorganism already resident in the lower gut (Gibson et al., 2010). Certain carbohydrates, oligoand poly-saccharides, occur naturally and meet the criteria of prebiotics. These consist mainly of
non-digestible fructooligosaccharades and inulin –type fructans, which are linked by β (2-1) bonds
that limit their digestion by intestinal enzymes. Crittenden and Playne (1997) describe food-grade
oligosaccharides in commercial production which include fructooligosachharides (FOS),
galactooligosaccharides (GOS), gentiooligosaccharides, inulin, isomaltooligosaccharides (IMOS),
maltooligosaccharides, palatinose-oligosaccharides
lactulose, glucosyl sucrose,
lactosucrose,
soybean-oligosaccharides (SOS), xylooligosaccharides (XOS) and cyclodextrins ( Gibson et al.,
2010). Most of them are present in significant amounts in many edible fruits and vegetables
including wheat, onion, chicory, garlic, leeks, artichokes and bananas (Salvatore and Vandenpalas,
2010). Because of their chemical structure, prebiotics are not hydrolyzed and absorbed in the small
intestine but are fermented in the colon by endogenous bacteria to act as energy and metabolic
substrates, with lactic and short chain carboxylic acid as end products of fermentation (Salvatore
and Vandenpalas, 2010). Some non-digestible carbohydrates have a number of functional effects on
the GIT which have been used to validate functional and health claims. The carbohydrate group that
has received the most attention and research is the oligosaccharides – FOS may selectively
stimulate the growth of bifidobacteria in the large intestine, increase calcium absorption, and fecal
weight, shortening of gastrointestinal transit time and possibly, lowering the blood lipid level
(JoMay, 2002).
When prebiotics are added in combination with probiotic strains, the combination referred
to as “synbiotic”, can selectively stimulate the growth of probiotic bacteria, especially, but not
exclusively lactobacilli and bifidobacteria. Prebiotics can serve as a selective growth substrate for
the probiotic strain during fermentation. Combining probiotics and prebiotics could beneficially
affect the host by improving the survival and of live microbial dietary supplements into the
gastrointestinal flora and by improving the microbial balance of the gastrointestinal tract (Chen. and
Walker, 2005).
21
2. MATERIAL AND METHODS
2.1 Growth substrates
Prebiotics used in the present study are listed in Table 5.
Table 5. Carbohydrate source list used in this study
Carbohydrate source
Purity
Company
Fructooligosaccharides (FOS)
93.7% Orafti (Tienen, Belgium)
Galactooligosaccharides (GOS)
96.2% Friesland Campina Domo (The Netherlands)
Lactulose
95% Sigma-Aldrish (Canada)
Xylooligosaccharides (XOS)
95% Sweet Town Bio-tech (Taiwan)
Isomaltooligosaccharides (IMOS) 60% Wako Pure chemical, Ltd (Germany)
2.2 Bacterial strains
Probiotics and enteric pathogens strains used in this study are listed in a Table 6.
Table 6. List of bacterial strains used in the present investigation
No.
1
2
3
4
5
6.
7.
8
9.
1.
2.
3.
4.
Name of strain
Probiotic strains
L. crispatus LMG 12003
L. paracasei F8
L. rhamnosus LС 35
L. plantarum F44
L. plantarum F17
B. breve 46
B. lactis 88
B. longum 618
B. pseudocatenulatum JCM 1200
Enteropathogens
S. typhimurium (Gram Negative/resistant)
ESBL (Gram Negative/resistant)
MRSA-S. aureus (Gram Positive/resistant)
B. cereus (Gram Positive /resistant)
Source
Infant, feaces
Biobank, LUH
Lyocentre, France
Biobank, LUH
Biobank, LUH
Biobank, LUH
Biobank, LUH
Biobank, LUH
Feces of infant
Biobank, LUH
Biobank, LUH
Biobank, LUH
Biobank, LUH
2.3 Bacterial growth conditions
Bifidobacteria and lactobacilli strains were screened for antimicrobial properties
against Gram-negative and Gram-positive bacteria. Lactic acid
bacteria were grown
microaerobically in 5ml of De Man, Rogosa, Sharpe (MRS) broth (Difco Laboratories, Detroit, MI)
24h at 37 oC and bifidobacteria strains were grown in 5 ml MRS broth supplemented with 0.05 %
22
L- cysteine hydrochloride (MP Biomedicals, LLC, France) 24h at 37 oC under anaerobic conditions,
achieved using the Anoxomat system (Mart Microbiology, The Netherlands).
Enteropathogens used were E. coli, S. typhimurium, S. aureus and B .cereus were
grown aerobically in 5 ml Muller-Hinton broth (Becton Dickinson company Cockeysville,
MD21010, USA) or on agar (OXOID AB LTD, Sollentuna, Sweden).
2.4 Antibacterial activity of selected bacteria
2.4.1 A time course of antimicrobial activity of cell-free supernatant of probiotic
bacteria against enteric pathogens
AMA of LAB and bifidobacteria was determined against E. coli, S. typhimurium, S.
aureus and B. cereus using microtiter plate method.
Preparation of cell free culture filtrates: Briefly, a cell free culture filtrate was
obtained by harvesting 24 h old cultures of lactobacilli and bifidobacteria grown on MRS and
MRSC broth by centrifugation (15.000 x g for 15 min at 4 oC in an eppendorfs centrifuge 5810,
Hamburg). The supernatants were collected, pH determined and adjusted to pH 6.0 with 2 M.
NaOH (MERCK, Germany) and filter sterilized by passing through a sterile syringe and 0.2- µm
pore size with cellulose acetate membrane filter (VWR INTERNATIONAL AB, Stockholm,
Sweden). To determine if the antimicrobial activity recorded was bacteriostatic or bactericidal, the
cell free culture filtrates were added on pathogens in a sterile 96 well micro titer plates (TPP,
92696, Switzerland) (Catherine and Rolfe., 2000). Dilutions 1:1 and 1:10 (in separate experiment
1:100 and 1:500) were prepared in Muller-Hinton broth. One hundred of the culture filtrates of
lactic acid bacteria and bifidobacteria was added to the microtiter plate wells and incubated
overnight at 37oC.
Preparation of enteric pathogens for antimicrobial activity: E. coli, S. typhimurium, S.
aureus and B. cereus cells were grown in Muller-Hinton broth (MHB) for 24 h in aerobic
conditions at 37 oC (cells were harvested by centrifugation and washed twice in sterile phosphate
buffer saline 0.015 M. PBS, pH 7.2) and suspended in PBS. Ten micro liters of E. coli, S.
typhimurium, S. aureus and B. cereus
cells with 0.5 OD (optical density) at 620 nm was prepared
in PBS and 10 µl of the suspension was added to 100 µl of test supernatants in the microtiter plates.
After 24 h, the growth in microtiter plates was measured using microtiter plate reader ( iMarkTM
Microplate Absorbance Reader catalog number 168-1130, BIO-RAD Laboratories AB,
23
Sundbyberg, Sweden). The antimicrobial activity was expressed as the percent inhibition of the
growth of pathogen compared to control.
2.4.2 Effect of prebiotics on the AMA of probiotic strains against E. coli and S.
typhimurium, S. aureus and B. cereus
The lactic acid bacteria and bifidobacteria were grown in MRS and MRSC for 24 h.
Cells were centrifuged and washed twice in PBS and the biomass density was determined by
measuring the optical density at 620 nm. Pre-reduced MRS and MRSC broth supplemented with
filter sterilized (0.2 µm) 1% each of fructooligosaccharides (FOS), galactooligosaccharides (GOS),
lactulose, xylooligosaccharide (XOS), isomaltooligosaccharides (IMOS) was inoculated with
probiotic strains to get an OD of 0.5 in 5 ml broth. After 24 h of growth at 37oC, the cells were
washed with sterile phosphate buffer saline (PBS, pH 7.2, 0.015) and cell –free culture filtrates
were adjusted to 6.0 pH and tested for antimicrobial activity against E. coli , S. typhimurium, S.
aureus and B. cereus cells using 96 well sterile microtiter plates (Brink et al., 2006).
2.4.3 Effect of bile stress on AMA of probiotic strains against E. coli and S.
typhimurium, S. aureus and B. cereus
Effect of bile was determined as described by Brink et al., 2006 with a slight
modification. Briefly, LAB and bifidobacteria were grown in MRS and MRSC broth supplemented
with 5% porcine bile instead of oxbile (Brink et al., 2006). After 24 h of growth at 37oC, the cell
free culture filtrates were obtained by centrifugation. The pH was adjusted to 6.0 and was used to
determine antimicrobial activity against cells of the E. coli, S. typhimurium, S. aureus and B. cereus
strains using microtiter plate.
2.4.4 Effect of probiotic strain combinations on AMA against E. coli, S. typhimurium,
S. aureus and B. cereus
LAB and bifidobacteria strains were checked for compatibility and various mixed
cultures (Co-cultures) of different lactic acid bacteria and bifidobacteria were performed in 5 ml
MRSC broth and incubated under anaerobic condition. After 24 h of growth at 37 oC, cell free
culture filtrates were obtained by centrifugation and neutralized to pH 6.0 with 2 M NaOH, filter
sterilized and was tested for antimicrobial activity against E. coli and S. typhimurium S. aureus and
B. cereus cells using micro titer plate and listed in Table 7.
24
Table 7. List of co-culture mixes tested for antimicrobial activity
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Strain
combinations
F44+F8+88
F44+F8+46
F8+46+88
F17+F8+88
F17+46+88
F8+88
F44+46
F17+46
F17+88
F8+46
8:8+618
46+88
46+618
46+88+F8+F44
F44: L. plantarum; F8: L. paracasei; 46: B. breve; 618: B. longum & 88: B. lactis
LUH strain Biobank collection (Åsa Ljungh; to be published; www.qualvivo.eu)
A part of the work will be presented in “International conference on probiotics and probiotics
ICP2011” Slovakia during June 2011.
2.4.5 Partial purification of proteinecious antimicrobial substance
LAB and bifidobacteria strains were grown in MRS or MRSC broth for 24 h at 37 °C.
After incubation, the supernatants were collected by centrifugation at 5000 × g for 10 min. and used
for the precipitation of antimicrobial peptides with 80% saturation of ammonium sulphate (Joshi et
al., 2006). After stirring on a magnetic stirrer, it was kept undisturbed at 4 °C overnight. Precipitates
formed were collected by centrifugation at 10 000 × g for 10 min and redissolved in 10 mM sodium
phosphate buffer, pH 6.0. Crude protein extract was dialyzed using 1000 Dalton cutoff dialysis
membrane against 10 mM Phosphate buffer of pH 6.0 extensively. Similarly proteins precipitated
from MRSC broth uninoculated with probiotic strains served as a control. Antimicrobial activity of
the crude protein was determined against enteric pathogens at 1:1; 1:10; 1:100 and 1:500 dilutions.
25
3. RESULTS AND DISCUSSION
3.1 The antimicrobial activity of LAB and Bifidobacteria of growth of
enteric pathogens (screening)
The antimicrobial activity (AMA) of LAB and bifidobacteria and inhibition of growth
of enteropathogens is shown in Table 8. From a total 5 lactic acid and 4 bifidobacteria, the culture
supernatants of two Lactobacilli (L. paracasei F8, L. plantarum 44) and two bifidobacteria ( B.
breve 46, B. lactis 88) has shown strong antimicrobial activity when tested against the two Gram
negative and Gram positive pathogens. The inhibition of enteropathogens ranged from 70 to 83%.
The strongest inhibition (84%) was obtained with culture supernatant of strains L. paracasei on
ESBL. In contrast, B. cereus was the most resistant strain; only inhibited at pH 6 at 1:1 dilution.
In the present study, the acid supernatants (pH 3.5-4.9) of the L. paracasei, L.
plantarum, B. breve and B. lactis strains inhibited the growth of all tested enteropathogens. The
most sensitive pathogen was the Gram positive S. aureus to inhibitory substances produced by the
LAB and bifidobacteria. It was observed that four top probiotic cell-free supernatants at 1:1 and
1:10 dilutions suppressed the growth of S. aureus. We also found that the inhibitory effect of L.
paracasei, L. plantarum, B. breve and B. lactis on growth also decreased after adjustment of the pH
to 6.0.
Table 8a. Antimicrobial activity (AMA) of acid cell-free supernatants of probiotic strains against
enteropathoges by microtiter plate assay
Pathogens
Acidic cell free supernatants
Strains
S. typhimurium
ESBL
S. aureus
1:1
1:10
1:1 1:10 1:1 1:10
B. breve 46
3+
3+ dW 2+ 2+
B. longum 6:18
2+
3+ W+ 2+ 2+
B. lactis 8:8
3+
3+ dW 2+
B.pseudocatenulatum JCM 1200
2+
2+ dW 2+
+
L.paracasei F8
3+
3+
3+
+
L.plantarum 44
3+
3+ W+ 2+ W+
L.plantarum F17
2+
dW
3+
+
2+ W+
L.crispatus LMG 12003
2+
3+ dW 2+
+
L.rhamnosus LC35
2+
3+
+
2+
+
B. cereus
1:1 1:10
2+ W+
2+
2+
+
2+
2+
W+
2+
+
-
((3+) - 80% to 90% inhibition, (2+) –60% to 80% inhibition, (+) –30% to 60% inhibition, (W) –10% to 30%
inhibition, (dW) –5% to 10 % inhibition, (-) – no inhibition
26
Table 8b. Antimicrobial activity (AMA) of neutral cell-free supernatants of probiotic strains
against Enteropathoges by microtiter plate assay
Pathogens
Neutralized cell free supernatants
Strains
S. typhimurium
ESBL
S. aureus B. cereus
1:1
1:10
1:1 1:10 1:1 1:10 1:1 1:10
B. breve 46
2+
+
2+
dW
B. longum 618
+
+
+
B. lactis 88
+
+
+
B. pseudocatenulatum JCM 1200 dW
dW
+
L. paracasei F8
+
dW
2+
L. plantarum F44
+
W+
2+
L. plantarum F17
+
W+
+
L. crispatus LMG 12003
+
+
2+
L. rhamnosus LC35
+
+
2+
((3+) - 80% to 90% inhibition, (2+) –60% to 80% inhibition, (+) –30% to 60% inhibition, (W) –10% to 30%
inhibition, (dW) –5% to 10 % inhibition, (-) – no inhibition
3.2 A time course of antimicrobial activity of cell - free supernatants of probiotic
bacteria against enteric pathogens
Time course of AMA of cell free supernatants (acid and neutralized) of two lactobacilli
and two bifidobacteria strains against four enteric pathogens was evaluated by collecting the cell
free supernatants of the probiotic strains at 4, 8, 12 and 24 h of growth. (Fig. 5, 6, 7 and 8).
L. paracasei F 8
Acid supernatants of L. paracasei F8 obtained at 4 h of growth showed strong inhibition of
the 4 enteric pathogens at 1:1 dilution (Fig. 5). AMA of supernatants obtained at 8, 12 and 24h
showed a reduction against 4 enteric pathogens. Neutralized supernatants (pH 6.0) obtained at 8 h
of growth at 1:1 dilution showed highest AMA against S. aureus. However neutralized supernatants
showed a reduction in AMA against S. typhimurium, ESBL E. coli and B. cereus after 12 hours of
incubation.
27
Fig. 5 Time course of AMA of L. paracasei F8 against 4 enteropathogens (a) acid supernatants and
(b) neutralized supernatants (pH 6.0)
L. plantarum F44
Time course of AMA of L. plantarum F44 against 4 enteric pathogens is shown in Fig. 6.
Acid supernatants of L. plantarum obtained at 4h of growth at 1:1 dilution inhibited the growth of
enteric pathogens by more than 80%. However a decrease in AMA was observed with the
supernatants obtained at 8, 12 and 24 h of growth. Neutralized supernatants (pH 6.0) obtained at 4 h
of growth showed a reduction in AMA against ESBL, B. cereus and S. typhimurium, meanwhile S.
typhimurium was decreased by incubation with the supernatants of L. plantarum strain grown after
8 h of incubation. However AMA against S. aureus remained the same.
Fig. 6 Time course of AMA of L. plantarumF44 against enteropathogens (a) acid supernatants and
(b) neutralized supernatants (pH 6.0)
28
B. breve 46
Time course of AMA of cell free supernatants of B. breve 46 against 4 enteric pathogens is
shown Fig. 7. Acid extracts obtained at 4 h of growth of B. breve 46 showed strong inhibition of S.
aureus
and
S. typhimurium where as AMA against ESBL and B. cereus was highest with
supernatants obtained at 8 and 12 h of growth. AMA of acid supernatants obtained after 24 h
showed a slight reduction against the 4 enteric pathogens.
Neutralized extracts obtained at 4 h of growth at 1:1 dilution showed strong AMA against
S. aureus and stabilized after 12h and did not decrease after 24h of incubation, whereas AMA
against B. cereus was about 50% with the neutralized supernatants obtained after 12h of growt at
1:1 dilution. AMA against ESBL and S. typhimurium was highest
with neutralized extracts
obtained after 24 h of growth at 1:1 dilution.
Fig. 7 Time course of AMA of B. breve 46 against 4 enteropathogens (a) acid supernatants and (b)
neutralized supernatants (pH 6.0)
B. lactis 88
Time course of AMA of cell free supernatants of B. lactis 88 against 4 enteric pathogens is
shown Fig. 8. Acid supernatants obtained at 12 h of growth at 1:1 dilution showed highest inhibiton
of all the enteric pathogens. However the inhibition was decreased with the supernatants obtained at
24 h of growth.
Neutralized supernatants (pH 6.0) of B. lactis 88 obtained at 4 h of growth showed strong
inhibiton of S. aureus and the AMA was retained upto 12 h and was decreased with the supernatants
obtained at 24 h of growth.
29
Neutralized supernatants of B. lactis 88 obtained at 8 and 12 h of growth at 1:1 dilution
showed strong AMA against B. cereus and the AMA greatly reduced with the neutralized
supernatants obtained at 24 h of growth. However neutralized supernatants of B.lactis 88 obtained
after 24 h of growth at 1:1 dilution showed highest AMA against S. typhimurium and ESBL. It was
observed that AMA at 1:10 dilution was completely lost or imperceptible.
Fig. 8 Time course of AMA of B. lactis 8:8 against Enteropathogens (a) acid supernatants and (b)
neutralized supernatants (pH 6.0)
In conclusion, the results on time course of AMA of probiotic strains against the 4 enteric
pathogens showed that the inhibition was highest with acid supernatants. When the supernatants
were neutralized to pH 6.0 there was a decrease in AMA. This could be due to the synergistic action
of acid metabolites like lactic, acetic, propionic and butyric acids and also the possible secretion of
proteinecious bacteriocins (and bacteriocin like substances or non-proteinecious substances (H2O2)
by probiotic strains in acid supernatants which further needs to be investigated).
3.3 AMA studies of LAB and Bifidobacteria in presence of prebiotics
The ability of FOS, GOS, IMOS, LACT and XOS to enhance lactobacilli and
bifidobacteria antimicrobial activities against enteric pathogens were analyzed (Fig. 9, 10, 11, 12,
13). We have observed that addition of prebiotics to MRS or MRSC resulted to increased
antimicrobial activity, but differed from strain to strain. B. breve 46 and B. lactis 8:8 growth with
FOS, GOS, IMOS, LACT and XOS (B. lactis 8:8), were effective in inhibiting growth of all tested
enteric pathogens with acid supernatants. Cell free supernatants consistently conferred a
significantly greater inhibitory effect than neutralized supernatant fractions. It was observed that B.
30
breve 46 cell free supernatants in presence of all tested prebiotic sources showed stronger
antimicrobial activity at pH 4 compared with B. lactis strain. In contrast, B. breve was most
effective with FOS, GOS and XOS imparting high inhibition levels against all four enteropathogens
(Fig. 9, 10 and 13). No AMA or very slight was recorded with neutralized cell free supernatants of
B. breve and B. lactis in presence of IMOS, LACT and XOS (Fig. 11, 12, 13). Highest inhibition of
the pathogens by the neutralized cell free supernatants of B. breve strain against S .aureus was
obtained with FOS and GOS (Fig. 9 & 10) meanwhile GOS was the only carbohydrate source
where inhibition of B. cereus was observed with AMA of neutralized cell free supernatants of B.
lactis strain at pH 6 (Fig. 10)
AMA of cell-free supernatants obtained from L. plantarum and L. paracasei cultured in
MRS broth with 1 % each of GOS, IMOS, LACT was determined (Fig.10, 11 & 12). L. plantarum
grown with GOS was the most effective at inhibiting pathogen growth at no adjustment pH (Fig. 9)
while no antimicrobial activity or very slight was recorded in the cell free supernatant of
L.planatrum in present of lactulose (Fig. 12). It was observed that at neutralized pH of strain
supernatants (1:1) in the presence of GOS and IMOS were ineffective against S. aureus and B.
cereus) (Fig. 10 & 11). In contrast, L. paracasei in presence of IMOS has shown high AMA at acid
pH only against Gram-positive pathogens (Fig. 16).
Fig. 9 Inhibition of 4 enteropathogens by B. breve and B. lactis in presence of FOS
31
Fig. 10 Inhibition of 4 enteropathogens by B.breve, B. lactis and L. plantarum in presence of GOS
Fig. 11 Inhibition of 4 enteropathogens by B. lactis, B. breve, L. plantarum and L. paracasei in
presence of IMOS
32
Fig. 12 Inhibition of 4 enteropathogens by B. lactis, B. breve and L. plantarumin presence of LACT
Fig. 13 Inhibition of 4 enteropathogens by B. lactis in presence of XOS
33
3.4 Effect of bile on AMA of Probiotic strains
Bile is reported to enhance cell surface hydrophobicity of probiotic strains and certain
pathogens for better adhesion to the mucus lining. Therefore the effect of 5% porcine bile on AMA
of probiotic strains was evaluated and compared with AMA of supernatants obtained from
unstressed cells. As data shown in Fig. 14 a & b, the acid supernatants of L. plantarum in presence
of bile and without bile at 1:1 dilution are able to inhibit the growth of 4 enteric pathogens by more
than 60%. Growth was much more vigorous at a neutralized pH in the presence of bile cell-free
supernatant at 1:1dilution inhibited the growth of all target pathogens.
Fig. 14 AMA of L. plantarum cell free supernatant against 4 enteropathogens (a) grown in presence
of 5% (w/v) porcine bile and (b) grown without bile in the growth medium
AMA of bile stressed cell-free supernatant of L.paracasei against 4 pathogens are
represented in Figure 15. Acid supernatants of L. paracasei at 1:1 dilution showed slight or
moderate inhibition of the growth of enteropathogens meanwhile inhibition of pathogens was much
more inhibited in the presence of bile. It was observed that among the strains Gram-negative
pathogens are able to survive bile conditions at neutral pH, whereas Gram-positive bacteria (S.
aureus and B. cereus) were significantly suppressed with the neutralized supernatants of bile at 1:1
dilutions.
34
Fig. 15 AMA of L. paracasei cell free supernatants against 4 enteropathogens (a) grown in
presence of 5% (w/v) porcine bile and (b) grown without bile in the growth medium
The inhibition of growth of four target pathogens in presence of B.breve cell-free
supernatant with 5% bile was also determined. Data obatined and sumarised in Figure 16. In this
study it was observed that AMA of acid extract at 1:1 dilution showed strong AMA against all
enteric pathogens meanwhile AMA of supernatant obtained from bile grown cells against
S.typhimurium and ESBL was lost completely. It was determine that Gram-negative strains
displayed good survival at pH 6 whereas larger differences were observed between Gram-positive
strains such as S.aures and B.cereus where neutralized supernatants of B. breve caused maximum
inhibition at 1:1 and 1:10 dilutions of S. aureus and B. cereus.
Fig. 16 AMA of B. breve cell free supernatants against 4 enteropathogens (a) grown in presence of
5% (w/v) porcine bile and (b) grown without bile in the growth medium
35
Simmilar results were obtained in presence of B. lactis cell-free supernatant with 5% of
bile (Fig. 17b). Results contrasted without bile showed that B.lactis cell-free supernatant at pH 4 at
1:1 dilution can completely supressed the growth of three target enteric pathogens, B. cereus was
was the most resistant strain. AMA of B. lactis extract in presence of bile showed highest inhibition
of B. cereus.
Fig. 17 AMA of B. lactis cell free supernatants against 4 enteropathogens (a) grown in presence of
5% (w/v) porcine bile and (b) grown without bile in the growth medium.
In conclusion, bile stress did not abolish the AMA of LAB supernatants meanwhile AMA
against ESBL and S. typhimurium by bifidobacteria cell free supernatants was abolished when
grown in presence of 5% porcine bile. However B. cereus was inhibited by supernatant of
bifidobacteria strains grown in presence of 5% porcine bile.
3.5 Effect of probiotic strain combinations on AMA against E. coli, S.
typhimurium, S. aureus and B. cereus
AMA of cell-free culture supernatants collected from 14 different combinations of
Lactic acid bacteria and bifidobacteria against E. coli and S. typhimurium S. aureus and B .cereus
were examined.
Acid cell free supernatant of probiotics strains combinations at 1:1 dilutions inhibited
the growth of E. coli and S .typhimurium, S. aureus and B. cereus more than 80%. At higher
dilutions (1:10, 1:100 &1:500), cell free supernatants did not show antimicrobial activity or it was
much less on S. typhimurium, E. coli and B. cereus.
Neutralized cell free supernatants of tested probiotics strain combinations did not
show antimicrobial activity on the Gram-positive and Gram-negative bacteria tested except S.
aureus. Generally, acid cell free supernatants of 14 probiotics strain combinations were more
36
effective in inhibiting the growth of S. aureus than S. typhimurium, B. cereus and E. coli. However
B. cereus was found to be the most resistant strain
Fig. 18 AMA of cell free supernatant of co-cultured probiotic strain ombination of F44+F8+88 (a)
acid supernatants (b) neutralized supernatants
As shown in Fig. 18a, the acid supernatants of F44+F8+88 combinations at 1:1 dilution
inhibited the growth of E. coli and S. typhimurium, S. aureus and B. cereus by more than 80%. At
higher dilutions (1:10, 1:100, 1:500), cell free supernatant did not show antimicrobial activity or it
was very less except S. aureus which growth has been suppressed at all dilutions by more than 30%.
When pH was adjusted to 6, inhibitory effect on S. typhimurium, E. coli and B. cereus pathogens
was detected except S. aureus which growth was suppressed (Fig. 18b).
Fig. 19 AMA of cell free supernatant of co-cultured probiotic strain combination of F44+F8+46 (a)
acid supernatants (b) neutralized supernatants
37
As shown in Fig. 19a, the acid supernatants of F44+F8+46 probiotic strain combination at
1:1 dilution inhibited the growth of E. coli, S. typhimurium. S. aureus and B. cereus by more than
80%. At higher dilutions (1:10, 1:100, 1:500), cell free supernatants showed slight bactericidal
effect on the growth of the E. coli and the most observable of the S. aureus, whereas no inhibitory
effect of supernatant of B. cereus was noticeable . In contrast to the supernatant of the F44+F8+46,
the bactericidal effect on tested pathogens was also detected on S. aureus (in all dilutions) even
when the pH of the supernatants has been adjusted to pH 6.0 (Fig. 19b).
Fig. 20 AMA of cell free supernatants of co-culture of probiotic strain combinationF8+46+88 (a)
acid supernatants (b) neutralized
As shown in Fig. 20a, the acid supernatants of F8+F88+46 strain combinations at 1:1
dilution inhibited the growth of E. coli, S typhimurium. S. aureus and B. cereus by more than 80%.
At higher dilution (1:10, 1:100 & 1:500), the cell free supernatants showed slight bactericidal effect
on the growth of all tested pathogens however the highest against S. aureus. In contrast when the
supernatants of the F8+88+46 was adjusted to pH 6 the bactericidal effect on tested pathogens was
also detected on 1:1 dilution, meanwhile on the growth of S. aureus it has been retained in all
dilutions (Fig. 20b).
38
Fig. 21 AMA of cell free supernatants of co-cultured probiotic strain combination F17+F8+88 (a)
acid supernatants (b) neutralized supernatants
As shown in Fig. 21a, the
acid cell free supernatant of
F17+F8+88 strain
combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium, S. aureus and B. cereus
by more than79 %. At higher dilution (1:100 and 1:500), the cell free supernatants did not show (or
it was very less) strong antimicrobial activity against Gram-positive and Gram-negative bacteria. It
was observed that pH adjustment to 6.0 of F17+F8+88 supernatant enhanced the growth (except 1:1
unlisted S. aureus) of all tested pathogens.
Fig. 22 AMA of cell free supernatants of co-cultured probiotic strain combination F17+46+88 (a)
acid supernatants (b) neutralized supernatants
As shown in Fig. 22a, the acid cell free supernatants of F17+46+88 probiotic strain
combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium. S. aureus and B. cereus
by more than 80 %. Three pathogens ( E. coli-39%, S. aureus 86% and B.cereus-35%) were
significantly decreased by incubation with the supernatant of F17+46+88 at 1:10 dilution. When
39
pH was adjusted to 6 the inhibitory effect of cell free supernatants of probiotic strain combination
on the 4 enteropathogens was disappeared except S. aureus (Fig. 22b).
Fig. 23 AMA of cell free supernatant of F8+88 strain co-culture (a) acid supernatants (b)
neutralized supernatants
As shown in Fig.
23a, the acid cell free supernatants of
F8+88 probiotic strain
combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium, S. aureus and B. cereus
by more than 80%. In this experiment it was observed that the acid supernatant of F8+88 first time
suppressed growth of B. cereus in all serial dilutions and it has been also showed a quite strong
bactericidal effect on other pathogens growth in spite that it was lost after adjustment of the pH to 6
(except S.aureus) (Fig. 23b).
Fig. 24 AMA of cell free supernatants of 44+46 co-culture (a) acid supernatants (b) neutralized
supernatants
40
As shown in Fig. 24a, the acid cell free supernatants of 44+46 probiotics strain
combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium, S. aureus and B. cereus
by more than 74%. In 1:100 and 1:500 dilutions the effect of non-pH adjusted supernatant did not
show a bactericidal effect on the growth of tested enteropathogens. Neutralized CFS did not inhibit
the pathogens (Fig. 24b).
Fig. 25 AMA of cell free supernatants of F17+46 co-culture (a) acid supernatants (b) neutralized
supernatants
As shown in Fig. 25am, the acid cell free supernatants of
F17+46 probiotic strain
combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium, S. aureus and B. cereus
by more than 76%. Tested strain combination supernatants at acid pH were able to inhibit the
growth at original cell free extract meanwhile neutralized supernatant did not show antimicrobial
activity or it was very less in all higher dilution cases (Fig. 25 b).
Fig. 26 AMA of cell free supernatant of F17+88 co-cultured probiotic strain (a) acid supernatants
(b) neutralized supernatants
41
As shown in Fig. 26a, the acid cell free supernatants of F17+88 probiotic strain
combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium, S. aureus and B. cereus
by more than 79%. At higher dilution (1:10, 1:100& 1:500), cell free supernatants showed slight
bactericidal effect on the tested enteropahogens. We found that inhibitory effect of F17+88 was also
disappeared after adjustment of the pH (Fig. 26b).
Fig. 27 AMA of cell free supernatants of F8+46 co-cultured probiotic strains (a) acid supernatants
(b) neutralized cell free supernatants
As shown in Fig. 27a, acid cell free supernatants of F8+46 probiotics strain
combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium. S. aureus and B. cereus
by more than 77 %. At higher dilution (1:10, 1:100 & 1:500) cell free supernatants showed
bactericidal effect on the growth of S. aureus. In contrast when pH value of the F8+46 supernatant
was adjusted to pH 6.0, the bactericidal effect on tested pathogens was also detected on 1:1 dilution
meanwhile the growth of S. aureus has been retained at all dilutions (Fig. 27b).
Fig. 28 AMA of cell free supernatant of 88+618 co-culture (a) acid supernatants (b) neutralized cell
free supernatants
42
As shown in Fig.28a, the acid cell free supernatants of 88+618 probiotic strain
combination at 1:1 dilution inhibited the growth of E .coli, S. typhimurium. S. aureus and B. cereus
by more than 80%. AMA of serially diluted supernatants from 88+618 showed antimicrobial
spectrum just on S. aureus. In contrast to the supernatant of the 88+618, the bactericidal effect on
tested pathogens was also detected on S. aureus growth (in all dilutions) even when the pH of the
supernatants has been adjusted to pH 6.0 (Fig. 28b).
Fig. 29 AMA of cell free supernatant of 46+88 co-culture (a) acid supernatants (b) neutralized
supernatants
As shown in Fig. 29a, the acid cell free supernatants of 46+88 probiotics strain
combination at 1:1dilution inhibited the growth of E. coli, S typhimurium, S. aureus and B. cereus
by more than 80 %. At higher dilution (1:10, 1:100 & 1:500), cell free supernatants did not show
antimicrobial activity (or it was much less except for S. aureus growth suppressed in all serial
dilution more than 35%). When pH was adjusted to 6, there was no inhibitory effect on S.
typhimurium, E. coli and B. cereus pathogens was detected except S. aureus which growth was
suppressed (Fig. 29b).
Fig. 30 AMA of cell free supernatant of 46+618 co-culture (a) acid supernatants (b) neutralized
supernatants
43
As shown in Fig. 30a, the acid cell free supernatants of 46+618 probiotic strain
combination at 1:1dilution inhibited the growth of E .coli, S. typhimurium, S. aureus and B. cereus
by more than 80 %. S. aureus (77% ) were significantly decreased by incubation with the
supernatants of 46+618 at 1:10 dilution. When pH was adjusted to 6.0, the inhibitory effect of
probiotic strain combination on S. aureus did not decrease significantly (Fig. 30b).
3.6 AMA of partially purified bacteriocins from LAB and Bifidobacteria strains
against enteric pathogens
Ammonium sulphate precipitated crude proteins from two of the 4 candidate probiotic
strains showed strongest antimicrobial activity against target pathogens (Fig. 31 & 32). Partially
purified proteins from L. plantarum and B. lactis at 1:10, 1:100 dilution showed maximum AMA of
92 % against E. coli, S. typhimurium, S. aureus and B. cereus.
Fig. 31 AMA of partially purified proteins from L. plantarum against 4 enteric pathogens
Fig. 32 AMA of partially purified proteins from L. plantarum against 4 enteric pathogens
44
CONCLUSIONS
1. In this study it was observed that AMA of a collection of candidate probiotic strains
(different time course) against 4 enteric pathogens showed inhibition of all 4 target enteric
pathogens was highest with acid supernatants. When the supernatants were neutralized to pH
6.0 there was a decrease suggesting the possible synergistic action of secreted acids and
other proteinecious or non-proteinecious metabolites by the probiotic strains inhibited the
growth of pathogens.
2. Probiotics grown with different prebiotics tested in a microtiter plate assay showed
inhibition of each of the pathogenic strains, S. typhimurium, ESBL, S. aureus and B. cereus.
The extent of inhibition was dependent on the probiotic strain and on the prebiotic source
used.
3. AMA of top probiotics strains cell free supernatant in presence of 5% porcine bile was also
determined since it has been shown that bile stress enhanced AMA of some probiotic
strains. In the present investigation it was observed that bile stress did not abolish the AMA
of LAB supernatants meanwhile AMA against Gram negative pathogens (ESBL and S.
typhimurium) by bifidobacteria cell free supernatant was abolished when grown in presence
of 5% porcine bile.
4. In conclusion
acidic supernatants all of probiotic strain combinations at 1:1 dilution
inhibited the growth of S. typhimurium ESBL, S. aureus and B. cereus by more than 80%
meanwhile neutralized cell free supernatant did not show antimicrobial activity or it was
much less.
5. This study has lead to the identification of 4 potential probiotic strains (2 lactobacilli and 2
bifidobacteria) that can inhibit some important Gram positive or Gram negative
enteropathogenic bacteria and could be potential candidate strains for in vivo studies in
animal models.
6. Ammonium sulphate precipitated crude proteins from L. plantarum and B. lactis showed
highest inhibition against four target enteric pathogens at a lower dilution of 1:10 and 1:100.
No AMA at a higher dilution of 1:1 could be due to the potential protein aggregation.
45
ACKNOWLEDGEMENT
This Master’s thesis could not have been written without Assoc. Prof. Åsa Ljungh, Prof.
Torkel Wadström and Dr. Kanthi Kiran Kondepudi, Prof. Limas Kupčinskas who encouraged
and challenged me through my learning program. They never accepted less than my best efforts.
Thank you.
I would also like to warmly acknowledge Dr. Kanthi Kiran Kondepudi who went out of
his way and invested time on me. Thank you for your help and support in guiding me. Without you,
this work would have taken years of my life.
I would also like to express my gratitude to Ingrid Nilsson, Padma Ambalam, Peren
Karagin, Oksana Poltavska and Praveen Babu for their time and valuable feedback.
In addition, a special thanks to my family and all my friends for their consideration and
motivation and especially to God, for making difficult things possible.
46
REFERENCES
1. Bernet-Camard M-F., Lievin V., Brassart D., Neeser J-R., Servin A-L. and Hudault S.
(1997) The Human Lactobacillus acidophilus Strain LA1 secretes a Nonbacteriocin
Antibacterial Substances(s) Active In Vitro and In Vivo. Environ. Microbiol., 2747-2753.
2. Biavati B and Mattarelli P. (2006) The family Bifidobacteriaceae. Dworkin M., Falkow S.,
Rosenberg E., Scheilfer K.H. and Stackebrandt E (2006) The Prokaryotes. A handbook of
the biolofy of bacteria: Archea. Bacteria: Firmicutes, Acntinomycetes, Third edition, chapter
1.1.2.
3.
Biavati B., Vescovo M., Torriani S., Bottazzi V. (2000) Bifidobacteria: history, ecology,
psychology and applications. Annals of Microbiol., 50, 117-131.
4. Boekhorst J., Siezen R.J., Zwahlen M-C., Vilanova D, Pridmore RD., Mercenier A,
Kleerebezem M, Willem M. de Vos., Bru¨ssow H. and Desiere F. (2004) The complete
genomes of Lactobacillus plantarum and Lactobacillus johnsonii reveal extensive
differences in chromosome organization and gene content.Microbiol., 150, 3601–3611.
5. Bolotin A., Wincker P., Mauger S., Jaillon O., Malarme K., Weissenbach J., Ehrlich S.D.
and Sorokin A (2001) The Complete Genome Sequence of the Lactic Acid Bacterium
Lactococcus lactis ssp. lactis IL1403. 11:731–753 ©2001 by Cold Spring Harbor
Laboratory Press ISSN 1088-9051/01.
6. Brink M., Todorov S.D., Martin J.H., Senekai M. and Dicks L.M.T. (2006) The effect of
prebiotics on production of antimicrobial compounds, resistance to growth at low pH and in
the presence of bile, and adhesion of probiotic cells to intestinal mucus. Microbiol., 100,
813-820.
7. Cai H., Thompson R., Budinich M.F., Broadbent J.R. and Steele J.L (2009) Genome
Sequence and Comparative Genome Analysis of Lactobacillus casei: Insights into Their
Niche-Associated Evolution. Published by Oxford University Genome. Biol. Evol., 1:239–
257.
8. Catherine, S. M and R.D. Rolfe. (2000) In vitro and In vivo Activities of Nitazoxanide
against Clostridium difficile. Antimicrob Agents Chemother., 44: 2254–2258.
9. Chen. C.C and Walker W.A (2005) Probiotics and Prebiotics: Role in Clinical disease
States. a handbook Advance in Pediatric, Volume 52, Chapter 5, 77-113.
10. Claesson M. J., Sinead Leahy Y.L., Canchaya C., Pijkeren J.P., Cerden˜ o-Ta´ rraga A.M.,
Parkhill J., Flynn S., O’Sullivan G.S., Collins J.K., Higgins D., Shanahan F., Fitzgerald
47
G.F., Douwe van Sinderen, and O’Toole P.W (2005) Multireplicon genome architecture of
Lactobacillus salivarius. PNAS vol. 103 no. 17.
11. Claesson M.J., Douwe van Sinderen, and O’Toole P.W (2007) The genus Lactobacillus – a
genomic basis for understanding its diversity. FEMS Microbiol. Lett., 269; 22-28.
12. Coconnier M-H., bernet M-F., Kerneis S., Chauviere G., Fourniat J. And Servin A.L. (1993)
Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by
Lactobacillus acidophilus strain LB decrease bacterial invasion. FEMS Microbiol., 110;
229-306.
13. Coconnier M-H., Lievin V., Lorrot M. and Servin A.L (2000) Antagonistic Activity of
Lactobacillus
acidophilus
LB
against
Intracellular
Salmonella
enterica
Serovar
Typhimurium Infecting Human Enterocyte-Like Caco-s-TC-7 Cells. Environ. Microbiol.,
1152-1157.
14. Connolly E. and Lonnerdal E. (2004) D (-)- Lactic acid-producing bacteria/ Safe to use in
infant formulas. Nutra foods, 3(3) 37-49.
15. Eric Altermann E., Russell M.W.,. Azcarate-Peril M. A., Barrangou R., Buck
L.B.,McAuliffe O., Souther N., Dobson A., Duong T., Callanan M., Sonja L., Hamrick A.,
Cano R and Klaenhammer T. R (2005) Complete genome sequence of the probiotic lactic
acid bacterium Lactobacillus acidophilus NCFM. PNAS vol.102, no.11.
16. Fayo-Messaoudi D., Berger C.n., Coconnier-polter m-H., Lievin-Le Moal V. and Servin
A.L. (2005) pH-, lactic Acid-, and Non-Lactic Acid-Dependent Activities of probiotic
lactobacilli against Salmonella enterica Serovar Typhimurium. Environ. Microbiol., Vol 71.
No 10.
17. Felis G.E. and Dellaglio F. (2007) Taxonomy of Lactobacilli and Bifidobacteria. Online
journal at www.ciim.net. Sep;8(2):44-61.
18. Felis G.E., Dellaglio F.
and Torriani S. (2009) A handbook: Taxonomy of Probiotic
Microorganisms. 591-637, DOI: 10.1007/978-0-387-79058-9_15.
19. Fernandez M.F., Boris S. and Barbes C. (2003) Probiotic properties of human lactobacilli
strains to be used in the gastrointestinal tract. Microbiol., 94, 449-455.
20. Fuller R. (1989) Probiotics in man and animals. Appl. Bacteriol., 66:365–78
21. Fuller R. and Gibson G.R (1997) Modification of the intestinal microflora using probiotics
and prebiotics. Scand. Gastroenterol., 32, 28-31.
22. Gibson G.R., Scott K.P., Rastall R.A., Tuohy K.M., Hotchkiss A., Dubert-Ferrandon A.,
Gareau M., Murphy E-F., Saulnire D., Loh G., Macfarlane S., Delzenne N., Ringel Y.,
Kozianowski G., Dickmann R., Lenoir-Wijnkopp I., Walker C. and Buddington R. (2010)
Dietary prebiotics: current status and new definition. Food Scien. And Tenchnol.,7(1) 1-19.
48
23. Guarner F., Khan G.A., Garisch J., Eliakim R., Gangl A., Thomson A., Krabshuis J., Mair
T.L., Kaufmann P., Paula J.A., Fedorak R., Shanahan F., Sanders M.E., Szajewskaja H.
(2008) Probiotics and prebiotics. World Gastroenterology Organization Practice Guideline.
24. Hammes W.P and Hertel C. Genus Lactobacillus. Beijerinck 1901 // Bergey’s Manual of
Systematic Bacteriology 2edition (2004) Volume 3 (2009) /Paul De Vos, George Garrity,
Dorothy Jones, Noel R. Krieg, Wolfgang Ludwig, Fred A. Rainey, Karl-Heinz Schleifer and
William B. Whitman ISBN 0-387-95041-9.
25. Helander I.M., von Wright A., Mattila-Sandholm T.M. (1997) Potential of lactic acid
bacteria and novel antimicrobials against Gram-negative bacteria. Trends Food Sci. Tech., 8,
146e150.
26. Horie M., Ishiyama A., fujihira-ueki Y., Sillanpää J., korhonen T.K. and Toba T. (2002)
Inhibition of the adherence of Escherichia coli strains to basement membrane by
Lactobacillus crispatus expressing an S-layer. Microbiol., 92, 396-403.
27. Jamuna M. and Jeevaratnam K. (2004) Isolation and partial characterization of bacteriocins
from Pediococcus species. Microbiol. Biotechnol., 65, 443-439.
28. Jime´nez E., Langa S., Martín V., Arroyo R., Martín R., Ferna´ndez L. and Rodríguez J.M
(2010) Complete Genome Sequence of Lactobacillus fermentum CECT 5716, a Probiotic
Strain Isolated from Human Milk. J. Bacteriol., 4800 vol. 192, no. 18.
29. Jin l.Z., Ho Y.W., Abdullah N., Ali M.A. and Jalaludin S. (1996) Antagonistic effects of
intestinal Lactobacillus isolates on pathogens of chicken. Lett. in Appl. Microbiol., vol23,
issue 2, pages 67–71.
30. Johnson-Henry K.C., Hagen K.E., Gordonpour M., Tompkins T.A and Sherman P.M.
(2007) Surface-layer extract from Lactobacillus helveticus inhibit enterohaemorrhagic
Escherichia coli O157:H7 adhesion to epithelial cells. Cellular Microbiol., Vol.9 no.2, 356367.
31. JoMay C., (2002) Probiotics and Prebiotiocs: A Brief Overview. J. Renal Nutr., 12, 76-86.
32. Joshi, V. K., Sharma, S and Rana, N. S (2006) Production, purification, stability and
efficacy of bacteriocin from isolates of natural Lactic acid fermentation of vegetables. Food
Technol. Biotechnol., 44 (3) 435-439.
33. Juarez M.S.T., Ocana V.S., Wiese B and Nader-Macias M.E. (2003) Growth and lactic acid
production by vaginal Lactobacillus acidophilus CRL 1259, and inhibition of uropathogenic
Eschericia coli. Microbiol., 52, 1117-1124.
34. Kim J.F., Jeong H., Yu D.S., Choi S-H., Hur C-G., Park M-S., Yoon S.H., Kim D-W., Ji
G.E., Park H-S. and Oh T.K. (2009) Genome Sequence of the Probiotic Bacterium
Bifidobacterium animalis subs. lactis AD01. J. Bacteriol., doi:10.1128/JB.01515-08.
49
35. Lee J-H. and O’Sullivan D.J. (2010) Genomic Insights into Bifidobacteria. J. American
Society for Microbiology, vol 74, no. 3.
36. Lee Y-K., Puong K-Y., Ouwehand A.C and Salminen S. (2003) Displacement of bacterial
pathogens from mucus and Caco-2 cell surface by lactobacilli. J. Med. Microbiol., 52,925930.
37. Lee. Y-K. and Puong K-Y. (2002) Competition for adhesion between probiotics and human
gastrointestinal pathogens in the presence of carbohydrate. J. Nutr., 88, S101-S108.
38. Lievin V., Peiffer I., Hudault S., Rochat F., Brassart D., Neeser J-R. and Servin A.L. (2000)
Bifidobacterium strains from resident infant human gastrointestinal microflora exert
antimicrobial activity. GUT, 47, 646-652.
39. Lilly D. M. and Stillwell R. H. (1965) Probiotics. Growth promoting factors produced by
micro-organisms. Science, 147:747–8.
40. Linden SK., Sutton P., Karlsson NG., Korolik V. and McGuckin MA (2008). Mucin in the
mucosal barrier to infection Nat. Mucosal Immuno. 1:183-197.
41. Ljungh A. and Wadstrom T. (2009) A handbook – Lactobacillus Molecular biology From
Genomics to Probiotics.
42. Magnusson J., Stro¨m K., Roos S., Sjo¨gren J., Schnu¨rer J. (2003) Broad and complex
antifungal activity among environmental isolates of lactic acid bacteria. FEMS Microbiol.
Lett., 219, 129e135.
43. Mayo B and Sinderen vD. (2010) A handbook : Bifidobacteria/ Genomic and Molecular
Aspects.
44. Medellin-Pena MJ., Wang H., Johnson R., Anand S. and Griffiths MW. ( 2007) Probiotics
effect virulence-related gene expression in Escherichia coli O157:H7. Environ. Microbiol.,
73:4259-267.
45. Miyazaki Y., kamiya S., Hanawa T., Fukuda M., Kawakami H., Takahashi H. and Yokota
H. (2010) Effect of probiotic bacterial strains of Lactobacillus, Bifidobacetrium, and
Enterococcus on enteroaggregative Escherichia coli. J. Infect. Chemother. 16, 10-18.
46. Morita H., Toh H., Fukuda S., Horikawa H.,
Oshima K., Suzuki T., Murakami M.,
Hisamatsu S., KatoY., Takizawal T., Fukuoka1 H., Yoshimura T., Itoh K., O’sullivan D.J.,
Mckay L.L., Ohno H., Kikuchi J., Masaoka T. and Hattori M. (2008) Comparative Genome
Analysis of Lactobacillus reuteri and Lactobacillus fermentum Reveal a Genomic Island for
Reuterin and Cobalamin Production.DNA RESEARCH, vol.15, 151–161.
47. Nesser J´-R., Granato D., Rouvet M., Servin A., Teneberg S. and Karlsson K-A. (2000)
Lactobacillus jonhsonni La1 shares carbohydrate-binding specificities with several
enteropathogenic bacteria. Glycobiol., vol 10. No.10. 1196-1199.
50
48. Ng S.C., Hart A.L., Kamm M.A., Stagg A.J and Knight S.C ( 2009) Mechanism of action of
Probiotics: Recent Advances. Inflamn Bowel Dis., vol. 15. No.2.
49. Ogawa M., Shimizu K., nomoto K., tanaka R., Hamabata T., Yamasaki S. , Takeda T. and
Takeda Y. (2001) Inhibition of in vitro growth of Shiga toxin-producing Escherichia coli
O157-H7 probiotic Lactobacillus strains due to production of lactic acid. Food Microbiol.,
68, 135-140.
50. Orla-Jensen S. (1919) The Lactic Acid Bacteria. , Fred Host and Son, Copenhagen
51. Parker R. B. (1974) Probiotics, the other half of the antibiotic story. Anim. Nutr. Health,
29:4–8.
52. Quegley E.M.M and Quera R. (2006) Small Intestinal bacterial Overgrowth: roles of
Antibiotics. Prebiotics, and Probiotics. Gastroenterol., 130:S78-S90.
53. Quigley E.M.M. (2010) Prebiotics and probiotics; modifying and mining the microbiota.
Pharma. Research, 213-218.
54. Rastall R.A., Gibson G.R., Gill.S.H., Guarner F., Klaenhammer T.R., Pot B., Reid G.,
Rowland I.R. and Sanders M.E (2005) Modulation of the microbial ecology of the human
colon by probiotics, prebiotics and synbiotics to enhance human health: An overview of
enabling science and potential applications. FEMS Mircobiol. Ecol., 52; 145-152.
55. Reddy G., Altaf Md., Naveena B.J., Venkateshwar M. and Vijay Kumar E. (2008)
Amylolytic bacterial lactic acid fermentation — A review. Biotech. Advanc., 26; 22–34.
56. Reid G., Sanders
M.E., Gaskins
H.R., Gibson
G.R., Mercenier
A., Rastall R.A.,
Roberfroid M.B., Rowland I., Cherbut C. and Klaenhammer T.R. (2003) New scientific
paradigms for probiotics and prebiotics. J. Clin. Gastroenterol., 37, 10-118.
57. Salvatore S. and Vandeplas Y. (2010) Handbook: Bioactive foods in Promoting health.
Probiotics and Prebiotics. Chapter 13. 656 pages.
58. Sanders, M.E. (1993) Effect of consumption of lactic cultures on human health. Food.Nutr.
Res., 37, 67-130.
59. Santos A., Mauro M.S., Sanchez A., torres J.M. and Marquina D. (2003) The antimicrobial
properties of Different Strains of lactobacillus spp. Isolated from Kefir. Microbiol., 26, 434437.
60. Saulnire D.MA., Spinler J.K., Gibso R.G.and Versalovic J. (2009) Mechanism of probiosis
and prebiosis: considerations for enhanced functional foods. Curr. Opinion in Biotech.,
20:135-141.
61. Servin
A.L. (2004) Antagonistic activities of lactobacilli and bifidobacteria against
microbial pathogens. FEMS Microbiol. Rev., 28, 405e440.
51
62. Sgouras D., Maragkoudakis P., Petraki K., Martinez- Gonzalez B., Eriotou E., Michpoulus
S., Kalatzpoulus G., Tsakalidou E. and Mentis A. (2004) In vitro and in vivo inhibition of
Helycobacter pylori by Lactobacillus casei strain Shirota. Envirom. Microbiol. 70, 518-526.
63. Sherman P.H., Ossa J.C. and Johnson-Jenry K. (2009) Unraveling Mechanisms of Action of
Probiotics. Nutrn.in Clinic. Pract., Vol12; no.1.
64. Sperti G. S. (1971) Probiotics. West Point, CT: Avi Publishing Co.
65. Stiles M.E. and Holzapfel W.H. (1996) Lactic acid bacteria of foods and their current
taxonomy. Food Microbiol., 36, 1-29.
66. Suskovic J., Kos B., beganovic J., Pavunc A.L., Habjanic K.H and Matosic S. (2010)
Antimicrobial activity- The most important property of probiotic and starter lactic acid
bacteria. Food Technol. Biotechnol., 48 (3) 296-307.
67. Thomas. Dan W., Greer F.R. and committee on nutrition: section of gastroenterology,
hepatology, and nutrition (2010) Clinical report – Probiotics and Prebiotics in Pediatrics. J.
Pediatrics vol.126; no. 6.
68. Tuomola E.M., Ouwehand A.C and Salminen S. (1999). The effect of probiotic bacteria on
the adhesion of pathogens to human intestinal mucus. Immuno and Med. Microbiol.,
26,137-142.
69. Turoni F., Sinderen D and Ventura M. (2011) Genomic and ecological overview of genus
Bifidobacterium. Food Microbiol., doi:10.1016/j.ijfoodmicro.
70. Valerio F., Lavermicocca P., Pascale M., Visconti A. (2004) Production of phenyl lactic
acid by lactic acid bacteria: an approach to the selection of strains contributing to food
quality and preservation. FEMS Microbiol. Lett., 233, 289e295.
71. Ventura M., Connell-O’Mortherway M., Leahy S., Moreno-Munoz J.A., Fitzgerald G.F. and
Sinderen D. (2007) From bacterial genome to functionality; case bifidobacteria. Food
Microbiol., 120; 2-12.
72. Ventura M., Sinderen D., Fitzgerald G.F. and Zink R. (2004) Insight into the taxonomy,
genetic and physiology of bifidobacteria. Antonie van Leeuwenhoek, 86:205-223.
73. Walker W.A and Duffy L.C. (1998) Diet and bacterial colonization: Role of probiotics and
prebiotics. Nutr. Biochem., 9:668-675.
74. Walker W.A. (2008) Mechanism of Action of Probiotics. J. CID Doi:10.1086/523335.
75. Watanabe T., Nishio H., Tanigawa T., Yamagami H., Okazaki H.,Watanabe K., Tominaga
K., Fujiwara Y., Oshitani N., Asahara T., Nomoto K., Higuchi K., Takeuchi K.
and
Arakawa1 T. (2009) Probiotic Lactobacillus casei strain Shirota prevents indomethacininduced small intestinal injury: involvement of lactic acid. Physiol. Gastrointest. Liver
Physiol., 297: G506–G513.
52
76. Wult M., Johansson-Hagslatt M-L. And Odenholt I. (2003) Lactobacillus plantarum 299 v
for a treatment of reccurent Clostridium difficile-associated diarohea: A double-blind,
placebo –controlled trail. Infect Dis. 35, 365-367.
53